THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 241, No. 21, Issue of November 10, PP. 4923-4930, 1966

Printed in U.S.A.

Rat Liver Lactate Dehydrogenase

AMIKO-TERMINAL AND ACETYLATION STATUS*

(Received for publication, April 11, 1966)

LEWIS D. STEGINK~ AND CARL S. VESTLING

From the Department of Biochemistry, University of Iowa, Ioua City, Iowa 62241

SUMMARY

The amino-terminal sequence of rat liver M4 lactate dehy- drogenase has been investigated in an attempt to determine the number of polypeptide chains in this protein. Quantita- tive dinitrophenylation and phenylthiocarbamylation experi- ments carried out under a wide variety of conditions yielded only submolar quantities of amino-terminal residues. The conditions used included those which lead to the dissocia- tion of lactate dehydrogenases and those which cause ex- tensive unfolding of the polypeptide chains.

The acetylation status of rat liver Md lactate dehydrogenase was investigated by means of a microenzymatic method de- veloped during this study. The presence of 6 to 8 moles of acetate per mole of rat liver lactate dehydrogenase was shown. Bovine heart Hd and bovine heart MH3 lactate dehydrogenases also contained 7 to 8 moles of acetate per mole. Chicken heart Hd lactate dehydrogenase contained 3 to 4 moles of acetate per mole. All the acetyl groups in rat liver Md lactate dehydrogenase are N-acetyl residues. Rat liver M4 lactate dehydrogenase also contains a quantity of a carbohydrate-like material which is not easily removed by dialysis, precipitation with ammonium sulfate, or repeated Bio-Gel chromatography.

These data, combined with the lack of reactivity of rat liver M4 lactate dehydrogenase toward leucine aminopep- tidase (63, lead to the conclusion that the amino-terminal residues of rat liver M4 lactate dehydrogenase are not readily available for reaction with amino-terminal labeling reagents and are probably acetylated. The large number of acetyl residues suggests the presence of more than one polypeptide chain per subunit.

Numerous investigators have demonstrated the molecular heterogeneity of extracted lactate dehydrogenase, and it is now thought that lactate dehydrogenase exists in at least five forms in the organs of most vertebrates. A useful hypothesis to ac- count for this information has been advanced by Appella and

* This work has been supported by Grant CA 07617 from the National Institutes of Health.

$ Present address, Children’s Research Unit, Department of Pediatrics, University of Iowa, Iowa City, Iowa.

Markert (1)) Markert (2), and Cahn et al. (3). This hypothesis states that the lactate dehydrogenase molecule in its native form is a tetramer, which can be composed of two diierent kinds of subunits: A or M (so-called muscle type) and B or H (so-called heart type) .I This allows for the possibility of five lactate dehy- drogenase isozymes: Mh, MIH, M2H2, MH,, and Hh types. The biosynthesis of each of these subunits is apparently controlled by a separate gene. This hypothesis is supported by hybridiza- tion studies (2) and by a large amount of immunochemical, ki- netic, and “fingerprint” data, as well as by amino acid composi- tion studies (3-S). These data show a steady progression of values ranging from the Mb lactate dehydrogenase through the various hybrids to the H4 lactate dehydrogenase.

However, not all of the information available completely fits this tetramer hypothesis. Fritz (9) has reported that crystalline rabbit muscle lactate dehydrogenase can be separated into I2 enzymatically active bands by polyacrylamide electrophoresis, and it is well known that there are considerably more than five bands of lactate dehydrogenase activity in crude homogenates separated by this electrophoretic procedure. other evidence (10) indicates that the pattern of the sub-bands of lactate dehy- drogenase during electrophoresis may be altered by varying con- centrations of 2-mercaptoethanol. In view of the work by Calm (ll), in which he was able to change the mobility of serum albu- min by variations in borate buffer concentrations, care must be taken that electrophoretically separable forms are distinct chemi- cal entities, and not the same molecule in diierent conformations as a result of the binding of inorganic ions or changes in the sulf- hydryl status.

With these reservations in mind, we have attempted to deter- mine quantitatively the NHz-terminal residues of rat liver M4 lactate dehydrogenase in an effort to provide a chemical basis for the number of polypeptide chains in the lactate dehydrogenase monomer. If lactate dehydrogenase is composed of four poly- peptide chains, it should be possible to use NHt-terminal labeling techniques, such as dinitrophenylation or phenylthiocarbamyla- tion, under a variety of conditions and to determine the number and identity of the residues found. These conditions should include those known to dissociate lactate dehydrogenase into subunits or to cause extensive unfolding of the polypeptide chains.

Rat liver lactate dehydrogenase was chosen for this study since its properties have been investigated in this laboratory for some

1 The M,H subunit designation will be used in this paper.

4923

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4924 Amino-terminal Status of Lactate Dehydrogenase Vol. 241, No. 21

time. This enzyme fits the general definition of an Md lactate dehydrogenase.

MATERIALS AND METHODS

Fluorodinitrobenzene and phenyl isothiocyanate were pur- chased from Distillation Products Industries. Bio-Gel resins were purchased from Bio-Rad. Biodryex was purchased from Liivdalens Industri Aktiebolag, Centralpalatset, Stockholm. Dowex 50 resin was purchased from Calbiochem. Adenosine triphosphate, L-malic acid, diphosphopyridine nucleotide, re- duced diphosphopyridine nucleotide, standard dinitrophenylated amino acids, and standard phenylthiohydanatoin derivatives of amino acids were purchased from Sigma. The redistilled hydra- zine and the Duolite A-4 resin were gifts of Professor Rex Mont- gomery of the Department of Biochemistry, University of Iowa.

