J. Mol. Biol. (1965) 11, 12-22

Multiple Molecular Forms of fi-Galactosidase

S. H. ApPELt, D. H. ALPERst AND G. M. TOMKINS

Laboratory of Molecular BiologyNational Institute of Arthritis aud Metabolic Diseases

National Institutes of H eaUhBethesda, Maryland, U.s.A

(Received 13 July 1964)

In uninduced lactose-fermenting strains of Escherichia coli, ,a-galactosidasemigrated as a single rapidly moving electrophoretic component which, on sucrosegradient centrifugation, has a sedimentation coefficient of 16 s, On induction, atleast 7 electrophoretic forms of the enzyme appeared, the slowest of which had asedimentation coefficient of 34 s. Values for the Michaelis constant of the 16 and34 s forms were similar. Evidence is presented that this pattern is stable andreproducible in vitro, and reversible in vivo.

Nearly identical patterns of galactosidase activity were seen in constitutivebacteria and in various hybrid strains containing an episome derived fromE. coli. It is concluded that these forms represent higher molecular weightaggregates of ,a-galactosidase.

A significant portion of the thiogalactoside transacetylase also existed as highmolecular weight forms as detected by electrophoresis and density-gradientcentrifugation, and could be disaggregated by dialysis or the presence of iso­propyl-ji-n-thiogalaetoside.

1. Introduction

Multiple forms of various enzymes have been reported from many sources (Wroblewski,1961). In Escherichia coli, several electrophoretic forms of alkaline phosphatase havebeen demonstrated as products of a single gene (Levinthal, Signer & Fetherolf, 1962),although the basis for their separation is not known.

In the present communication, multiple forms of the enzymes of the lactoseoperon of E. coli, ,B-galactosidase and thiogalactoside transacetylase, have beendemonstrated. These differ from each other at least by molecular weight. The stabilityand reproducibility of the enzyme pattern of ,B-galactosidase suggests that this multi­plicity is not the result of experimental manipulations but represents the intracellularstate of the enzyme.

t Present adress: Department of Neurology, Duke University School of Medicine, Durham,North Carolina, U.S.A.

~ Present address: Department of Medicine, Massachusetts General Hospital, Harvard MedicalSchool, Boston, Massachusetts, U.S.A.

12

MULTIPLE MOLECULAR FORMS OF ,8-GALACTOSIDASE 13

2. Materials and MethodsBacterial strains

The following bacterial strains were used: E. coli K12, strains 200P Flac (i+z+y-jF i+z+y+)t, ABll05 (i+z+y-), W8092 (i+z+y+, galactokinase-Iess), 2Z01c (i-z+y-),2EOIc (i-z+y-), W3876 (lacd e l ) ; E. coli B (i+z+y+); S. marcescens Flac (lac-/F i+z+y+),Salmonella typhimurium Flac (lac- jF i+z+y+); P. mirabilis Flac (lac- jF i+z+y+)t.

Preparation of extracts

Bacteria were grown at 37°C in a New Brunswick rotatory shaker in a synthetic medium(Fraser & Mahler, 1957) 0-9% in glycerol and containing 5 p.gjm!. thiamine hydrochloride.Inducible bacteria were also grown in the presence of 5 X 10- 4 M-IPTG. Most of theexperiments to be described were performed with extracts of E. coli 200P Flac, the doublingtime of which was 50 min under the above conditions. Bacterial extracts were preparedfrom log phase cells grown to a density of 1 to 3 X 109 jm!. The bacteria were centrifugedat 12,000 g for 15 min, resuspended in one-tenth the original volume of 0'05 M-tris pH 7·8(measured at 20°C), chilled to 4°C, and sonicated with a Sonifier (Branson Instrument00., Stamford, Conn.) at a setting of 3 A for 30 sec. The sonicated extract was centrifugedat 30,000 g for 20 min and the pellet discarded. Protoplasts were prepared from E. coli200P Flac, using lysozyme and EDTA (Fraser & Mahler, 1957).

