BIOCHEMICAL
MEDICINE
AND
METABOLIC
Characterization
BIOLOGY
43, 147-158 (1990)
of a Protein:Glucosyltransferase in Human Platelets
Activity
PROVIDENCIA RODRIGUEZ AND PAULA DURANTE Laboratorio
de Trombosis Experimental, Centro de Biojikica y Bioquimica, Institute Venezolano de Investigaciones CientQicas, Apartado 21827, Caracas 1020-A, Venezuela Received July 10, 1989, and in revised form November 27, 1989
The presence of glucosyltransferase in human blood platelets has been established by several authors (l-6). Collagen:glucosyltransferase was found in the plasma membrane and has been implicated in the mechanism of platelet:collagen adhesion (1,2). Several reports have described the enzyme glycogen synthase (glycogen:glucosyltransferase) responsible for the synthesis of the polysaccharide in platelets (3-5). With the help of this enzyme, the platelets are able to build up the required glycogen stores necessary as an energy source. The first evidence of an enzymatic activity that transfers glucose from uridine diphosphate glucose (UDP-Glc) to an endogenous protein acceptor in platelets was reported by Greenberg et al. (5). They did not characterize the enzyme and concluded from hydrolytic experiments with P-amylase, trypsin, and collagenase that the glycogen present in their platelet preparation may be complexed to protein, causing it to precipitate with trichloroacetic acid together with the protein. Previous results from our laboratory showed the existence of a protein:glucosyltransferase in platelets and demonstrated that this enzyme was present in a higher amount than the protein:galactosyltransferase reported (6). On the other hand, from its properties it was concluded that the protein:glucosyltransferase and protein:galactosyltransferase were two distinct enzymes in terms of their substrate specificity and solubilization with Triton X-100. On the formation of glycogen, it has been suggested that a glucoprotein is synthesized as a precursor of the polysaccharide for several tissues, and two distinct enzymes are required for this (7-l 1). Since platelets contain glycogen and protein:glucosyltransferases, we have tried to characterize the enzyme responsible for the formation of the glucoprotein and at the same time determine the synthesis of glycogen under such conditions. In this report, we present evidence that these glucosylation reactions in platelets are carried out by two different enzymes. In addition, a partial separation of both activities is shown. 147 0885-45OY90 $3.00 Copyright 0 1990 by Academic Press. Inc. All rights of reproduction in any form reserved.
148
RODRiGUEZ
MATERIALS
AND
DURANTE
AND METHODS
Concentrated platelets were provided by the Municipal Blood Bank. UDP-[U“C]glucose (sp act 223 mCi/mmole) was obtained from New England Nuclear Corp. (Boston, MA). Sephadex G-25 and G-100 were obtained from Pharmacia Fine Chemical (Upsala). Bovine liver glycogen, sodium dodecyl sulfate (SDS), and all other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Preparation of Platelets and Platelet Homogenates Platelets from two concentrates of human platelets were isolated and homogenized according to the procedure described previously (6). The homogenate was centrifuged at 105,000g for 60 min at 4”C, and the pellet was resuspended in 30 mM Tris-HCl buffer, pH 7.5. The demonstration of glucosylation of platelet proteins and the characterization of the enzyme reported here were conducted with this fraction. The 105,OOOg supernatant was chromatographed on Sephadex G-100. Protein was measured by the procedure described by Peterson (12) using bovine serum albumin as the standard. Glucosylation of Endogenous Platelet Proteins and Enzyme Assays The standard incubation mixture contained in 100 ~1 total volume, 15 PM UDP[‘4ClGlc (20,000 dpm), 50 mM Tris-HCl, pH 7.5 (standard buffer), and 20 pg of protein. The incubation was carried out at 37°C for 20 min and stopped by addition of 0.5 ml 10% chilled trichloroacetic acid. The reaction mixture was cooled in ice for 30 min and centrifuged at 10,OOOg. The precipitate was considered as the trichloroacetic acid-insoluble protein (fraction I). The resulting supernatant (fraction II) was retained in order to assay the trichloroacetic acidsoluble glycogen. Controls with denatured enzyme were run with each incubation and the values obtained were subtracted from those of the test samples. Chromatography
of Reaction
Product
Fraction I was washed with 10% trichloroacetic acid, and the pellet was dissolved with 1% SDS in 0.1 N acetic acid. This sample was then run through a Sephadex G-25 column (0.7 x 5 cm) equilibrated with standard buffer. The column was eluted with the same buffer, and aliquots of the fractions were counted in 3 ml of Aquasol. Untreated fraction II was also chromatographed on the same column. Endogenous Protein: Glucosyltransferase Activity Fraction I was washed free of acid-soluble radioactivity with 0.5 ml portions of 10% trichloroacetic acid and rinsed twice with 95% ethanol. The pellet was digested in 200 ~1 of Soluene-350. The radioactivity was measured in 3 ml of Aquasol containing 200 ,ul of 0.5 N HCl. Enzyme activity was expressed as dpm of [‘4C]glucose transferred to endogenous acceptor protein.
