‘I‘HE .JOURNAL OF BloLoClCAL C:HEMISTRY Vol. 2.51. No. 4, Issue of February ‘5. pp. 950-961. 1976

Printed in 1J.S.A.

A Major Polypeptide Component of Carbamyl Phosphate Synthetase”

.t Liver Mitochondria:

(Received for publication, May 9, 1975)

STEVEN CLARKE+

From the Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138

One of the major components of rat liver mitochondria detected by gel electrophoresis in sodium dodecyl sulfate is a 165,000 molecular weight polypeptide that makes up 15 to 20% of the total mitochondrial protein. This component appears to be a single molecular species. Evidence is presented here for the identification of this protein with the poiypeptide chain of a urea cycle enzyme, carbamoylphosphate synthetase I (EC 2.7.2.5).

The 165,000 molecular weight polypeptide was solubilized from mitochondria with Triton X-100 and purified to 90% homogeneity by DEAE-cellulose chromatography. This component co-migrated with

carbamyl phosphate synthetase activity when mitochondrial proteins were separated by gel filtration or sucrose gradient centrifugation. The identification of the 165,000 molecular weight polypeptide with this activity was also supported by the presence or absence of this protein in a variety of rat tissue mitochondria, in liver and kidney mitochondria from various ureotelic and nonureotelic species, and in fetal rat liver mitochondria.

This protein is contained completely within the inner mitochondrial membrane. When isolated in Triton X-100, it has a calculated molecular weight of 191,000 and exists preferentially as a monomer in solution, although some evidence is presented for monomer-dimer equilibrium. The polypeptide chain molecular weight of 165,000, determined by gel electrophoresis in dodecyl sulfate, is confirmed by gel filtration in 6 M guanidinium chloride and in 0.2% dodecyl sulfate. The chemical composition of this protein has also been determined.

Rat liver mitochondria contain a high molecular weight polypeptide chain in large amounts which migrates as a single component at a molecular weight of 165,000 on dodecyl sulfate gel electrophoresis (1). This component is absent from rat kidney mitochondria and therefore presumably has no role in electron transport, CaZ+ transport, or other functions common to these mitochondria. The distribution of this polypeptide in kidney and other types of mitochondria suggested that the protein was involved in the urea cycle, which involves both cytoplasmic and mitochondrial proteins for nitrogen excretion in ureotelic species (2, 3). Evidence presented here shows that

the 165,000 molecular weight component copurifies with one of these proteins, N-acetyl glutamate-dependent carbamyl phos- phate synthetase, and is present in various types of mitochon- dria in amounts comparable with the enzyme activity. This enzyme, described in the mitochondria of various species, should be distinguished from the extramitochondrial carbamyl phosphate synthetases, which generally do not require N- acetyl glutamate as a cofactor, and can use glutamine as a nitrogen source (see Ref. 4 for a review; cf. also Ref. 5).

Several purifications &mammalian mitochondrial carbamyl

*This research was supported by Grants GB 36827 from the National Science Foundation and by Grant HL-08893 from the National Institutes of Health to Guido Guidotti, Harvard [Jniversity.

$ Predoctoral Fellow of the National Science Foundation.

phosphate synthetase have been reported and the low fold

purification generally obtained suggests that a ,major polypep- tide may be responsible for this activity (6-8). In the present

study, the assignment of this activity to the mitochondrial polypeptide component with a molecular weight of 165,000 has allowed a reinvestigation of the native molecular weight and the subunit structure of the enzyme. In addition, chemical studies to characterize the polypeptide have been performed, including experiments designed to determine if the protein is modified by glycosylation or phosphorylation.

This protein is not only important in terms of its enzymatic

function in nitrogen excretion, but because it is one of the major protein components of the mitochondrion, both in terms of mass and the number of molecules, it provides a suitable example to study the biosynthesis, transport, and membrane interaction of a mitochondrial protein. As a first step in this study, the localization of this major component has been reinvestigated to determine whether the protein is contained completely within the inner membrane or if some of its residues are exposed on the outside of the inner membrane, as previ-

ously suggested (1).

EXPERIMENTAL PROCEDURES

Materials-Triton WR-1339 was obtained from Ruger Chemical Co., Irvington, N.J.; Triton X-100 was purchased from Sigma. Purified Tris base (Trizma) and sodium dodecyl sulfate (95%) were Sigma

950

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Carbamyl Phosphate Synthetase from Rat Liver Mitochondria 951

products, while “ultrapure” guanidinium chloride was obtained from H&co. Acrylamide, NJ-methylenebisacrylamide, mercaptoethanol, mercaptoacetic acid, and N,N,N’,N’-tetramethyleneamine were East- man products. [SzP]Orthophosphate was obtained from New England Nuclear and dimethylsuberimidate was a gift of G. Guidotti. Sodium tetrathionate (K&K Laboratories) was stored desiccated at 4”, while imidazole (Eastman) was recrystallized from 95% ethanol.

Chromatography was performed on Bio-Gel A-1.5m, a Bio-Rad product, Sepharose 4B (Pharmacia), and on the DE52 brand of DEAE-cellulose (Whatman). Escherichia coli RNA polymerase was a gift of K. Weber, and bovine lactoperoxidase was purchased from Sigma. The source of the other proteins for column and gel calibration was described previously (9).

Preparation of Rat Liver Mitochondria and Mitoplasts-Mito- chondria were prepared by differential centrifugation from 200. to 500-g albino rats (Charles River, CD strain) that had been fasted overnight. Excised livers were minced and homogenized in 2-g portrons with 20 ml of ice-cold medium (280 rn~ sucrose/4 rn~ Tris/0.12 rn~ EGTAIO.1’1 bovine serum albumin (Sigma, Fraction V), adjusted to pH 7.6 with H,SO,). In some preparations the albumin was replaced by 1 rn~ sodium tetrathionate. Homogenization was carried out in 50.ml centrifuge tubes (inside diameter, 2.65 cm) with two up and down strokes of a motor-driven Teflon pestle (outside diameter 2.54 cm). The homogenate was centrifuged at 700 x g for 10 min. and the supernatant was then spun at 14,500 x g for 12 min. The supernatant and fluffy layer from this step were discarded, along with the white material adhering to the sides of the centrifuge tube. The pellet was resuspended (using the “cold finger” technique recommended by Chappell and Hansford (10)) in fresh medium and centrifuged 14,500 x g for 12 min. The final mitochondrial pellet was resuspended as before in medium to give a protein concentration of approximately 100 mg/ml. When mitochondrial preparations were to be analyzed by dodecyl sulfate gel electrophoresis directly, the serum albumin was removed by two additional centrifugation cycles in medium free of this protein. All centrifugations were performed in the SS 34 rotor of a Sorvall RCB-B centrifuge at 4’.

This preparation contains mostly mitochondria, but also significant amounts of lysosomes, peroxisomes, and microsomes. To eliminate these contaminants, along with the outer mitochondrial membrane and the contents of the intermembrane space, the digitonin fractiona- tion procedure of Schnaitman and Greenawalt was employed to obtain a mitoplast fraction of inner membrane/matrix vesicles (11). A rapid protein assay was devised for this procedure based on the absorbance at 280 and 310 nm of solubilized mitochondria in 50 rn~ Tris .H,SO,, pH 8/0.2% sodium dodecyl sulfate. The net A,,, “~ (sample minus buffer blank) was subtracted from the net A,,, “~ and divided by 1.05 to give values for protein concentration (in milligrams/ml) equivalent to those obtained by the Lowry or alkaline ninhydrin method. Digitonin, obtained from Calbiochem or Sigma, was recrystallized from absolute ethanol and was dissolved in the isolation medium (minus albumin) to a concentration of 20 mg/ml by gentle warming over a boiling water bath. The fractionation was performed exactly as described (ll), generally using 0.15 mg of digitonimmg of mitochon- drial protein. The final mitoplast pellet was resuspended in medium free of albumin to a protein concentration of 10 to 15 mg/ml. The yteld of protein during this fractionation was between 55 and 65%. En- zymatic assay for the outer membrane marker monoamine oxidase indicated that less than 5% of the original activity remained in the mitoplast fractions. This assay was done routinely by measuring the increase in A,,, “~ in a 0.02% solution of benzylamine (Eastman) in 0.04% Triton X-100 and 50 rn~ Tris.H,SO,, pH 7.6, after the procedure of Tabor et al. (12).