Assays

,a.Galactosidase was assayed at 25°C by a modification of the method of Pardee, Jaco b &Monod (1959) using 0·003 M-ONPG as substrate in a solution which was 0·1 M in sodiumphosphate (pH 7,0), 0·001 M in MgCl2 and 0'01 Min ,a.mercaptoethanoI. The reaction wasterminated by the addition of t vo!. of M-sodium carbonate and the color read at 420 mp:in 10-rom light path cells in a Beckman DU spectrophotometer. One unit of enzyme isdefined as producing 1 mumole of o-nitrophenoljmin at 25°C, pH 7·0. The units of enzymecan be calculated from the E m ax of 3000 for o-nitrophenol obtained under the aboveconditions.§

Thiogalactoside transacetylase was assayed in 0·5-m!. samples containing 25 p.molestris (pH 7'8); 0·5 pmole DTNB; 0·05 p.mole acetyl CoA; 25 p.moles IPTG; and bacterialextract, 0·01 ml. The liberation ofthionitrobenzoic acid was followed at 412 mp' in 10-mmlight path cells, in a Gilford recording spectrophotometer model 2000. One unit of enzymeis defined as that amount which produces 1 mumole of thionitrobenzoic acidjmin at 25°C,pH 7·8. The Em.... for the sulfanion of thionitrobenzoate under the above conditions is15,700. This assay will be described in greater detail elsewhere (Alpers, Appel & Tomkins,1965).

Oatalase was assayed by the method of Beers & Sizer (1952).

Electrophoresis

Electrophoresis in 5% polyacrylamide gel was performed by a modification ofthe methodof Ornstein (1961, in a preprint, Disc Electrophoresis, Canal Industrial Corp., Bethesda).5% rather than 7'5% gels were used to effect a wider separation of the various proteinspecies. In 0-5 X 10 em glass columns, 1 ml. of lower gel was polymerized with ammonium

t Abbreviations used: i, the gene controlling inducibility in the lactose operon; Z, the structuralgene for ,8.galactosidase; y, the structural gene for galactoside permease; IPTG, isopropyl-S.n­thiogalactoside; ONPG, orthonitrophenyl-,8.D-galactoside; DTNB, 5,5 dithio-bis-z-nitrobenzoioacid; acetyl-CoA, acetyl coenzyme A; BNG, 6-bromo-2-naphthyl-,8-D.galactopyranoside; TMG,thiomethyl-,8-D-galactoside.

t We would like to thank the following persons for these bacterial strains: Drs F. Jacob, A. J.Clark, C. B. Anfinsen, A. Weissbach, W. Dreyer, L. Baron and C. Willson.

§ This value was obtained both by determining the optical density of a known concentration ofo-nitrophenol, and by observing the change in optical density produced by complete enzymichydrolysis of a known concentration of ONPG. The values obtained by both methods agreed witheach other and with the value reported earlier by Lederberg (1950), but were lower than that givenby Pardee et al, (1959).

14 S. H. APPEL, D. H. ALPERS AND G. M. TOMKINS

persulfate and then overlaid with 0·4 ml. upper gel which was photopolymerized. Theupper layer was overlaid with 0·001 ml. to 0·05 ml. of crude extracts made 0·15 Min sucroseto prevent mixing of the sample with the buffer. A small amount of bromphenol blue wasadded as a tracking dye, and tris-glycine buffer, pH 8'3, was carefully layered over theextract. Twelve vertical columns were run simultaneously at 200 V for 1 hr at 4°C, atwhich time the tracking dye was near the bottom of the column. The gels were thenremoved from the columns and assayed histochemically for ,8-galactosidase with BNG asdescribed by Burstone (1962). The samples were incubated for 10 to 20 min at 37°C (longerwith extracts from uninduced bacteria), after which they were washed with distilled waterand exposed to an alkaline solution of Diazo Blue B (1,0 mgjml.) for 5 min or until acolored precipitate appeared within the gel. The reaction was terminated by washing withdistilled water and then adding 5 to 10% acetic acid. The samples were stored indefinitelyin 5 to 10% acetic acid with minimum diffusion of the original precipitate lines.

Preparative runs were performed on larger columns measuring 1 em X 15 em and con­taining 5 ml. of polymerized lower gel, 1 ml. of polymerized upper gel, and 1 ml. of enzymeextract. These were electrophoresed for 2 hr at 220 V. After removal of the gels from thecolumns, a longitudinal segment was cut and stained histochemically for ,8-galactosidase.The remainder of the gel was cut into twelve 5-mm thick slices. Each slice was homo­genized with a tight pestle in a Dounce tissue homogenizer (Blaessig Co., Rochester, N.Y.)in 3 ml. of 0·05 M-tris (pH 7·8), and centrifuged at 10,000 g for 5 min. The supernatantfractions were then assayed for ,8.galactosidase and transacetylase as described above.With 5% polyacrylamide gels, only 20 to 25% of the ,8.galactosidase originally appliedwas recovered. With 3% gels, however, adequate separation of the multiple forms presentwas achieved and 65% of the ,8-galactosidase and 30% ofthe transacetylase were recovered.

