JOURNAL OF BACTERIOLOGY, Oct. 1974, p. 466-474Copyright 0 1974 American Society for Microbiology

Vol. 120, No. 1Printed in U.S.A.

f3-Galactosidase from Termination and DeletionMutant Strains

MERNA R. VILLAREJO AND IRVING ZABIN

Department of Biological Chemistry, School of Medicine, and Molecular Biology Institute, University ofCalifornia, Los Angeles, California 90024

Received for publication 19 July 1974

,B-Galactosidase fragments were isolated from strains of Escherichia coli withmutations in the lacZ gene. The polypeptide obtained from a terminationmutant (lacZNG125) appeared to be the intact gene product, containing the firsthalf of the ,3-galactosidase amino acid sequence. From an internal deletionmutant strain (lacZU163), an aggregate was obtained of several partially de-graded polypeptides. Each of these was smaller than predicted from geneticdata for the fragment. Introduction of the lacZU163 mutation into a proteindegradation-deficient strain (Deg-) resulted in the protection of the amino-ter-minal region of the protein. Some of the BrCN peptides from the U163polypeptides were separated and identified. From such experiments it was shownthat in both Deg- and Deg+ strains the COOH-terminal region is rapidlydegraded. This indicates that the complete gene product of lacZU163 has notbeen detected. The use of genetically defined enzyme fragments in studyingstructure-function relationships and in determination of primary structure isdiscussed.

The Z gene (specifying ,-galactosidase, EC3.2.1.23) of the lac operon of Escherichia colihas been studied extensively by genetic tech-niques (1). An additional approach to fine-structure mapping of the gene is through thedetermination of the structure of proteins pro-duced by mutant strains. The protein productof the wild-type Z gene is an enzyme composedof four identical subunits of molecular weight135,000, each containing an active site (21).Many terminator and deletion mutant strainsare available that produce only a portion of the,B-galactosidase polypeptide chain. These mu-tant strains produce polypeptides which, al-though enzymatically inactive, retain some as-pects of native conformation. For example, theability to cross-react with antibody preparedfrom the wild-type enzyme (6) and the presenceof a substrate binding site in certain incompletepolypeptides (19) indicate that some of thesefragments have a tertiary structure similar tothat of ,3-galactosidase itself. Isolation of a vari-ety of such mutant proteins will contribute to anunderstanding of structure-function relation-ships of the enzyme.

f,-Galactosidase polypeptides from two termi-nation mutant strains have been isolated inpure form (2, 3). Genetic mapping of both theX90 and NG200 mutations indicates that thesites of mutation are in the COOH-terminal

third of the Z gene (Fig. 1). The molecularweights of the enzyme polypeptides (122,000and 89,000, respectively) were found to be ingood agreement with the size predicted from theposition of the mutation. Using a site-specificreagent and affinity chromatography, we havedemonstrated a normal substrate binding site ineach of these enzyme fragments (19). Theselong-chain polypeptides are relatively stable toin vivo proteolysis in contrast to short-chaintermination and deletion fragments, which arerapidly degraded in vivo (11).

In this report, we describe the isolation of,B-galactosidase fragments from two additionalmutant strains. The NG125 polypeptide is de-rived from a strain with an amber mutationnear the middle of the Z gene and should,therefore, contain the amino acid sequence ofthe NH2-terminal half of the ,8-galactosidasemonomer. The U163 fragment is the product ofan internal deletion mutation that has resultedin the loss of approximately 50% of the geneticmaterial (Fig. 1). We have shown previouslythat these two polypeptides are retarded on anaffinity column, suggesting that they have weaksubstrate binding sites (19). An important prop-erty of these particular fragments is their rela-tive stability. Nonetheless, we encountered dif-ficulties owing to degradative processes. Buk-hari and Zipser (4) have identified mutations in

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V,-GALACTOSIDASE FROM TERMINATION MUTANTS

P 0 z

NG 125 NG 200 X90IUU 163

FIG. 1. Genetic map of the lac Zgene. Terminatingmutations are shown above the line. The extent ofdeletions is given below the line.

the deg locus that result in reduced rates ofproteolysis of,-galactosidase mutant proteins.We have utilized a Deg- strain to aid in theisolation of one of the mutant polypeptides.

