Cross-Linking of the Components of Lactose Synthetase with ... · as soluble protein in milk (14)....

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 250, No. 4,Issue of February 25, pp. 1434-1444, 1975

Printed in U.S.A.

Cross-Linking of the Components of Lactose Synthetase with

Dimethylpimelimidate*

(Received for publication, July 11, 1974)

KEITH BREW,$ JOEL H. SHAPER,~ KENNETH W. OLSEN,~ IAN I'. TRAYER,/~ AND ROBERT L. HILL

From the Department of Biochemistry, Duke University Mkdical Center, Durham, North Carolina 27710

SUMMARY

The cross-linking of the two components of lactose synthetase, a-lactalbumin and a galactosyltransferase, with dimethylpimelimidate was examined. The extent of the cross- linking at pH 8.1 was found to be dependent upon the presence of substrates or inhibitors for the galactosyltransferase. N-acetylglucosamine and mixtures of either N-acetylglucosamine, Mn2+ and UDP, or UDP-galactose and Mn2+ promoted the formation of cross-linked species. Glucose or a mixture of UDP and Mn*+ were much less effective in promoting cross-linking. Two types of intermolecularly cross- linked species of a-lactalbumin and the galactosyltransferase were obtained. Each was a 1: 1 cross-linked complex of cu-lactalbumin and either of the two forms of the transferase with molecular weights of about 42,000 and 48,000, respectively. Cross-linked complexes were not observed with more than 1 molecule each of cY-lactalbumin and the transferase.

The cross-linked complexes were obtained in homogeneous form by gel filtration on Sephadex and absorption of uncross- linked enzyme by affinity chromatography on Lu-lactalbumin- Sepharose in the presence of N-acetylglucosamine. They migrated on gel electrophoresis in sodium dodecyl sulfate with mobilities in accord with their predicted molecular weights as 1: 1 complexes of a-lactalbumin and the transferase. The amino acid composition of the cross-linked complex was in reasonable agreement with the expected composition of a 1: 1 mixture of Lu-lactalbumin and galactosyltransferase.

The enzymic properties of the cross-linked and uncross- linked enzymes were compared. The cross-linked complex had a much higher intrinsic lactose synthetase activity than did uncross-linked enzyme although only about 1% of the potential activity of uncross-linked enzyme in the presence

* These studies were supported by Research Grants HE-06400 from the Nationa! Heart and Lung Institute and GB-12676 from the National Science Foundation.

1 Recipient of a European Molecular Biology Organization Re- search Travel Grant. Present address, Department of Biochem- istry, University of Miami School of Medicine, Miami, Florida 33152.

5 Present address, Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520.

7 Present address, Department of Biological Sciences, Purdue University, Lafayette, Indiana 47907.

11 Present address, Department of Biochemistry, University of Birmingham, Birmingham B15, England.

of optimal concentrations of cy-lactalbumin. The lactose synthetase activity of the cross-linked complex, however, was unaffected by exogenous a-lactalbumin. In addition, the complex readily catalyzed the transfer of galactose from UDP-galactose to xylose in the absence of exogenous a-lactalbumin. The N-acetyllactosamine synthetase activity of the complex was low compared to its activity with other monosaccharides. Ovalbumin, which is a good acceptor for the uncross-linked transferase, was not an acceptor for the cross-linked complex. Kinetic studies of the complex suggest that its modified catalytic activity is not the result of the modification by dimethylpimelimidate but reflects the expected effects of cr-Iactalbumin as an activator of monosaccharide binding to the galactosyltransferase, a function consistent with previous kinetic studies with the uncross-linked enzyme system.

Lactose synthetase (EC 2.4.1.22) consists of two components, a galactosyltransferase (UDP-galactose :N-acetylglucosamine galactosyltransferase) (1) and the regulatory protein, ac-lactalbumin (2). The galactosyltransferase is widely distributed among animal tissues and catalyzes the following reaction.

UDP-galactose + N-acetylglucosamine ---f

N-acet,yllactosamine + UDP (1)

The acceptor, N-acetylglucosamine, may be either the free sugar or the terminal, nonreducing end of a carbohydrate prosthetic group of a glycoprotein (3). Thus, the transferase in the absence of cr-lactalbumin functions in many tissues to catalyze the formation of gdlactosyl-(61+4)-N-acetylglucosaminyl bonds in glycoprotein biosynthesis. The enzyme will also transfer galactose to glucose in the absence of ac-lactalbumin, but this is un- likely to be an important physiological function of the transferase because the K,,, for glucose is 1 to 2.5 M (4-6). In the presence of a-lactalbumin, the K, for glucose and other acceptors is re- duced by up to 1000.fold, so that lactose synthesis can proceed at physiological concentrations of glucose. The lactating mammary gland is the only tissue that synthesizes cu-lactalbumin; thus lactose synthesis occurs uniquely in this organ. The transferase from many tissues, however, catalyzes lactose synthesis in vitro when cu.lactalbumin from different animal species is present in addition to UDPgalactose and glucose (1, 3).

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cr-Lactalbumin has been characterized structurally in con- DuPhar, Petten, Holland) diluted with unlabeled compound.

siderable detail and the complctc amino acid sequences of bovine The final specific activity of the labeled compound used in these

(7)) human (8), and guinea pig (9) cy-lactalbumins have been de- studies was 0.975 X lOa cpm per mmol under standard conditions.

termined, as well as a partial sequence for a kangaroo a-lactalbu- Other organic compounds were reagent grade unless specified.

min (10). The transferasc has not been examined as thoroughly but it is known to be bound to membrane of the Golgi apparatus in the mammary gland and other tissues (11-13) and is present as soluble protein in milk (14). The milk enzyme has been purified to constant specific activity by affinity chromatography and at least three species with molecular weights of about 42,000, 48,000, and 52,000, as determined by sodium dodecyl sulfate gel electrophoresis, have been found (15, 16). It appears that the enzymically active lower molecular weight forms result from pro- teolytic degradation of the higher molecular weight species (16).

Several studies have suggested that the interaction of cr-lactalbumin and the transferase is influenced by its substrates. For example, the transferase binds to cY-lactalbumin-Sepharose in the presence of either glucose (17), N-acetylglucosaminc (18), UDl and &2f (19), or UDl’-galactose and 1Wf (19). A 1: 1 complex appears to be formed between the transferasc and (Y- lactalbumin in the presence of N-acetylglucosamine (18, 20). The nature of the complex to which a-lactalbumin binds in the course of the lactose synthetase reaction has been suggested by Morrison and Ebner (2 I), from kinetic studies, to be a quaternary

complex of hW+, UDPgalactose, enzyme, and glucose. More recent kinetic studies by Rrew and co-workers (6, 22) could not confirm this conclusion but obtained evidence that during cataly- sis, cr-lactalbumin binds to a ternary complex formed by AIn2+, enzyme, and IJDl’-galactose.