Preparation of Mitochondria from Other Tissues-Livers and kid- neys were obtained from freshly slaughtered domestic chickens, albino rabbits, calves, and dogs. Mature Xenopus laeuis (South African clawed frog) were a generous gift of Dennis Hourcade (Harvard University). Live specimens of Opsanus tan (toadfish), Anguilla rostrata (American eel), and Fundulus heteroclitus (mummichog) were obtained from the Marine Biological Laboratory (Woods Hole, Mass.).

Liver and kidney mitochondria were prepared from the species above by the same procedure used for rat liver mitochondria, except that the mitochondria were washed an additional time in medium containing serum albumin and then were washed free of the albumin in two more resuspension-centrifugation cycles. The preparation of Xenopus liver mitochondria required two additional low speed (700 x g) centrifugations after the initial pelleting of the mitochondria to remove the large amount of pigment granules.

Skeletal muscle mitochondria were prepared from the leg muscula- ture of the rat after the procedure of Ernster and Nordenbrand (13). Homogenization was performed as described for rat liver in the KCl/MgSO,/ATP/EDTA medium made up to 1 rn~ in sodium tetrathionate. Rat heart mitochondria were prepared essentially by the method of Holton et al. (14) in 6.21 M sucrose/l0 rnrvt EDTA/l rn~ imidazole/l mM tetrathionate, adjusted to pH 7.5 with KOH. A crude preparation of rat brain mitochondria was made as described by Lovtrup and Zealander (15) in a medium of 0.44 M sucrose/l rn~ imidazole/l rn~ tetrathionate, adjusted to pH 7.5 with H,SO,.

Fetal livers were obtained from the litter of timed pregnant rats (Charles River, CD strain) and were pooled for homogenization and the preparation of mitochondrial fractions. Because of the small amount of tissue obtained from the 16. and l&day fetuses. the homogenization was done by hand in a glass tube (0.8 (inside diameter) x 7.5 cm) with a Teflon pestle. The time of centrifugation was adjusted for the shorter path lengths in these samples, but otherwise the preparation was identical with adult rat liver mitochondria. The age of the fetuses was calculated from the day on which the mothers were bred.

Polyacrylamide Gel Electrophoresis m Sodium Dodecyl Sul- fate-The borate buffer system of Davies and Stark was used (16), with gels containing 7.5’/, acrylamide, 0.1% methylenebisacrylamide, and 0.1% dodecyl sulfate unless otherwise stated. Samples were prepared for electrophoresis by incubation in borate buffered 1°C dodecyl sulfate/30 rn~ P-mercaptoethanol for 3 min at 100’ (9). Gels were stained with Coomassie brilliant blue, destained by diffusion in 40°C methanol, 7.5L70 acetic acid, and spectrophotometrically scanned as described (9). Polypeptide mobilities were calculated relative to the tracker dye, bromphenol blue (3’,3”,5’,5’‘-tetrabromophenolsulfone- phthalein).

Assay of Carbamyl Phosphate Synthetase and Marker Entymes- Enzyme acttvity was measured either by the formation of citrulline in a coupled assay mixture including ornithine and ornithine transcar- bamylase, or by the N-acetyl glutamate dependent production of ADP. The latter assay was performed exactly as described by Elliott and Tipton (8). Two variants of the first procedure were used. One of these involved the calorimetric determination of citrulline after the proce- dure of Brown and Cohen (l’i), but could not be used in samples containrng sucrose. A more useful assay was a modification of the protocol of Kerson and Appel (18) where the incorporation of [l’C]bi- carbonate into citrulline was measured by the nonvolatile radioactivity remaining after an acid treatment. Samples were incubated at 37” in a total volume of 200 ~1 in 5 rn~ Na,ATP, 10 mM MgSO,, 50 mM ammonium acetate, 20 mu [“C]sodium bicarbonate (100 cpm/nmol), 3 rn~ ornithine, 6.5 rn~ N-acetyl glutamate, 20 mM glycylglycine. 10 mM P-mercaptoethanol, and 0.8 fig/ml of omithine transcarbamylase (specific activity, 625 ~mol/min/mg) at pH 7.5. The reaction was quenched after 10 to 30 min with 100 ~1 of 30% trichloroacetic acid, and 25 ~1 of concentrated HCl were added. The open samples were heated over a hot plate 15 min, and 3.5 ml of Aquasol (New England Nuclear) were added after cooling. Ornithine transcarbamylase from Strepto- coccus faecalis was obtained from Sigma, [“Clbicarbonate from ICN. It was convenient to perform all of the operations in 4.ml glass counting vials.

Protein was quantitated by the alkaline ninhydrin method described previously (9). A unit of enzymatic acttvity is defined as the amount of enzyme causing the formation of 1 pm01 of citrulline (or 2 mnol of ADP)/min at 37”, pH 7.5.

Catalase, fumarase, and malate dehydrogenase were assayed as described (9). Ornithine transcarbamylase was determined by the method of Marshall and Cohen (19), except that the dilithium salt of carbamyl phosphate (Sigma) was used. Glutamate dehydrogenase was assayed spectrophotometrically after Strecker (20), while citrate syn- thase was assayed by the calorimetric determinatron of coenzyme A as described by Srere (21).

Amino Acid and Carbohydrate Composition-Amino acid analysis was performed on a high sensitivity Beckman model 120 C analyzer by the method of Spackman et al. (22) of samples hydrolyzed in uecuo in 6 M HCl/O.l% mercaptoacetic acid at 106 to 108”. Tryptophan was determined by the method of Edelhoch (23) on the purified 165,090 molecular weight polypeptide in 6 M guanidinium chloride using a Cary 15 spectrophotometer. Protein was quantitated by amino acid analysis. Cysteine and methionine were determined as cysteic acid and methio- nine sulfone after performic acid oxidation (24) of the polypeptide purified by dodecyl sulfate gel filtration, freed of detergent, and dialyzed into 5% formic acid. Color constants for cysteic acid and methionine sulfone were calculated as 1.005 and 0.991 times the color

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952 Carbamyl Phosphate Synthetase from Rat Liver Mitochondria

constant of aspartic acid.’ Cysteine was also determined as carboxy- methylcysteine, with a color constant 0.991 times that of aspartic acid and corrected for 90% recovery after hydrolysis2 Total carboxylic acids were determined by the reaction of the purified polypeptide in 6 M guanidinium chloride with 1 M glycine methyl ester and 0.1 M 1-ethyl-3-dimethylaminopropylcarbodiimide.HCl (Ott Chemical, Muskegon, Mich.) for 2 hours at pH 4.75 after the procedure of Hoare and Koshland (25). Controls were performed without carbodiimide present. Samples were dialyzed exhaustively and hydrolyzed for amino acid analysis, and the amount of excess glycine above the control without carbodiimide was taken as the amount of free carboxylic acid.

Amino sugars were determined after 6. and 24.hour hydrolysis in 6 M

HCl on the 15.cm analyzer column with pH 4.25 citrate buffer according to Waxman (26). Neutral sugar was analyzed on trichloro- acetic acid precipitates of the 165,000 molecular weight polypeptide from dodecyl sulfate gel filtration columns using the method of Dubois et al. (27) with human serum transferrin as a standard (neutral sugar content assumed to be 2.5% (28)).

