©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Catalytic Activities of Glycogenin Additional to Autocatalytic Self-glucosylation (*)

Miriam D. Alonso , Joseph Lomako , Wieslawa M. Lomako , William J. Whelan (§)

From the (1)Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida 33101

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Glycogenin is the autocatalytic, self-glucosylating protein that initiates glycogen synthesis in muscle and other tissues. We have sequenced the cDNA for rabbit muscle glycogenin and expressed and purified the protein in high yield as well as two mutant proteins in which Phe or Thr replaces Tyr-194, the site of glucosylation. While the wild-type protein can self-glucosylate, the mutants cannot, but all three utilize alternative acceptors by intermolecular glucose transfer for which the mutants have altered specificity. Tyr-194 is therefore not essential for the catalytic activity of glycogenin. All three proteins also hydrolyze UDP-glucose to glucose at rates comparable with the rate of self-glucosylation. The hydrolysis is competitive with glucose transfer to p-nitrophenyl -maltoside. Self-glucosylation, glucosylation of other acceptors, and hydrolysis all appear to be catalyzed by the same active center. In the absence of peptidase inhibitors, the homogenous recombinant proteins of M 37,000 break down to equally active species having M 32,000. The kinetics of self-glucosylation catalyzed by the wild-type enzyme suggest that the reaction could be intermolecular rather than, as previously reported, intramolecular. The wild-type recombinant enzyme and native muscle glycogenin, which is phosphorylated, are inhibited quite differently by ATP at physiological concentration.


INTRODUCTION

Glycogenin is a protein first obtained from rabbit muscle glycogen, to which it is covalently bound in one molecular proportion via the novel glucose-tyrosine bond, where its presence in this form suggested that it was the long sought primer for glycogen synthesis (for reviews, see Refs. 1 and 2). Glycogen-free proteins with similar properties were detected in muscle and other tissues. When purified to homogeneity they were found to be autocatalytic, undergoing self-glucosylation in the presence of µM UDP-glucose and Mn, creating a maltosaccharide chain of eight glucose residues that functioned as a primer for glycogen synthesis.

We are interested in the functionality of glycogenin, such questions as the nature of its active site, where and how it binds UDP-glucose, where ATP, a powerful inhibitor(3) , binds, how the first glucose residue is attached to tyrosine(4, 5) , and whether the phosphorylation of serine (6) and tyrosine (7) residues plays a role in the regulation of self-glucosylation. Molecular cloning, mutagenesis, and the expression of recombinant proteins are the preferred methodologies with which to pursue such questions. Accordingly, we have cloned the cDNAs for glycogenin from human and rabbit muscle, compared the deduced amino acid sequences, and expressed these proteins. We report here only on the rabbit muscle enzyme, which we have purified to homogeneity and tested for functionality. The similar cloning, expression, and purification of the rabbit protein and two mutant proteins have already been reported by Roach and co-workers(8, 9) . Asking whether it is necessary for Tyr-194 to be present for glycogenin to function as a transglucosylase, we have expressed the same recombinant proteins as Roach and co-workers, in which Phe and Thr replace Tyr-194. Contrary to their report that these two mutants are enzymically inactive because Tyr-194 is ``essential for activity''(9) , we have already reported elsewhere that both mutants display intermolecular transglucosylase activities(4) . That of the Phe-194 mutant is comparable in its catalytic efficiency with that of wild-type glycogenin. Here we report a much more detailed study, with different substrates, of the ability of wild-type and mutant glycogenins to carry out intermolecular transglucosylation. What this has revealed is that the self-glucosylation, previously claimed to be intramolecular, could be intermolecular and that the transglucosylase activity of glycogenin functions irrespective of whether Tyr-194 is fully glucosylated or is replaced by Phe or Thr. A new catalytic activity of glycogenin that we report here is the hydrolysis of UDP-glucose at a rate significant in comparison with that at which glycogenin uses UDP-glucose in autocatalysis, while the homogeneous recombinant enzymes break down spontaneously to 32-kDa species. We also note a difference between native and recombinant glycogenin in respect to inhibition by ATP.