Starch gel electrophoresis was performed at pH 8·3 according to the method of Smithies(1955)·t

Other methodsAgar gel chromatography was performed according to the method of Andrews (1962),

using 3% purified agar prepared to a final mesh size of 60 to 100. Following extensivewashing of the gel particles with 0·05 M-tris (pH 7'8), 30 ml. of crude bacterial extract wasapplied to a 2·5 em X90 em column and eluted with 1000 ml. of 0·05 M-tris, pH 7·8.

Sucrose density-gradients were performed as described by Martin & Ames (1961).Protein was determined by the method of Lowry, Rosebrough, Farr & Randall (1951);

and RNA by the orcinol technique (Schneider, 1957).

ReagentsDTNB was purchased from the Aldrich Chemical Co., Milwaukee, Wisconsin. Acetyl

CoA, IPTG, TMG, and melibiose were obtained from Mann Research Laboratories (NewYork). ONPG and BNG were purchased from the Sigma Chemical Co. (St. Louis, Mo.),Catalase was obtained from Worthington Biochemical Co. (Freehold, N. J.), and DiazoBlue B was purchased from Nutritional Biochemicals Co. (Cleveland, Ohio). The reagentsfor the polyacrylamide gel electrophoresis were obtained from the Canal Industrial Corp.(Bethesda, Md.). Sephadex G25 was purchased from Pharmacia Co., Uppsala, Sweden.Purified agar was purchased from Difco Laboratories, Detroit, Michigan.

3. ResultsElectrophoretic patterns of (3-galactosidase

When a crude extract of uninduced E. coli 200P Flac was electrophoresed inpolyacrylamide gels and stained with ENG, a single band of (3-galactosidase activitywas seen, while an extract from bacteria, grown in the presence of IPTG, run simul­taneously, exhibited at least seven bands of activity. In fact, in Plate I, nine bandsare apparent in the electrophoresis of extracts from induced cells. The lowest bandwas the most dense and corresponded exactly to the position of the band of basalenzyme from uninduced organisms.

t We should like to thank A. Gottlieb and E. Norris for performing these experiments.

Uni ndu ced Indu ced

PLATE 1. Polyacrylamide gel electrophoresis pattern of f1-galactosidase from induced and un­induced E. coli. Crude extracts were prepared from E. coli 200P Flac induced with IPTG (5 X 1O-4 M)

for 1 hr at 37°C, and from uninduced organisms. They were then applied in O·05-ml. portions to thegel column, electrophoresed for 1 hr at pH 8'3, and stained for f1-galactosidase using BNG assubstrate and Diazo Blue B as post-coupling dye. The migration of the proteins in the gel wastoward the anode, and the lowest band represents the most rapidly migrating component.

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PLATE II. Sedimenta­t.ion pattern of ,a-galacto­sidase in a sucrose gradientand an electrophoreticpattern of enzyme fromvarious fractions. A crudeextract of 0·3 ml. frominduced E. coli 200P Flaccontaining 0·015 ml. cry­stalline catalase (5 mg/ml.)was layered on 4'7 ml. ofa 5 to 20% linear sucrosegradient in 0·05 M-tris,pH 7,4, and centrifugedat 22,000 rev./min for 15 hrat - 8°C in an SW40 rotorusing a Spinco model Lultracentrifuge. Sampleswere collected dropwise,and assayed spectrophoto­metrically for ,a-galacto­sidase and catalase. Thecontents of individualtubes were then appliedto 5% polyacrylamidecolumns, olectrophoresedand stained for ,a-galacto­sidase. The gel patterns oftho rapidly moving andslowly moving compo­nents refer to the tubesencompassed by thobrackets. Tho centralphotograph depicting alls vven bands was obtainedfor gel electrophoresis oftubes 12 to 14 taken fromthe peak of ,a-galacto­sidase activity. -e-e-,,B.galactosidase;

0-- 0--, catalase.