Information on the primary structure of thesepolypeptides would be of significance in thedetermination of the amino acid sequence of,8-galactosidase (now in progress in this labora-tory) (5, 7, 22). We have used cyanogen bromideto obtain peptides that can be compared withthose from the complete enzyme. The BrCNpeptides present in the mutant proteins can beassigned to a genetically defined region of theamino acid sequence. This approach may alsolead to a precise definition of the point ofmutation in these strains.

MATERIALS AND METHODS

Bacterial strains. Bacterial strains used are deriv-atives of E. coli strain K-12: (i) E. coli 8049, (LacPro)del Deg- StrR (kindly supplied by David Zipser;4); (ii) M15/F'M15 (DZ291), StrR lacZM15/F'lacZM15; (iii) NG125/F'NG125 (DZ404), StrRlacZNG125/F'lacZNG125; (iv) U163/F'U163 (DZ439),StrR His- Arg- lacZU163/F'lacZU163; (v) DZ444, E.coli 8049/F'lacZU163. Strains with DZ numbers were

constructed and genetically characterized by PatriceJ. Zamenhof of this department. Standard tech-niques of homogenote formation and genetic analysisof merodiploids were employed (14, 23).

Growth of strains and preparation of extracts.The E. coli partial diploid strains DZ291, DZ404, andDZ439 were grown on a minimal salts-glycerol me-dium and induced with 5 x 10-4 M isopropyl thi-ogalactoside as described previously (11). StrainDZ444 grows poorly on minimal medium and was

therefore grown on antibiotic medium 3 (Difco) andinduced with 5 x 10-4 M isopropyl thiogalactoside.The cells were harvested by centrifugation while stillin the exponential phase of growth. The cell pelletswere suspended in a solution of 0.05 M NaPO4 and0.02 M mercaptoethanol (pH 7.2) and were broken bysonic oscillation. The extract was centrifuged at48,000 x g for 30 min to remove cell debris. Nucleicacids were precipitated from the supernatant solutionby addition of a 40% solution (wt/vol) of streptomycinsulfate to a final concentration of 5%. After overnightincubation at 4 C, the streptomycin precipitate was

removed by centrifugation and discarded. A saturatedsolution of ammonium sulfate (pH 7.0) was thenadded slowly to a final concentration of 40%. After 2days at 4 C, the protein precipitate was collected bycentrifugation and redissolved in a solution of 0.05 MNaPO4 and 0.02 M mercaptoethanol (pH 7.2).

Enzyme and protein assays. ,B-Galactosidase wasassayed by hydrolysis of o-nitrophenyl-fl-D-galactosi-dase (Calbiochem) in 0.1 M NaH2PO4-2 x 10-3 MMgSO4-2 x 10-4 MnSO4 (pH 7.0; PM-2 buffer)containing 0.05 M 0-mercaptoethanol (10). Proteinwas measured by the Lowry method (12).Enzyme a-complementation assays. A polypep-

tide to be tested for a-donor activity in the com-plementation test was treated with 10 times its weightof cyanogen bromide in 70% formic acid. After 20 h atroom temperature, the reaction mixture was diluted10-fold with water and lyophilized. The dried powderwas thoroughly suspended in PM-2 buffer by briefsonic oscillation, and samples were removed for com-plementation. A saturating amount of M15 a-accep-tor was used. This was prepared from DZ291 asdescribed above. After incubation at 28 C for 3 h, thesamples were assayed for ,8-galactosidase activity.SDS polyacrylamide gel electrophoresis. Large

polypeptides were separated, by the method of Weberand Osborne (20), on 10% polyacrylamide gels con-taining 0.1% sodium dodecyl sulfate (SDS). Disc gels(0.5 by 9.0 cm) were run at 8 mA/gel for 5 or 6 h.

Recovery of a-donor complementation activityfrom polytcrylamide gels. Electrophoresis was car-ried out on SDS polyacrylamide gels as describedabove. The gels were removed from the tubes, frozen,and sliced into 1-mm thick sections with a Mickle GelSlicer. Each 1-mm section was minced in 0.21 ml offormic acid in a conical centrifuge tube, 0.09 ml of aBrCN solution (40 mg/ml) was added, and the cov-ered tubes were incubated at 37 C for 15 h. Water (2ml) was added to each tube, the contents were mixedthoroughly, and the pulverized gel was removed bycentrifugation. The supernatant solution was care-fully decanted and lyophilized. The peptides werethen dissolved in 0.4 ml of PM-2 buffer, and 20 zlitersof the M15 cell extract was added. After incubation at28 C for 3 h, the fractions were assayed for ,-galactosi-dase activity.SDS-urea polyacrylamide gel electrophoresis.