EXPERIMENTAL PROCEDURES

The commercial sources of biochemical reagents and kits for molecular cloning and expression are mentioned at their point of use. Otherwise the supplier was Sigma. Oligonucleotides were synthesized by the Biotechnology Facility at the University of Florida, Gainesville, and by Dr. R. Werner of our department. 1 SSC()was used in more concentrated or less concentrated forms such as 2 SSC, 0.5 SSC. Glycogenin self-glucosylating activity was measured by incorporating glucose from UDP-[C]glucose (DuPont) into the protein and precipitating it with trichloroacetic acid(10) . For Western blotting we used an antibody to native rabbit muscle glycogenin(10) . Transglucosylase activity was measured with p-nitrophenyl saccharides (Ref. 11, and see below) or with n-dodecyl -maltoside (DBM) as acceptor substrate (4, 12) and UDP-[C]glucose as donor. Treatment of glycogenin with insoluble -amylase prior to UDP-[C]glucose () was carried out with cell lysates in 100 mM acetate buffer, pH 7.0, 10 mM CaCl and 2 units of insoluble enzyme for 1 h at room temperature with shaking. The enzyme was removed by centrifugation through SpinX filter units (Costar), and the filtrate was used for self-glucosylation in comparison with a corresponding volume of untreated lysate.

When the E. coli cultures containing expressed proteins (see below) were tested for the presence of glycogenin, 1-ml portions of the cultures were centrifuged, and the pellets were resuspended in 500 µl of lysis buffer (see below), sonicated, and centrifuged at 12,000 rpm for 10 min. Portions (65 µl) were used for self-glucosylation reactions (10) in 100-µl digests where the C incorporated was measured by trichloroacetic acid precipitation and counting () or by SDS-PAGE and radioautography (Fig. 1). The C counts/min in represent the total [C]glucose that can be incorporated, not the relative rates. Western blotting (10) was carried out on 10-µl aliquots of the lysates.


Figure 1: Properties of recombinant glycogenins as shown by SDS-PAGE, radioautography, and immunoblotting. A, Coomassie Blue-stained gel (M markers on the left) of E. coli lysates in which human muscle glycogenin (induced (lane 1) or uninduced (lane 2)) rabbit muscle glycogenin (induced (lane3) or uninduced (lane4)) had been expressed. B, radioautograph of the gel in A. C, Western blot of the same lysates after SDS-PAGE. PanelD contains the Coomassie Blue-stained purified wild type (lane1), Phe-194 (lane2), and Thr-194 mutants (lane3) studied here.



Cloning of Rabbit Muscle Glycogenin cDNA and Expression of the Protein

The cloning procedures were performed independently of and prior to the appearance of the publications by Viskupic et al.(8) and Cao et al.(9) . A 728-base pair probe was used to screen a rabbit muscle ZAP II cDNA library (Stratagene). The probe was obtained by PCR utilizing two oligonucleotide primers derived from the primary sequence of rabbit muscle glycogenin (2) and the library itself as the DNA template. The sense primer (5`-AACGACGCCTA(C/T)GC(C/G)AA(G/A)GG-3`) originated from residues 11-17, while the antisense primer (5`-GTGGTGAAGATGTC(C/A)CACC-3`) was derived from amino acids 248-254. The probe, with its sequence confirmed by dideoxy sequencing (13) using Sequenase version 2.0 (U.S. Biochemical Corp.), was labeled by the PCR incorporation of digoxigenin 11-dUTP (Boehringer Mannheim)(14) . Digoxigenin is an intermediary in chemiluminescent signal emission, detected by the procedures specified by the manufacturer. Approximately 10 phage particles from the cDNA library were screened by standard techniques(15) . After three rounds of plaque purification, putative positive clones were excised in vivo from ZAP II to yield recombinant pBluescript phagemid. The in vivo excision protocol provided by the manufacturer (Stratagene) was followed. The clones were confirmed by dideoxy sequencing of both strands(13) . The subcloning of the full-length cDNA into the prokaryotic expression vector was done essentially as by Viskupic et al.(8) , except for the use of a different expression vector: pET-11d (Novagen). The resulting expression construct was designated pET-R. The expression of the wild-type and mutagenized forms of glycogenin was done as by Studier et al.(16) . Small samples of the culture expressing the wild-type protein were subjected to self-glucosylation assays, SDS-PAGE, and Western blotting (see above) (Fig. 1, ). The purification of the three forms of glycogenin has been described elsewhere(4) .

