α-glucan synthesis on a protein primer : a reconstituted system for the formation of protein-bound...

Eur. J. Biochem. 162,609-614 (1987) 0 FEBS 1987

a-Glucan synthesis on a protein primer A reconstituted system for the formation of protein-bound a-glucan

Silvia MORENO, Carlos E. CARDINI and Juana S. TANDECARZ

Iiistituto de Investigaciones Bioquimicas 'Fundacion Campomar' and Facultad de Ciencias Exactas y Naturales, Buenos Aires

(Received July 25/0ctober 23, 1986) - EJB 86 0799

Reconstitution experiments with the DEAE-cellulose-treated enzymes, engaged in a two-step mechanism of synthesis of a-glucan bound to protein, are performed. Urea/sodium dodecyl sulfate/polyacrylamide gel electrophoretic analysis of the radioactive products synthesized by the reconstituted system shows highly glucosylated, labeled bands, whose apparent molecular masses change with the acrylamide concentration in the gels. The long carbohydrate chains synthesized during the second step arise from the sequential addition of glucosyl moieties to the glucoprotein formed during the first step. A deglucosylation experiment confirms that the product of the reconstituted system originates from the 38-kDa glucosylated component of the reaction 1 product by the addition of P-amylase-sensitive glucosyl moieties. Our data suggest that specific phosphorylases and starch synthetases are found in potato tuber, which are capable of utilizing reaction 1 product as primer for the synthesis of protein-bound glucan.

The occurrence of a glucoprotein intermediate in polysaccharide biosynthesis has been postulated for several tissues [l-61. A two-step mechanism for the synthesis of a-1,4- glucosidic chains, covalently bound to protein in a particulate preparation of potato tuber, was postulated in earlier reports [7,8]. The enzymatic system involved in the proposed mechanism, which does not require the addition of an exogenous acceptor, could be solubilized by increasing the ionic strength of the medium [9]. This mechanism involved at least two reactions: reaction (1) and reaction (2) [lo], that can be schematically expressed as follows:

UDP-Glc + acceptor protein (1) --f acceptor protein-Glc + UDP .

Acceptor protein-Glc + n UDP(ADP)-Glc or n glucose-1-P -+ acceptor protein-Glc-(Glc), (2) + n UDP (ADP) or n P i .

During reaction 1 an endogenous acceptor is glucosylated and serves then as primer for phosphorylase and starch synthetase (reaction 2). The major functional and physical properties of

Correspondence to J. S. Tandecarz, Instituto de Investigaciones Bioquimicas, 'Fundacion Campomar', Antonio Machado 151, RA- 1405 Buenos Aires, Argentina

Dedicated to Prof. Dr. Luis F. Leloir on the occasion of his 80th anniversary.

The data in this report have been submitted as partial fulfillment of the requirement for the Ph.D. degree by S.M.

Abbreviations. ADP-Glc, adenosine 5'-diphosphoglucose; glucose-I -P, glucose 1 -phosphate; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate.

Enzymes. a-Amylase (EC 3.2.1.1); j-amylase (EC 3.2.1.2); amyloglucosidase (EC 3.2.1.3); phosphorylase or 1,4-a-glucan: ortho- phosphate glucosyltransferase (EC 2.4.1.1); starch synthase or ADPglucose: starch 4-a-glucosyltransferase (EC 2.4.1.21); UDPglucose: protein 4-a-~-glucosyltransferase or UDPGlc: protein transglucosylase I (EC 2.4.1.112).

the enzyme catalyzing reaction 1, isolated from a solubilized potato tuber preparation, were recently reported [lo]. This enzyme was shown to catalyze the formation of a covalent linkage between a hydroxyamino acid of the endogenous acceptor protein and ['4C]glucose from UDP-[14C]Glc, giving rise to a glucoprotein. Only one residue of the monosaccharide is transferred to the specific aminoacyl residue of the endogenous acceptor during reaction 1 [lo]. The reaction 1 product was found to be a glucoprotein of the 0-glycosidic type. Upon denaturing PAGE of the 14C-labeled reaction 1 product, a 38 000-Da radioactive component was obtained, indicating that this would be the subunit of the endogenous acceptor protein that became glucosylated. A common mechanism for glucan chain elongation, independent of whether exogenous polysaccharide or endogenous glucoprotein were the primers, was proposed, based on the behaviour of the engaged enzymes towards 6-gluconolactone [8]. In order to investigate if any of the classical elongating enzymes, i.e. phosphorylase and starch synthetase, is able to catalyze reaction 2, utilizing the product of reaction 1 as acceptor, a reconstituted system with partially purified enzymes was assayed. The ability of reaction 1 product to be used as primer by specific starch synthetases and phosphorylases is being reported here.