We wish to report here the nature of the interaction between bovine cu-lactalbumin and the bovine transferasc from milk as

judged by covalent cross-linking of the two proteins with the bifunctional reagent dimethylpimelimidate. This diimidoester and its homologue, dimethylsuberimidate, have been shown to

cross-link effectively the subunits of oligomeric proteins by reaction with lysyl residues in different subunits (23, 24). It has been found that the extent of cross-linking of ar-lactalbumin and the transferase by dimethylpimelimidate is dependent on substrates, and in the absence of substrates, little cross-linking oc-

curs. In the presence of substrates, 1: I cross-linked complexes of cY-lact’albumin and the two major forms of the transferase are formed. The cross-linked complexes have been isolated and

their chemical and enzymic properties have been determined. The results of these studies give additional insight into the mechanism of lactose synthetase.

EXPERIMENTAL PROCEI)URE

Materials

Galactosyltransferase from bovine milk was isolated by affinity chromatography on UDP-Sepharose and cu-lactalbumin-Sepharose adsorbents as described by Barker et al. (15). The pure enzyme was hctcrogeneous with respect to molecular weight as reported by these workers and others (15, 16). On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, two major species wit,h molecular weights of 42,000 and 48,000 were observed in addition to a third component in minor amount with a molecular weight of about 51,000.

Bovine a-lactalbumin was prepared as described earlier (7). u-Lactalbumin-Sepharose (17) and UDP-Sepharose (15) were prepared by published methods. Ovalbumin (once crystallized) was obtained from Pentex, Inc. Dimethylpimelimidate dihydrochloride was synthesized from 1,5-dicyanoheptane (Aldrich) by the method of Davies and Kaplan (24). Dimethyl[l,‘l-‘“C]pimelimidate dihydrochloride was synthesized in the same manner from 1,7-dicyano[l,7-‘%]hexane (4.5 mCi per mmol; N. V. Philips-

Methods

Enzyme Assays-Galactosyltransferase activities were assayed with monosaccharide acceptors by following the transfer of [l-l%]- galactose from UDP-n-[l-i4C]galactose to the acceptors as described by Brew et al. (unless otherwise indicated, the concentrations of UDP-galactose and Mn2f used in the assavs were 0.15 mM I~ -~ and 40 mM, respectively). Reactions were stopped by adding a solution of EDTA at a concentration sufficient to combine with all Mnzf in the assay mixture (10 mM EDTA for routine mixtures containing 40 mM Mn2+). Identical assays were used with ovalbumin as substrate except that monosaccaride acceptors were omitted. The final ovalbumin concentration was 1 mg per ml. The reaction was stopped by adding 1 ml of IO’% trichloroacetic acid; 1 ml of ovalburnin (5 my per ml) was added as carrier. After mixing, the precipit’ate was collected on a filter (Whatman glass paper, GF/A, 25 mm) and washed three times with 15 ml of 5% trichloroacetic acid. After drying, the filter disc was added to 10 ml of Aquasol scintillation fluid (New Eneland Nuclear) and the counted (Pack- ard Tri-Carb spectrometer, model 3320). A unit of enzyme activity represents nanomoles of galaetose transferred per min under the above assay conditions.

Protein Concentrations-Galactosyltransferase concentrations were estimated spectrophotometrically assuming an extinction co- efficient EO.‘% van “7” = 1.61 (17). a-Lactalbumin concentrations were

_“ I “ . - I

also estimated spectrophotometrically using the extinction co- efficient (E!&itrn = 2.05) reported earlier (25).

Gel Electrophoresis-Electrophoresis was performed on 7.5% polyacrylamide gels in 1% sodium dodecyl sulfate as described previously (17). The same molecular weight standards and methods reported earlier (17) were used for estimation of molecular weights of protein mixtures analyzed on these gels.

Amino Acid Analysis-Protein samples (0.1 to 1.0 mg) were hydrolyzed with 6 N hydrochloric acid (1 to 2 ml) at 110” in evacu- ated, sealed tubes for 24 hours. The resulting hydrolysates were analyzed on a Beckman-Spinco model 120C automatic amino acid analyzer (equipped with a high sensitivity range card) by slight modification (26j of the standard methodology (27).

Cross-linking with Dimethylpimelimidate-Solutions of the galactosyltransferase (0.3 to 0.5 mg per ml) and a-lactalbumin (5 to 10 mg per ml) were made 0.2 M in triethanolamine.HCl, pH 8.1, by gel filtration at 5” through columns (2.5 X 20 cm) of Sephadex G-25 equilibrated with 0.2 M triethanolamine, pH 8.1. Dimethyl- pimelimidate.HCl was dissolved in 0.2 M triethanolamine, pH 8.1, immediately prior to use, and the solution was adjusted to pH 8.1 with 1 M sodium hydroxide. Solutions of the transferase, a-lactalbumin, and dimethylpimelimidate were then mixed to give a final concentration of reactants as described under “Results.” All reactions were performed at 22-24”. Reactions were initiated by addition of dimethylpimelimidate and terminated by the addition of an equal volume of 1 M glycine, pH 8.5. If the samples were to be analyzed on gels, they were exhaustively dialyzed against water and lyophilized prior to analysis. Cross-linked samples which were used for enzyme and chemical characterization of the complex were purified as described under “Results.”

RESULTS

Determination of Conditions for Cross-Linking and Isolation and Characterization of Cross-Linked Complex

Conditions for Cross-Linking-Optimal cross-linking of protein subunits with diimidoesters has been reported to occur at pH 8.5 (23, 24) but the present studies were performed at pH 8.1 in

order to minimize the formation of manganese dioxide from Mn2f, which was present in several cross-linking experiments. In addition, a-lactalbumin was always present in about a IO-fold excess, by weight, over the transferase. Optimal formation of complexes of the two proteins will occur, as judged from kinetic analysis of lactose synthetase (1, 6, 21) only when a-lactalbumin is present at substrate concentration levels.


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- 28,000) bLA),

c 14,500 - ULA

a b c d e a b c d

FIG. 1 (left). Analysis of the cross-linking of a-lactalbumin (&A) and galactosyltransferase by dimethylpimelimidate in the presence of N-acetylglucosamine by gel electrophoresis in sodium dodecyl sulfate. a-Lactalbumin (1 mg per ml), galactosgltrans- ferase- (0.1 mg per ml), N-acetylglucosamine (56 mM), and dimethylpimelimidate (1 mg per ml) in 0.2 M triethanolamine, pH 8.1, were incubated at 23”. Samples were removed at intervals and mixed with an equal volume of 1 M glycine, pH 8.5, exhaustively dialyzed against water, lyophilized, and analyzed on the gels. The gel patterns shown were after reaction for 3 min (a), 30 min (b), 60 min (c), and 120 min (d). Gel e is the pattern after cross-linking for 120 min exactly as described above, except that N-acetylglucosamine was not present in the reaction mixture.