GLUCOSYLTRANSFERASE
Glycogen:
Glucosyltransferase
ACTIVITY
149
Activity
Fraction II was precipitated with 95% ethanol (1.5 vol) in the presence of 1.2 mg of glycogen carrier. The tube was heated in a bath of boiling water for 1 min and then stored for 16 hr at 4°C. To separate the glycogen the sample was centrifuged at 12,OOOg; the pellet was washed free of ethanol-soluble radioactivity with 0.5 ml portions of 95% ethanol and recentrifuged. The precipitate was digested and the radioactivity counted as described above. Enzyme activity was expressed as dpm of [‘4C]glucose incorporated into glycogen. Sephadex
G-100 Chromatography
The 105,OOOg supernatant was applied to a Sephadex G-100 column (1.5 x 25 cm) which had been equilibrated and eluted with the standard buffer. Enzymatic activities and absorbance at 280 nm were determined in the fractions. RESULTS
A representative partial purification of protein and glycogen:glucosyltransferase from human platelets is shown in the Table 1. Both enzymes were present in the crude homogenate in an approximate ratio of 7: 1 and after Sephadex G-100 this ratio varied to 36: 1. The protein:glucosyltransferase was recovered with an approximate 50% yield after an 1l-fold purification and glycogen:glucosyltransferase was obtained in this procedure with a purification factor of 5 and a yield of 10%. In every preparation the activities for both glucosyltransferases in the 105,OOOg pellet were significantly higher than in the homogenate, and in consequence the aggregate yield of activities of supernatant and pellet obtained from the homogenate was always greater than 100%. When the 105,OOOg supernatant fraction was applied to a Sephadex column, the enzymatic activities were eluted as shown in Fig. 1. A major protein peak I (fractions 25-55) showed protein:glucosyltransferase activity. Contamination with glycogen:glucosyltransferase activity was less than 3% in this step of fractionation and did not increase when the enzyme was assayed in the presence of exogenous glycogen. A minor peak II (fractions 83-100) contained protein:glucosyltransferase only, but at a very low concentration (Fig. 1). To test if peak II behaved as an endogenous acceptor for the protein:glucosyltransferase, the enzymatic activity of the first peak was assayed with aliquots of the second one as substrate. No stimulation of glucosyltransferase was observed. Incubation of UDP-[14C]Glc with a platelet preparation which sediments at 105,OOOg led to incorporation of radioactivity into a fraction precipitable by 10% trichloroacetic acid (fraction I) and solubilized by SDS. The elution profile obtained on Sephadex G-25 gel filtration (Fig. 2) of the product formed under these conditions showed that one peak of radioactivity eluted with the V,,; 1.2 of the total radioactivity was incorporated into protein. When the supernatant (fraction II) was filtered on the same column, the peak eluting with VT (Fig. 2) corresponded to unreacted UDP[‘4C]Glc added to the assay mixture or radioactive sugar liberated from the sugar nucleotide. In a parallel incubation we determined
52 15.6
24.9 2.2
0.9
6 2.3
1
Protein (mg)
6.5 5.2
Volume (ml)
5
1217 234
468 393
5.5
48.9 106.4
9 25.2
Specific activity (KU/mg protein)
1
260 50
100 84
Yield (%)
a One unit is the amount of enzyme which transfer 1 dpm of [‘4C]glucose per 20 min at 37°C. b No detectable enzymatic activity was found.