MULTIPLE MOLECULAR FORMS OF f./-GALACTOSIDASE 15

When extracts from induced cells were electrophoresed and stained with a solutioncontaining ONPG (similar to that described above for the f./-galactosidase spectro­photometric assay), yellow bands appeared which were faint and diffused rapidly.However, those bands which appeared immediately corresponded to the densestbands seen with the BNG method, indicating that the histochemical reagent measuredthe same activity as that measured by ONPG hydrolysis. To obtain further evidencethat the histochemical and spectrophotometric assays were comparable in theirspecificity, gels were split longitudinally following electrophoresis of an extract.One portion was stained with BNG, and the other portion was sliced into 12 sectionsand f./.galactosidase was eluted and assayed as described above. The activity in theeluates corresponded closely to that of the bands stained histochemically. Whensimilar extracts prepared from E. coli W3876 (lac deletion), were electrophoresedand stained, no f./.galactosidase activity appeared in the gels.

Similar extracts from induced and uninduced bacteria were electrophoresed instarch gel and stained with BNG. A single band of activity was found in the uninducedextract, while at least seven bands were found after induction.

Stability of electrophoretic patterns

The electrophoretic pattern of f./-galactosidase was the same whether sonicatedextracts or osmotic lysates of protoplasts were examined. These patterns were stablewhen the extracts were stored at 4 or at - 20°0 for several days, or when passedthrough Sephadex G25.

The fastest moving component was isolated by elution from the gel, concentrated100·fold by lyophilization, and re-run on polyacrylamide. This revealed the fastcomponent, together with a small amount of the third most rapidly moving band.However, when the intracellular enzyme concentration increased 100-fold on in­duction in vivo, the full pattern of bands was seen.

Neither the volume of extract applied to the gel nor the number of enzyme unitsused significantly affected the pattern seen. Dilution of the enzyme 1000-fold in0·05 M-tris pH 7·8 (with or without 0·001 M-MgOlz, 0·1 M-,8-mercaptoethanol, or0·01 M-IPTG), rapid freezing and thawing, pH changes from pH 6 to 10, and lyo­philization did not alter the pattern of the multiple electrophoretic forms.

Sucrose density-gradient patterns of f./.galactosidase

Extracts from induced E. coli 200P Flac were sedimented in a 5 to 20% sucrosegradient and fractions were collected and assayed spectrophotometrically for ,8­galactosidase. As seen in Plate II, there was a single asymmetric peak of activitywith an S value of 16 s, which was skewed in the direction of the more rapidlysedimenting material. In fact, 30% of the total activity sedimented with an S valuegreater than 20 s.

Samples were taken from various portions of the gradient, and polyacrylamideelectrophoretic patterns for f.l-galactosidase activity developed. Samples from themore rapidly sedimenting portion of the gradient yielded only the slowest movingelectrophoretic bands, whereas samples from the most slowly sedimenting portions ofthe gradient yielded only the fastest electrophoretic band (Plate II). The peak ofactivity from the gradient and all tubes between the two extremes demonstratedthe complete pattern of seven bands. However, between these extremes of heavyand light components, there existed a continuum with the heavier material giving a

16 S. H. APPEL, D. H. ALPERS AND G. M. TOMKINS

higher density of slow-moving electrophoretic bands and the lighter material givinga higher density of fast-moving electrophoretic bands.

Equal portions from sucrose gradient fractions which possessed only either therapidly or slowly migrating electrophoretic components were recentrifuged to deter­mine their sedimentation coefficients. The peak of activity from the heavy fractions,which showed the three slow electrophoretic bands, sedimented asymmetrically withan S value of about 34 s. The peak of activity from the light fractions, which showedonly a single fast-moving electrophoretic band, sedimented symmetrically with an8 value at about 16 (Fig. 1). When the heavy and light fractions were combined

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and centrifuged in the same tube, only the two peaks, 16 and 34 s, were obtained.When the 34 s peak was electrophoresed in polyacrylamide, the protein staincorresponded exactly to the stain for enzyme activity, suggesting that the heaviestcomponent was relatively pure. A third centrifugation of both the 16 and 34 s formsgave sedimentation patterns identical to those of the second centrifugation. Whenthe 34 s form, isolated by centrifugation, was stored at 4°0 for a week or longer andelectrophoresed, a small amount of the band, corresponding to the 16 s form of theenzyme, could be detected.

These experiments showed that the separation of the various forms of fj-galacto­sidase by electrophoresis was, at least partly, on the basis of differences in molecularweight. It also appeared that the presence of multiple forms of fj-galactosidase was

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PLATE. III. j3-galactosidase activity of eluates from agar gel column, and electrophoretic patternof enzvrne from various fractions. The conditions were as indicated in Materials and Methods.Samples were applied to polyacrylamide gel columns, electrophoresed, and stained for f3-galacto­sidase.