FIG. 2 (center). Analysis of the cross-linking of a-la&albumin

The extent of cross-linking with time, and as a function of substrates for either lactose synthetase or the galactosyltransferase, was examined by electrophoresis on polyacrylamide gels in sodium dodecyl sulfate. Figs. 1 to 3 show the patterns of cross- linking of ar-lactalbumin and the transferase by dimethylpimelimidate as infiuenced by Mn 2+, UDP-galactose, UDP, N-acetylglucosamine, or mixtures of these substances. All gel patterns showed several species, including uncross-linked a-lactalbumin (molecular weight 14,500), a minor contaminant of the cr-lactalbumin preparation (molecular weight 20,000), and two species of the transferase (molecular weight 42,000 and 48,000). 1 he protein contaminating the cu-lactalbumin is not observed when 5 to 10 mg of ac-lactalbumin are applied to each gel but is seen only when the gels are “overloaded.” Overloading with respect to a-lactalbumin was necessary in order to observe the transferase and the complexes. The protein contaminant does not appear to influence the results of the cross-linking studies since it is present in about the same amount throughout the course of the reaction.

Figs. 1 to 3 show that as reaction with dimethylpimelimidate proceeds, new species with molecular weights greater than those of either cY-lactalbumin or the transferase appear, and increase in amount with time. Two major species, with molecular weights of about 57,000 and 61,OOOr appear during the cross-linking reaction. Their apparent molecular weights are close to those expected if 1: 1 complexes were formed between cu-la&albumin and the two forms of transferase, which differ in molecular weight by

r The molecular weights of the cross-linked complexes cannot be judged exactly by gel electrophoretic analysis, since they may contain branched-like structures which may not bind dodecyl sulfate in the same manner as single, unbranched polypeptide chains (28). Thus, the apparent molecular weights of the complexes differ somewhat from those expected on the basis of the sum of the weights of &a&albumin and either of the two species of the transferase.

-2E,ooOj-(ff LA),

- 14,500 - CI LA

\ a b c cj “’

(c&A) and galactosyltransferase by dimethylpimelimidate in the presence of N-acetylglucosamine, Mn2+, and UDP. Reaction mixtures were the same as described in Fig. 1 except that Mn2+ (2 mM) and UDP (2 mM) were also nresent. The reaction was analyzed as indicated in Pig. 1. Reaction times were: (a) 3 min, (b) 30 min, (c) 60 min, and (d) 120 min.

FIG. 3 (right). Analysis of the cross-linking of ol-lactalbumin (&A) and galactosyltransferase by dimethylpimelimidate in the presence of UDP-galactose and Mn2+. Reaction mixtures were exactly the same as given in Fig. 1 except that UDP-galactose (2 mM) and Mn2+ (2 mM) were present and N-acetylglucosamine was absent. The reactions were analyzed as indicated in Fig. 1. Reaction times were: (a) 3 min, (b) 30 min, (c) 60 min, and (d) 120 min.

about 6,000. The other species, which increases in amount during cross-linking, has a molecular weight of about 28,000 and has been tentatively identified as a cross-linked complex containing 2 molecules of cr-lactalbumin. Exact characterization of this species awaits further study. The species with molecular weights corresponding to 1: 1 complexes of cr-lactalbumin and either of the two forms of the transferase were subsequently identified as such in further experiments (see below).

The gel patterns shown in Figs. 1 to 3 indicate that formation of cross-linked complexes is dependent upon the presence of substrates for lactose synthetase, in accord with predictions based on kinetic analyses (1, 6, 21). Fig. 1 shows that N-acetylglucosamine promotes complex formation and that the higher molecular weight complexes (Gels a to d) are not observed even at the longest reaction time in the absence of Iv-acetylglucosarnine (Gel e). Figs. 2 and 3 show that a mixture of either N-acetylglucosamine 9 Mn2f and UDP, or UDP-galactose plus Mn2+, respectively, also promote formation of cu-lactalbumin-transferase complexes. The maximal extent of cross-linking, under the conditions shown in Figs. 1 to 3, was about 50% as judged by the intensity of staining of the newly formed cross-linked species and the decrease on staining of the two major forms of the transferase. Electrophoretic analyses (not shown) of the cross-linking reaction under the same conditions as given in Figs. 1 to 3, except that either MI? (2 mM) plus UDP (2 mM), or glucose (20 mM),

replaced the other combinations of substrates, indicated that much less cross-linking had occurred.

Isolation of Cross-Linked Transferase-a-Lactalbuma’n Complex- In order to characterize more thoroughly the cross-linked complexes, they were isolated as follows. A reaction mixture (5 ml final volume) containing galactosyltransferase (0.2 mg per ml), Lu-la&albumin (1.2 mg per ml), MnC12 (2 mM), UDP (2 mM), N-acetylglucosamine (20 mM), and dimethylypimelimidate (1 mg per ml) in 0.2 M triethanolamine, pH 8.1, was incubated


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at 23” for 2 hours. The reaction mixture was then applied di- rectly to a column of Sephadex G-100. The effluent fractions were assayed for lactose synthetase activity in the absence of a-lactalbumin with 4 mM glucose, as well as for N-acetyllactosamine synthetase activity with 50 mM N-acetylglucosamine. The conditions chosen for measuring lactose synthetase activity would show no activity for uncross-linked enzyme, but there was detectable activity for the cross-linked enzyme. The effluent fractions were also monitored at 280 mm to detect protein. The chromatographic pattern obtained is shown in Fig. 4. Sev-

0 IO 20 30 40 50 60 70 80 Fraction Number

FIG. 4. Chromatography of a reaction mixture of a-lactalbumin cross-linked with galactosyltransferase on Sephadex G-100. The reaction mixture (see text) was applied to a column (2.5 X 60 cm) of Sephadex G-100 equilibrated with 0.01 M sodium cacodylate buffer, pH 7.4, containing 1 mM mercaptoethanol. The column was developed at 5” with the equilibration buffer at a flow rate of 13 ml per hour and 4.25-ml fractions were collected. The effluent fractions were monitored at 280 nm to estimate protein concentration (- - -), for lactose synthetase (o), and N-acetyllactosamine synthetase (0) activities. The fractions indicated by the bar were pooled and applied to the column shown in Fig. 5.

I I. I I = E \ \

.$ 60-

.2 2 :z 50 - 2 $ f 40- f 5

; 30- ,I

.- E $ g 20-

B = 2 IO-

=i

A I

0 5 IO 15 20 Fraction Number

FIG. 5. Purification of cross-linked ol-lactalbumin-galactosyltransferase on cu-lactalbumin-Sepharose. The fractions indicated in Fig. 4 were made 20 mM in N-acetylglucosamine and applied to a column (1 X 5 cm) of cu-lactalbumin-Sepharose. The column was equilibrated and developed at 5’ with 20 mM sodium cacodylate buffer, pH 7.4, containing 20 mM N-acetylglucosamine. The N-acetylglucosamine was removed from the developing buffer after Fraction 10 was collected. The column was developed af a flow rate of 10 ml per hour and 4-ml fractions collected. The fractions were assayed for N-acetyllactosamine synthetase activity.