Proteins-Highly purified rat liver Me lactate dehydrogenase was prepared by a modification of the procedure of Hsieh and Vestling (12). This procedure yields an Mk lactate dehydrogen- ase which is homogeneous upon ultracentrifugation and electro- phoresis. The modification in this procedure was made follow- ing the carboxymethyl cellulose column fractionation to avoid the use of Biodryex as a means of concentrating the enzyme frac- tions. This is necessary because of an apparent oligosaccharide, derived from the Biodryex, which contaminates the enzyme at this step. The fractions of suitable specific activity from the carboxymethyl cellulose column were collected, diluted with one- third their volume of cold water, and dialyzed for 2 to 3 hours against 0.01 M phosphate buffer-O.001 M 2-mercaptoethanol, pH 6.0. The dialyzed enzyme fraction was then applied to a short carboxymethyl cellulose column (2.5 x 1 cm) which had been extensively washed with 0.02 M phosphate buffer-O.001 M P-mer- captoethanol (pH 6.0) to remove any soluble carbohydrate mate- rials. The lactate dehydrogenase was completely absorbed on the column at a flow rate of 200 ml per hour. The column was washed with 50 ml of 0.02 M phosphate buffer-O.001 M 2-mercap- toethanol (pH 6.0), and the enzyme was eluted from the column with 0.1 M phosphate buffer-O.001 M 2-mercaptoethanol, pH 7.0. Fractions (4 ml) from t,he column were collected and assayed for protein at 280 ml*. Fractions possessing an absorbance of at least 0.400 were collected and dialyzed overnight against 0.02 M

phosphate buffer-O.001 M 2-mercaptoethanol, pH 7.0. The enzyme was then fractionated on a diethylaminoethyl cellulose column as described (12). The enzyme fractions collected from the diethylaminoethyl cellulose column were not concentrated on Biodryex, but were used directly in an ammonium sulfate precipitation step. These modifications had no effect on the electrophoretic or the catalytic properties of the enzyme, but markedly reduced the carbohydrate content. The protein con- centration of rat liver lactate dehydrogenase was measured by its absorbance at 280 rnp based on a molar extinction coefficient of 1.585 x lo5 (13) and a molecular weight of 126,000 (14).

Samples of chicken heart Ha, bovine heart Hq, and MH3 lac- tate dehydrogenases were the gift of Dr. Sonia Anderson, The University of Illinois, Urbana. Ovalbumin was the gift of Professor H. B. Bull, or was purchased from Sigma. Bovine serum albumin and horse heart cytochrome c were purchased from Sigma. Citrate-condensing enzyme .and pig heart malate dehydrogenase were purchased from Calbiochem. Acetyl-CoA synthetase was purified from yeast by the method of Berg (15).

NH&erminal Amino Acid Studies with Fluorodinitrobenzene- Quantitative dinitrophenylation studies of the NH&erminal

amino acid residues of rat liver Md lactate dehydrogenase were carried out by the modified Sanger technique described by Fraenkel-Conrat, Harris, and Levy (16). Protein samples were dialyzed against 0.005 M phosphate buffer-O.1 M EDTA, pH 7, to remove traces of heavy metals, which have been reported to interfere with this determination in certain cases (17, 18).

Two samples of rat liver Mq lactate dehydrogenase (0.2 pmole) were used for each determination. In each case the duplicate sample of the dinitrophenylated protein, containing 0.2 pmole of each of the standard DNP2-amino acids expected to be found, was carried through the appropriate steps to determine the cor- rection factor for the loss of DNP-amino acids through hydrolysis and manipulation of the sample. These correction factors are shown in parentheses in Table I. The dinitrophenylated pro- teins were hydrolyzed for 18 to 20 hours at 110” with constant boiling hydrochloric acid, which had been redistilled from glass three times to remove trace impurities damaging to the DNP- amino acids (19). This is sufficient time for complete hydrolysis of rat liver Mq lactate dehydrogenase (20). Ether extractions of the hydrolyzed dinitrophenylated proteins were carried out in micro liquid-liquid extractors. Protein standards of known NHz-terminal structure were analyzed in parallel studies to pro- vide reference controls.

Dinitrophenylation in the presence of sodium dodecyl sulfate was carried out with bicarbonate, but without ethanol, after the lactate dehydrogenase samples were brought to a final concen- tration of 4 x 1OW M sodium dodecyl sulfate by the addition of the solid salt. Previous work has shown that sodium dodecyl sulfate in concentrations up to 0.1 M does not interfere with the dinitrophenylation reaction (19).

Dinitrophenylation experiments in the presence of urea and sodium chloride were carried out as follows. The lactate dehy- drogenase samples were dialyzed for at least 12 hours against a solution which was 8 M with respect to urea and 1 M with respect to NaCl, after which fluorodinitrobenzene and bicarbonate were added. The samples were rapidly frozen in Dry Ice and allowed to thaw slowly at room temperature. This freeze-thaw proce- dure was repeated four times, and the dinitrophenylation reaction was allowed to proceed at room temperature for 6 hours. The urea was dialyzed from the precipitated protein, and hydrolysis and extraction were carried out as usual.