RESULTS

Purification of 165,000 Molecular Weight Component. Comi- gration with Carbamyl Phosphate Synthetase-The 165,000 molecular weight polypeptide was extracted from mitochon- drial inner membrane/matrix preparations (mitoplasts) by incubation with the nonionic detergent Triton X-100. Mito- plasts were suspended at a protein concentration of 5 to 10 mg/ml in the starting buffer used for DEAE-cellulose chroma- tography which contained 0.5 to 1% Triton X-100. After 30 min on ice, the preparation was spun at 60,000 rpm for 15 min/cm path length in the A321 rotor of an IEC B60 ultracentrifuge to remove the membrane fragments. The supernatant was frac- tionated by DEAE-cellulose chromatography as shown in Fig. 1. The 165,000 molecular weight polypeptide eluted in the gradient at an approximate ionic strength of 0.05 and was contaminated with small amounts of lower molecular weight polypeptides. The measured activity of carbamyl phosphate synthetase paralleled the amount of the 165,000 molecular weight polypeptide across the column and suggested that this component was in fact the polypeptide chain of this enzyme. When the DEAE-cellulose chromatography was performed as in Fig. 1, but using imidazole buffers at a slightly higher pH, samples of the 165,000 molecular weight polypeptide of greater purity (and lower yield) could be obtained. Fig. 2 shows a densitometric scan of a gel of such a purified fraction as well as a scan of a gel containing the polypeptides of entire mitochon- drial inner membrane/matrix complex. The specific activity of carbamyl phosphate synthetase purified by DEAE-cellulose chromatography in this way ranges from 0.5 to 1.3 hmol of citrulline formed/min/mg of protein at 37”, which represents an approximate 4- to 6-fold purification over the initial Triton X-100 extract.

Additional steps of purification were attempted to remove the lower molecular weight contaminants of the 165,000 molecular weight component seen on dodecyl sulfate gel electrophoresis of the purified enzyme (Fig. 2). However, the residual proteolytic activity of this preparation (see “Discus- sion”) effectively prevented such further purification because the build-up of proteolytic products of the large polypeptide obscured the removal of the small amounts of contaminants. Repeated DEAE-cellulose chromatography, hydroxylapatite chromatography, gel filtration, and reverse ammonium sulfate chromatography (29) in each case led to a decrease in the specific activity of the enzyme and the appearance of lower molecular weight polypeptides coupled to the loss of material

’ G. Guidotti, personal communication. z L. Waxman, personal communication.

at 165,000 molecular weight on dodecyl sulfate gels. The first intermediate in this degradation is a 155,000 dalton compo- nent, followed by the rapid accumulation of material at a molecular weight of 50,000 on dodecyl sulfate gels. All of these newly formed polypeptides copuri’fied with the 165,000 molecu- lar weight component, suggesting that the small proteolytic fragments are retained in the native structure of the protein.

Because the 165,000 molecular weight component (carbamyl phosphate synthetase) could not be purified to homogeneity by these methods (using the criterion of one polypeptide band on dodecyl sulfate gel electrophoresis), it was important to dem- onstrate that the enzymatic activity and this polypeptide behaved as a single component in other separation systems. Fig. 3 shows the fractionation of Triton-extracted mitoplast proteins by gel filtration. Aliquots were taken for carbamyl phosphate synthetase assay and for dodecyl sulfate electropho- resis. The distribution of the 165,000 molecular weight poly- peptide and the activity was identical. Fig. 4 shows the separation of mitoplast proteins on the basis of their sedimen- tation coefficient in 10 to 30% glycerol gradients. Again, the distribution of the polypeptide and the enzymatic activity was identical. An attempt was made to show that the apparent sedimentation coefficient of both the enzymatic activity and the polypeptide decreased in the presence of N-acetyl gluta- mate (Fig. 4B) based on the observation of Guthohrlein and Knappe (6) that the enzyme disaggregates in high concentra- tions of the cofactor. However, little or no change in the sedimentation rate of either the activity or the polypeptide was found (see below).

Correlation of Presence of 165,000 Molecular Weight Poly- peptide with Carbamyl Phosphate Synthetase in Other Tissues-In general, carbamyl phosphate synthetase I (am- monia, N-acetyl glutamate-dependent) is found only in the liver mitochondria of ureotelic species (although its presence in the small intestine has been documented in the rat (30)). The specific cofactor for the reaction, N-acetyl glutamate, is found only in liver (30 nmol/g of tissue) and in small intestine (10 nmol/g of tissue) (31), and the absence of this compound in other tissues suggests that the failure to measure carbamyl phosphate synthetase activity in these tissues is not due to endogenous inhibition.3 If the 165,000 molecular weight com- ponent is the polypeptide of carbamyl phosphate synthetase, then this polypeptide should be present on dodecyl sulfate gels of liver mitochondria from all ureotelic species, and absent from nonureotelic species. In addition, the polypeptide should be absent from nonhepatic tissues of ureotelic species. That this is in fact the case is shown below.

Fig. 5 shows the results of dodecyl sulfate gel electrophoresis of mitochondria from two urea-excreting species, rat and rabbit, and from one uric acid-excreting species, the domestic chicken. Large amounts of the 165,000 molecular weight polypeptide were demonstrated in rat and rabbit liver mito- chondria, while no significant amount of this component was detected in chicken liver mitochondria or in any of the kidney mitochondrial fractions. This large polypeptide was also ab- sent from rat heart and skeletal muscle mitochondria, and was present at most in minute amounts in brain mitochondria. Fig. 6 shows dodecyl sulfate gels of liver mitochondria from more diverse species. The distribution of the 165,000 molecular

sLow levels of carbamyl phosphate synthetase activity have been reported from kidney, salivary gland, brain, and pancreas (32). It is not clear whether this activity is mitochondrial or represents cytoplasmic enzyme activity.

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Carbamyl Phosphate Synthetase from Rat Liver Mitochondria

5 IO 15 20 25 30 35 40

953

Fraction No. FIG. 1. Purification of carbamyl phosphate synthetase from rat liver

mitochondria by DEAE-cellulose chromatography; coelution with the 165,000 molecular weight polypeptide. A Triton X-100 extract of mitoplasts (40 mg of protein) in 10 rnMO Tris.acetate/lO% glyc- erol/0.5% Triton X-100, pH 7.59, was applied to a column (1.1 x 20 cm) of DEAE-cellulose previously equilibrated in the same buffer 0.05% in Triton X-100. After the unbound proteins were washed through (Fractions 1 to 5), a linear gradient of 280 ml of 0 to 0.15 M

sodium acetate in this buffer was applied. At Fraction 38, the column was washed with a saturated solution of sodium acetate. Fractions (7 ml) were collected at 4” and aliquots were assayed for carbamyl phosphate synthetase activity by the [“CJbicarbonate method

weight polypeptide in these species (present in Xenopus, toadfish, dog, and calf, and absent in the American eel and the mummichog) is in accord with the presence or absence of carbamyl phosphate synthetase activity (3).’ It is of interest

that, although teleosts in general do not have urea cycle activity, the toadfish is exceptional (3).

Fig. 7 shows the development of the 165,000 molecular weight component in fetal rat liver. Since it has been shown that the synthesis of a given liver enzyme in the developing animal is not a gradual process, but occurs over a rather short,

specific period of time, and because functionally related groups of enzymes tend to appear in “clusters,” analysis of the time course of the appearance in mitochondria of a polypeptide can provide information on its possible function (34). It has been well documented (32,35,36) that the enzymes of the urea cycle develop in rat liver in the 3 to 4 days before birth. Densitomet- ric scans of the dodecyl sulfate gels in Fig. 7 indicate that the 165,000 molecular weight polypeptide also appears in mito-

chondria in this period. This component makes up 0.5% of the total protein in the 16-day and B-day fetus, but is present at near adult levels (12% of mitochondrial protein; adult level is

‘The low relative activity of carbamyl phosphate synthetase in Xenopus (3) may be related to the lability of this activity and the fact that this organism can also excrete nitrogen as ammonia (33).

(O-O) and protein by the alkaline ninhydrin procedure (O-O). Samples were also taken for polyacrylamide gel electrophoresis in dodecyl sulfate as described under “Experimental Procedures” (insets). The amount of the 165,000 molecular weight component in each fraction (A--A) was calculated from the amount of stained material (integrated A,,, ,,,,, of Coomassie in the gel band marked) and is plotted on an arbitrary scale. A dodecyl sulfate gel of the polypep- tides present in the material applied to the column is shown on the left. The specific activity of the enzyme in the peak fractions (where the 165,000 molecular weight polypeptide makes up 80 to 85% of the total protein) is 1.14 units/mg of protein; the activity of the initial Triton X-100 extract is 0.27 units/mg.