Small peptides obtained by BrCN reaction was sepa-rated, by the method of Swank and Munkres (17), on12.5% polyacrylamide gels containing 0.1% SDS and 8M urea. Disc gels (0.5 by 9.0 cm) were run at 2.5mA/gel for 15 to 20 h.

RESULTSPurification and characterization of a

polypeptide from termination mutant strainNG125/F'NG125. A selective adsorbent foraffinity chromatography was prepared by cou-pling a ,B-galactosidase substrate analogue to anagarose matrix (16). The dialyzed extract wasapplied to the affinity column equilibrated with0.05 M NaPO4-0.02 M mercaptoethanol (pH7.2) at room temperature, and the column waswashed with the same buffer. Under these con-ditions, the polypeptide produced by the NG-125 termination mutant strain was retarded, butnot retained, by the affinity column as describedpreviously (Fig. 2). The proteins in the active

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fractions were concentrated by precipitationwith (NH4)2SO4, dialyzed, and reapplied tothe same affinity column, resulting in a nearlytwofold further purification (Table 1).The purified NG125 polypeptide was exam-

ined by SDS acrylamide gel electrophoresis (20)and found to be greater than 90% pure. Themolecular weight obtained by this method was

60,000. The NH2-terminal amino acid, deter-mined by dansylation of the intact protein (9),was threonine, confirming the homogeneity ofthe isolated polypeptide. An amino acid analy-

sis of the NG125 polypeptide is shown in Table2.The interaction of the NG125 polypeptide

with the deletion mutant protein from M15 toform complemented enzyme is illustrated inTable 3. Fragmentation into smaller peptidesby reaction with BrCN results in a 25-foldincrease in the extent of enzyme complementa-tion, as compared to the intact protein.

Purification and characterization of a

polypeptide from deletion mutant strainU163/F'U163. An extract containing the U163

1.5

1.0

I

EC

4c

0.5

0

1.0

0.5

0

I

0.4 Eo06N

0.2 41

0

0

4

zw

aw

ILa

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0.4w

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50 100 150FRACTION NUMBER

FIG. 2. Affinity chromatography of enzyme fragments. A 5-ml volume of extract was applied to column (0.8by 55 cm) in a solution of 0.05 M NaPO4, and 0.02 M mercaptoethanol (pH 7.2) and eluted with the same bufferor with buffer plus 0.1 M NaCl, as indicated. Fraction volume was 1.5 ml; flow rate was 35 ml/h.

TABLE 1. Isolation of NG125 and U163 polypeptides

Enzyme a-comple-Purification step mentation Protein Sp act Recovery

(,-galactosi- (mg) (U/mg of protein) (%)dase units)

NG125 polypeptideCrude extract 9,500,000 4,230 1,530 100Ammonium sulfate precipitation 7,400,000 630 11,700 78First affinity chromatography 2,485,000 36 82,500 26Second affinity chromatography 1,478,000 10 145,450 16

U163 polypeptideCrude extract 4,650,000 5,180 900 100Ammonium sulfate precipitation 3,090,000 670 4,600 67First affinity chromatography 1,020,000 68 15,000 22Second affinity chromatography 630,000 6 105,000 14

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1-GALACTOSIDASE FROM TERMINATION MUTANTS

TABLE 2. Comparison of the amino acid compositionsof f3-galactosidase and mutant enzyme fragments