MATERIALS AND METHODS

Materials

UDP-['4C]Glc (268 Ci/mol) and ADP-['4C]Glc (262 Ci/ mol) were obtained according to Thomas et al. [ll]. ['4C]Glucose-l-P (156 Ci/mol) was from New England Nu- clear. Sweet potato p-amylase, bacterial a-amylase (type II- A), amyloglucosidase and a-D-glucose-1-P (dipotassium salt, grade I) were obtained from Sigma Chemical Co. Acrylamide and bisacrylamide were purchased from Bio-Rad.

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Enzyme preparations

The solubilized preparation from potato tuber was isolated as in previous work [9]. UDPGlc: protein transglucosylase I (peak I) as well as phosphorylase and ADP(UDP)Glc : starch synthase activities (peaks 11, and IIb) were obtained by DEAE-cellulose chromatography [ 101.

Assays

Unless otherwise specified, the assay conditions for UDPGlc : protein transglucosylase I, primed and unprimed phosphorylase as well as for ADP(UDP)-Glc: starch synthase were as described in [lo].

Assay for the reconstituted system Fig. 1. Size determination of the ‘‘C-labeled glucosylated polypeptides of products of reaction ( 1 ) + reaction ( 2 ) . The labeled products

This was determined with the DEAE-cellulose partially @ O pg protein; 10 300 cpm) were submitted to urea/SI>S gel electrophoresis. Fluorograms of the labeled polypeptides on 10% (lane 1) purified protein components, in two-step experi- or 12.5% (lane 2) acrylamide gels are shown, The corresponding

merits. Conditions for each of the reconstituting experiments positions of protein molecular standards are indicated on each are detailed under each Of the corresponding tables and figures. At least four enzyme preparations were tested to ensure reproducibility.

side in kDa. Exposure time was 10 days for the 10% gel and 20 days for the 12.5% gel

Analytical non-denaturing PAGE

This was carried out using 7% gels according to Davis [I21 with slight modifications [13]. Location of primed and unprimed phosphorylase activities on the gels, following electrophoresis, was done as already reported [14].

Urea-SDS-PAGE

The electrophoretic system of Ziegler et al. [15] was used at 10% and 12.5% acrylamide in the gels. Protein standards were identified by staining with 0.25% Coomassie blue, and radiolabeled proteins by fluorography [16].

Other procedures

Protein was extracted from the gels by macerating the gel slices with a Potter-Elvehjem homogenizer in 100 mM Tris/ HCl buffer pH 7.4, containing 5 mM 2-mercaptoethanol. Gel fragments were removed by centrifugation.

Conditions for 8-amylase, a-amylase and amyloglucosidase digestions were as in [l]. Sucrose density gradient centrifugation was carried out as previously reported [lo]. Proteins were measured by the procedure of Lowry et al. [17].

RESULTS

In accordance with previous work on potato tuber mem- branous preparations [7, 81, incubation of the potato tuber solubilized preparation [9] with 4 pM UDP-[‘4C]Glc led to a plateau in the radioactivity incorporation in about 30 min. The addition of unlabeled UDP-Glc, ADP-Glc or glucose-l- P at 4 mM concentration (1000-fold) after 30 min gives rise to protein-bound ~c-Z,6glucan (product of reaction 1 + reaction 2). The reaction products were characterized by urea/ SDSjPAGE at two acrylamide concentrations. Fig. 1 shows

the fluorogram obtained by such a two-step incubation, in which unlabeled UDP-Glc was added during the second step. In addition to the 38-kDa labeled band of reaction 1 product [lo] a component of 50 kDa was obtained at 12.5% acrylamide concentration in the gel (Fig. 1, lane 2). The mobility of this band decreased when a 10% gel was used and an apparent molecular mass of 70 kDa was found (Fig. 1, lane 1). On occasions additional broad bands may appear on the fluorogram caused by variable amounts of glucan bound to protein. The high content of sugar is responsible for the anom- alous behavior of glycoproteins during SDS-PAGE and thus, different apparent molecular masses were obtained at different acrylamide concentrations in the gel [18]. Similar gel patterns were obtained with the other unlabeled sugar donors used as chain-lengthening substrates. Therefore, both reactions can be defined by denaturing PAGE product analyses.