Dinitrophenylation in the presence of 2 M sodium chloride was carried out with bicarbonate but without ethanol after the pro- tein had been dialyzed against 2 M sodium chloride. Dinitro- phenylation in the presence of 6 M guanidine was accomplished after dialysis of the protein against 6 M guanidine.HCl-0.1 M

2-mercaptoethanol and sufficient NaHC03 to bring the pH to 7 to 8. Dinitrophenylation was carried out with bicarbonate, but without ethanol, as usual; the excess guanidine was removed by dialysis, followed by hydrolysis and extraction of the DNP- amino acids.

Phenyl Isothiocyanate Studies-The phenyl isothiocyanate technique of Edman (21, 22) as modified by SjGquist (23) and Eriksson and Sjijquist (24) was used to check the dinitrophenyla- tion studies. Samples containing 0.2 pmole of lactate dehydro- genase were allowed to react for li and 6 hours with phenyl isothiocyanate in the aqueous pyridine-triethylamine solvent as described, Known phenylthiohydantoin amino acid derivatives were carried through the required procedure to allow calculation

2 The abbreviation used is: DNP-, 2,4-dinitrophenyl-.

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Issue of November 10, 1966 L. D. Stegink and 6. 8. Vestling 4925

of a correction factor for each amino acid, and proteins of known NHz-terminal structure were again analyzed as a check on the method. Our correction factors were in good agreement with those published by Sjoquist (23) and Eriksson and Sjiiquist (24).

ildicroenzymatic Assay for Acetate-Several assays are availa- ble for the determination of acetate (2533), but t,he procedures which have been used for the determination of acetyl groups in proteins (25-28) require fairly large quantities of protein, 0.5 to 10.0 pmoles. Accordingly, the microenzymatic method for acetate originally described by Von Korff (29) was modified for this purpose. Von Korff’s original conditions involved the use of a crude heart extract and did not yield 1 mole of DPNH per mole of acetate added. His data showed, at best, a 26% con- version of acetate to citrate. This required a standard curve to be run with each determination to ascertain the variable response of the assay system to acetate. In addition, this procedure re- quired treatment with 2,4-dinitrophenylhydrazine and a micro- distillation step to remove pyruvate from the samples because of the presence of lactate dehydrogenase in the crude enzyme sys- tem. By considering the equilibrium constants of the enzymes involved, as described by Pearson (34) (see “Discussion”), and by the use of highly purified enzymes, we have demonstrated that added acetate is quantitatively converted to citrate with the production of an equivalent quantity of DPNH. The enzymatic reactions used in this assay are shown in Scheme 1.

Mg++ Acetate + ATP +CoA Q acetyl-CoA + AMP + PPi

L-Malate + DPN+ - <p oxalacetate + DPNH + H+ Acetyl-CoA + oxalacetate + Hz0 - citrate + CoA

SCHEME 1. Microenzymatic assay for acetate

In this method, acetate, after release from the protein by acid hydrolysis, is converted to acetyl-CoA in the presence of ATP, CoA, MgC12, and acetyl-CoA synthetase. The acetyl-CoA formed is coupled with oxalacetate to form citrate in the presence of citrate-condensing enzyme. Oxalacetate in turn is formed from n-malate in the presence of malate dehydrogenase and DPNf, and the appearance of DPNH is followed spectrophoto- metrically with the Gary model 15 spectrophotometer at 340 rnp. If the precautions recently described by Pearson (34) are fol- lowed, the amount of DPN+ reduced to DPNH is equivalent to the amount of acetate added. The optical assay was carried out in l.O-ml silica cuvettes with a l-cm light path. The complete reaction mixture contained 200 pmoles of Tris buffer (pH 8.0), .5 pmoles of magnesium chloride, 10 pmoles of adenosine triphos- phate, 50 pmoles of potassium fluoride, 0.10 Mmole of coenzyme A, 2.5 pmoles of potassium n-malate, 1.4 pmoles of DPNf, 0.10 pmole of DPNH, malate dehydrogenase (5 pg of protein; specific activity, 39), citrate-condensing enzyme (20 pg of protein; spe- cific activity, 30), acetyl-CoA synthetase (100 pg; specific activ- ity, 39), and the aliquot to be assayed for acetate, in a total vol- ume of 1.00 ml. A cuvette without CoA or without acetate served as a control. Coenzyme A was added last, to initiate the reaction. The rate of the reaction was followed at 340 rnp until the system reached equilibrium, at which time the change in absorbance was noted and the quantity of DPNH produced was calculated from its molar extinction coefficient (35). This method exhibits a linear response to added acetate (0 to 0.15 pmole) and is reproducible within the limits &1095, in this range.

This assay is quite specific for acetate, and neither formate nor propionate is active in the over-all system. Acrylate and propi-

onate are substrates of acetyl-CoA synthetase, although only one-third as active as acetate (36), but propionyl-CoA, butyryl- CoA, malonyl-Co,4, and glycolyl-CoA are not substrates for the citrate-condensing enzyme.3 Thus the specificity of the assay is not surprising.