era1 fractions (Fractions 21 to 28) with lactose synthetase activity appeared slightly before those with N-acetyllactosamine synthetase activity (Fractions 24 to 39) and were judged to contain the cross-linked complex. The materials emerging between Fractions 35 to 50 and 60 to 80 were judged t.o be a cross-linked dimer of cr-lactalbumin and uncross-linked cY-lactalbumin, respectively, and were devoid of either synthetase activity. The fractions with lactose synthetase activity were pooled, made 20 InM in N-acetylglucosamine, and chromatographed on a column (1 X 5 cm) of cr-1actalbuminSepharose as shown in Fig. 5. This column was expected to separate cross-linked and uncross- linked species, because the cross-linked enzyme containing a- lactalbumin should not bind to cr-lactalbumin-Sepharose in the presence of N-acetylglucosamine, whereas the uncross-linked transferase should bind. The effluent fractions were assayed for N-acetyllactosamine synthetase activity. Those fractions emerging unretarded from the column had a low synthetase activity, which is expected for a cross-linked complex, in view of the well established inhibition of this synthetase activity by cr-lactalbumin (1). When elution was continued with buffer devoid of N-acetylglucosamine after the emergence of the unretarded fractions, additional material was eluted with higher synthetase activity. Fractions 1 to 9 and 12 to 17 were pooled separately and dialyzed against 10 mM sodium cacodylate buffer, pH 7.2. From the chromatographic properties and the activity of these fractions, Fractions 1 to 9 were judged to contain the cross-linked complex and Fractions 12 to 17 to contain uncross- linked galactosyltransferase. These identifications were con- firmed by the observation that Fractions 1 to 9 had lactose synthetase activity in the absence of added cr-lactalbumin at 4 mM glucose, whereas under the same conditions Fractions 12 to 17 showed insignificant activity except when assayed in the presence of cY-lactalbumin. Further evidence supporting this identifica- tion of the two fractions was obtained by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate as shown in Fig. 6. Fractions 12 to 17 had two major components with molecular weights of 42,000 and 48,000 and were electro- phoretically indistinguishable from uncross-linked galactosyltransferase or intramolecularly cross-linked complex. Fractions 1 to 9 contained two major components with molecular weights of about 57,000 and 62,000, the size expected for 1: 1 intermolecularly cross-linked complexes of cr-lactalbumin and either of

a b

FIG. 6. Gel electrophoresis of sodium dodecyl sulfate of the uncross-linked galactosyltransferase (%Z a) and the cross-linked ol-lactalbumin-galactosyltransferase (Gel b).


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the two major forms of the galactosyltransferase. On the basis of the intensity of staining of the two cross-linked species on these gels, each species represents about 50% of the total cross-linked preparation. The cross-linked material was stored at -10” and used for subsequent characterization of the catalytic properties of the cross-linked complex.

Preparation of c+Lactalbumin-Transjerase Complex Cross- Linked with Dimethyl[l , 7J4C]pimelimidate-A mixture (80 ml total volume) containing galactosyltransferase (0.2 mg per ml), cr-lactalbumin (1.2 mg per ml), UDP (2 RIM), MnClz (2 m&r), N-acetylglucosamine (20 mM) and dimethyl[l,7-14C]pimelimidate (1 mg per ml; specific activity 0.975 X lo8 cpm per nmol) was incubated at 23” for 1 hour. The mixture was then applied to a column (5 x 150 cm) of Sephadex G-100. The column was developed and the effluent fractions were monitored for protein, lactose synthetase, and N-acetyllactosamine synthet#ase activities as described for the experiment in Fig. 4. Fractions were also monitored for radioactivity. The protein and ensymic activity profiles were essentially identical with those shown in Fig. 4. Radioactivity was associated with all protein-containing fractions including the cr-lactalbumin peak, which suggests that a large amount of monovalent modification and perhaps internal divalent modification of ar-lactalbumin had occurred with the diimidoester. The fractions with lactose synthetase activity were pooled and rechromatographed on a column (2 x 25 cm) of cY-lactalbumin-Sepharose as described in the experiments shown in Fig. 5. Material with intrinsic lactose synthetase activity in the absence of added a-lactalbumin emerged unretarded from the column. Fractions containing this material were pooled, concentrated by ultrafiltration (Amicon membrane FM-30) at 4”, and dialyzed against 10 RIM sodium cacodylate buffer, pH 7.4. This material had the expected enzymic activities (see above) and gel electrophoretic mobilities (Fig. 6) for a 1: 1 cross-linked complex of a-lactalbumin and galactosyltransferase.

The specific activity of the cross-linked complex was found to be 6800 cpm per mg of protein and, assuming an average molecular weight of 60,000 for the complexes, they were calculated on the basis of the specific activity of the [‘4C]diimidoester to contain 4.2 molecules of diimidoester per molecule of complex. The amounts of divalently and monovalently incorporated cross- link were not estimated, but at least 1 molecule of divalently cross-linked ester must have been incorporated to give the cross- linked complexes with molecular weights of about 56,000 and 61,000 (Fig. 6).

Amino Acid Composition of Cross-Linked a-Laclalbumin and Galactosyltransjerase-The amino acid composition of the cross- linked complex prepared with dimethyl[14C]pimelimidate was estimated by analysis of 24.hour acid hydrolysates. Table I lists the composition obtained and those of the galactosyltransferase, cu-lactalbumin, and the predicted composition of a 1: 1 complex of a-lactalbumin and the transferase. The composition of the 1: I complex is in reasonable agreement with the expected composition calculated from the compositions of cr-lactalbumin and the transferase except for the proline, glycine, valine, and lysine contents. The high value for proline and the low values for glycine cannot be explained readily, although the amounts of these two amino acids may vary considerably from preparation to preparation of the transferase (17). This variation may result from the heterogeneity of the transferase, which contains species of different molecular weights presumably as the result of pro- teolytic action on the transferase either in the mammary gland or in milk (19). Indeed, some preparations of the transferase with a molecular weight of about 42,000 (17) were found to have gly-

tine and proline contents in closer accord to those found in the cross-linked complex. The low valine content in the complex may result from incomplete release of this amino acid after only 24-hour hydrolysis. The low lysine content may be expected on the basis of modification of lysyl e-amino groups by the diimidoester. Because about 4 residues of lysine in each protein should react with the diimidoester, the lysine content should be low by a total of 8 residues. With the exception of these residues, however, it is reasonable to conclude that the composition of the complex is close to that expected. Clearly, more exact determinations of the composition of thz complex based on analyses of samples hydrolyzed for different time periods may clarify the few discrepancies.

Catalytic Properties

Substrate XpeciJicity-The effects of varying the concentrations of a-lactalbumin, N-acetylglucosamine, glucose, and xylose on the synthetase activities of the cross-linked a-lactalbumin-galactosyltransferase complex are compared with uncross-linked transferase in Figs. 7 to 9. Because of the lower specific activity of the cross-linked complex, in each of the studies shown in Figs. 7 to 9, the amount of cross-linked enzyme in each assay was about 90 times that of uncross-linked enzyme.