Differentiation of phosphorylase activities by their primer requirement

As recently reported [lo] different molecular forms of glucosyl transferase (peaks 11, and IIb) occurring in the solubilized preparation were separated from the enzyme catalyzing reaction 1 (peak I) by DEAE-cellulose chromatography. While analyses in that paper concerned only peak I, some properties of the enzymes in peaks TI, and in IIb were studied in order to determine the ability of reaction 1 product to be used as primer by some specific enzyme.

Phosphorylase activities in peaks 11, and Ilb are dis- tinguishable by their primer requirement [lo]. Fig. 2 illustrates phosphorylase activity in both peaks as a function of time, in the presence or in the absence of added polysaccharide. In the latter case a lag period in the incorporation of [14C]glucose from labeled glucose-1-P into trichloroacetic-acid-insoluble material, catalyzed by the enzyme in peak 11, can be observed (Fig. 2A). No incorporation was found when peak IIb was used as source of enzyme (Fig. 2B). On the other hand, phosphorylase activity in both peaks, measured in the presence of polysaccharide, has a linear dependence on time

61 1

8

I!? 6

4

n

x

E a * 2

0

I I A ' 9

T i m e ( m i n ) T i m e ( m i n l

Fig. 2. Time course of phosphorylase activity in the absence (0-0) or in thepresence (0-0) of an exogenous acceptor. (A) Peak 11, (4 pg protein); (B) peak 1Ib (6 pg protein). Radioactivity incorporation in the trichloroacetic acid precipitate (0-0) or in the methanol-insoluble polysaccharide (0-0) was measured as described in Materials and Methods. Amylopectin (45 pg) was used as the exogenous acceptor

Fig. 3. Gel electrophoretic patterns of'phosphorylase activity in peaks IZ, and IIb. Samples containing 57 pg protein from peak 11, (lanes 1 and 2) or 58 pg protein from peak IIb (lanes 3 and 4) were submitted to non-denaturing gel electrophoresis on 7% acrylamide gels. Unprimed (lanes 1 and 3) or primed (lanes 2 and 4) activities were localized on the gels after the run, as described in [I41

(Fig. 2A, B). In the presence of added polysaccharide, at the conditions of incubation employed (micromolar concentrations of ['4C]glucose-l-P), no incorporation of radioactivity was found in the trichloroacetic-acid-insoluble fraction [19]. Peaks 11, and IIb were submitted to non-denaturing PAGE and the enzyme patterns are shown in Fig. 3. Primed and unprimed phosphorylase activities coexisting in peak 11, were coincident (Fig. 3, gels 1 and 2), while only primed phosphorylase was present in peak IIb (Fig. 3, gels 3 and 4). In addition to the major band coincident with that present in peak II,, a slow-moving minor band was observed (Fig. 3, gel 4).

Centrifugation of peak 11, through gradients of 5 - 20% sucrose shows that the unprimed phosphorylase activity cosediments with the primed activity with an experimental sedimentation coefficient of 8.2 - 8.4 S (data not shown). A similar result was reported for a soluble potato tuber phosphorylase [19]. Therefore, these activities appear to be associated with the same protein and possibly to the same polypeptide chain. However, the possibility that the two activ-

Table 1. Reconstitution of the synthesis of protein-bound a-l,l-glucan The first step of incubation was for 30 min in the presence of peak I (34 pg protein), 4 (IM UDP[14C]Glc (75000 cpm) and 10 mM MnCI,. The additions listed in the table were done after 30 min and the second step of incubation lasted 5 h. Reaction mixtures contained 40 pg and 27 pg protein from peak 11, or peak IIb respectively. Radioactivity incorporation was measured as described in Materials and Methods. Values given in parenthesis are the amount of glucose incorporated into the trichloroacetic acid precipitate. The average difference between the individual reconstitution experiments was * 20%

Additions during Radioactivity in the the second step trichloroacetic acid

precipitate enzymatic glucosyl fraction donor

(2 mM)

cpm (pmol) - - 6460 (13.3)

- UDP-Glc 6430 (13.2)

Peak 11, - 6510 (13.4) UDP-Glc 9330 (740) UDP-Glc 120" - 140"

Peak IIb UDP-Glc 6350 (13.1)

a Peak I was omitted during the first incubation.

ities exist on highly similar proteins, or on non-identical sub- units of a heterodimer cannot be completely ruled out [20].