Substitution of Tyr-194 by Phe or Thr

The ``megaprimer'' method of PCR-mediated site-directed mutagenesis was employed(17) . The sequences of the primers for the Phe and Thr substitutions of Tyr-194 in glycogenin were, respectively, 5`-GCATTTCTATATTCTCCTACCTCCCAGC-3` and 5`GCATTTCTATAACCTCCTACCTCCCAGC3`, spanning residues 190-199. The oligonucleotide primers employed in the subcloning of the wild-type cDNA into pET-11d (see above) were used along with the mutagenic primers and a preparation of pET-R plasmid DNA in the two-step PCR procedure to generate the mutant molecules. These were subcloned into pET-11d as above to yield the pET-RF (Phe-194 mutant) and pET-RT (Thr-194 mutant) expression vectors. Sequence confirmation was done by the dideoxynucleotide method(13) .

Glucosylation of p-Nitrophenyl -Saccharides by Wild-type and Mutant Forms of Glycogenin

In order to measure the glucosyltransferase activities of the wild type and its mutagenized forms, p-nitrophenyl -glucoside, -maltoside, -maltotrioside, -maltotetraoside, and -maltohexaoside were used as glucose acceptor substrates(11) . The digests (100 µl each) contained 1 µM homogeneous recombinant protein (Fig. 1D), 10 mM UDP-glucose, 10 mMp-nitrophenyl -saccharide, 5 mM MnCl, and 50 mM Tris-HCl buffer, pH 7.4. After incubation for 18 h at room temperature, 5 µl or 10 µl of each digest was loaded onto a C18 column and fractionated by reverse phase high performance liquid chromatography using an ascending gradient of acetonitrile (10-20%; 1 ml/min for 20 min). The increased concentration of UDP-glucose and the time of incubation were designed to ensure that a sufficient mass of product was formed for accurate measurement. The wild-type enzyme was completely stable during the 18-h incubation. The formation of p-nitrophenyl saccharide species containing n + 1 glucose residues with respect to the acceptor substrate from which they originated, was monitored and quantitated by absorbance at 320 nm (). The results in were calculated from the relative areas under the curves for each peak, determined by the integrator.

[C]Glucose arising from hydrolysis of the [C]UDP-glucose substrate (see below) was measured concomitantly with intermolecular transfer by the Phe-194 mutant enzyme of [C]glucose to p-nitrophenyl -maltoside at concentrations of the latter ranging from 0.1 µM to 10 mM. The reaction mixtures (100 µl) were as above, except that the enzyme concentration was 0.2 µM and the concentration of UDP[C]glucose was 2 µM. Incubation was for 2 h at room temperature, after which excess UDP-glucose was removed by applying the digest to a column of mixed bed resin (Bio-Rad AG 501-X8(D), 0.8 ml) and eluting with 1 ml of water. The effluent (200 µl) was fractionated by reverse phase high performance liquid chromatography as above, p-nitrophenyl -maltoside and -maltotrioside being mixed in to permit their appearance to be detected by absorbance at 320 mn. [C]Glucose was collected in the fractions immediately postinjection. The C-labeled p-nitrophenyl -maltotrioside, formed from the maltoside, was also collected (see ).

Hydrolysis of UDP-Glucose by Recombinant Proteins

Each of the three recombinant proteins (20 pmol, wild type, Phe-194, and Thr-194 mutants) was incubated in a 100-µl digest containing 2 µM UDP-[C]glucose, 50 mM Tris-HCl, pH 7.4, in the presence and absence of 5 mM Mn. After 2 h at room temperature, the digests were applied to mixed bed resin columns (0.5 ml, Bio-Rex RG 501-X8 resin, Bio-Rad) and eluted with 2 ml of water, collecting 0.5-ml fractions in which [C]glucose released by hydrolysis was counted. In the case of wild-type protein, an additional column was used to remove self-[C]glucosylated protein from free [C]glucose. The mixed-bed resin column was connected to a Sep-Pak C cartridge (Millipore) prewetted with acetonitrile. Elution was done with 5 ml of water in 0.5-ml fractions in which the [C]glucose was counted. Control digests omitting enzyme were processed in the same way to ensure that in the absence of enzyme no [C]glucose was released. The results are shown in I.