14 to 18%) by the time of birth, correlating well with the appearance of carbamyl phosphate synthetase activity.”

Is 165,000 Molecular Weight Polypeptide Involved in Other Urea Cycle Activities?-All of the data presented on the development of the large component in fetal rat liver mito- chondria, and its distribution in the tissues of various species are also consistent with the hypothesis that the polypeptide has some role in urea biosynthesis other than that of the synthesis of carbamyl phosphate. There are at least three other activities associated with urea production in mitochondria, ornithine transcarbamylase, ornithine transport, and citrulline transport (37). Ornithine transcarbamylase activity is clearly not associated with the 165,000 molecular weight protein; the peak of activity is well separated from the large polypeptide on gel filtration (see Fig. 3 and Ref. B), and by sucrose gradient centrifugation (data not shown). In all of these respects, the rat liver ornithine transcarbamylase activity chromatographed as has been shown for the bovine liver enzyme by Marshall and

Cohen (19), who proposed a trimeric structure of 38,000

“There is, however, some difficulty in making exact correlation of the activity (units/g wet weight of liver) and the amount of 165,000 molecular weight polypeptide (per cent of total protein) because the ratio of parenchymal to non-parenchymal cells changes rapidly in the developing liver, and because the newly synthesized enzyme may be in an inactive form (34).

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954 Carbamyl Phosphate Synthetase from Rat Liver Mitochondria

Tracker Dye

FIG. 2. Polypeptide composition of purified carbamyl phosphate synthetase and mitochondrial inner membrane/matrix (mitoplast). Densitometric traces of Coomassie-stained dodecyl sulfate gels of the purified enzyme (A) and mitoplasts (B) are shown. The enzyme was purified as in Fig. 1 but m a buffer system of imidazole.H,SO,, pH 7.65, with gradient elution in sodium sulfate and subjected to electrophoresis as described under “Experimental Procedures.” The 165,000 molecular weight polypeptide makes up 89% of the Coomassie- stained material of this preparation, and minor impurities at 55,000, 47,000, 36,000, and 28,000 molecular weight make up the other 11%. The lower trace shows the polypeptide composition of the inner membrane/matrix complex; the 165,000 molecular weight polypeptide accounts for 15.9% of the total stain on the gel. The position of the marker proteins is shown in B: a, myosin (220,000); b, P-galactosidase (135,000); c, serum albumin (68,000); d, fumarase (48,500); e, carbonic anhydrase (30,000); and f, cytochrome c (11,500).

FIG. 3. Co-elution of carbamyl phosphate synthetase and the 165,000 molecular weight polypeptide during gel filtration chromatog- raphy. A Triton X-100 extract of rat liver mitoplasts, containing 10 mg of protein, was prepared as m Fig. 1 but m a buffer containing 10 rnM Tris H,SO,, 100 mM Na,SO,, 10% glycerol, and 2% Triton X-100 at pH 7.5. This sample was mixed with blue dextran 2000 (Pharmacia) and P-mercaptoethanol (B-ME), applied to a column (2 x 72 cm) of Bio-Gel A-1.5m (8% agarose), and eluted in the same buffer containing 0.05% Triton X-100 at 4”. Thirty-minute fractions containmg 2.5 ml were collected and analyzed for enzymatic activity and the 165,000 molecular weight polypeptide as in Fig. 1. The void volume of the column was marked by the A,,, nm from the blue dextran and the total volume by the A,,, nm after the addition of 5,5’-dithiobis-(2-nitroben- zoic acid). Column fractions were also analyzed for glutamate dehydro- genase (a), catalase (b), fumarase (c), and malate dehydrogenase (d) activity, and the peak of each activity is shown by an arrow. ‘I’he elution position of the peak of ormthine transcarbamylase activity (OTC) is also indicated.

60

0 10 12 14 16

- ti Fractm No.

i P o)--

3

b E 3 - P

r"

FIG. 4. Co-sedimentation of the 165,000 molecular weight polypep- tide and carbamyl phosphate synthetase activity in 10 to 30% glycerol gradients. In A, 150 ~1 of a dialyzed Triton X-100 extract of mitoplasts prepared as in Fig. 3 were layered over a 4.ml gradient of glycerol in 10 mM Tris .H,SO,/lOO mM Na,S0,/0.05% Triton X-100, pH 7.5, and centrifuged for 7 hours at 60,000 rpm in the SB 405 rotor of the IEC B-60 ultracentrifuge at 2’. Twenty-three fractions were collected from the bottom of the tube and aliquots were taken for enzymatic assays and were subjected to electrophoresis on dodecyl sulfate gels. The amount of the 165,000 molecular weight polypeptide was determined from quantitative scans of these gels as in Fig. 1. Carbamyl phosphate synthetase was assayed by the spectrophotometric technique. .In B, the procedure was exactly as in A, but the sample and gradient included 10 mM N-acetyl glutamate. Twenty-four fractions were collected and assayed for the large component as above and for enzyme activity by the [“Clbicarbonate technique (values shown were multiplied by a factor of 2.5). Marker proteins include: a, catalase (11.3 S); b, fumarase (9.1 S); c, citrate synthase (6.1 S); and d, malate dehydro- genase (4.3 S).

molecular weight polypeptide chains for the latter enzyme. Citrulline transport has been described in rat liver mitochon-

dria by Gamble and Lehninger (37). Transport of this com- pound and of a variety of other neutral amino acids was found in liver but not heart mitochondria. I have been able to show

that the 165,000 molecular weight polypeptide is probably not involved in this transport because a similar system for the transport of citrulline and other neutral amino acids was shown to occur in rat kidney mitochondria where the large polypep- tide is not found. These data are given in Table I, where the permeability of each of the amino acids is measured by light scattering changes when mitochondria are incubated in iso- tonic solutions of the amino acids (37). Because kidney mitochondria transport citrulline as well as liver mitochondria, the efflux of citrulline during urea synthesis may involve a general neutral amino acid transport system present in mito- chondria and not a specific citrulline carrier. Further evidence against a transport role for the 165,000 molecular weight polypeptide is that this protein is at most loosely attached to the inner membrane and does not span the membrane (see below). This criterion also suggests that this component is not involved in the energy-linked uptake of ornithine into mito- chondria. In fact, the transport of ornithine into rat liver

mitochondria may also utilize a general transport system (in this case specific for basic amino acids), such as has been de-

scribed for arginine in dog kidney mitochondria (38). Chemical Composition of Carbamyl Phosphate Syn-

thetase-The chemical composition of the enzyme was deter- mined on preparations of the 165,000 molecular weight poly- peptide separated from the lower molecular weight contami- nants seen in Fig. 2. Fractions from DEAE-cellulose chroma-

tography (80 to 90% pure) were concentrated by ultrafiltration,

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Carbamyl Phosphate Synthetase from Rat Liver Mitochondria 955

FIG. 5 (top). Electrophoresis of mi- tochondrial polypeptides from various tissues on dodecyl sulfate-polyacrylam- ide gels. Mitochondria were prepared as described under “Experimental Procedures,” washed free of serum al- bumin, and subjected to electrophoresis on 7.5% acrylamide/O.l% methylene- bisacrylamide/O.l% dodecyl sulfate gels. From left to right, the Coomassie bril- liant blue stained gels of rabbit liver and kidney mitochondria, chicken liver and kidney mitochondria, and rat liver, kid- ney, brain, skeletal muscle, and heart mitochondria are shown. The arrow on each gel marks the calculated migration position for a 165,000 molecular weight polypeptide, based on the polypeptide molecular weight standards shown in Fig. 6, and corrected for the mobility of the tracker dye, bromphenol blue, in each gel. The major high molecular weight polypeptide of chicken liver mi- tochondria has a calculated molecular weight of 140,000.