As previously reported [lo], Peaks 11, and IIb also contained primed ADP(UDP)Glc : starch synthetase.

Requirements for the reconstituted system

As a consequence of the separation of the enzyme catalyzing reaction 1 by means of DEAE-cellulose column chromatography [lo], we were unable to bring about the synthesis of protein-bound glucan in the remaining fractions of the column, since the enzymes which catalyze it (reaction 2 ) require the product of reaction 1 (the glucosylated endogenous acceptor) as primer. Unprimed phosphorylase constitutes an exception, since the presence of reaction 1 product does not represent an absolute requirement for the synthesis of a protein-bound glucan [19].

In order to reconstitute the original system of a-glucan synthesis bound to protein with the protein components partially purified on DEAE-cellulose, two-step incubation experiments were undertaken. During the first step of incubation the endogenous acceptor protein and the enzyme catalyzing reaction 1 were provided by peak I. The enzymatic activities required for the elongation of reaction 1 product were assayed for in peaks 11, and 1 I b (second step of incubation), since different phosphorylase and starch synthetase activities were previously found [lo].

As shown in Table 1, a significant increase in the incorporation of glucose was observed in assays containing peak 11, and 2 mM UDP-Glc during the second step of incubation, only if the first step of incubation was done in the presence of peak I. The addition of either unlabeled UDP-Glc or peak 11, were without effect. Peak IIb cannot replace peak 11, (Table 1).

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Table 2. Reconstitution of the synthesis of protein-bound a-l,l-glucan Conditions of incubation were as in Table 1, except for the amounts of peak I (13 pg protein) and peak 11, (16 pg protein) employed. The average difference between the individual reconstitution experiments was &20%

Additions during Radioactivity in the the second step trichloroacetic acid

precipitate enzymatic glucosy 1

(3 mM) fraction donor

CPm - - 2200 - ADP-Glc 2250 Peak 11, - 2190

ADP-Glc 3260 ADP-Glc 160" - 130"

Peak IIb ADP-Glc 21 50 ~~~~ ~

a Peak I was omitted during the first step.

Table 3. Reconstitution of the synthesis of protein-bound a-I ,I-glucan Conditions of incubation were as in Table 1 , except for the amounts of peak I (1 3 pg protein) and peak 11. (86 pg protein) employed. The average difference between the individual reconstitution experiments was +20%

Additions during the second step

Radioactivity in the trichloroacetic acid precipitate

enzymatic glucosyl

(4 mM) fraction donor

CPm - - 21 30 - Glc-1-P 2030 Peak 11, - 2050

- 60 a

Glc-1 -P 4110" Glc-1 -P 16870

Peak IIb Glc-1-P 2200

a Peak I was omitted during the first step.

A similar experiment, in which ADP-Glc was used as the glucosyl donor during the second step, also leads to an enhanced incorporation of radioactivity when peak 11, was utilized as source of the enzyme catalyzing reaction 2 (Table 2). Results similar to those shown in Table 1 were obtained.

Phosphorylase in peak 11, can also catalyze reaction 2, giving rise to an increase in the radioactivity incorporation in the trichloroacetic-acid-insoluble fraction, as depicted in Table 3. A point that requires an explanation is the origin of a great proportion of radioactivity in the trichloroacetic acid precipitate when peak 11, and glucose-1-P were present in the second step of incubation, but peak 1 was omitted during the first step. This is due to the formation of a-1,4 glucan chains catalyzed by the unprimed phosphorylase activity in peak 11, in the presence of unlabeled glucose-1-P. This unlabeled glucan served as primer for UDP-Glc: starch synthetase, also