MULTIPLE MOLECULAR FORMS OF ,B-GALACTOSIDASE 17

not an artifact of the electrophoretic technique, since single bands of differentelectrophoretic mobility could be detected after separation by gradient centrifugation.The experiments further indicated that the multiple forms of the enzyme were stable.

Agar gel chromatography

In order to confirm the correlation of electrophoretic mobility with sedimentationcharacteristics, crude bacterial extracts were subjected to agar gel filtration. Thistechnique separates primarily on the basis of molecular weight, and as a result thelarge components emerge from the column before the smaller ones. Plate III demon­strates the elution of ,B-galactosidase activity from the agar gel column and theelectrophoretic patterns of various eluates. When higher molecular weight fractionswere electrophoresed, only the slowly moving components were present, while lighterfractions from the trailing edge yielded only the most rapidly moving component.Fractions from the peak yielded the entire range of seven bands.

DEAE-cellulose chromatography

An extract of E. coli 200P Flac was treated with spermine, fractionated withammonium sulfate, and passed through a column of Sephadex G200. It was thenapplied to a column of DEAE.cellulose and eluted by a 0·1 to 0·5 M-KCI gradientin 0·5 M-tris buffer (pH 7·4). Thisprocedureproducedapeakof,B-galactosidaseactivitywith a purification of 20-fold over the crude extract. Fractions from the leading edge,the peak, and the trailing edge of the elution pattern of ,B-galactosidaseactivity werethen electrophoresed in polyacrylamide. The entire pattern of at least seven bandswas seen in all fractions examined.

Properties of forms of ,B-galactosidase

Since the sedimentation coefficient of the heaviest isozyme was close to that of the30 s ribosomal subunit, the possibility was considered that the heavy ,B-galactosidasecomponents were associated with ribosomal particles. This did not appear to be thecase, since these heavy components were detected both in crude bacterial extractsfrom which nucleic acids had been precipitated by spermine and in an RNA-freeextract purified 20-fold with respect to ,B-galactosidaset. In addition, when sucrosegradient fractions containing heavy forms of ,B-galactosidase were assayed by theorcinol method, no RNA was detected.

Despite the differences in sedimentation properties, the 34 s and the 16 s forms ofthe enzyme were found to be similar in various other properties. Figure 2 showsthat the K m values for ONPG using the crude bacterial extract and the two isolated'16 sand 34 s components were the same, 1·3 X10- 4 M.

Studies of enzyme induction

E. coli 200P Flac was induced at 25°C with 5 X 10-4 M·IPTG so that the develop­ment of the enzyme pattern could be studied as a function of time. Figure 3, adiagrammatic representation of the patterns at different times, illustrates theappearance of slowly moving forms of the enzyme. Beginning with the slowest band,the pattern demonstrates the third most rapid band, then the second most rapid band,

t We should like to thank Dr G. Craven for a sample of purified ,B-galactosidase. Subsequentexperiments were performed with enzyme purified by ammonium sulfate fractionation, SephadexG200 filtration, and DEAE chromatography.

2

18 S. H. APPEL, D. H. ALPERS AND G. M. TOMKINS

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!(mMr'FIG. 2. Kinetics of ,8-galactosidase. The crude extract and heavy (34 s) and light (16 s) forms of

,8.galactosidase prepared by sucrose density-gradient centrifugation were assayed with ONPG asindicated in Methods, and the velocity (v) and substrate concentration (8) values plotted asreciprocals to obtain K m (Lineweaver & Burk, 1934). The differences in V max reflect differentamounts of enzyme activity in the various forms used in the assay, and do not reflect differencesin the properties of the enzyme forms.

0' 10' 20' 30' 60' 120'

Length of induction

FIG. 3. Induction of ,8-galactosidase with IPTG at 25°0. E. coli 200 Flac was induced at 25°0with IPTG for the times indicated and extracts prepared. The activity was determined speetro.photometrically and ,8-galactosidase dilutions of the extracts were made with 0·05 M-tris, pH 7·4.Equal amounts of enzyme activity (in 0·001 ml. to 0·5 ml.) were applied to the polyacrylamide gels,electrophoresed, and stained histochemically for ,8.galactosidase. The conditions of migration andpH were similar to those in Plate 1.