FIG. 6 (center). Electrophoresis of liver mitochondrial polypeptides from ureotelic and nonureotelic species on dodecyl sulfate-polyacrylamide gels, as in Fig. 5. From left to right, rat, dog, calf, Xenopus laeuis (South African clawed frog), Opsanas tao (toadfish), Anguilla rostrata (American eel), and Fundulus heteroclitus (mummichog). The gel on the far right contains: a, myosin; b, p-galactosidase; c, catalase (60,000); d, ovalbumin (43,500); e, mal- ate dehydrogenase (33,000); and f, cyto- chrome c. (The polypeptide molecular weight of these standards is given in parentheses; see also Fig. 2). The arrow on each gel marks the expected migra- tion position for a protein of 165,000 molecular weight based on these stan- dards and the relative migration of the tracker dye in each gel. The calculated molecular weight for the highest band on the gels of eel and mummichog mito- chondria is 135,000.

FIG. 7 (bottom). Electrophoresis of the polypeptides of fetal rat liver mito- chondria in dodecyl sulfate gels, as in Fig. 5 and Fig. 6, demonstrating the appearance of the 165,000 molecular weight component. From left to right, 16-day embryo, l&day embryo, 20.5- day embryo, newborn, and 7 days after birth. The arrow marks the expected position of a polypeptide of 165,000 molecular weight (see Figs. 5 and 6).

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956 Carbamyl Phosphate Synthetase from Rat Liver Mitochondria

TABLE I

Demonstration of citrulline transport actiuity in rat liuer and kidney mitochondria

Rotenone-treated mitochondria (0.5 mg of protein) were added to 0.9-ml solutions containing 10 mM Tris, 0.5 mM EGTA, and 250 mM specific solute (pH adjusted to 7.6 with H,SO,) in the cuvette of a Cary 15 spectrophotometer at room temperature. The absorbance at 560 nm was recorded agamst time using the expanded scale slide wire (0 to 0.1 A). Inrtial rates are given below.

Solute

Sucrose Glycine Glycylglycine Glycylglycylglycine L-Alanine or,-Alanine n-Alanine L-Isoleucine L-Valine L-Citrulline

Lwer Kldney

M,.mrnhn

0.000 0.000 0.138 0.064 0.008 0.031 0.001 0.000 0.061 0.041 0.046 0.031 0.035 0.029 0.046 0.028 0.116 0.052 0.011 0.012

denatured in 1 to 3% dodecyl sulfate/30 mM @-mercaptoethanol (3 min at 100°) and chromatographed on a Sepharose 4B column containing 0.2% dodecyl sulfate. As shown in Fig. 8, the 165,000 molecular weight polypeptide was eluted from the column with a K, of about 0.33 and was essentially free of the lower molecular weight material that contaminated the DEAE-cellulose preparation. For applications where the deter- gent might interfere with subsequent analysis, it was removed by the procedure of Weber and Kuter (39). Alternatively, the 165,000 molecular weight polypeptide was prepared in 6 M

guanidinium chloride by gel filtration in Sepharose 4B. The amino acid composition of the 165,000 molecular weight

polypeptide is given in Table II. These data are compared with published compositions of the enzyme from calf liver and the large subunit of Escherichia coli enzyme, which functions as an ammonia dependent synthetase. The largest difference in the composition of the calf and rat enzyme is the greater percent- age of basic residues in the former. The number of free carboxylic acids was determined as described in “Experimen- tal Procedures” on the denatured polypeptide in guanidinium chloride. The results indicate that 191 to 197 of the 299 Asx and Glx residues in the protein are present as aspartic and glutamic acid.

The number of cysteine residues in the large polypeptide, determined as 21 residues/m01 from the cysteic acid content after performic acid oxidation, was confirmed by alkylation of the protein with iodoacetamide. The results indicate that essentially all of the cysteine residues could be alkylated on the native protein in Triton X-100 (20.8/mol), and that denatura- tion in dodecyl sulfate, with or without prior reduction with 0.1 M P-mercaptoethanol, exposed no further residues (21.0 and 19.6 residues reacted /mol, respectively). This evidence suggests that there are no disulfide bonds and no “buried” sulfhydryl groups on the enzyme.

No glucosamine or galactosamine was detected by amino acid analysis of hydrolysates under conditions where 0.2 mol of glucosamine or 0.5 mol of galactosamine per mol would have been observed. Furthermore, the neutral sugar content was determined to be less than 0.5% of the total protein, or less than 4 residues/165,000 molecular weight chain.

Only a single dansylated NH,-terminal residue, 5-dimethyl-

035-

E =030- 0 % zo25-

8 rozo- x b

$j O.l5- Q

O.IOM , / I ( , , , j 35 50 70 90 110 130

Fraction No. FIG. 8. Isolation of the denatured 165,000 molecular weight poly-

peptide from purified preparations of carbamyl phosphate synthetase by gel filtration in dodecyl sulfate. DEAE-cellulose purified material (10 mg of protein), concentrated by XM-50 ultrafiltration (Amicon), was incubated for 3 min at loo”, followed by 2 hours at 37” in 2.5% dodecyl sulfate/50 rnM imidazole.H.$0,/30 mM &mercaptoethanol, pH 7.8. This sample was applied to a column (2.5 x 72 cm) of Sepharose 4B equilibrated and eluted with 0.2% dodecyl sulfate/50 mM

imidazole .H,SO,, pH 7.8. Thirty-minute fractions were collected at 20” containing 2 to 3 ml and were monitored for absorption at 280 nm. Material collected from the leading half of Fraction A contained the 165,000 molecular weight component as shown in the photograph of a dodecyl sulfate gel on the left. The small amount of polypeptides of lower molecular weight on this gel are degradation products of the 165,000 molecular weight polypeptide. Fraction B contains the lower molecular weight polypeptides of the DEAE-cellulose preparation of carbamyl phosphate synthetase and some 50,000 molecular weight degradation product of the large polypeptide. The absorbance at 280 nm in Fraction C is due to residual Triton X-100 present in the dodecyl sulfate micelle fraction. The void volume of this column was 140 ml (fraction 42); the total volume was 370 ml (fraction 150).

aminonaphthalene-1-sulfonyl (dansyl)-leucine, was detected for the 165,000 molecular weight polypeptide using the method of Weiner et al. (41).

No phosphorylated form of the large polypeptide could be detected in an experiment designed to find a covalent interme- diate in the enzymatic catalysis. Intact mitochondria were given a pulse of $*P inorganic phosphate while respiring on glutamate. Dodecyl sulfate was used to “trap” enzyme.phos- phate covalent complexes as has been done with the (Na+ + K+)-dependent adenosine triphosphatase (42), and the mito- chondrial polypeptides were immediately subjected to electro- phoresis in dodecyl sulfate gels. No counts were detected in the 165,000 molecular weight of region of carbamyl phosphate synthetase, while a large radioactive peak, probably corre- sponding to the phosphorylated intermediate of succinyl CoA synthetase (43), was found at a calculated molecular weight of 31,000. Experimenm were also conduct.ed to determine whether the 165,000 molecular weight component might be phosphoryl- ated independently of the active site in a serine-phosphate or threonine-phosphate linkage. After a 60-min incubation of mitochondria in s2P,, and the separation of polypeptides by dodecyl sulfate gel electrophoresis, acid-stable counts were incorporated into two lower molecular weight polypeptides (at 41,000 and 48,000) but not into the 165,000 molecular weight component.