RESULTS

Cloning and Sequencing the cDNAs for Rabbit Muscle Glycogenin

A full-length clone of the cDNA for rabbit muscle glycogenin was obtained and sequenced by standard procedures. The clone contained 1809 base pairs encoding a protein of 332 residues. The sequences were identical both with respect to the nucleotide coding and the amino acid sequence with those already reported by Viskupic et al.(8) . We simultaneously cloned and expressed the human muscle protein. The results, which will be reported elsewhere, showed the lengths of both proteins to be the same, with 34 differences between the amino acid sequences.

Expression and Purification of Recombinant Proteins

We expressed both the human and rabbit proteins and proceeded with study of the latter because of a much greater level of expression. The wild-type expression vectors were introduced into an Escherichia coli BL 21/DE 3 host. Protein synthesis was induced with isopropyl -thiogalactopyranoside. Glycogenin was detected in lysates of cultures by means of Mn-dependent self-glucosylation with a UDP-[C]glucose donor. After SDS-PAGE, prominent bands at M 37,000 could be seen on the stained gel. Fig. 1is a composite picture of the properties of lysates of cultures of recombinant glycogenins. In Fig. 1A, stained for protein, the high level of expression of induced rabbit protein (lane3, M 37,000) can be seen. These lysates had been preincubated with UDP-[C]glucose, and Fig. 1B is a radioautograph of Fig. 1A. Radioglucosylated protein was seen in all cases. A qualitative comparison of relative amounts of glycogenin in each lysate is afforded by Fig. 1C, a Western blot using antibody to glycogenin from rabbit muscle glycogen(10) . Compared with the induced lysates (Fig. 1C, lanes 1 and 3), the amounts of glycogenin in the uninduced lysate (Fig. 1C, lanes 2 and 4) were much smaller, although active glycogenin was certainly present (Fig. 1B, lanes 2 and 4). By contrast, there was a much greater relative degree of radiolabeling of the uninduced versus induced proteins (compare Fig. 1B and Fig. 1C). This is shown quantitatively in , where the relative amounts of [C]glucose incorporated by rabbit protein lysates are compared. The lysates were also treated with insolubilized -amylase before incubation with UDP-[C]glucose, and the results are compared.

Given the strong expression of the induced wild-type rabbit muscle protein, we decided to examine this protein and mutants derived from its cDNA where Phe and Thr replaced Tyr-194. All three proteins were purified to homogeneity by a two-step column procedure that we have described elsewhere(4) . A comparison with the data reported by Cao et al.(9) for their rabbit protein expression system reveals the superiority of ours in terms of level of expression. So much glycogenin was expressed in our system that only a 2.5-fold purification was necessary to obtain a homogeneous product in a two-step process ( Fig. 1and Ref. 4) in a yield of 49%. Previously (9) a five-step process was necessary to achieve an 8.4-fold purification to homogeneity in a 14.4% yield. (Compare Fig. 1A, lane1, in Ref. 9 with our Fig. 1A, lane3). Fig. 1D depicts the Coomassie Blue stains of the purified proteins after SDS-PAGE. There is a clear difference in M between the wild-type protein and the mutants, which can be related to the presence (wild type) or lack (Phe-194 and Thr-194 mutants) of glucose carbohydrate. In this respect, the wild-type enzyme was able to glucosylate itself and, when homogeneous (Fig. 1), different preparations added glucose equivalent to 0.8-2 mol.proportions, suggesting that 6-7 mol.proportions of glucose were already present. The mutant enzymes did not appear to self-glucosylate. During storage of the lysate or during concentration of the homogeneous proteins, each was converted into a product having M 32,000, but no change in activity occurred. When a mixture of peptidase inhibitors such as is used to prevent breakdown of muscle glycogenin during purification (10) was included at all stages of purification, and during subsequent storage, no breakdown occurred. We report here that the same behavior has been noted in wild-type carbohydrate-free glycogenin expressed in a different strain of E. coli(5) , but we did not observe breakdown with purified native rabbit muscle glycogenin(10) . The recombinant enzymes, stored with peptidase inhibitors, were stable for up to 3 months when refrigerated.

We measured the initial rate of self-glucosylation by wild-type glycogenin under the standard conditions (10) over a range of enzyme concentration from 0.01 to 1 µM. The rate was uniform during the 5-min incubation. The results are shown in Fig. 2.