MULTIPLE MOLECULAR FORMS OF ,8-GALACTOSIDASE 19

and then the fourth band. The fifth, sixth, and seventh bands appear simultaneously.When fully induced bacteria were washed and allowed to grow in the absence of in­ducer, the slowly moving bands disappeared as the intracellular ,8-galactosidaseconcentration diminished, and the sequence noted in Fig. 3 was reversed. Thus, theformation of heavier forms of the enzyme was readily reversible in vivo, in contrastto the apparent lack of reversibility in vitro. The same pattern of seven electrophoreticforms of ,8-galactosidase was present in extracts from E. coli 200P Flac when inducedwith IPTG or TMG at 5 X 10- 4 M, or lactose or melibose at 10- 3 M. It was alsoidentical in constitutive (2Z01c) and in other inducible strains (ABll05, W8092,E. coli B). Extracts from induced S. marcescens Flac, where the episome was derivedfrom the 200P Flac strain, demonstrated the identical pattern of ,8-galactosidase.The same pattern was also found in extracts from induced S. typhimurium Flac andP. mirabili8 Flac, where the episome differs from thl}t in the 200P Flac strain. Itwas also of interest that the uninduced P. mirabili8 Flac had a high basal level of,8-galactosidase which increased only twofold on induction with IPTG, but this in­crease was associated with a definite increase in the activity of heavier forms of theenzyme.

Thiogalacto8ide tran8acetyla8e

The presence of heavy forms of ,8-galactosidase raised the possibility that multipleforms of thiogalactoside transacetylase might also occur. A crude enzyme extractfrom induced E. coli 200P Flac was sedimented in a sucrose gradient and portionswere assayed for both ,8-galactosidase and thiogalactoside transacetylase. Figure 4illustrates a major peak of transacetylase with an S value of 7, but with a markedasymmetry. In fact, 38% of the acetylase sedimented more rapidly than 15 s.

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FIG. 4. Sedimentation pattern of ,8-galactosidase and thiogalactoside transacetylase activity insucrose. A crude extract of 3·0 ml. of induced E. coli 200P Flac was layered on 27 ml. of a 5 to20% linear sucrose gradient in 0·05 M-tris (pH 7,4) and centrifuged at 22,500 rev./min for 15 hr ina SW25·1 rotor at - goC in a Spinco model L ultracentrifuge. Fractions were collected dropwise,and assayed for (-e-e-) ,8-galactosidase and (-- 0-- 0--) thiogalaetoside transacetylase.

20 S. H. APPEL, D. H. ALPERS AND G. M. TOMKINS

"Heavy" transacetylase was also found with polyacrylamide gel electrophoresis.After electrophoretic separation, fractions of the gel (prepared as described inMaterials and Methods) were eluted and assayed for fJ-galactosid~se and transacetyl­ase. Although these were performed in a 3% gel, the peak of transacetylase activitymigrated more rapidly than the fJ-galactosidase. However, a significant fraction of thetransacetylase activity migrated more slowly than the major galactosidase com­ponent, consistent with the results from sucrose gradient experiments (Fig. 5).

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FIG. 5. fJ-Galadosidase and thiogalactoside transacetylase activity of eluates from polyacryl­amide gel. Crude extracts of induced E. coli 200P Flae were electrophoresed in large columns asdescribed in Materials and Methods. They were then eluted and assayed for fJ·galactosidase andthiogalactoside transacetylase.

When an extract of induced E. coli 200P Flac was dialyzed against 0·05 M-tris,pH 7·8, for 12 hours, there was no change in its total acetylase activity. However, thisprocedure promoted disaggregation of the heavy enzyme since no transacetylaseactivity sedimented with an S value greater than 10 s. Sedimentation of a bacterialextract in the presence of 0·1 M-IPTG also resulted in disaggregation of the heavytransacetylase, whereas the presence of IPTG had no effect on the distribution offJ·galactosidase activity.

4. DiscussionThe results presented above indicate that in bacterial cells where the lactose operon

is uninduced, the basal fJ-galactosidase migrated as a single rapidly moving electro­phoretic component which sediments with a velocity of 16 s. On induction, multiplestable electrophoretic forms of the enzyme appeared. This might be related to thereport (Cohn, 1957)that highly purified galactosidase sediments as several componentsin the analytical ultracentrifuge. However, the stability and reproducibility of the

MULTIPLE MOLECULAR FORMS OF ,8.GALACTOSIDASE 21

pattern seen in the present study strongly suggest that such forms exist in vivo.This also seems likely since the induced pattern returned toward the basal patternwhen inducer was removed.