Fig. 7 shows the activity of the cross-linked complex and the galactosyltransferase as a function of cr-lactalbumin concentration. The cross-linked complex has a higher intrinsic activity for lactose synthesis, than uncross-linked enzyme in the absence of exogenous cx-lactalbumin and it is completely unaffected by a-lactalbumin. This behavior of the complex contrasts markedly with that of the uncross-linked enzyme which, in the absence of exogenous a-lactalbumin, was virtually inactive for lactose synthesis at the glucose concentrations (4 mM) used, but showed progressively increasing activity with increasing (Y- lactalbumin concentrations. Fig. 7 also shows that the cross- linked complex has a low but significant N-acetyllactosamine synthetase activity which was very slightly inhibited by cu-lactalbumin, in contrast with the uncross-linked enzyme which is

TABLE I

Amino acid compositions of galactosyltransjerase, a-lactalbumin, and cross-linked complex of &actalbumin and transjerase

/ I

Amino acid

Cm-cysteine. Aspartic acid. Threonine Serine. . . . Glutamic acid . Proline. Glycine, Alanine. Valine Methionine. Isoleuciue Leucine Tyrosine Phenylalanine Histidine Lysine. Arginine

-

Galactosyl- transferase

Cross-linked complex a-lactalbumin

Theparti- Found

2.5 8 10.5 8.7 26 21 47 47 27.5 7 34.5 32 39.5 7 46.5 46 39 13 52 46.5 17 2 19 30.2 32.5 6 38.5 28 22.5 3 25.5 25 34.5 6 40.5 31 3 1 4 3

10 7 17 17.1 28 13 41 39.5 10.5 4 14.5 16.6 9 4 13 14.8 6 3 9 8.4

21.5 12 33.5 25 12.5 1 13.5 15.9


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Y ’ I I I I I I 0 0.1 0.2 0.3 0.4 0.5 0.6

o -Lactalbumin concentration (mg/ml)

FIG. 7. Lactose synthetase activity of cross-linked and uncross-linked galactosyltransferase and the N-acetyllactosamine synthetase activity of cross-linked enzyme as a function of LY- lactalbumin concentrations. Lactose synthetase activity for cross-linked (0) and uncross-linked (0) transferases, and N- acetyllactosamine synthetase activity of cross-linked transferase

(0).

I I I ~ylos~ mM

100 200 300 4oc

I I I I

FIG. 8 (left). N-acetyllactosamine synthetase activity of cross- linked and uncross-linked galactosyltransferases as a function of N-acetylglucosamine concentrations. 0, uncross-linked enzyme; 0, cross-linked enzyme.

FIG. 9 (center). Rate of synthesis of lactose and galactosyl- (p l--14)-xylose by the cross-linked and uncross-linked galactosyltransferases as a function of glucose and xylose concentrations.

TABLE II

Comparison of apparent kinetic parameters associated with different monosaccharides us substrates of galactosyllransjerase and

cross-linked complex

Monosaccharide Galactosyltransferase I Complex

N-Acetylglucosamine Glucose. ._....._..__,,I.

Xylose -

for N-acetyllactosamine synthesis than the uncross-linked enzyme, but that its rate of synthesis is almost unaffected by increasing the concentrations of N-acetylglucosamine above 1 mM.

In contrast, the uncross-linked transferase shows the typical velocity increase with increasing concentrations of N-acetylglucosamine.

Fig. 9 shows the relationship between the rate of synthesis of lactose and galactosyl-@+4)-xylose2 as a function of the concentrations of glucose and xylose, respectively. Earlier studies had shown that the activity of uncross-linked enzyme for trans-

fer of galactose from UDP-galactose to either glucose or xylose was a function not only of the concentration of these acceptor substrates but also of the concentration of a-lactalbumin (6, 21). The cross-linked enzyme had the ability to transfer galactose to both glucose and xylose in the absence of added cY-lactalbumin, unlike the uncross-linked enzyme which, under the conditions of the assays, was unable to transfer galactose to either glucose or xylose.

Fig. 10 shows the activities of the cross-linked and uncross- linked enzymes for transfer of galactose from UDPgalactose to

Enzyme

ApK=t

rnM

7.35 1000

00 -

milliunits/ mg

15.000

Apparent &I

0.036 0.35

24

Vftl

milliunilr/ w

42 137

66

strongly inhibited for N-acetyllactosamine synthctase activity by cY-lactalbumin, at the monosaccharide concentration used in these experiments.

Fig. 8 shows that the cross-linked complex has a lower activity

I I I I I

ovalbumin (mg/ml)

Cross-linked enzyme with glucose ( l ), and xylose (0); uncross- linked enzyme with glucose and xylose (0).

FIG. 10 (right). Rate of galactosyl transfer from UDP-galactose to ovalbumin by cross-linked and uncross-linked galactosyltransferases as a function of ovalbumin concentrations. l , uncross- linked enzyme; 0, cross-linked enzyme.

ovalbumin, which contains terminal Wacetyiglucosamine in its polysaccharide prosthetic group (30, 31), and will serve as an acceptor for galactose. The uncross-linked enzyme transferred galactose readily to ovalbumin but the cross-linked enzyme was inactive in catalyzing this reaction.

The apparent values for K, and V,,, for the substrates examined in Figs. 7 to 9 determined from Lineweaver-Burk plots of the appropriate data for the cross-linked and uncross-linked enzymes are listed in Table II. The most significant differences between the two forms of enzyme are the markedly lower values of K, for the monosaccharide acceptors and the considerably decreased Vmax with N-acetylglucosamine for the cross-linked enzyme.

The specific activity of the cross-linked complex for lactose synthesis was found to be 137 units per mg with 4 mM glucose,

2 This product was isolated from react,ion mixt,ures and was indistinguishable on paper chromatography from authentic o-galactosyl-(P, 1+4)-n-xylose (29) in two solvent, systems (ethyl acetate- acetic acid-water, 3: 1: 1 and butanol-l-ethanol-water, 10:3:5). We thank Dr. Lennart Roden for performing these analyses.


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0.15 mM UDP-galactose, and 40 mM MnCla in the assay mixture. Under comparable conditions the activity of the uncross-linked enzyme for synthesis of N-acetyllactosamine was found to be 15,000 units per mg. When the lactose synthetase activity of the the cross-linked enzyme is extrapolated to saturating concentrations of substrates (see below) a V,,, value of 174 units per mg was obtained. Therefore, the turnover number for lactose synthesis by the cross-linked enzyme is about 1% that of the uncross-linked enzyme for N-acetyllactosamine synthesis.

Kinetics of Lactose Synthesis by a-Lactalbumin-Transferase Complex-Uncross-linked galactosyltransferase isolated from the reaction mixture after incubation with [14C]diimidoester showed incorporation of radioactivity to a level comparable with that of the cross-linked material. Despite this degree of modification, the catalytic activity of the enzyme was unimpaired, as evidenced by an unchanged turnover number for N-acetyllactosamine synthesis. It is therefore reasonable to suggest that the differences in the catalytic properties between the cross- linked complex and normal galactosyltransferase are a direct result of the covalent attachment of a-lactalbumin to the complex. A more detailed examination of the kinetic properties of the complex would therefore be expected to yield information relevant to the effects of a-lactalbumin on the functional prop-

erties of the galactosyltransferase, and hence on the mechanism of lactose synthesis. The steady state kinetics for lactose synthesis by the cross-linked complex was therefore examined in greater detail.