Figure 2: Dependence of glycogenin self-glucosylation on protein concentration. The initial rate of glucose incorporation was measured at 5 min under standard conditions (10) with 20 µM UDP-[C]glucose in the presence of bovine serum albumin at 1 mg/ml over the range 0.01-1 µM enzyme. The rates with 0.01-0.3 µM enzyme were also measured at 0.1 mg/ml bovine serum albumin. The results for 0.01-0.3 µM enzyme are the averages of four determinations, and the results for 0.5 and 1 µM enzyme are the averages of duplicates.



Transglucosylation by Mutant Proteins

Although the two mutants could not self-glucosylate, there was the possibility that they could use an alternative glucose acceptor. p-Nitrophenyl -glucoside and -maltosaccharides act as alternative glucose acceptors for the muscle enzyme, competing with self-glucosylation (11), and proved to be acceptors not only for the recombinant wild-type enzyme but also for the Phe-194 and Thr-194 mutants (). The mutant proteins were even superior to the wild-type enzyme in respect to their relative abilities to glucosylate p-nitrophenyl -glucoside, but not so for the maltoside, which is the preferred substrate for the wild-type enzyme ().

UDP-Glucose Hydrolysis

All three recombinant proteins hydrolyzed UDP-glucose in an Mn-dependent manner (I). The Thr-194 mutant protein was most active. When the Phe-194 mutant protein was allowed simultaneously to glucosylate p-nitrophenyl -maltoside, the hydrolytic release of glucose was inversely related to the maltoside concentration ().

Inhibition by Nucleoside Triphosphates

In agreement with Cao et al.(9) , the wild-type glycogenin was powerfully inhibited by UTP (98.5% in 10 mM UTP) and less so by ATP (53% in 10 mM ATP). Cao et al.(9) reported 95% inhibition by UTP and 46% by ATP at the same inhibitor concentrations. These results for ATP stand in marked contrast to those for the inhibition exerted by ATP on native rabbit muscle glycogenin(3) , where the glycogenin is virtually inactive in 5 mM ATP.


DISCUSSION

Rabbit muscle glycogenin was expressed and detected in lysates of the E. coli host, where induced and uninduced cultures were compared (Fig. 1, ). Both lysates contained immunologically positive, active glycogenin of the correct M as shown by Mn-dependent [C]glucosylation and Western blotting (Fig. 1, B and C). The induced lysate contained much more glycogenin protein than the corresponding uninduced lysate (Fig. 1C). That in the induced lysate was such that it could easily be seen after SDS-PAGE and protein staining (Fig. 1A, lane3), amounting to almost half the total soluble protein. Therefore, we used this system to obtain purified enzyme and to construct and express the Phe-194 and Thr-194 mutants. The relative amounts of [C]glucosylation seen in the induced lysates of the wild-type enzyme in three experiments varied 4-fold ().

This variability reflects not so much the amount of glycogenin protein, but instead reflects the level to which the protein is already glucosylated and how much more glucose is to be added to bring the maltosaccharide chains to an average of eight glucose units. Roach and co-workers (8, 9) had noted for the recombinant rabbit protein that glucosylation had already occurred in the expression system. We agree, noting the higher M, presumably due to carbohydrate, of the wild type versus the Phe-194 and Thr-194 mutants (Fig. 1D) and the fact that prior -amylolysis of the induced rabbit protein resulted in its ability to incorporate much more [C]glucose (). Therefore, -amylase-degradable maltosaccharide was already present in the expressed protein. An unexpected feature of the comparison between lysates of the wild-type enzyme was the relative degree of [C]glucosylation as between the induced and uninduced proteins. In different sets of lysates the uninduced lysate incorporated 4-10 times as much [C]glucose as did the corresponding induced lysate (). This occurred even though there was less glycogenin present in the uninduced lysate (Fig. 1C). The induced glycogenin was therefore already glucosylated to a greater degree than the uninduced protein, meaning that in vitro the latter would self-glucosylate with UDP[C]glucose to a greater extent. This comparison suggested that the uninduced glycogenin was largely unglucosylated, and this is borne out by comparing the M values of the proteins (Fig. 1C). An unexpected feature of the ability of the two types of protein to self-glucosylate was seen when they were first treated with -amylase to shorten the preexisting maltosaccharide chains. The induced glycogenin now incorporated 5-7 times more C than before -amylolysis. The uninduced glycogenin, however, almost lost its capacity to glucosylate (). This phenomenon requires further examination.