No. of amino acid residuesper molecule

Amino acid 0-galac-tosidase NG125 DegU

(7) U163

Lysine .............. 27 13 17Histidine ............ 36 16 13Arginine ............. 73 39 30Aspartic acid ........ 122 64 69Threonine ........... 70 23 29Serine.69 17 34Glutamic acid ...... . 145 68 69Proline.71 34 29Glycine.84 42 47Alanine.94 44 50Half-cystine.17 8Valine .............. 75 45 44Methionine.21 13 12Isoleucine.44 24 22Leucine.112 54 48Tyrosine.34 16 17Phenylalanine.45 21 19Tryptophan . . 32

polypeptide was applied to an affinity columnunder the same conditions as described above.The U163 polypeptide was retained on the col-umn, indicating that it binds more tightly to asubstrate analog than NG125 protein (Fig. 2).Elution from the affinity column was achievedby a step increase in ionic strength. Considera-ble further purification was obtained by reap-plication to the selective adsorbent and elutionwith an ionic strength gradient (Table 1).The ,B-galactosidase fragment isolated from

strain U163/F'U163 by affinity chromatographyappeared to be homogeneous by several criteria.Sedimentation equilibrium studies of the nativemolecule indicated a single species with a

molecular weight of 98,000. When the nativestructure was subjected to gel filtration on

SephadeX G-150, it emerged in a sharply de-fined zone exactly corresponding to the peak ofcomplementing activity. However, under dis-sociating conditions, three major componentswere found to be present. The molecularweights of the dissociated components deter-mined by SDS acrylamide gel electrophoresiswere approximately 65,000, 52,000, and 12,000.The polypeptide of molecular weight 52,000 wasthe major component (Fig. 3). Direct dansyla-tion of the U163 polypeptides showed thatalanine was the NH2-terminal amino acid pres-ent in largest amount. Threonine was alsopresent in smaller quantity.

Further analysis of the isolated fragments wasundertaken to determine whether the polypep-tide of molecular weight 65,000 represents theundegraded gene product and whether all of thefragments observed were actually derived fromthe mutant,-galactosidase.The presence of an intact NH2-terminal re-

gion of a ,B-galactosidase derived polypeptidecan be detected by using enzyme a-complemen-tation as an assay. For this test, the polypep-tides derived from strain U163/F'U163 wereseparated by SDS acrylamide gel electrophore-sis. The gel was sliced and treated with BrCN,and the eluted peptides were incubated with anM15 extract. The main component (52,000) didnot contain the enzyme complementing activity(Fig. 4). The NH2-terminal region was presentprimarily in the peptide of molecular weight12,000. A small amount of activity was alsopresent in the position corresponding to thelargest peptide (65,000).To confirm this result, the peptides of the

aggregate were separated by gel filtration. Thepeptides were dissolved in a solution of 30%acetic acid, 8 M urea, and 0.1 M mercaptoetha-nol and applied to a Sephadex G-150 column, in30% acetic acid (Fig. 5). More than 70% of thecomplementing activity was found in the small-est peptide. Electrophoretic analysis of thepooled fractions indicated that the two largepolypeptides (65,000 and 52,000) were presenttogether in first 20 fractions; the amount ofcomplementation activity in these fractions wasproportional to the amount of the larger peptidepresent.The fractions containing the two larger en-

zyme fragments were treated with BrCN, andthe resulting peptides were examined by theSDS-urea polyacrylamide gel system of Swankand Munkres (17). fl-Galactosidase was treatedin the same manner to compare the two peptidepatterns (Fig. 6). All bands from the fragmentscorresponded in position to bands from ,B-galac-tosidase, indicating that the peptides in theisolated aggregate were derived from the U163 Zgene product.

Purification and characterization of a

TABLE 3. Effect of BrCN treatment on enzymea-complementation activity of mutant polypeptides

Relative a-comple-Treatment mentation activity

NG125 U163

None ........................ 1 1BrCN ............. 25 85

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65,000

52,000°

1 2,'000tjjy. .

A S CFIG. 3. Comparison of U163 and Deg- U163 polypeptides by SDS-polyacrylamide gel electrophoresis.

Approximate molecular weights for the polypeptides are indicated. (A) Deg- U163 polypeptides (30 Ag), (B)U163 polypeptides (40 jg), (C) Deg- U163 and U163 polypeptides were mixed before being applied to the gel.