Fig. 4. PAGE (12.5% acrylamide) and fluorography of labeled products synthesized by the reconstituted system. Peak I was incubated in separate tubes in the presence of UDP-[14C]Glc as described in [lo]. After 30 min of incubation, the additions indicated below were carried out and the reaction continued for 5 h. Proteins were then precipitated with trichloroacetic acid and analyzed by PAGE. (1) No additions; (2) peak 11, + unlabeled 2 mM UDP-Glc; (3) peak 11, + unlabeled 3 mM ADP-Glc. Exposure times were 20 days for lanes 1 and 2 and 10 days for lane 3. Numbers indicate molecular mass in kDa

present in peak II,, which catalyzes the transfer of labeled glucose from UDP-['4C]Glc added during the first step of incubation. Similar results were already reported with a non- sedimentable preparation of potato tuber [19, 211. However, when the first step of incubation gives rise to the formation of reaction 1 product, a fourfold increase in radioactivity in the trichloroacetic acid precipitate was obtained after incubation with glucose-1-P and peak 11, during the second step (Table 3). Peak I was without effect on the unprimed phosphorylase activity found in peak 11, (data not shown). Again, peak IIb cannot replace peak 11,.

The radiolabeled products, synthesized by each of the reconstituted systems described in Tables 1 and 2, were identified by fluorography after denaturing PAGE on a 12.5% acrylamide gel. Two major components of 43 kDa and 50 kDa apparent molecular mass (Fig. 4, lanes 2 and 3) were obtained with the reconstituted system using UDP-Glc and ADP-Glc, respectively, as glucosyl donors during the second step of incubation. These components migrate as rather broad bands, as compared with the other specifically labeled band of 38 kDa apparent molecular mass, characteristic of reaction 1 product (Fig. 4, lane 1). An explanation of that might reside in the possible microheterogeneity of the slower moving components. Microheterogeneity is often the manifestation of a variable extent of glucosylation. Analysis by urea/SDS/ PAGE and fluorography of controls, in which peak 11, or unlabeled UDP-Glc or unlabeled ADP-Glc was omitted during the second step of incubation, showed only the presence of the 38-kDa component of reaction 1 product (data not shown). The radiolabeled product, synthesized by the reconstituted system described in Table 3, was also identified by fluorography after denaturing PAGE on a 10% acrylamide

61 3

Fig. 5. PAGE (10% acrylamide) andfluorography ofthe labeledprod- ucts synthesized by a reconstituted system. As legend to Fig. 4, but peak 11, + unlabeled 4 mM glucose-1-P were added during the second step of incubation, which lasted 180 min. (1) No additions; (2) peak 11, + unlabeled 4 m M glucose-I-P; (3) as in (2), but peak I was omitted during the first step of incubation

gel. An apparent molecular mass of 70 kDa (Fig. 5 , lane 2) was obtained, while a 50 kDa polypeptide was observed when the same product was electrophoresed on a 12.5% gel (data not shown). In addition, the 38-kDa component could also be seen on the fluorogram, showing analogous behavior at 10% as well at 12.5% acrylamide in the gels (Fig. 5, lane 1 and Fig. 4, lane 1). On the other hand, only one labeled band of about 70 kDa could be seen on the fluorogram of the product of a control experiment in which the formation of reaction 1 product was omitted during the first step (Fig. 5, lane 3 ) . In the 70-kDa species the product of reaction 1 + reaction 2 as well as a product of unprimed phosphorylase both coexist. This enzyme was shown to be able to synthesize an a-glucan bound to protein using an acceptor, the nature of which is still unknown [22].

In order to confirm that the 38-kDa component and at least part of the 70-kDa polypeptide share the same protein core, a deglucosylation experiment was undertaken. The 70- kDa component was excised from a 10% gel (Fig. 5, lane 2) and submitted to /?-amylase short-term treatment. This brief treatment was chosen with the aim of following up the labeled 70-kDa component afterwards. A long-term treatment only showed the appearance of the 38-kDa band (data not shown). After precipitation with trichloroacetic acid and washing off the released sugar, the resulting product was resubmitted to denaturing PAGE and the fluorography is shown in Fig. 6. Part of the 70-kDa component was converted into a com- pound possessing the same electrophoretic mobility as the labeled component of reaction 1 product (38 kDa). These results support the view that part of the 70-kDa species originate from the 38-kDa glucosylated polypeptide and the increase in molecular mass from 38 kDa to 70 kDa is due to the addition of /?-amylase-sensitive glucosyl moieties. When the same treatment was performed on the product of the control experiment, in which formation of reaction 1 product