Because unidentified inhibitors of the enzymatic reactions were present in the hydrolyzed protein samples, it was necessary to extract the acetate from the hydrolysis mixture before assay. The following method was used in these experiments. The pro- tein sample to be studied is assayed before hydrolysis for the presence of acetate remaining from the purification procedure. If this quantity is large, the sample must be extensively dialyzed and reassayed for acetate. Protein samples calculated to con- tain 0.1 to 0.2 pmole of acetyl groups (although larger samples may also be used) are lyophilized upon the surface of a hydrolysis vial. The protein is suspended in 0.7 ml of 6 N HCl, the vial is sealed, and the sample is hydrolyzed at 110” for 12 hours. The lyophilization step may be omitted if the protein sample is con- centrated, and an equal volume of concentrated HCl may be added directly to the protein sample, which is hydrolyzed as usual. After hydrolysis the vials are thoroughly chilled in ice before opening to prevent the loss of acetic acid, and the protein hydrolysate is removed to a large glass-stoppered conical test tube. The vial is carefully washed with a quantity of cold water, and the hydrolysis mixture is diluted with enough cold water to bring the final HCl concentration to 1 M. The chilled tube is carefully extracted with a 2-ml portion of diethyl ether on a Vor- tex mixer. The ether layer is removed to a second glass- stoppered test t.ube containing 0.80 ml of M Tris buffer, pH 8.6, and 0.2 ml of 5 N NaOH. The acetate is extracted from the ether into the aqueous layer by careful mixing with the Vortex mixer. The ether layer is removed and discarded, and the procedure is repeated five times. A known quantity of acetate may be sub- jected to the above procedure in the presence of 0.2 pmole of bovine serum albumin and carried through the extraction and assay to determine a correction factor. The recovery should be at least 90 to 95%. The alkaline layer containing the acetate is titrated to pH 7 to 8 by the addition of concentrated HCI (about. 0.04 ml), and a suitable aliquot (usually 0.05 to 0.10 ml) is re- moved and assayed. The aliquots assayed should be of such a size that the response of the assay system is linear with respect to increasing or decreasing amounts of sample. With increasing sample size the quantity of inhibitor carried into the assay sys- tem from the extracted solution increases and markedly lowers the amount of acetate converted to citrate.

The hydrazinolysis technique of Phillips (25) was used for the analytical differentiation between 0-acetyl and N-acetyl groups in rat liver Mh lact,ate dehydrogenase.

Carbohydrate-reacting Material in Lactate Dehydrogenase-The phenol-sulfuric acid method of Montgomery (37) was used to determine the quant,ity of hexose-reacting material present in lactate dehydrogenase preparations. It should be noted that this method does not detect hexosamines. Separation of phenol- sulfuric acid-positive material from lactate dehydrogenase was partially obtained by chromatography of 72 mg of lactate dehy- drogenase on a Bio-Gel P-100 column (3.5 x 50 cm) previously equilibrated with 0.1 M phosphate buffer, pH 7.6. The column was eluted with the same buffer and lo-ml fractions were collected and assayed for lactate dehydrogenase activity, absorbance at

3 P. Srere, personal communication.

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4926 Amino-terminal Status of Lactate Dehydrogenase

Substituted amino acids found in rat liver

lactate dehydrogenase

Glutamate. ........ Valine. ............ Threonine ......... Alanine. ........... Glycine ............ Serine .............

Total ............ :I -

Vol. 241, No. 21

TABLE I NHz-terminal dinitrophen$ation studies

Conditions of reaction

HCOr-ethanol Sodium dodecyl sulfate (4 x lo-a?&)

Urea (8 x)-N&l (1 IX); freeze-thaw NaCl (2 M) Guanidine (6 M)-

mercaptoethanol (0.1 M)

moles/1~6,000 g lactde dehydrogenose

0.32 (1.33) 0.24 (1.36) 0.42 (2.22) 0.13 (1.35)

0.28 (1.35)” 0.26 (1.35)” 0.07 (1.35p

0.74 I

0.98

0 Not experimentally determined.

280 rnp, phenol-sulfuric acid-positive material, and sulfhydryl compounds (38). The lactate dehydrogenase placed on the column contained 0.001 M 2-mercaptoethanol, and the elution of this material 2 column volumes after the lactate dehydrogenase activity gives a measure of the separation of the large lactate dehydrogenase molecule from smaller carbohydrate molecules. The peak lactate dehydrogenase tubes were collected and rechro- matographed on a similar column after the lactate dehydrogenase was brought to a final concentration of 0.001 M 2-mercapto- ethanol. The peak lactate dehydrogenase fractions from the second P-100 column were collected and assayed as before, and the lactate dehydrogenase was concentrated by precipitation with ammonium sulfate. The precipitated lactate dehydro- genase was suspended in 10.0 ml of ammonium sulfate solution (0.5 g per ml) and centrifuged, and the supernatant liquid was discarded. This process was repeated five times in a further attempt to remove phenol-sulfuric acid-positive material.

Lactate dehydrogenase was also assayed for the presence of “neutral carbohydrate” as follows. Samples of rat liver Mb lactate dehydrogenase (0.25 pmole) were hydrolyzed in 1.0 N

HCl for 6 hours at 110”. The hydrolysate was passed in succes- sion over a Dowex 50 column and then a Duolite A-4 column to remove ions. The eluate containing neutral carbohydrate was collected and concentrated to dryness, dissolved in a small quan- tity of water, and subjected to descending chromatography with suitable standards on Whatman No. 1 paper in a solvent system of ethyl acetate-acetic acid-formic acid-water (18:3:1:4) for 44 hours (39). The hexose components were detected with the alka- line silver method (40). Samples of ovalbumin and bovine serum albumin were also carried through the same procedure.