polypeptide from a degradation-deficientmutant strain U163. Because the active ag-gregate was partially degraded, an episome car-rying the U163 mutation was introduced into arecipient (strain 8049) carrying a deg- mutation,which renders the cell deficient in protein deg-radation. The U163 polypeptide was isolatedfrom the Deg- strain in the manner describedabove. Sedimentation equilibrium analysis ofthe purified material indicated that it was a ho-mogeneous protein of molecular weight 63,000±i 3,000. Electrophoresis on SDS acrylamidegels showed that the polypeptide of approxi-mately 65,000 molecular weight was the maincomponent of the Deg- U163 strain (8049/F'lac-ZU163), but reduced amounts of the other pep-tides were also present (Fig. 3). When a-com-plementing activity was eluted from an SDSpolyacrylamide gel as described above, onecomponent with activity was found correspond-ing to the mobility of the larger chain of molecu-lar weight 65,000 (Fig. 4).The BrCN peptide active in the a-com-

plementation reaction was isolated from theDeg- U163 protein. After treatment of theprotein with 50 times its weight of BrCN in 70%formic acid, the resulting BrCN peptides wereseparated by using Sephadex G-25 and carboxy-methyl cellulose columns. The complementingpeptide from the Deg- U163 strain, whichrepresents residues 3 through 92 in the fl-galac-tosidase sequence, was identified by its activityin enzyme complementation, by its characteris-tic position in the elution pattern (Figure 7), byits mobility in urea and in SDS acrylamide gelelectrophoresis, and by identification of itsNH2-terminal amino acid, isoleucine, by dansy-lation (K. Langley, A. V. Fowler, and I. Zabin,manuscript in preparation).

If the Deg- U163 polypeptide represents theundegraded, properly translated gene product,it should contain the COOH-terminal BrCNpeptide as well. The expected position (A. J.Brake, A. V. Fowler, and I. Zabin, unpublisheddata) of the COOH-terminal peptide in a car-boxymethyl cellulose-elution pattern is shown

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V,-GALACTOSDDASE FROM TERMINATION MUTANTS

by the arrow in Fig. 7. The peptides from thatportion of the column are shown separated onurea gels in Fig. 7. This result shows that theDeg- U163 polypeptide of molecular weight65,000 is not the complete, properly translatedgene product because it does not contain theCOOH-terminal sequence of ,-galactosidase.An amino acid analysis of Deg- U163 is shownin Table 2.The ability of Deg- U163 polypeptide to

donate its amino-terminal region for in vitro

135,000

z'60

0

402w-ja.

0u

w 65,000~20

zw I12,000

00.2 0.4 0.8

MOBILITYFIG. 4. Estimation of the molecular weight of the

a-complementing fragments by recovery of activityafter SDS polyacrylamide gel electrophoresis. ,-Galactosidase (50 pg), U163 (25 Mg), and Deg- U163polypeptide (20 Mg) were run on separate gels. Molec-ular weights shown above the peaks were estimated bycomparison of mobilities with standard proteins ofknown molecular weight. Symbols: , ,j-galactosi-dase; 0, U163; -----, Deg- U163.

v0 20 30 40

FRACTION NUMBER

FIG. 5. Separation of polypeptides of the U163 ag-gregate on Sephadex G-150. The polypeptides wereseparated on a column (1.5 by 80 cm) in 30% aceticacid. Fractions (1 ml) were collected and pooled asindicated by the arrows. Pooled fractions were testedfor a-complementation as shown in the bar graph.

enzyme complementation is illustrated in Table3. The amino terminal region reacts very poorlywhen present in the intact molecule; the reac-tion is enhanced 85-fold by treatment withcyanogen bromide. This effect is also observed

A SFIG. 6. Diagram of BrCN peptides from ,8-galac-

tosidase and U163 separated by SDS-urea polyacryl-amide gel electrophoresis. (A) j-galactosidase; (B)U163.

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FIG. 7. Separation of BrCN peptides from Deg- U163 and ,8-galactosidase on carboxymethyl cellulose. Thepeptides were applied to a column (1.5 by 20 cm) of carboxymethyl cellulose in a solution of 0.02 M ammoniumacetate and 8M urea (pH 4.7) and eluted with a linear ionic-strength gradient of NaCI. The arrows indicate theexpected position of the COOH-terminal BrCN peptide of ,B-galactosidase. The insert (upper left) showsseparation of the peptides from fractions A and B on 8 M urea, polyacrylamide disc gels (pH 8.6) (13).

in vivo. We prepared the partial diploid lac-ZU163/F'lacZM15 and found it to have a Lac-phenotype. This indicates that no functionalcomplemented enzyme is formed, despite thepresence of both necessary enzyme fragments inamounts exceeding 1% of the soluble cell protein(18).