Fig. 6. Effect of P-amylase treatment on the electrophoretic mobility of the labeled product from the reconstituted system. The protein-bound glucan, synthesized by the reconstituted system as in Fig. 5 lane 2 (band of low mobility), was excised from the gel and submitted to 8-amylase, as described in [I]. After B-amylase digestion the radioactive material was precipitated with trichloroacetic acid and resubmitted to denaturing gel electrophoresis on a 10% acrylamide gel. The photograph shows the fluorogram of the urea/SDS gel; the arrow marks the 38-kDa band

was omitted during the first step (Fig. 5, lane 3), most of the radioactivity was released and no additional band appeared on the fluorogram. The occurrence of a-1 ,Cglucosidic linkages in the saccharide portion of the products of the three reconstituted systems (Tables 1, 2 and 3) was verified by the following amylolytic treatments. 85% of the incorporated radioactivity was recovered as [14C]maltose after /?-amylase digestion. Amyloglucosidase and a-amylase treatments yielded ['4C]glucose and radioactive glucose and maltose respectively. These results agree with those obtained with the solubilized and particulate preparations [7, 91.

DISCUSSION In this report we describe a reconstitution assay for protein- bound glucan synthesis in potato tuber, utilizing partially purified components. The synthesis of protein-bound glucan was obtained through two-step incubation experiments (Tables 1 ,2 and 3). The formation of a covalent linkage of the 0-glycosidic type between the amino acid of the endogenous acceptor protein and ['4C]glucose from labeled UDP-Glc (micromolar concentrations) takes place during the first step, giving rise to reaction 1 product [lo]. This unique glucose residue added to the protein later becomes the reducing end of the nascent glucosidic chain. Then, during the second step of incubation and using the product of reaction 1 as primer, the consecutive covalent addition of glucosyl moieties from millimolar concentrations of UDP-Glc, or ADP-Glc or glucose-1-P to the already-linked first glucose requires the enzymes present in peak 11, (Tables 1 ,2 and 3). It is interesting to point out that labeled UDP-Glc in excess from the first

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step of incubation is still present in the reaction mixture. Since the affinity of UDPG1c:starch synthase (peak 11,) for the sugar nucleotide is very low with inefficient primers like reaction 1 product. ['4C]glucose is incorporated once the carbohydrate chain has acquired an appropriate length with unlabeled ADP-Glc or glucose-1-P as chain-lengthening donor substrates. This would account for the increase in radioactivity observed in Tables 2 and 3.

Some aspects of this work deserve consideration. The first point to be considered is the absolute requirement for the enzymes found in peak 11, to bring about the synthesis of this protein-bound a-glucan during the second step of incubation. Phosphorylase and starch synthetase present in peak IIb cannot utilize reaction 1 product as primer (Tables 1, 2 and 3) . Therefore, although both peaks in DEAE-cellulose chromatography have phosphorylase and synthetase activities, they differ basically in primer specificity.

The second consideration stems from Table 3, where formation of labeled trichloroacetic-acid-insoluble material in the absence of reaction 1 product is shown. This means that the phosphorylase activity present in peak 11, also synthesizes an a-glucan bound to protein in which the previous formation of reaction 1 product is not an absolute requirement (see Fig. 5, lane 3). This unprimed phosphorylase-catalyzed reaction has been recently analyzed by Sivak et al. [19, 221 with a non-sedimentable preparation of potato tuber. However, phosphorylase in peak 11, can also utilize the product of reaction 1 as primer, as illustrated in Table 3. Additional evidence, providing support for the interpretation of the results described here, comes from a deglucosylation experiment carried out on the 70000-Da highly glucosylated component of the product of the reconstituted system shown in Fig. 5, lane 2. Although the P-amylase treatment was incomplete, its action on the 70-kDa polypeptide excised from the gel (Fig. 5, lane 2) gave rise to a 38-kDa species on the fluorogram (Fig. 6). It is worth pointing out the recent report by Rodriguez and Whelan [23] and the one by Rodriguez et al. [24] showing that rabbit muscle glycogen contains a covalently bound protein of 37 kDa. The authors claim that this protein is to be seen as the biosynthetic origin of muscle glycogen [23, 241.