In order to explore the mechanism of self-glucosylation of glycogenin, we constructed mutants in which Phe or Thr replaced Tyr-194 and tested them with UDP-glucose. No self-glucosylation occurred, but the mutants could still be capable of transglucosylation. This proved to be the case. The p-nitrophenyl -maltosaccharides and the -glucoside are efficient competitive acceptors of glucose transferred from UDP-glucose by the rabbit muscle enzyme(11) . The mutants also used these acceptors (). In other words, the removal of Tyr-194 only prevents the glucosylation of glycogenin. The catalytic activity is retained in the mutants, but their acceptor substrate preferences are changed. The mutants use the glucoside as the preferred substrate, whereas wild-type glycogenin prefers the maltoside. What the mutants share in common is the absence of a carbohydrate chain. Therefore, the mutant proteins are capable of intermolecular transglucosylation, with altered specificity but comparable catalytic activity. The ability to transglucosylate does not reside to any important extent in Tyr-194. These findings contradict the claim by Cao et al.(9) , who also expressed the Phe-194 and Thr-194 mutants and, finding them unable to self-glucosylate, or to glucosylate a 30-residue synthetic peptide surrounding Tyr-194, stated that ``Tyr-194 is essential for function [and] essential for activity.''

The ability of the mutant proteins to transglucosylate but not self-glucosylate should be of assistance in exploring the catalytic mechanism of transglucosylation. Hitherto only the wild-type enzyme has been available, acting both to donate and to accept transferred glucose. Now, in the mutant proteins, we have an enzyme that is no longer a substrate. Correspondingly, the availability of low molecular weight saccharide acceptors shows that the aglycone does not have to be a protein but can be a very small molecule. Thus, glucose has no detectable acceptor activity(11) , while p-nitrophenyl -glucoside is a powerful acceptor ().

The discovery that the Phe-194 and Thr-194 mutants are capable of intermolecular transglucosylation made us realize that this property, which is shared by the wild-type enzyme, is independent of self-glucosylation. The question arises whether there is an endogenous receptor substrate, other than glycogenin itself, for such intermolecular transglucosylation. It is clear that maltose is such a substrate(9, 11) . Second is the question whether the activity is significant. It is certainly capable of being more rapid than self-glucosylation. The rate of transfer of glucose to DBM at 2 mM UDP-glucose is 0.92 µmol of glucose transferred/min/mg of protein, some 200 times greater than the maximal rate of self-glucosylation shown in Fig. 2. Whether self-glucosylation also occurs by intermolecular transfer has not previously been decided, despite claims to this effect. Both Pitcher et al.(18) and Cao et al.(9) reported first-order kinetics for self-glucosylation and concluded that it is intramolecular. However, the Pitcher et al. laboratory had reported that glycogenin is a dimer(19, 20) , while Cao et al.(9) did not comment on the size of their recombinant enzyme. When we found that homogeneous muscle glycogenin underwent self-glucosylation, we also noted that it was oligomeric (10) and did not attempt to draw a conclusion as to intra- or inter-molecular transfer. When the intermolecular glucosylation of the p-nitrophenyl saccharides was discovered, we pointed out the implication that this had for the self-glucosylation reaction, suggesting that glycogenin, in its autocatalysis, ``may act by intermolecular glycosylation between aggregated protein molecules and that the aggregation may be purposeful''(11) .

Using wild-type recombinant enzyme we reexamined the self-glucosylation reaction kinetics, paying particular attention to the reaction rates at the lower end of the range of glycogenin concentration that Cao et al.(9) employed (Fig. 2). The reaction only assumes first-order kinetics above 0.5 µM enzyme concentration. Below this, the rate falls away, suggestive of the dissociation of an interactive complex. Therefore, previous conclusions, based on kinetics, that the reaction is intramolecular (9, 20) are not justified on that evidence alone, and the question remains open.

With respect to the mass of the glycogenin monomer, we noted that unless peptidase inhibitors were added to the E. coli lysates during purification and subsequent storage of the purified enzyme, the wild-type and mutant proteins each broke down to active species with M about 32,000.()This occurred even after the M 37,000 proteins had been purified to apparent homogeneity. The breakdown occurred without change of activity. This phenomenon was not noted by Cao et al.(9) .