An attempt to exchange 14C-glucose into the %arbohydrate” fraction of lactate dehydrogenase was carried out as follows. A sample of rat liver M4 lactate dehydrogenase (0.38 pmole), which had been passed over two successive Bio-Gel P-100 columns to remove the loosely associated carbohydrate material, was incu- bated with 0.4 pmole of uniformly labeled 14C-glucose (123 mC per mmole) in 0.1 M phosphate buffer-O.001 M 2-mercaptoethanol, pH 7.6. The sample was frozen slowly, then allowed to thaw at room temperature, and finally incubated for 36 hours at 4’. The lactate dehydrogenase was precipitated by the addition of solid ammonium sulfate (0.5 g per ml), and collected by centrifu- gation, and the supernatant solution was discarded. The protein precipitate was suspended in 10 ml of 0.1 M phosphate buffer, pH

0.44 (1.40) 0.38 (1.52) 0.24 (1.47) 0.14 (1.37) 0.14 (1.38) 0.08 (1.47) 0.35 (2.04) 0.13 (1.27) 0.15 (1.25) 0.31 (1.66) 0.17 (1.23) 0.15 (1.54) 0.16 (2.18) 0.25 (3.34)

0.13 (2.61) 1.38 1.05 0.80

7.6, containing ammonium sulfate (0.5 g per ml) and stirred for 2 min, after which the lactate dehydrogenase precipitate was collected by centrifugation. This washing was repeated four more times to remove excess 14C-glucose. The final precipitate was dissolved in 0.01 M phosphate buffer and an aliquot was removed and counted in a Packard Tri-Carb liquid scintillation counter.

RESULTS

NH&erminal Amino kid Residue Studies-Amino-terminal labeling techniques (dinitrophenylation and phenylthiocarba- mylation) have been used to determine the number and identity of the residues found. Under the usual dmitrophenylation con- ditions (bicarbonate, ethanol) DNP-valine and DNP-glutamate are found, but in quantities substantially less than 1 mole per mole, and certainly far below the 4 moles per mole required by the tetramer hypothesis. Since the NHz-terminal residues of rat liver M4 lactate dehydrogenase may be largely unavailable for reaction with fluorodinitrobenzene, the dinitrophenylation reaction was carried out under a variety of conditions reported to dissociate lactate dehydrogenase into subunits or cause extensive unfolding of the polypeptide chains (I, 2, 41-43). The results of these studies, shown in Table I, indicate that no single DNP- amino acid appears in a quantity approaching 1 mole per mole of enzyme, although some peptide bond cleavage seems to occur during the reaction process.

The phenyl isothiocyanate technique of Edman (21, 22), as modified by Sjijquist (23) and Eriksson and Sjijquist (24), was used to confirm the dmitrophenylation studies. The results of these experiments are shown in Table II. Again, no single amino acid appeared in a quantity approaching 1 mole per mole. In the extended reaction with phenyl isothiocyanate (6 hours), consid- erable peptide bond cleavage appears to occur in the alkaline, aqueous pyridine-triethylamine solvent.

Acetate Analysis-The possibility of NH*-terminal acylation, in particular acetylation, must be considered in view of such residues found in ovalbumin (44)) cytochrome c (45)) enolase (46)) myosin (47), and several other proteins (48-55).

The isolation of N-acetylamino acids from protein hydroly- sates requires large quantities of protein, which were not readily available. Accordingly, rat liver M4 lactate dehydrogenase and several other proteins were assayed for the presence of acetate after acid hydrolysis by the microenzymatic method described

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Issue of November 10, 1966 L. D. Xtegink and C. X. Vestling 4927

in this paper. The quantities of acetate found after acid hy- drolysis in rat liver Mk lactate dehydrogenase, beef heart Md and M1H3 lactate dehydrogenases, chicken heart Hb lactate dehydro- genase, ovalbumin, horse heart cytochrome c, and bovine serum albumin are shown in Table III. Ovalbumin was found to con- tain 3.7 acetyl residues, in good agreement with the expected 4; horse heart cytochrome c contained 1 residue as expected, while bovine serum albumin contained no detectable acetate. Chicken heart Hq lactate dehydrogenase contained 3 to 4 acetyl residues per mole, substantially less t’han the other lactate dehydro- genase enzymes assayed.

The acetate residues could arise from either an 0-acetyl or an N-acetyl linkage to the protein. Accordingly, the hydrazinoly- sis technique of Phillips (25) was used to differentiate between these linkages in rat liver lactate dehydrogenase. The results of 0-acetyl and N-acetyl determinations with this technique are also shown in Table III. These data show the absence of any 0-acetyl grouping in rat liver lactate dehydrogenase. The N- acetyl hydrazinolysis experiment yields 7.3 acetyl groups per mole of rat liver Mq lactate dehydrogenase, in good agreement with the enzymatic assay.