The last point that merits a comment is the fact that although reaction 1 product serves as primer for the elongating enzymes present in peak II,, no perceptible decrease in the amount of the 38-kDa polypeptide was observed on the fluorograms (Fig. 4, lanes 2 and 3 and Fig. 5, lane 2). A single-chain mechanism of elongation [25] could explain this observation. Once a few molecules are sufficiently elongated they become more efficient primers than reaction 1 product, thus being preferentially utilized by the chain- lengthening enzymes present in peak 11,.

The authors are indebted to the members of the Znstituto de In-

cussions and criticism. J.S.T. is a Career Investigator of the Consejo Nacional de Investigaciones Cientficas y Tecnicas, Argentina.

REFERENCES

1. Lavintman, N., Tandecarz, J., Carceller, M., Mendiara, S. &

2. Krisman, C. R. & Barengo, R. (1975) Eur. J . Biochem. 52, 117-

3. Barengo, R., Flawia, M. M. & Krisman, C. R. (1975) FEBS Lett.

4. Takahara, H. & Matsuda, K. (1977) J . Biochem. (Tokyo) 81,

5. Blumenfeld, M. L., Whelan, W. J. & Krisrnan, C. R. (1983) Eur.

6. Tomos, A. D. & Northcote, D. H. (1978) Biochem. J . 174,283 -

7. Tandecarz, J. S. & Cardini, C. E. (1978) Biochim. Biophys. Acta

8. Tandecarz, J. S. & Cardini, C. E. (1979) Plant Sci. Lett. 15,151 -

9. Moreno, S. & Tandecarz, J. S. (1982) FEBS Lett. 139, 313-316. 10. Moreno, S., Cardini, C. E. & Tandecarz, J. S. (1986) Eur. J .

11. Thomas, J. A,, Kaith, K., Schlender, K. & Larner, J. (1968) Anal.

12. Davis, B. J. (1964) Ann. N.Y. Acad. Sci. 121, 404-427. 13. Tandecarz, J. S., Lavintman, N. & Cardini, C. E. (1973)

14. Tandecarz, J. S., Sivak, M. N. & Cardini, C. E. (1978) Biochem.

15. Ziegler, A., Harrison, S. C. & Leberman, R. (1974) Virology 59,

16. Chamberlain, J. P. (1970) Anal. Biochem. 98, 132-135. 17. Lowry, 0. H., Rosebrough, J. J., Farr, A. L. & Randall, R. J.

(1951) J . Biol. Chem. 193,265-275. 18. Segrest, J. P. & Jackson, H. L. (1972) Methods Enzymol.

(Ginsburg, V., ed.), vol. 28, pp. 54-63, Academic Press, New York.

19. Sivak, M. N., Tandecarz, J. S. & Cardini, C. E. (1981) Arch. Biochem. Biophys. 212, 525 - 536.

20. La Porte, D. C. & Koshland Jr, D. E. (1982) Nature (Lond.) 300,

21. Tandecarz, J. S., Lavintman, N. & Cardini, C. E. (1975) Biochim. Biophys. Acta 399, 345 - 355.

22. Sivak, M. N., Tandecarz, J. S. & Cardini, C. E. (1981) Arch. Biochem. Biophys. 212, 537 - 545.

23. Rodriguez, I. R. & Whelan, W. J. (1985) Biochem. Biophys. Res. Commun. 132, 829 - 836.

24. Rodriguez, I. R., Tandecarz, J. S., Kirkman, B. R. & Whelan, W. J. (1986) Proc. 18th Miami Winter Symp. pp. 96-99 (Puett, D., ed.) ICSU Press/Cambridge University Press.

25. Whelan, W. J. & Bailey, J. M. (1954) Biochem. J . 58, 560-

Cardini, C. E. (1974) Eur. J . Biochem. 50, 145-155.

123.

53,274-278.

1587 - 1594.

J. Biochem. 135,175-179.

290.

543,423 -429.

158.

Biochem. 157, 539 - 545.

Biochem. 25,486-499.

Carbohydr. Res. 19, 385 -392.

Biophys. Res. Commun. 282, 157 - 164.

509 - 51 5.

458 - 460.

vestigaciones Bioquimicas 'Fundacion Campomar ' for helpful dis- 569.

α-glucan synthesis on a protein primer : a reconstituted system for the formation of protein-bound...

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