Finally, we report two additional properties of glycogenin, the first that it hydrolyzes UDP-glucose. The second is the only difference between native and recombinant glycogenin so far noted. Homogeneous wild-type glycogenin and the two mutants hydrolyze UDP-glucose (I). The Thr-94 mutant is the most active. The rate of hydrolysis is comparable with the rate of self-glucosylation. Thus, in a 20-min period during which the wild-type enzyme would have utilized 2.0 mol of UDP-glucose in self-glucosylation(4) , the enzyme would have hydrolyzed 0.6 mol of UDP-glucose. Therefore, the hydrolytic capacity of glycogenin has to be considered of potential significance, and in light of the fact that the concentrations of UDP-glucose employed for its assay are in the low micromolar range, investigators who have previously engaged in prolonged incubation of glycogenin with micromolar UDP-glucose in order to learn how much glucose the enzyme would add to itself have not known that the enzyme was simultaneously hydrolyzing the substrate. Therefore, reported extents or rates of glucosylation may have been in error because the substrate had disappeared.()In contrast to our findings, others have stated, but without providing evidence, that ``we have excluded the possibility that glycogenin itself has any form of glycosidase activity''(9, 24) . We disagree. No mention was made in these reports of the substrates tested.

The hydrolysis of UDP-glucose was also seen in experiments in which the Phe-194 mutant protein was allowed to transglucosylate p-nitrophenyl -maltoside over a range of concentrations of the acceptor from 0.1 µM to 10 mM. As the acceptor concentration increased and more p-nitrophenyl -maltotrioside was formed, the amount of glucose, formed by hydrolysis, decreased (). When the native enzyme is incubated with -maltoside, there is also an inverse relation between the degree of self-glucosylation and -maltoside concentration(11) . Therefore, we may conclude that self-glucosylation, which may be intermolecular (Fig. 2), intermolecular transglucosylation to simple saccharides, and UDP-glucose hydrolysis are each catalyzed by the same active center.

Lomako et al.(3) reported that ATP is a powerful inhibitor of native rabbit muscle glycogenin such that at physiological ATP concentration (5 mM) there was almost complete inhibition. Cao et al.(9) however, found that in 10 mM ATP, recombinant muscle glycogenin is inhibited only 45%, while UTP is much more inhibitory. We find similar levels of inhibition of recombinant glycogenin by ATP and UTP. A reason for the difference between the two forms of glycogenin, native and recombinant, could be protein phosphorylation. Since it was not exposed to mammalian protein kinases, we would not expect the recombinant protein expressed in E. coli to be phosphorylated; but native glycogenin from rabbit muscle glycogen contained 0.8 mol.proportions of phosphate, and additional phosphate could be introduced at Ser-43 by protein kinase and [P]ATP(6) .

  
Table: [C]Glucosylation of proteins in lysates of E. coli expressing rabbit-muscle glycogenin


  
Table: Intermolecular transglucosylation of p-nitrophenyl saccharides by wild-type and recombinant glycogenins


  
Table: Hydrolysis of UDP-glucose by recombinant proteins


  
Table: Simultaneous hydrolysis of UDP-glucose and glucose transfer to p-nitrophenyl -maltoside by Phe-194 mutant glycogenin



FOOTNOTES

*
This research was supported by National Institutes of Health Grant DK 37500. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology (M823), University of Miami School of Medicine, P.O. Box 016129, Miami, FL 33101-6129. Tel.: 305-243-6266; Fax: 305-324-5665.

The abbreviations used are: SSC, 17.5% sodium chloride, 8.82% sodium citrate, pH 7.0; DBM, n-dodecyl -maltoside; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction.

A self-glucosylating rat-kidney protein with some properties similar to glycogenin also has M 32,000 (21, 22), but it should be noted that the kidney protein failed to give a positive immunoblot with two antibodies to muscle glycogenin, one of which gave a positive blot with the M 32,000 breakdown product of muscle glycogenin (23) and, we now report, with the M 32,000 breakdown product of wild-type recombinant glycogenin as described here or when carbohydrate-free (5).

In a report on the specific activity of homogeneous recombinant rabbit muscle glycogenin, a range of values is given, from 7.5 to 15.4 pmol glucose transferred/min/µg of protein (9).


ACKNOWLEDGEMENTS

We thank Erik Lagzdins and John Rodriguez for carrying out the initial studies with DBM, Michele Montejo and Michael Muench for the molecular sieving of recombinant glycogenin, and Dr. Lennart Roden and colleagues for providing information about DBM prior to publication(12) .


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