Homogenate 105,OOOg supematant 105,OOOgpellet Sephadex G-100 fractions 33-37 Fractions 96-97
Step
Total activity (KU)
4.9 6.7 -
NDh
1.6 1.3
Specific activity (KU/mg protein)
Glycogen:glucosyltransferase
122 6.5
25 68
Total activity (KU)”
from Human Platelet
Protein:glucosyltransferase
TABLE 1 Protein and GlycogenXilucosyltransferase
-
179 10
37 loo
Yield @)
z $ 3 m
:
E, ? i-t
g
GLUCOSYLTRANSFERASE
Fraction
ACTIVITY
Number
FIG. 1. Gel filtration on Sephadex G-100. Supematant (105,OOOg) was applied to the Sephadex G-100 column (1.5 x 25 cm) equilibrated in the standard buffer. Fractions (0.5 ml) were eluted with the same buffer. Aliquots were assayed for protein:glucosyltransferase activity (0); glycogen:glucosyltransferase activity (0); and protein absorbance at 280 nm (A). Enzymatic activities are plotted per fraction.
that 0.1% of the total radioactivity was incorporated into glycogen (fraction II) synthetized by the 105,OOOg pellet. The denatured protein routinely yielded blank values of less than 0.05% of the radioactivity incubated. The effect of time, UDP-Glc, and protein concentration on the incorporation of [‘4C]glucose into fractions I and II were determined (Fig. 3). Glucosylation of the protein was linear with time for 20 min (Fig. 3A), at 60 min of incubation the activity was 83% of the maximum observed. The incorporation of glucose into glycogen was not linear with time and was completed in approximately 15 min (Fig. 3A). The formation of glucoprotein was dependent on the protein concentration and reached a maximum between 0.3 and 1.0 pg/ml (Fig. 3B). At protein concentrations above this level the radioactivity decreased slightly and the activity remained constant between 2.0 and 200 pg/ml. Figure 2 also shows that no significant variation of radioactivity in fraction II was obtained with increasing protein concentration. The curve obtained for the glucosylation of the protein with various UDP-Glc concentrations showed a biphasic pattern (Fig. 3C). As can be seen, a first plateau was reached between a substrate level of 7 and 10 PM and a second one at a substrate concentration of 15 PM. This maximal
I-
152
4.c
T 0 .-g t .I-e ‘;
vo 4
RODRIGUEZ I
AND DURANTE I
I VT i
)-
2.c
0.1
,”
!
I
IO Fraction
20 Number
30
40
FIG. 2. Chromatography on Sephadex G-25 of fractions I and II. Incubation of the lO5,OOOg pellet was performed under standard assay conditions, and fractions I and II were separated as described under Materials and Methods. 14C-fraction I (trichloroacetic acid-insoluble) dissolved in 0.1% SDS and 14C-fraction II (trichloroacetic acid-soluble) were subject to gel filtration on Sephadex G-25. The column (0.7 x 5 cm) was equilibrated with the standard buffer. Fractions (0.5 ml) were collected, and radioactivity was measured for each fraction.
incorporation of radioactivity into fraction I was maintained even with lOOO-fold higher UDP-Glc concentration. The synthesis of glycogen was very low and invariable to 10 PM UDP-Glc. Concentrations of substrate greater than 10 PM progressively increased the radioactivity and became constant between 15 PM and 15 mM (Fig. 3C). As indicated in Table 2 the enzyme responsible for the formation of the glucoprotein was found to be inhibited 76% by Mn2+ (5 mM) whereas at lower concentrations Mn2+ had no effect on the activity. Ca2’ and Mg2+ (5 mM) ions increased the activity 23 and 1l%, respectively. Glycogen:glucosyltransferase activity was stimulated 43, 80, and 200% with Mn2+, Ca2+ , and Mg2+, respectively. The effect of Triton X-100 on protein and glycogen:glucosyltransferase was studied between 0 and 0.75% v/v concentration (data not shown). The activities
GLUCOSYLTRANSFERASE
Time
Protein
(min)
60
I
c
UDP-Glc
concentmtion
153
ACTIVITY
concentration
@g/ml)
4
(PM)
FIG. 3. Glucosyltransferase activity as a function of (A) time, (B) protein concentration, and (C) UDP-Glc concentration. Incubations were carried out under the conditions described under Material and Methods, except with the variations indicated. Protein:glucosyltransferase (0) and glycogen:glucosyltransferase (0) activities were measured with the 105,OOOgpellet.