There are three substrates to be considered in such a kinetic study: Mn2+, UDP-galactose, and glucose. Previous studies with galactosyltransferase in the presence and absence of a-lactalbumin have shown that in the uncross-linked system these are bound to the galactosyltransferase in an ordered sequential manner: Mn2+, then UDP-galactose, and then glucose. Line- weaver-Burk plots for t,he variation of initial velocity with concentrations of two substrate pairs: VW+ and UDP-galactose, UDP-galactose and glucose, at fixed concentrations of glucose (4 mM) and RI@ (40 mM), respectively, are shown in Fig. 11. The R/lnzi-UDP-galactose pattern is of a normal intersecting type at low concentrations of Mn2+ changing to a pattern of parallel lines at higher concentrations of Mn2+. The biphasic

I I I I I I I I

I I I I I I I I 0 4 6 12 I6 20 24 28 32

IAJDP - golactose CrnM-‘I

FIG. 11. The effect of UDP-galactose concentrations on the rate of lactose synthesis by the cross-linked complex at different fixed concentrations of Mn2+. The concentration of glucose was held constant at 4 mM. The concentrations of Mn*+ were: l , 0.5 mM; 0,l mM; A, 2 mM; q ,4 mM; X, 10 mM; A, 20 mM; n , 40 rnM.

nature of the Mn2+ effect is clearly shown in the replot of the intersects from Fig. 11 versus l/Mn*+, as shown in Fig. 12. This is closely similar to the effects of II&P+ observed with human galactosyltransferase under conditions similar to those used here (6, 22) and have been attributed to the fact that MnZ+ binds to both the galactosyltransferase and to the substrate UDP-galactose. Stimulation of activity by Mn2+ continues up to a concentration of at least 40 mM, under which conditions approximately 90% of the UDY-galactose is present as the Mn*+ complex (6). It is clear that the galactosyltransferase must accept the R/Ins+-UDP-galactose as substrate, but the precise reaction mechanism at high Mn2+ concentrations remains obscure. The Lineweaver-Burk plots for the data shown in Fig. 11 with Mn2+ as variable substrate are nonlinear with pronounced downward curvature at high Mn2+ concentrations (i.e. low l/M&) and are not shown here.

The double reciprocal plots of initial velocity and concentration for UDP-galactose and glucose as variable substrates at 40 mM Mn*+ are parallel lines. The plot with UDP-galactose as the variable substrate at a series of fixed concentrations of glucose is shown in Fig. 13. Although this type of pattern is frequently diagnostic of a ping-pong mechanism (see Ref. 32), in the case of human galactosyltransferase where parallel lines are obtained in equivalent plots, more detailed studies have eliminated such a mechanism for the enzyme (6).

I I

t/r&l++

FIG. 12. A replot of the intersects from Fig. 11 versus ~/MI@.

6

0 4 8 12 16 20 24 28 32 I/UDP -galoctose (mM-‘1

FIG. 13. The effect of UDP-galactose concentrations on the rate of lactose synthesis by the complex at different fixed concentrations of glucose. The concentration of Mn2+ was held constant at 40 mM. The concentrations of glucose were: 0, 0.8 mM; 0, l.OmM; q ,1.33mM; A, 2mM; n ,8mM.


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The possible reaction product UDP binds Mn2+ strongly (stability constant of 8700 M-’ (33)), and it is therefore not possible to examine the inhibitory effects of free UDP on the enzyme as it has an absolute requirement for Mn2+. The inhibitory effects of the Mnz+ complex of UDP were therefore examined by conducting the experiments at 40 mM Mn2+, under which conditions the small proportion of the total Mn*+ bound by UDP should not have any significant effect on the reaction rate. Previous kinetic studies with human galactosyltransferase have indicated that UDP may be released from the enzyme as the Mn*+ complex (6). As shown in Figs. 14 and 15, MnUDP appears to act as a competitive inhibitor with respect to UDP- galactose (apparent Ki of 0.017 mM) and an uncompetitive inhibitor with respect to glucose (apparent Ki of 0.13 mM).

DIscussIoN

The results presented here provide further insight into the conditions required for interaction between the galactosyltransferase and cu-lactalbumin.

Clearly, for optimal interaction, which is assumed to be re- flected by the extent of cross-linking, the presence of substrates is required. In their absence, cross-linked species were not de- tected after a 180-min reaction whereas, in the presence of appropriate combinations, approximately 50% of the enzyme be- came cross-linked during this time period. N-Ac&ylglucosamine (20 mM) and combinations of either UDP-galactose (0.6 mM) and Mn2+ (2 mM), or UDP (2 mM), Mn2+, and N-acetylglucosamine,

I I I

0 IO 20 30 I/UDP-Golactose (mM-‘1

FIG. 14. Inhibition of lactose synthesis by MnUDP with UDP- galactose as the variable substrate. The concentrations of Mn2+ and glucose were 40 rn~ and 4 mM, respectively, and the concentrations of MnUDP were: l , 0; 0, 0.17 mM; A, 0.34 mM; n , 0.51 rnM.

16-

I I I I I I 0 02 04 06 0.0 I 0 1.2

I/Glucose (mM-‘1

FIG. 15. Inhibition of lactose synthesis by MnUDP with gIu- case as the variable substrate. The concentrations of Mn2+ and UDP-galactose were 40 mM and 0.15 mM, respectively, and the concentrations of MnUDP were: 0, 0; 0, 0.17 mM; A, 0.34 mM; 0, 0.51 rnM.

1441

promoted maximal cross-linking. In contrast, a combination of Mn2+ (2 mM) and UDP (2 mM), and either UDP (2 mM) or glucose (20 mM) alone, are less effective in promoting cross-linking. Although it would be more satisfactory to try a range of concentrations of the various substrates these results are in good agreement with the findings of Trayer and Hill (17), An- drews (IS), and Mawal et al. (19), who studied the retardation of galactosyltransferase on columns of a-lactalbumin-Sepharose in the presence of different substrate and product combinations. N-Acetylglucosamine and combinations of either UDP-galactose and Mnzf, or N-acetylglucosamine, UDP, and Mn2+ caused complete retention of enzyme, whereas glucose was not quite as effective.

Under the conditions of cross-linking (pH 8.1) it is apparent that a 1: 1 combination of galactosyltransferase and Lu-lactalbumin is formed and that higher order complexes are absent. At pH 8.5, with dimethylsuberimidate, the reaction appears less specific and complexes corresponding in molecular weight to galactosyltransferase-(a-1actalbumin)z and perhaps galactosyltransferase dimer are formeda. It is possible that the former complex results from the cross-linking of a-lactalbumin dimer (which forms rapidly at pH 8.5) with a molecule of galactosyltransferase.