The different forms of the enzyme could be partially separated from one anothereither by disc electrophoresis, starch gel electrophoresis, agar gel filtration, or sucrosegradient centrifugation, but they could not be resolved by chromatography onDEAE­cellulose. Therefore, the forms of the enzyme differed from one another by molecularweight and probably not on the basis of charge, or other differences.

The evidence indicates that ,8-galactosidase aggregated with itself rather than non­specifically with other cell proteins, since the same pattern was seen in a crude extractand in a preparation purified 20-fold with respect to enzyme activity. In addition,the 34 s form ofthe enzyme can break down to yield the 16 s form, and the K m valuesfor ONPG of the 16 sand 34 s components were the same. The same galactosidasepatterns were observed in z+y- bacteria lacking both galactoside permease andthiogalactoside transacetylase, indicating that the association of fi-galactosidase didnot require even other proteins of the lactose operon, at least in their wild typeconfiguration. Finally, the same electrophoretic forms appeared whether the ,8­galactosidase structural gene was functioning in E. coli or in the hybrid strains,S. marcescens Flac, S. typhimurium Flac, or P. mirabilis Flac, where presumablyother cell proteins would be different. These considerations indicate that galactosidaseaggregates only with itself.

The complex pattern observed suggests that ,8-galactosidase components smallerthan the smallest active form seen (16 s) are involved in the aggregation. This followsfrom the observations that the molecular weight of the lightest and the heaviest formsof ,B-galactosidase differed by a factor of about three, based on differences in sedimen­tation coefficients (assuming that the frictional coefficients of the two forms are notgrossly different). Therefore, simple aggregation of the 16 s form could not accountfor the large number of bands seen, since three would be the maximum numberpossible.

It may be pertinent that the first heavy component to appear during inductionwas the third, rather than the second, most rapidly migrating band (Fig. 3). Further­more, it was this band, only, which appeared when the 16 s component was con­centrated. These observations, together with its intermediate electrophoretic mobility,may indicate that the third band represents a dimer of two 16 s components. Theremainder of the pattern could be generated by addition of units smaller than 16 seither to the 16 s or to larger forms.

Recently, it has been found that active ,B-galactosidase is composed of severalchemically different types of subunits (Craven, Steers & Anfinson, 1964 and manu­script in preparation). It is possible, therefore, that the enzyme pattern represents,B-galactosidase molecules with different proportions of the different polypeptidechains (in addition to the differences in molecular weight). A similar explanationhas been proposed for the isozyme pattern of mammalian lactic dehydrogenase(Appella & Markert, 1961; Cahn, Kaplan, Levine & Zwilling, 1962).

Since no histochemical method was available for the transacetylase, the existenceof multiple discrete forms of the enzyme could not be established. However, fromsucrose gradient and disc electrophoresis experiments, about a third of the activitymigrated differently from the principal component. Since dialysis or treatment withIPTG caused the heavier forms to sediment with the principal component, the

22 S. H. APPEL, D. H. ALPERS AND G. M. TOMKINS

aggregates of acetylase are more labile than those of galactosidase. Since IPTG is asubstrate for the transacetylase, aggregation-disaggregation changes in the enzymemay be associated with the enzymic reaction.

Several forms of alkaline phosphatase in E. coli have been observed, but thismultiplicity does not appear to be due to differences in molecular weight between thevarious forms (Levinthal et al., 1962). Multiple forms of other bacterial enzymes,as well, have been observed in the course of the present work, e.g. glutamic dehydro­genase, oc-glycerophosphate dehydrogenase, lactic dehydrogenase, malate de­hydrogenase, and glucose-6-phosphate dehydrogenase (Appel, Alpers & Tomkins,unpublished observations). However, unlike ,a-galactosidase, no more than threebands were seen for any given activity. The basis for these multiple forms is not known.

The most intriguing question about these observations concerns their biologicalsignificance. The fact that an identical pattern of activity was seen with constitutivebacteria grown in the absence of inducer and with inducible bacteria shows that themultiplicity of forms was not the result of the induction process itself. Their presencemay be related to stabilizing enzyme molecules in vivo or to the facilitation of lactosemetabolism. Although the significance of these forms is uncertain, understanding themechanism of their formation may contribute to an understanding of the assemblyof macromolecular structures.

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