It is possible that lactate dehydrogenase contains a heretofore undetected carbohydrate residue containing either N-acetyl- glucosamine or N-acetylgalactosamine. Preliminary tests in which the phenol-sulfuric acid method of Montgomery (37) was used were carried out to investigate this possibility. Table IV gives the results of such studies. Ovalbumin contains the ex- pected 5 mannose residues. Rat liver Mh lactate dehydrogenase consistently gives a distinct positive reaction, although the values fluctuate. The central problem is the presence of phenol-sulfuric acid-positive material leached from the diethylaminoethyl and carboxymethyl cellulose columns used in the purification proce- dure for the enzyme. This error was minimized by using a dupli- cate column technique. The level of the phenol-sulfuric acid- positive material could be decreased by chromatography on Bio-Gel P-100 columns, but only to a threshold level. No addi- tional decrease was found after additional chromatography, ex- tensive dialysis, or repeated- ammonium sulfate precipitations, as shown in Table IV. A sample of rat liver Mq lactate de- hydrogenase was hydrolyzed and the neutral carbohydrate frac- tion was examined by paper chromatography in ethyl acetate- acetic acid-formic acid-water (18 : 3 : 1:4). Only glucose was detected, but no mannose or galactose.

TABLE II

NH=-terminal studies-phenylthiocarbamylation

Substituted amino acids found in rat liver lactate debydrogenase

Length of reaction

I 14 hours

I 6 hours

mole/lZ6,000 g lactate dehydrogenose

Glutamate.. . Valine. Threonine Alanine. Glycine. Serine......... Aspartic acid. Methionine. Leucine

Total.

.

. .

.

0.10 0.20 0.07 0.15 0.25

.I 0.20

0.97

0.55 0.10

0.40 0.50 0.45 0.60 0.20 0.20 3.00

TABLE III Amount of acetyl groups found in various proteins

Material studied

Bovine serum albumin. . . . Ovalbumin. . . . . Rat liver M4 lactate dehydro-

genase.................... Chicken heart Hb lactate de-

hydrogenase. . . . Bovine heart Hd lactate de-

hydrogenase . . . . . . . Bovine heart MHa lactate de.

hydrogenase . . . Horse heart cytochrome c Ethyl acetate. . . . . . . . . . .

Microenzymatic

(totz:etyl residue)

0.0; 0.0 3.6; 3.7

5.8;8.0; 7.7

2.8; 4.4

7.3

8.3 0.97

.-

Differential bydrazinoIysisD

0-Acetyl residues

N-Acetyl residues

<O.l <O.l

<O.l

1.0

0.2 3.9

7.3

TABLE IV Amount of phenol-sulfuric acid-reactive material in various

protein preparations

Protein Galactose-mannose equivalents -

??&S/?PZ&

Bovine serum albumin . . . 0.32; 0.40; 0.24; 0.20 Ovalbumin............................. 5.1; 4.8; 4.7” Rat liver Mq lactate dehydrogenase . 4.0; 8.7; 3.1, 6.1 Rat liver Mq lactate dehydrogenase

Isolated.............................. 6.1 After Bio-Gel P-100 column. . 3.6 After 2 times over Bio-Gel P-166 col-

umn............................... 3.8 After 2 times Bio-Gel P-106 and 5 times

precipitated with ammonium sulfate 3.7

a Expressed as mannose equivalents since only mannose is present.

An attempt was made to exchange uniformly labeled “C-glu- case into the phenol-sulfuric acid-positive material present in rat liver Mb lactate dehydrogenase. However, only 2 x 10-b mole of 14C-glucose per mole of lactate dehydrogenase was found, indi- cating no exchange with the phenol-sulfuric acid-positive material bound to this enzyme.

DISCUSSION

The tetramer hypothesis of the structure of lactate dehydro- genase is consistent with an orderly progression of values ranging from the M4 lactate dehydrogenase through the various hybrids to the H4 lactate dehydrogenase. Absolute chemical values are lacking such as might be obtained with quantitative NHrtermi- nal labeling techniques or by COOH-terminal studies. In view of the continuing studies on rat liver M4 lactate dehydrogenase being carried out in this laboratory, we hoped to use quantitative NHz-terminal labeling techniques to supplement an original ob- servation by Hsieh (20), who noted the presence of 14C-DNP- valine and 14C-DNP-glutamate after treatment of a small quan- tity of lactate dehydrogenase with “C-fluorodinitrobenzene. Our quantitative studies also indicate the presence of DNP-

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4928 Amino-terminal Xtatus of Lactate Dehydrogenase Vol. 241, No. 21

valine and DNP-glutamate under ordinary dinitrophenylation conditions, but only in submolar amounts, and far less than 4 moles per mole. These results indicate that the NHz-terminal residues are largely unavailable for reaction with the reagents used. If this is the case, it should be possible to carry out the dinitrophenylation under a variety of conditions reported to dis- sociate lactate dehydrogenase into subunits, and determine the type and quantity of the residues found.

Di Sabato and Kaplan (41) have reported the dissociation of chicken heart and bovine heart lactate dehydrogenase in the presence of sodium dodecyl sulfate. The results of our dinitro- phenylation studies in the presence of sodium dodecyl sulfate indicate the absence of any DNP-amino acid in a quantity ap- proaching 1 mole per mole, although several new amino acids appeared in submolar quantities. The appearance of additional submolar quantities of NHz-terminal amino acids in sodium dodecyl sulfate is compatible with the studies of Colacicco (19) who observed similar behavior in studies with ovalbumin and various y-globulins.