Gel electrophoresis in sodium dodecyl sulfate of the purified cross-linked complex formed at pH 8.1 shows the presence of components which are closely similar in molecular weights to those expected for 1: 1 complexes of cY-lactalbumin with the two major galactosylt’ransferase components. The failure of the cross-linked species to attach to a-lactalbumin-Sepharose in the presence of N-acetylglucosamine, and the lack of stimulation of their activity for lactose synthesis by exogenous a-lactalbumin, confirms that 1: 1 complexes of the two proteins are the maximal and the only active complexes formed in the catalytic mechanism of the lactose synthetase system. Previously, Andrews (18) and Klee and Klee (20) have shown that 1:1 complexes form between the two proteins in the presence of either N-acetylglucosamine or UDl’-galactose and hln2+. Our results show that this complex is both necessary and sufficient for the maximum effects of a-lactalbumin on lactose synthesis.

Several aspects of the enzymic properties of the cross-linked complex are of interest. The intrinsic lactose synthetase activity at low concentrations of glucose (4 m&I) and the low N- acetyllactosamine synthetase activity are expected properties of such a complex since uncross-linked enzyme has virtually no lactose synthetase activity at these concentrations of glucose but much higher N-acetyllactosamine synthetase activity under the assay conditions employed. It is known that Lu-lactalbumin stimulates the lactose synthetase activity of uncross-linked enzyme and inhibits N-acetyllactosamine synthetase activity. Al- though the lactose synthetase activity is essentially unaffected by the addition of cY-lactalbumin, significant inhibition of N- acetyllactosamine synthesis was obtained on addition of cY-lactalbumin (Fig. 7). This could possibly reflect the addition of further molecules of a-lactalbumin to the complex to form higher order inhibitory complexes, or alternatively, a low level of contamination of the cross-linked complex with uncross-linked enzyme. As the specific activity of the complex is about 1% for lactose synthesis or 0.3 y0 for N-acetyllactosamine synthesis when compared with the activity of unmodified protein for N-acetyllactosamine synthesis, a level of contamination of 0.05% would account for the observed inhibition. This can obviously not be excluded by the criteria of purity used here.

3 K. W. Olsen, I. P. Trayer, and R. L. Hill, unpublished observa- tions.


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Ml?+ UDP-gol plUCOSe loctoss MnUDP

kl kz ka b ks k6 k7 k. ko ho

E E Bhln EMrluDP-gal EM”UDP-gobglc E.MrvUDP

Jr EAWJDP~ lactose

SCHEME 1

The initial examination of the kinetic properties of the cross- linked complex indicated that the most striking functional differences from untreated galactosyltransferase are a lowered maximum velocity for all reactions combined with very marked decreases in the apparent values for the K, (determined at fixed concentrations of Mn2+ and LJDP-galactose) of the acceptor substrates N-acetylglucosamine, glucose, and xylose (see Table II). Galactosyltransferase, in the absence of a-lactalbumin, showed insignificant activity for galactose transfer to xylose and virtually no activity for transfer to glucose at the concentrations used.

The kinetic properties of the cross-linked complex with respect to the obligatory reactants (MnZ+, UDP-galactose, and monosaccharide) were examined in order to ascertain the nature of any changes in the true values of the kinetic parameters that occurred on cross-linking. Previous studies of uncross-linked bovine and human galactosyltransferases have shown that the reaction mechanism involves an ordered sequential addition of R/In*+, UDP-galactose, and monosaccharide (6, 34, 35). cY-Lactal- bumin does not affect the order of binding of substrates, but attaches to enzyme-substrate complexes to produce enhancement or inhibition of the reaction rate, depending on the nature of the complex formed.

The kinetic properties of the cross-linked complex can be in- terpreted in terms of a mechanism similar to that of the uncross- linked galactosyltransferase as shown in Scheme 1.

,4t high concentrations of MnZf a slightly different mechanism must predominate in which the MnZf complex of UDP-galactose is accepted by the enzyme. As seen in Fig. 12 (6), this is re- flected in the biphasic effects of Mn2+ on the activity of the enzyme .

The parallel lines obtained for double reciprocal plots with UDP-galactose (Fig. 13) and glucose (Fig. 15) as variable substrates with the cross-linked enzyme are closely similar to those previously obtained with human galactosyltransferase under conditions similar to those used in the present study (pH 7.4, 37” (6)). In general, such patterns can be attributed to the in- tervention of an irreversible step in the reaction mechanism between the attachment of the two substrates (see Ref. 32) and often indicates a ping-pong mechanism (i.e. release of product before attachment of second substrate). However, detailed studies of the kinetics of human galactosyltransferase have shown that the pattern results from the irreversibility of the binding of UDP-galactose to E e Mn (i.e. the dissociation constant of E. Mn. TJDP-galactose is zero). The rate equation for the Ter-Bi mechanism in Scheme 1, including terms for product inhibition by MnUDP is given below (taken from Ref. 6).

VABC

’ = KiaKibKcO + Q/Ki,l + KiaK,A + &JX’(l + Q/f&) + KcAB + &AC

(2)

+ KaBCO + Q/KiJ + ABC

Where Ti is the maximum velocity in the forward direction of the reaction, A, B, C, and Q are the concentrations of Mn2+, UDP-

TABLE III Comparison of kinetic constants associated with substrates for lactose

synthesis by cross-linked complex and galactosyltransferase

Mn2+”

UDP-galactose

Glllcose

KC3 Kia Kib & K

n%M

0.15 0.16 1.64 1.64 0 0 0.025 0.027 0.33 0.33

i

HIllllall galactosyl- transierase

(Ref. 6)

mBf

0.083 1.42 0 0.024

2260

a Not determined for lactose synthesis by galactosyltransferase. The value given is for N-acetyllactosamine synthesis.

galactose, glucose, and MnUDP, respectively, K, is the Michaelis constant for XIn2+, Kg for UDP-galactose, and K, for glucose. Ki,, Kib and Ki, are dissociation constants for Mn2f from E. Mn, for UDPgalactose from E. Mn .UDP-galactose, and for MnUDP from E. MnUDP.

Assuming that t,he binding of UDP-galactose is virtually irreversible (Kzb = 0), the equation becomes

VABC

’ = &K&(1 + Q/Ki,) + K,AB + KbAC (3) + K,BC(l + Q/&J + ABC

Rearrangement of this in reciprocal form with A, B, or C varying and Q = 0 shows the dependence of slopes and intercepts in reciprocal plots for a particular substrate on the concentrations of the other two substrates and permits the evaluation of K,, Kia, Kb, and K, (see Ref. 6). These are shown in Table III in comparison with the corresponding parameters previously determined for human and bovine galactosyltransferases (6, 34).

These results clearly confirm the provisional conclusions drawn from the results summarized in Table II. Thus, the kinetic parameters for the complex associated with Mn2+ (K, and Ki,) and UDP-galactose (Kb and Kib) are quite similar to those previously found for uncross-linked galactosyltransferases, whereas K,, the Xchaelis constant for glucose, is decreased by 3 to 6 x lo3 and the turnover number is also decreased.

Rearranging Equation 2 in reciprocal form to predict the inhibition patterns for MnUDP with respect to UDP-galactose and glucose gives Equations 4 and 5.