DISCUSSIONThe product of the Z gene, ,B-galactosidase,

has a monomer molecular weight of 135,000.Isolation and characterization of geneticallydefined incomplete enzyme polypeptides result-ing from mutations can contribute to fine-struc-ture mapping of the Z gene and constitute anapproach to analyzing structure-function rela-tionships in the wild-type enzyme.The molecular weight of the purified NG125

polypeptide is 60,000, in good agreement withthe length predicted by genetic data. Dansyla-tion of the intact protein indicated that threo-nine was the amino terminal amino acid, as it isin the wild-type enzyme. The high specificactivity obtained in the a-complementation

reaction after BrCN treatment is a furtherindication that the amino-terminal region of themolecule was undegraded. It is, therefore, likelythat the polypeptide isolated from a straincontaining the NG125 mutation is the unde-graded gene product, although the possibility ofsome hydrolysis of amino acids at the carboxyl-terminus of the molecule cannot be ruled out.

Strain U163/F'U163 is a deletion mutantmissing a large segment of deoxyribonucleicacid from the interior of the Z gene (Fig. 1).From genetic data, the beginning of the deletionshould correspond to a point in the polypeptidechain about 50,000 daltons from the amino-ter-minal end (15). An amber termination mutantat the end point of the deletion produces aprotein whose molecular weight has been esti-mated at 103,000 by sucrose density gradientcentrifugation (2). Therefore, the predicted mo-lecular weight of the U163 polypeptide is be-tween 75,000 and 80,000. No protein of this sizewas actually obtained from strain U163/F'U163.Instead, the protein aggregate isolated by affin-ity chromatography contained three polypep-

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V-GALACTOSIDASE FROM TERMINATION MUTANTS

tide chains of molecular weights 65,000, 52,000,and 12,000. The largest of these, present inlesser amount, was shown to be intact at theNH2-terminal end by its ability to form comple-mented, active enzyme. The 52,000-molecularweight peptide, which was the major compo-nent, lacked that region of the ,8-galactosidasesequence. Its NH2-terminal segment apparentlyhad been cleaved from the larger polypeptideand appeared in the 12,000-molecular weightpeptide.These results indicated that in vivo proteo-

lytic enzymes in exponentially growing E. colican complicate the task of obtaining geneticallydefined enzyme fragments. Bukhari and Zipser(4) have identified a class of mutations in thedeg locus that resulted in a substantial reduc-tion of proteolysis of mutant proteins. Theirmeasurements in the normal strain of E. coli(Deg+) showed a biphasic rate of proteolysis.The Deg- mutation eliminated the more rapidrate but had no effect on the slower proteolyticprocess (4).The introduction of the U163 mutation into a

degradation deficient strain (Deg-) resulted inan increased proportion of the 65,000-molecularweight peptide, so that it became the maincomponent of the aggregate. This shows thatthe Deg- mutation results in protection of theNH2-terminal end of the polypeptide from pro-teolysis. Characterization of the BrCN peptidesproduced by this mutant protein showed thatthe COOH-terminal sequence is not present,although this portion of the Z gene is intact.Therefore, the absence of the COOH-terminalsequence should be due to translational orpost-translational events. If the U163 deletionresulted in a frameshift, the latter portion of theZ messenger ribonucleic acid would be incor-rectly translated and could contain terminationsignals. If the deletion does not result in aframeshift and translation is accurate, it ispossible that the intact gene product is neverseen owing to rapid proteolysis at the COOH-terminal.The differential effect of the Deg- mutation

on the opposite ends of the U163 sequence couldbe explained if there were two different proteo-lytic systems with differing specificities, onlyone of which is affected by the Deg- mutation.Alternatively, differences in conformation maydetermine the degree of exposure of sensitivepeptide bonds to proteolysis.Data showing that the amino-terminal re-

gions of the NG125 and U163 polypeptides areunavailable for direct enzyme a-complementa-tion (Table 3) suggest that this portion of the

sequence is buried. Studies on degradation ratesin a longer termination mutant, X90, by Linand Zabin (11), indicated that the half-life ofthe amino-terminal region was about 30 min at37 C. Bukhari and Zipser (4) and Goldschmidt(8) determined the overall decay rate of the X90product to be 5 to 7 min under similar condi-tions. These data suggest that in the X90polypeptide the amino-terminal region is lesssusceptible to proteolysis than other parts of theprotein and that differences in conformationplay an important role in protein stability.Further work on the proteolytic enzymes of E.coli is needed to elucidate the mechanisms ofproteolysis and its relation to the cell economy.The identification of two key ,8-galactosidase