The failure to detect phosphorylation of the 165,000 molecu- lar weight component under the first set of experimental conditions suggests that if an enzyme phosphate linkage is involved in catalysis (see for example Rubio et al. (44)), it may

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Carbamyl Phosphate Synthetase from Rat Liver Mitochondriu 957

TABLE II

Amino acid composition of carbamyl phosphate synthetases

Rat liver Calf liver” Escherichia colP (large subunit)

mol%

Lysine Hi*,tidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrcsine Phenylalanine Tryptophan

residues1 165,Ooog

6.94 104

2.00 30 3.80 57

10.08 151

5.81’ 87 6.54’ 98

9.88 148

4.13 62 7.81 117

8.28 124

1.406 21

1.88 118

2.53d 38 6.21 93

9.21 138

2.34 35

3.80 51

1.33e 20

mole % mole 9%

8.37 5.08

2.55 1.40

5.31 6.43

9.60 9.11

5.11 5.65 5.62 4.36

10.32 12.07

4.70 5.01

7.46 8.21 1.97 10.53 1.63 1.65

7.35 8.26

1.43 2.58 5.82 6.14

8.78 7.54

2.66 2.34

3.98 3.22

1.33 0.44

“Data of Elliott and Tipton (8). *Data of Trotta et al. (40). c Determined by zero time extrapolation from 24-, 48-, and 72-hour

hydrolysates. d Determined as cysteic acid and methionine sulfone after performic

acid oxidation. ‘Determined by absorption spectrum in 6 M guanidinium chloride

according to Edelhoch (23).

not be a covalent one; the failure to detect phosphorylation

under the second set of conditions indicates that control of enzyme activity by phosphorylation/dephosphorylation proba- bly does not occur.

Subunit Structure of Carbamyl Phosphate Synthetase-The molecular weight of the denatured large polypeptide chain was determined by its mobility on dodecyl sulfate gels and its elution position after gel filtration in 6 M guanidinium chloride (Fig. 9). A polypeptide molecular weight of 165,000 was assigned to this component from these data; this value is, however, dependent upon the values of the polypeptide molec- ular weights assigned to the marker proteins. In this case, the calculated value is particularly dependent upon a value for the polypeptide chain molecular weight of E. coli P-galactosidase of 135,000 (47) and of the large chain of skeletal muscle myosin of 220,000 (48). Gel filtration experiments were also done in dodecyl sulfate as shown in Fig. 8; these experiments indicated

a polypeptide chain molecular weight of 155,000. The number of 165,000 molecular weight polypeptide sub-

units in the native structure of carbamyl phosphate synthetase was determined from the molecular weight of the protein in Triton X-100. This molecular weight was calculated from hydrodynamic measurements (s~~,~, D,,,, U) according to the Svedberg equation (49). A partial specific volume of 0.74 cm’/g was calculated from the amino acid composition (Table II) by the method of Cohn and Edsall (50). A similar value of U was

obtained experimentally by the method of Clarke (9), indicat- ing that little or no non-amino acid components were present. (The amount of Triton X-100 bound to the enzyme was also directly determined as described (9); the results indicate an upper limit of 0.014 g of detergent bound/g of protein,

0 02 04 06 OS IO 03 04 05 06 07 08 09 Mob,llty Relatlw lo BrompheW Blue

KD

FIG. 9. Determination of the polypeptide chain molecular weight of carbamyl phosphate synthetase. Left. calibration of electrophoresis in gels containing 7.5% acrylamide, 0.1% methylenebisacrylamide (MBA), and 0.1% sodium dodecyl sulfate (SDS). Standard proteins, and their polypeptide molecular weights are: a, myosin (226,000); b, j3 and 8’ subunits of RNA polymerase (155,000 and 165,000) (45)); c, &galactosidase (135,000); d, c subunit of RNA polymerase (90,000); e, bovine serum albumin (68,000); f, catalase (66,000); g, (Y subunit of RNA polymerase (39,000); h, carbonic anhydrase (30,000); and i, cytochrome c (11,500). The arrow marks the position of migration of the major polypeptide from purified preparations of carbamyl phos- phate synthetase; the interpolated molecular weight is 167,000. Right, calibration of a Sepharose column in 6 M guanidinium chloride by the method of Fish et al. (46). Protein samples were reduced with fi-mercaptoethanol and carboxymethylated with iodoacetamide prior to filtration. The resin was packed into a (1.8 x 55 cm) column at 20” and was eluted under a hydrostatic head of 10 cm. Thirty-minute frac- tions were collected, and protein was determined by the A,,, nm. The void volume was marked with blue dextran 2000 (see Fig. 3) and the total volume with potassium ferricyanide. K, was calculated as described (46). The interpolated molecular weight of the polypeptide of carbamyl phosphate synthetase is 163,000.

corresponding to less than 4 molecules bound/enzyme mole- cule.) Since the measured and calculated partial specific volumes did not deviate from that of normal “soluble” proteins, the method of Martin and Ames could be used to

determine the sedimentation coefficients by sucrose density centrifugation (9, 51). Although there have been reports that the value of s?,,, of various carbamyl phosphate synthetase preparations is dependent upon the temperature, huffer com-

position, or the presence of the cofactor, N-acetyl glutamate (6, 7, 52), no large changes in the sedimentation coefficient were found here under the conditions given in Table III. An average sZo+ of 8.4 S, based on the s20,w values of endogenous mitochondrial marker proteins, was calculated. The diffusion coefficient (4.1 x lo-’ cm*/s) was determined from the elution position of the enzyme on a column of 8% agarose in Triton X-100 as shown in Fig. 10.

These values of b, s%~,~, and D,,, were used to calculate a molecular weight of 191,000 and a frictional ratio of 1.36 for the native enzyme in Triton X-100 (49) (Table IV). This value of the native molecular weight is significantly greater than one would expect for a single polypeptide chain of 165,000 molecu-

lar weight, but is much less than the expected molecular weight of a dimer of these polypeptide chains. Since contribu- tions to the native molecular weight by carbohydrate and bound detergent have been shown to be less than 2%, and since the presence of a molar equivalent of a smaller polypeptide (as in an (~0 structure) does not seem to be indicated (Fig. 2), the molecular weight measured may reflect equilibrium between monomers and dimers. Under the conditions described here, this equilibrium must heavily favor monomer formation.

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958 Carbamyl Phosphate Synthetase from Rat Liver Mitochondria

TABLE III TABLE IV

Effect of temperature, buffer, and cofactor on sedimentation coefficient of carbamyl phosphate synthetase

Samples of a 1% Trition X-100 extract of rat liver mitoplasts (6 mg of

Summary of physical properties of 165,000 molecular weight componentlcarbamyl phosphate synthetase

protem/ml) were dialyzed into the indicated buffer at the temperature given for 8 hours. Aliquots (100 ~1) then were layered over 4 ml of 5 to 20% sucrose gradients in the same buffer and at the same temperature and spun for 4 hours at 20” or 7 hours at 2” at 60,000 rpm in the SB 405 rotor of the IEC B60 ultracentrifuge. Fractions were collected from the bottom of the tube and analyzed for carbamyl phosphate synthetase activity. Sedimentation coefficients were mterpolated from a plot of s20,w versus migration position for catalase (11.3 S), fumarase (9.1 S), citrate synthase (6.0 S), and malate dehydrogenase (4.4 S), assayed in the fractions of each grachent.

hwe D 2o.w li Native molecular weight in Triton X-100

flf0 Axial ratio of a prolate ellipsoid* Triton X-100 bound (mg/mg) Carbohydrate (mg/mg) Polypeptide molecular weight

Dodecyl sulfate gel electrophoresis Gel Filtration in guanidinium Cl Gel Filtration in dodecyl sulfate

NH,-terminal residue Buffer

Calculated slo.lu

2” 20”

10 mM Tris.H,SOJlOO mM Na,SOJ0.05% 8.5 s 8.5 S Triton X-100, pH 7.5

As above + 10 mM iV-acetyl glutamate 8.8 s 8.3 s 40 mM KH,PO,.KOH/O.OS% Trlton X-100, 8.2 s 8.3 s

pH 7.5 As above + 10 mM N-acetyl glutamate 8.6 s 7.9 s

9- CFS 165,ooO MW Polypeptlde

0.4 0.5 0.6 0.7 0.8

KD FIG. 10. Determination of the diffusion coefficient of carbamyl

phosphate synthetase in Triton X-100 by gel filtration. A column of 8% agarose (see Fig. 3) was calibrated with the following proteins (values of the diffusion coefficient (in cm”/s x 101) used to calculate the Stokes radii are given in parentheses): a, catalase (4.1); b, fumarase, (4.2); c, aldolase (4.54); d, malate dehydrogenase (6.1); and e, myoglobin (11.3). The distribution coefficient (KJ of the 165.000 molecular weight polypeptide, carbamyl phosphate synthetase, and the marker proteins was calculated from the elution position of the peak of protein or activity and the void and total column volumes are described (9). The &,,, of carbamyl phosphate synthetase from these data is 4.1 x lo-’ cm2/s.