Appella and Markert (1) have reported the dissociation of lactate dehydrogenase into subunits in high urea concentrations, and Epstein, Carter, and Goldberger (56) have reported reversi- ble denaturation of lactate dehydrogenase in urea. Markert (2) has also reported the dissociation of lactate dehydrogenase into subunits during a freeze-thaw procedure in 1 M NaCl. We used bot,h 8 M urea and 1 M NaCl with freezing and thawing in an at- tempt to obtain as much dissociation and chain unfolding as possible. No substantial difference from previous experiments was noted. Colacicco and Dawson (57) have shown that dini- trophenylation can be carried out in 8 M urea without effect on the quantitative recovery of DNP-amino acids from known pro- teins.

Jaenicke (43) has reported the dissociation of lactate dehy- drogenase at certain ionic strengths. Accordingly the dinitro- phenylation was carried out at an ionic strength of 2, but with no change in results.

Bppella and Markert (1) reported the dissociation of lactate dehydrogenase into subunits in the presence of 5 M guanidine-0.1 M 2-mercaptoethanol. Dinitrophenylation experiments in the presence of 6 M guanidine-0.1 M 2-mercaptoethanol again failed to reveal any significant quantity of NHzterminal amino acids.

The NHz-terminal residues of rat liver Mq lactate dehydrogen- ase were also determined by the modified Edman procedure (21, 22) described by Sjijquist (23) and Eriksson and SjGquist (24). Under the normal conditions no quantity of NH&erminal residue approaching 1 mole per mole could be found. If the react’ion with phenyl isothiocyanate is allowed to proceed for a longer time (6 hours) in the alkaline, aqueous pyridine-triethylamine solvent., some peptide bond cleavage takes place, but even under these conditions no single residue approaches a suitable value. From this study it becomes apparent that the NHz-terminal residues of rat liver Ma lactate dehydrogenase are not readily available for reaction with either fluorodinitrobenzene or phenyl isothiocyanate, under a wide variety of conditions. These results are in contrast to a brief report by Appella (58) on the presence of 8 NHz-terminal valines per mole of bovine heart Hk lactate dehydrogenase with the use of the 2,4,6&rinitrobenzenesulfonic acid method (59-61). Our results are consistent with the find- ings of Dabich (62) that rat liver Mq lactate dehydrogenase is markedly resistant to hydrolysis by leucine aminopeptidase,

while, in contrast, carboxypeptidase is able to hydrolyze up to’ 20 y0 of the molecule without loss of enzymatic activity.

The possibility of an NHrterminal acetyl group in rat liver Mq lactate dehydrogenase was considered in view of the negative results from the determination of NHz-terminal residues, the lack of reactivity with leucine aminopeptidase (62)) and the increasing number of proteins which have been shown to possess an NH2- terminal acetyl residue.

The method for the isolation of N-acetylamino acids from a- Pronase digest of a protein, as described by Narita (48)) requires large quantities of protein, which are difficult to obtain with this enzyme. In addition these determinations are difficult to esti- mate because of losses on handling. Thus to provide definite- information concerning the acetyl status of lactate dehydrogenase as well as the number of such residues, we assayed for acetyl residues in rat liver Mb lactate dehydrogenase. Several methods for acetyl determinations have been described in the literature (25-33), but those used for the study of acylated protein hy- drolysates (2528) require fairly large quamities of protein (0.5 to 10 pmoles) and, with the exception of the method of Ward and Coffey (28), are not convenient for routine use. The microen- zymatic method described in this paper has an advantage in sensitivity (0.1 to 0.2 Mmole of protein sample) over the other methods. Recovery of acetate is better than that reported by Phillips (25) in the only method of comparable sensitivity. The drawback to the present method lies in the presence of inhibitors of the enzymatic reactions which appear in acid hydrolysates of all proteins which we have studied. This requires the extraction of the acetic acid from the hydrolysate and necessitates several determinations on each extract to ensure a proportional response. Additional studies on this method are being carried out, and tentative results* indicate that a modification of this method will allow the analysis of protein hydrolysates directly. Various conditions for the acid hydrolysis of proteins have been tested, but all methods produce the inhibitor. Under alkaline hydroly- sis conditions (1 N NaOH, llO”), large quantities of acetate ap- pear even in bovine serum albumin, a protein which has no known acetyl groups. This acetate is identified on the basis of the specificity of the microenzymatic assay (see “Materials and Methods”). It presumably arises from the decomposition of one of the amino acids (possibly threonine) in the protein and also occurs in a mixture of amino acids treated in the same man- ner. This demonstrates the unreliability of determinations of acetate after alkaline hydrolysis.

Pearson (34) has recently described a source of error in the quantitative acetyl-CoA assay of Ochoa, Stern, and Schneider (63). This error arises because of the equilibrium state of the malate dehydrogenase-catalyzed reaction. The error may be eliminated by the addition of DPNH to the assay system at the outset so as to establish an initial ratio of DPNH to oxalacetate of about 10: 1. When Pearson’s paper (34) appeared, we had noted the requirement for added DPNH in our assay system to achieve quantitative results but had not uncovered the reason for this requirement. Further experiments with our coupled assay system involving acetate instead of acetyl-CoA have con- firmed Pearson’s calculations. Certainly his comments on the errors involved with coupled enzyme systems are most pertinent and must be considered in any use of a coupled enzyme system in

* L. Stegink, unpublished experiments.

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Issue of November 10, 1966 L. D. Stegink and C. S. Vestling 4929

which one product or reactant in a given equilibrium reaction is measured and another reacts further.