BrCN peptides, the a-complementing peptidenear the amino-terminus and the carboxyl-ter-minal peptide, has been useful in the determi-nation of the structure of partially degradedmutant polypeptides. Identification of otherBrCN peptides in the U163 and NG125 digestscan allow .the assignment of each peptide to agenetically defined region of the protein se-quence. Detailed comparison of the BrCN pep-tides derived from incomplete fragments withthose from the native enzyme is in progress inthis laboratory and should be an important aidin determining the primary structure of ,B-galac-tosidase. Conversely, when the complete pri-mary structure of f,-galactosidase is known, theisolation and characterization methods de-scribed here should enable us to define theprecise position of the mutation in many Z-strains.

ACKNOWLEDGMENTSThis investigation was supported by Public Health Service

grant AI-04181 from the National Institute of Allergy andInfectious Diseases.We are greatly indebted to P. J. Zamenhof for the

construction and characterization of bacterial strains. Wethank Douglas Brown for carrying out ultracentrifugal runs,and Audree V. Fowler for many helpful discussions.

LITERATURE CITED1. Beckwith, J. R. 1970. The genetic system, p. 5-26. In J.

Beckwith and D. Zipser (ed.), The lactose operon. ColdSpring Harbor Laboratory, New York.

2. Berg, A. P., A. V. Fowler, and I. Zabin. 19.70. ,-Galactosi-dase: isolation of and antibodies to incomplete chains.J. Bacteriol. 101:438-443.

3. Brown, J. L., D. M. Brown, and I. Zabin. 1967. ,8-Galac-tosidase: orientation and the carboxyl-terminal codingsite in the gene. Proc. Nat. Acad. Sci. U.S.A.58:1139-1143.

4. Bukhari, A. I., and D. Zipser. 1973. Mutants of Esche-richia coli with a defect in the degradation of nonsensefragments. Nature N. Biol. 243:238-241.

5. Fowler, A. V. 1972. The amino acid sequence of ,B-galacto-sidase. II. Tryptic peptides of the maleyated proteinand sequences of some tryptic peptides. J. Biol. Chem.

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247:5425-5431.6. Fowler, A. V., and I. Zabin. 1968. ,-Galactosidase:

immunological studies of nonsense, missense and dele-tion mutants. J. Mol. Biol. 33:35-47.

7. Fowler, A. V., and I. Zabin. 1970. The amino acidsequence of,-galactosidase. I. Isolation and composi-tion of tryptic peptides. J. Biol. Chem. 245:5032-5041.

8. Goldschmidt, R. 1970. In vivo degradation of nonsense

fragments in E. coli. Nature (London) 228:1151-1154.9. Gray, W. R. 1967. Dansyl chloride procedure, p. 139-150.

In C. H. W. Hirs (ed.), Methods in enzymology, vol. 11.Academic Press Inc., New York.

10. Horiuchi, T., J. I. Tomizawa, and A. Novick. 1962.Isolation and properties of bacteria capable of highrates of,-galactosidase synthesis. Biochim. Biophys.Acta 55:152-163.

11. Lin, S., and I. Zabin. 1972. #-Galactosidase: rates ofsynthesis and degradation of incomplete chains. J.Biol. Chem. 247:2205-2211.

12. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-.275.

13. Jovin, T., A. Chrambach, and M. A. Naughton. 1964. Anapparatus for preparative temperature regulated poly-acrylamide gel electrophoresis. Anal. Biochem.9:351-369.

14. Miller, J. H. 1972. Experiments in molecular genetics.Cold Spring Harbor Laboratory, New York.

15. Newton, W. A., J. R. Beckwith, D. Zipser, and S.Brenner. 1965. Nonsense mutants and polarity in thelac operon of Escherichia coli. J. Mol. Biol. 14:290-296.

16. Steers, jun., E., P. Cuatrecasas, and H. B. Pollard. 1971.The purification of ,B-galactosidase from Escherichiacoli by affinity chromatography. J. Biol. Chem.246:196-200.

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