The presence of a fraction of dimers in carbamyl phosphate synthetase was substantiated by the cross-linking experiment shown in Fig. 11. Reaction of purified carbamyl phosphate synthetase with dimethyl subermudate f’z& to cross-link the

majority of the molecules under conditions that result in fully cross-linked catalase. Nevertheless, a new cross-linked product is found on these gels at a calculated molecular weight of 320,000 which makes up about 20% of the total protein on the gel.

The physical properties of rat liver carbamyl phosphate synthetase are summarized in Table IV. These results indicate that the native molecule is a somewhat asymmetrical protein consisting of one (or two) 165,000 molecular weight polypeptide chains.

Intramitochondrial Location of Carbamyl Phosphate

8.4 s 4.1 x lo-‘cmz/s 0.74 cm”/g 191,000 1.36 4.6

<0.014 <0.005

167,000 163,000 155,000 Leucine

a All hydrodynamic values (s~,,,~, D,,,,, U) were determined in Triton x-100.

b Calculated assuming a hydration of 0.3 g/g after Edsall (49).

FIG. 11. Cross-linking of carbamyl phosphate synthetase in Triton X-100 with dimethyl suberimidate after the procedure of Davies and Stark (16). Purified protein from DEAE-cellulose chromatography was concentrated to 0.7 mg/ml and dialyzed into 0.2 M trietha- nolamine. H,SO,, pH 8.5. Samples of this protein were reacted with various concentrations of dimethylsuberimidate in the same buffer for 10 hours at 0”. Aliquots of the reaction mixture containing 25 pg of protein were subjected to electrophoresis on dodecyl sulfate gels containing 5% acrylamide, 0.07% methylenebisacrylamide. The con- centration of suberimidate in the reaction mixture (in milligrams/ml) is indicated above each gel. The arrow marks the position of the polypeptide’band migrating at a molecular weight of 320,000. The gel on the far right contains protein standards (Stds) including: a, myosin; b, P-galactosidase; c, catalase; d, fumarase; e, malate dehydrogenase; and f, cytochrome c.

Synthetase-It is clear from the results of digitonin fractiona- tion that the 165,000 molecular weight component is localized in the inner membrane/matrix complex of mitochondria, and that little if any of this polypeptide is present in the intermem- brane space or the outer membrane (1; see also Ref. 37). Previous studies (1) have also shown that most of the tyrosyl residues of the protein are not available for lactoperoxidase- catalyzed iodination from the outside of intact inner mem- brane/matrix vesicles prepared with digitonin (mitoplasts). These experiments have now been repeated using a modifica-

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Carbamyl Phosphate Synthetase from Rat Liver Mitochondria 959

tion of the procedure used to prepare the vesicles. This modification was the inclusion of bovine serum albumin in the digitonin fractionation medium, and this addition seems to be very important in obtaining a population of well sealed mitoplasts.

The results of these experiments show that the 165,000 molecular weight polypeptide, while making up 169 of the protein in the mitoplast, contains less than 1%’ of the total “Y

label after intact mitoplasts are iodinated with lactoperoxi- dase. In contrast, this component contains 15% of the total lZsI

label when mitoplasts are labeled in the presence of lytic concentrations of Triton X-100. As described previously (l), there is a great difficulty in directly comparing the sites available for iodination in intact and disrupted mitoplasts

because disrupted mitoplasts release factors which inhibit the lactoperoxidase labeling reaction. It is possible to correct for the extent of this inhibition using exogenous cytochrome c as an internal standard and estimate the relative number of available iodination sites in intact and disrupted mitoplasts. It was calculated in this way that less than 1.5% of the tyrosyl residues (or less than 0.5 residue/molecule) were exposed to the labeling reagent in intact mitoplasts.’ This calculation argues against the possibility that a part of this polypeptide extends

through the inner membrane to the exterior membrane surface. The small amount of label incorporated into the 165,000 molecular weight component in “intact” mitoplasts can result from the presence of a few disrupted vesicles or by labeling from activated free iodine which may penetrate the membrane (53).

The results from the lactoperoxidase labeling are confirmed by the insensitivity of the large polypeptide to digestion by exogenous trypsin, papain, chymotrypsin, or pronase in intact mitoplasts, although all of these proteases are capable of degrading the protein in Triton X-lOO.7 The most consistent interpretation of these results is that carbamyl phosphate synthetase is either a true matrix protein or is bound to the inner face of the inner membrane without penetrating it.

Gamble and Lehninger (37) have shown that this enzyme fits the operational definition of a matrix protein (11) because it can be solubilized from the inner membrane/matrix complex with low amounts of detergent. This type of procedure, however, may also remove loosely bound membrane proteins,

and it is very difficult to exclude the possibility that carbamyl phosphate synthetase is attached in this way to the membrane. It is clear that carbamyl phosphate synthetase does not interact strongly with the hydrophobic part of the phospholipid bilayer. This protein does not span the bilayer and can be solubilized by relatively mild procedures (freeze-thaw disrup- tion, osmotic shock) which may cause dislocations in the membrane, but do not release phospholipid or the more tightly bound membrane proteins.’ The fact that this enzyme does not bind Triton X-100 (see above) places it in a class of “soluble” proteins and distinguishes it from a class of “membrane” proteins which bind from 0.2 to 1.1 g of detergent/g of protein (9). If this protein is associated with the mitochondrial membrane, the interaction may be similar to the binding of

components of the erythrocyte membrane which can be ex-

‘This calculation was done by normalizing the specific activity of the 165,000 molecular weight component (counts per min/Fg of protein) in Triton-labeled samples by a factor which gave equivalent specific activities for the internal standard in the intact and disrupted labelled samples (see also Ref. 1).

‘Unpublished results.

tracted into aqueous buffers, such as glyceraldehyde-Zphos- phate dehydrogenase (54). In any case, it is likely that the present methods for determining the mode of attachment to the membrane of loosely bound proteins may not reflect the situation in viuo where the concentration of the components

may be very high and where the attachment may be controlled by interaction with specific metabolites.

DISCUSSION L

Polypeptide Composition of Carbamyl Phosphate Syn- thetase-Although two nominally pure preparations of car- bamyl phosphate synthetase have been reported from rat liver mitochondria with specific activities comparable to the ones measured in this work (6, 7), no precautions were taken to avoid proteolysis from lysosomal enzymes (see below) and it is possible that these preparations contained active proteolytic fragments of the enzyme. Neither of these preparations were analyzed by dodecyl sulfate gel electrophoresis to determine the polypeptide composition. However, a recent preparation of the enzyme from calf liver (8) has been analyzed in this way

and the results indicate that a high molecular weight polypep- tide may be involved in the activity. The purified enzyme gave a single band of calculated molecular weight of 215,000 on

dodecyl sulfate gel electrophoresis. When the preparation was reduced and carboxymethylated prior to electrophoresis, new bands at molecular weights of 165,000 (major) and 280,000 (minor) were observed.8

While the evidence presented here that the band seen on dodecyl sulfate gels of mitochondria at a molecular weight of 165,000 is the polypeptide chain of carbamyl phosphate synthetase is indirect, comparison of this preparation of the enzyme from rat liver with the enzyme from Escherichia coli and frog liver suggests that this large polypeptide, and not one of the minor components (Fig. 2), is associated with the enzymatic activity. The specific activities of the frog liver and bacterial enzymes (2.7 and 5.6 ~mol/min/mg, respectively (55, 56)) are comparable to those measured for the rat liver enzyme. If in fact the 165,000 molecular weight protein described here is the active species, then the turnover number of these enzymes

would also be similar. On the other hand, if one of the minor polypeptides in the rat liver preparation (Figs. 1 and 2) represented carbamyl phosphate synthetase, the turnover number of the rat enzyme would be much larger than that of the enzyme from the other sources.