$ (1 + Q/Kid + 1

(4)

% (1 + Q/&q) + 1

(5)

+ 9 (1 -t Q/K,) + 1

MnUDP (Q) should thus inhibit noncompetitively with respect to UDP-galactose and uncompetitively with respect to glucose. However, the intersect effect in the inhibition with respect to UDP-galactose is governed by the term KJA, which under the conditions of the experiment is extremely low (0.004). The intersect effects should therefore not be observed under these


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conditions, giving rise to the observed competitive inhibition. The replots of the slopes and intersects of Figs. 13 and 15 against [MnUDP] show a slight upward curvature at higher inhibitor concentrations (plots not shown). This can be attributed to the possibility that MnUDP may act as a dead-end inhibitor by attaching to E .Mn (possibly with the displacement of Mn*+) as well as a product inhibitor by attaching to E. Apparent Ki values of 0.017 mM and 0.13 mM for inhibition with respect to UDP-galactose and glucose, respectively, were determined by extrapolation of the replots at the lower MnUDP concentrations. Using Equations 4 and 5 and the values of the kinetic parameter of the complex shown in Table III, true values for K;, of 0.017 m&l and 0.020 mM, respectively, were obtained.

The internal consistency in the data supports the view that the mechanism shown in Scheme 1 is realistic.

The enzymic properties of the enzyme complex, including its low specific activity are best discussed in terms of two alternative proposals concerning the role of a-lactalbumin in the lactose synthetase system. Morrison and Ebner (34) have proposed that in the reactions catalyzed by bovine galactosyltransferase, ordered addition to the enzyme of Mn2+, UDP-galactose, and monosaccharide takes place and that the products are released in the order disaccharide, UDP, and Mn*f. As the mechanism appeared to be unchanged in the presence of cY-lactalbumin, and the enzyme is capable of lactose synthesis in the absence of a-lactalbumin, although with a high Km for glucose, they argue that cr-lactalbumin can only bind to the enzyme after addition of all substrates. In their scheme, cu-lactalbumin attaches to an enzyme-Mn*+-UDP-galactose-glucose complex, and is proposed to increase the concentration of central complexes in the reaction scheme by mass action. They were unable to explain satis- factorily the nature of the inhibition of N-acetyliactosamine synthesis by cr-lactalbumin (1).

More recently, Khatra et al. (6) have proposed an alternative mechanism for the lactose synthetase system. They observed kinetic effects with a-lactalbumin and glucose and other monosaccharides that can only be explained by rapid equilibrium binding of a-lactalbumin prior to glucose in the reaction mechanism. They propose that the binding of cr-lactalbumin to an enzyme- Mn2+-UDP-galactose complex prior to glucose produces an new enzyme form with an increased affinity for monosaccharides. These two alternative mechanisms are shown in simplified form in Schemes 2 and 3.

It is illuminating to compare the kinetic properties of the cross- linked complex with the previously observed effects of cu-lactalbumin on the activities of the galactosyltransferase. Increasing

SCHEME 2

SCHEME 3

concentrations of cY-lactalbumin progressively decrease the apparent K, values for glucose, N-acetylglucosaminc (4-6), aud xylose (6), resulting, on extrapolation to saturating concentrations of a-lactalbumin, in a new K, 2 to 3 orders of magnitude lower than that observed in the absence of cr-lactalbumin (6). At the same time, a-lactalbumin can act as an inhibitor of galactosyltransferase activities, an effect that is strongly dependent on the concentration of monosaccharide (1, 3, 6), and is most marked in the case of N-acetylglucosamine. The inhibition is uncompetitive with respect to Mn2+ and UDP-galactose (6) and at lower temperatures, where transfer to N-acetylglucosamine is not enhanced by cY-lactalbumin, inhibition is uncompetitive with respect to monosaccharide (35). The nature of this inhibition cannot easily be reconciled with Scheme 3, but is predicted by the complete form of Scheme 2 in which au-lactalbumin attaches after N-acetylglucosamine to produce dead-end inhibition (6).

The properties of the complex can be regarded as emphatically reflecting these effects of a-lactalbumin, in showing maximum activation of monosaccharide binding (lowered K, for monosaccharides) combined with maximum inhibition of reaction rates (lowered turnover number, particularly for transfer to N-acetylglucosamine). These properties do not appear to be consistent with the proposal of Morrison and Ebner (34) outlined in Scheme 3, for in the cross-linked complex, a mass action effect of cr-lactalbumin is not possible. These results do provide support for the hypothesis concerning the action of cu-lactalbumin put forward by Khatra et al. (6) in two main respects. First, the observation that a combination of M$+ and UDP-galactose is very effective in promoting the cross-linking reaction, demon- strating that the presence of monosaccharide in a complex is not required for the attachment of cY-lactalbumin. Second, the large decreases in the K, values for qlucosc, N-acetylglucosxmine, and xylose are more easily explained in terms of a large increase in the rate constants for the attachment of these monosaccharides to a complex which includes cr-lactalbumin. As no significant changes are found in the kinetic parameters associated with M& and UDP-galactose, it confirms the hypothesis that cY-lactalbumin exerts its effects at the stage of monosaccharide binding.

In Scheme I, if no isomerisation of central complexes occurs, the turnover number is given by the equation:

and is thus sensitive to changes in the rate constants for the release of either product. The lowered turnover number of the complex could therefore reflect a lowered rate of release of one or both products. Other possible explanations are that only a small proportion of the cross-linked complexes may be catalyti- cally active or that the covalent attachment of Lu-lactalbumin limits the rate of isomerisation of the central complexes in the reaction scheme (i.e. E. Mn . UDP-galactose glucose 6 E. MnUDP *lactose).

Although ovalbumin is an effective acceptor of galactose with normal galactosyltransferase, the cross-linked complex is completely inactive in transferring galactose to this acceptor. (Y- Lactalbumin is a potent inhibitor of galactose transfer to free N-acetylglucosamine (1, 3) but not to glycoprotein-bound N- acetylglucosdmine. In the inhibition of transfer to free N-acetylglucosamine, cr-lactalbumin appears to bind to au enzyme Mn2+.UDP-galactosea N-acetylglucosamine complex in the reaction mechanism giving dead-end inhibition. The large size of such a complex in the case of glycoprotein substrates would be expected to prevent the attachment of c+lactalbumin, and the


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fact that the binding of a-lactalbumin is at rapid equilibrium would prevent its being an effective competitive inhibitor with respect to glycoprotein substrates. However, the covalent attachment of cY-lactalbumin at or near to the site of binding of glycoprotein substrates would be expected to prevent sterically the approach and attachment of these substrates.

Finally, the apparently low extent of secondary chemical modification with dimethylpimelimidate in the complex (4.2 mol per mol of complex) suggests that radioactive labeling of the complex can be used as an appraoch to determining the site of the cross-links within the polypeptide chain of oc-lactalbumin and galactosyltransferase. This is currently under investiga- tion.

1.

2.

3.

4.

5. 6.

7.

8.

9. 10.

11.

12.

13.

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K Brew, J H Shaper, K W Olsen, I P Trayer and R L HillCross-linking of the components of lactose synthetase with dimethylpimelimidate.

1975, 250:1434-1444.J. Biol. Chem.

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