As can be seen from Table III, t.he acetyl values from bovine serum albumin, ovalbumin, and horse heart cytochrome c are in good agreement with the expected values (26,45). Rat liver Mh lactate dehydrogenase samples contained between 6 and 8 moles of acetate, usually closer to 8. If the tetramer hypothesis is correct, and if rat liver Md lactate dehydrogenase possesses N- acetylamino residues at its amino end, we would expect 4 moles of acetate per mole or a multiple. Samples of bovine heart iso- zymes (MH, a.nd H4) also show the presence of 7 to 8 moles of acetate per mole. The figure for rat liver lactate dehydrogenase has been confirmed by the method of Phillips (25). It is interest- ing to note that chicken heart Hq lactate dehydrogenase contains only 3 to 4 acetyl residues in contrast to the other lactate dehy- drogenases studied. Chicken heart Hq lactate dehydrogenase is somewhat unusual in other respects. It has a much slower elec- trophoretic mobility than would be expected of a Hq protein (5)) and resembles in this respect a M( lactate dehydrogenase. It is also capable of hybridizing with other Hd proteins (7,8,64,65). This enzyme has been tentatively assigned 3 to 4 moles of COOH- terminal leucine (8). Our results from chicken heart Hk lactate dehydrogenase are consistent with four polypeptide chains, but the results from bovine heart Hq, bovine heart MiH3, and rat liver Mq lactate dehydrogenase are more consistent with eight polypeptide chains if all the acetyl groups are NH9terminal. Appella (58) has made a preliminary report of fingerprint studies which are consistent with the presence of eight polypeptide chains in bovine heart lactate dehydrogenase, and he also reported the presence of 8 NH&erminal valine residues per mole. We have found 7 moles of acetyl groups per mole of bovine heart Hk lac- tate dehydrogenase; thus, either all the acetyl groups found are not NHz-terminal in this lactate dehydrogenase or the procedure used for NHz-terminal studies (59-61) in some way led to the hydrolysis of the acetyl residues.

There are other puzzling experiments which are not consistent with the tetramer hypothesis. Fritz (9) has reported t,he separa- tion of crystalline rabbit muscle lactate dehydrogenase into 12 enzymatically active bands on electrophoresis. Fritz and Jacobson (10) have also studied the effect of 2-mercaptoethanol on the pattern of lactate dehydrogenase isozymes and have dem- onstrated that 2-mercaptoethanol can affect the pattern of sub- bands as well as t.he rate of electrophoretic migration. On the basis of their results, they have questioned the tetramer theory.

One possible source of acetyl groups in rat liver Mh lactate .dehydrogenase could be the presence of a heretofore undetected carbohydrate fraction containing N-acetylgalactosamine or N-acetylglucosamine. Although rat liver lactate dehydrogenase gives a consistently positive result in the phenol-sulfuric acid method of Montgomery (37)) the values fluctuate widely. At the moment there appears to be a threshold level of positive material which cannot be readily lowered. Analysis of the “neutral car- bohydrate function” showed only the presence of glucose, but no mannose or galactose. Uniformly labeled 14C-glucose does not readily exchange into the phenol-sulfuric acid-positive material found in rat liver Mq lactate dehydrogenase. The glucose found in the neutral carbohydrate fra.ction after hydrolysis either is tightly bound or is present as oligosaccharide. Tanimura and Ishidate (66) have reported the binding of large amounts of glucuronic acid and smaller amounts of glucose to ovalbumin.

It is possible that lactate dehydrogenase contains some glucose bound in a similar manner, but the lack of binding of the 14C-glu- case makes this seem unlikely. After Bio-Gel P-100 chroma- tography, lactate dehydrogenase contains less phenol-sulfuric acid-positive material than is present initially. Such samples should bind 14C-glucose much more readily than lactate dehydro- genase which already contains 6 to 8 moles of galactose-mannose equivalents. This indicates to us that the carbohydrate mate- rial consists of oligosacchride derived from the cellulose columns used in purification. If lactate dehydrogenase were indeed a glycoprotein, we would expect to find either galactose or man- nose, or both, as has been found in nearly all the mammalian glycoproteins isolated to date (67, 68). In addition, the acetate values for rat liver Mh lactate dehydrogenase do not fluctuate with the carbohydrate content. For example, the lactate dehy- drogenase isolated following the Bio-Gel P-lOO-ammonium sul- fate treatment had a value of 7, while material not passed over the column had a value of 7.7.

These results lead us to the conclusion that the NHz-terminal amino acids of rat liver M4 lactate dehydrogenase are not availa- ble for reaction with either fluorodinitrobenzene or phenyl iso- thiocyanate under a wide variety of conditions. In addition, the presence of 6 to 8 moles of acetate per mole of lactate dehydrogen- ase, as well as the lack of reactivity of the enzyme toward leucine aminopeptidase, strongly suggests the presence of N-acetyl groups on the NHz-terminal residues of rat liver M4 lactate de- hydrogenase. The finding of 7 to 8 moles of acetate in the bo- vine heart H4 and MH, hybrids of lactate dehydrogenase and of 3 to 4 residues in the chicken heart H4 lactate dehydro- genase also suggests the presence of these residues in other lac- tate dehydrogenase molecules.

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Lewis D. Stegink and Carl S. VestlingACETYLATION STATUS

Rat Liver Lactate Dehydrogenase: AMINO-TERMINAL AND

1966, 241:4923-4930.J. Biol. Chem. 

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