The subunit structure of both the bacterial and the frog liver enzyme also suggest that the large polypeptide is associated with carbamyl phosphate synthetase in rat liver. Carbamyl phosphate synthetase from E. coli contains two polypeptide chains which can be separated in native conformation by KSCN treatment (40). The smaller polypeptide has glutamin- ase activity and a molecular weight of 42,000 to 48,000 (40, 57) but has no carbamyl phosphate synthetase activity. The larger subunit is made up of a 130,000 to 145,000 molecular weight polypeptide chain (40, 57), and has an ammonia-dependent carbamyl phosphate synthetase activity when isolated. The frog liver enzyme is made up of polypeptides which have a molecular weight of 156,000 (determined by sedimentation equilibrium in guanidinium chloride) to 143,000 (determined

by dodecyl sulfate gel electrophoresis) (52). That carbamyl

B Such behavior on dodecyl sulfate gel electrophoresis has not been observed in the present study of the rat liver enzyme; only one band at 165,000 molecular weight has been detected with or without reduction and alkylation.

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960 Carbamyl Phosphate Synthetase from Rat Liver Mitochondria

phosphate synthesis is carried out on such large polypeptides in both sources suggests some common origin for this enzyme

and supports the assignment of the 165,000 molecular weight component in rat liver mitochondria to this activity.

Stability of Enzyme Activity and Polypeptide Chain-The instability of solubilized carbamyl phosphate synthetase from mammalian liver is well known (7, 18, 58). The results obtained here suggest that one of the major causes of instabil- ity is proteolysis, and that the proteolysis of the 165,000 molecular weight component in vitro is done by lysosomal proteases, copurified with mitochondria during differential

centrifugation, and given access to mitochondrial proteins during the disruption of the membrane.’ That lysosomal enzymes are responsible for the proteolytic cleavage of this component was demonstrated by the relative stability of extracts from lysosome-free mitochondria, prepared by the density gradient fractionation of the mitochondrial fraction of Triton WR-1339 injected rats (59). No attempt has been made in other preparations to remove these proteases, but in the

present study the majority of lysosomal proteins were removed by the preliminary digitonin fractionation of the mitochondria

(1, 60). Two steps were taken during the purification to inhibit the

activities of known proteases: the inclusion of a sulfhydryl blocking reagent (which should inhibit the thiol-dependent activities of lysosomal cathepsins B,, B,, C, and IV (61-64)), and the replacement of chloride ions with sulfate or acetate (which should inhibit the halide-dependent cathepsin C (63)

and possibly cathepsin A, (65)). These steps probably have no effect on the remaining major lysosomal protease, cathepsin D (66). Because sulfhydryl blocking reagents inhibited carbamyl phosphate synthetase, and so that specific activities could be compared with previous preparations of this enzyme, no reagent was used in the experiments shown in Figs. 1, 3, and 4. However, in studies where it was important to maintain an intact polypeptide chain over extended periods, sodium tetra-

thionate (67) was employed. This sulfhydryl blocking reagent was useful because some recovery of enzymatic activity could be obtained by treatment with P-mercaptoethanol. Neverthe- less, the slow continued proteolysis of the 165,000 molecular weight polypeptide even under the most favorable conditions (estimated at one bond cleavedipolypeptide chain/96 hours at 5”) indicated that the removal of proteolytic activity was not complete. The tenacious copurification of proteases and enzymes has been well documented by Pringle (68).

Mitochondria do contain endogenous proteases which are thought to be involved with the turnover of matrix and inner membrane proteins (69, 70). Nevertheless, the instability of

carbamyl phosphate synthetase in vitro may not be related to the presence of these proteases because Fiala et al. (71) have recently measured the in uiuo turnover of two protein fractions of rat liver mitochondria and the one fraction that contained

the essentially pure 165,000 molecular weight component had a longer half-life (9.4 days) than that of the other lower molecular

weight polypeptides (5.3 to 7.7 days). Subunit Structure of Natiue Enzyme; Monomer-Dimer

8Released lysosomal phospholipases may be an important factor in protein degradation because new polypeptides are exposed for proteo- lytic attack when membrane structures are disrupted. In this work, the presence of serum albumin during the digitonin fractionation may not only be important in binding released proteases but also in binding the lytic products of phospholipase action, fatty acids and lysophospha- tides.

TABLE V

Comparison of sedimentation coefficients determined for carbamyl phosphate synthetase from rat and frog liver

sZO,,, Rat liver enzyme Temper- [N-acetyl Frog

ature glutamate] This Virden Guthohrlein liver” studyb & KnappeC

20” 0 8.4 9.ld 10.8’ 11.3 11.8

20 10 rnM 8.2 8.0 11.3 10.3 4 0 8.3 7.9 10.5 10.4

4 10 mh4 8.7 7.0 7.5 7.6 8.0

“Data of Strahler (52). Buffer or exact concentration of N-acetyl

glutamate not given. bResults from Table III in Tris and potassium phosphate buffers

were averaged. The low temperature actually used was 2” instead of 4’.

CData of Guthorhlein and Knappe (6). szO,U was determined by analytical ultracentrifugation at 30” (instead of 20”) and 10” (instead

of 4”). “Data of Virden (7). slO,w was determined by sucrose density

gradient centrifugation by comparison with marker proteins in a

companion tube (catalase, 11.3 S; IgG, 6.4 S). Concentration of N-acetyl glutamate was 12 mM, and these samples also contained the

other substrates for the enzyme. The results shown here are those

determined for an initial protein loading of 150 pg of purified enzyme.

‘Data of Virdin (7). s,O~,~ was determined by analytical ultracentrif-

ugation.

Equilibrium-Guthohrlein and Knappe (6) first described a temperature-dependent, cofactor-induced dimer-monomer transition for rat liver carbamyl phosphate synthetase by sedimentation velocity measurements, and Virden (7) con- firmed this observation using similar techniques. A similar pattern of dimer-monomer equilibrium has also been reported for the frog liver enzyme (52). The results presented in this

study, however, indicate that carbamyl phosphate synthetase exists mainly as a monomer of one 165,000 molecular weight polypeptide (Tables III and IV) under all of the experimental conditions employed. Some evidence of a monomer-dimer equilibrium was obtained, however. The measured molecular

weight was 15% larger than that of the polypeptide chain, and a small percentage of the molecules could be cross-linked to dimers with dimethyl suberimidate (Fig. 11).

Table V summarizes the available data on the sedimentation coefficients of the rat and frog liver enzymes. All of these data indicate that the enzyme exists as a monomer in the presence of N-acetyl glutamate in the cold, and although a transition to a predominately dimeric form has not been seen in this study, it has in several other ones. It is not clear whether this difference results from the specific conditions used (such as the presence of Triton X-100 in these experiments) or from

differences in the enzyme preparation (such as the degree of proteolytic modification of the polypeptide chain). It is also unclear what relevance these studies have to the in uiuo structure of the enzyme, which may be present at a concentra- tion of 70 mg/ml or higher, lo and may be attached to the inner surface of the inner membrane.

Carbamyl phosphate synthetase from E. coli has also been

“This calculation is based on a matrix located 165,000 molecular weight component that makes up 16% of the total mitochondrial protein, the data for the volume and protein content of isolated rat liver mitochondria of Glas and Bahr (72), and the assumption that the matrix makes up 60% of the total mitochondrial volume. The concen- tration of this component would be even higher in situations where the matrix volume was reduced, as in the “condensed” mitochondrial configuration (73).

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Carbamyl Phosphate Synthetase from Rat Liver Mitochondria 961

shown to undergo a monomer-dimer transition but in this case the dimerization is promoted by ammonium ions or ornithine (74).

Acknowledgment-This work was performed in the labora- tory of Guido Guidotti, whose kind support of these studies is greatly appreciated. Thanks are also due to Lloyd Waxman, Josh Farber, Bob Jackson, and Kurt Drickamer for valuable discussions.

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S Clarkesynthetase.

A major polypeptide component of rat liver mitochondria: carbamyl phosphate

1976, 251:950-961.J. Biol. Chem. 

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