were enhanced 37 and 30% respectively in the presence of O.Ol-0.04% Triton X-100. Concentrations above this level inhibited the enzymes, and when O.l0.75% of detergent was added 75 and 86% inhibition was observed. Effect of glycogen on [‘4Clglucose incorporation into protein and glycogen: The addition of 0.005-1.0% glycogen progressively inhibited the incorporation of [‘4C]glucose into glucoprotein from 65 to 95% (Fig. 4). However, 0.002, 0.005,
154
RODRiGUEZ
AND DURANTE
TABLE 2 Effect of Various Ions on Protein and GlycogenGlucosyltransferase Activity (% of control) Ion added
Protein: glucosyltransferase
None (control) Mn*’ Ca2+ MgZf
Glycogen: glucosyltransferase
100 24 123 111
100 143 180 300
Note. Protein and glycogen:glucosyltransferases were assayed as described under Material and Methods, with the fraction 105,OOOg pellet. The ions were added at 5 mM concentrations.
and 0.007% polysaccharide stimulated 2, 3, and 2.5fold respectively the incorporation into ethanol precipitate of the trichloroacetic acid-soluble fraction. Concentrations of glycogen greater than 0.007% severely inhibited the radioactivity incorporated into glycogen, which returned close to basal values observed in the absence of glycogen (Fig. 4).
40 i
I
0.025
: 0-
I
0.05 Percent Glycogen (W/VI
FIG. 4. The effect of glycogen on the protein and g1ycogen:glucosyltransferase activities. The enzymes were assayed as indicated under Material and Methods, except that the concentrations of glycogen added to the reaction mixture were increased as shown. Determinations of protein (0) and glycogen:glucosyltransferase (0) activities were made with the 105,OOOg pellet.
GLUCOSYLTRANSFERASE
ACTIVITY
155
When the effect of glycogen was assayed with the more purified Sephadex column (fractions 33-37) the protein:glucosyltransferase showed a response identical to that described earlier. However, the glycogen:glucosyltransferase activity did not exhibit any variation with added glycogen in the range studied (data not shown). DISCUSSION The results summarized in the preceding section demonstrate that human platelets can glycosylate endogenous protein acceptor. The synthesis of a glucoprotein is judged from the insolubility in 10% trichloroacetic acid, solubility in 1% SDS, and the gel filtration. These data are consistent with our earlier results (6,13). It is possible that the assay incubation used by Greenberg et al. (5) in the presence of the Mn*’ ion (0.22 M) inhibited substantially the formation of the glucoprotein. The partial purification of the enzyme (Table 1) has shown it to be distinct from that of the glycogen:glucosyltransferase. The change of the ratio from 7: 1 (protein:glucosyltransferase/glycogen:glucosyltransferase) in the homogenate to 36: 1 in peak I from the Sephadex column supports this conclusion. The fact that the yield and purification factor observed for protein:glucosyltransferase activity are greater than those shown by glycogen:glucosyltransferase (Table 1) could be explained by the fact that they are two different enzymes and are separable. A complete separation of both activities from rabbit muscle has been reported (11). The authors showed an increase in the specific activity of protein:glucosyltransferase and a decrease in glycogen:glucosyltransferase activity. Similar results were reported for two separable sialyltransferase activities in human platelets (14). Our results of the higher yield obtained by adding the activities in the supernatant and particulate fractions (Table 1) could reflect that the extent of glycosylation has no quantitative relation to the real transferase activities present in the homogenate. This could suggest the presence of an unknown inhibitor in the homogenate that produces an apparent lower activity for both enzymes in that fraction. With respect to protein:glucosyltransferase activity, it is also possible that the amount of acceptor present in the homogenate is limiting; the manipulation could provoke the appearance of more efficient endogenous substrate for the enzyme. In the case of glycogen:glucosyltransferase the higher yield after the centrifugation could be the result of the existence of two interconvertible forms of the enzyme in platelets, which change its activity. Concerning this point there have been considerable discussion and evidence for less active forms of the enzyme (15-17). The characterization of both enzymes (Fig. 3) indicated that protein:glucosyltransferase exhibits a definite dependence on the time of incubation, protein, and UDP-Glc concentration. This is in striking contrast to glycogen:glucosyltransferase, which did not significantly increase its activity, in response to those variables. This fact appears to reflect a lesser abundance of this enzyme in the mature platelet, or that our conditions are not optimal for glycogen synthesis. There are also differences between the two activities with respect to the metal ion requirements (Table 2). Mn*+ shows opposite effects on both activities, and Ca*+ and Mg*+ ions exerted a greater
156
RODRiGUEZ
AND
DURANTE
stimulation on glycogen:glucosyltransferase than that on protein:glucosyltransferase. It has been reported that in rabbit skeletal muscle the protein:glucosyltransferase, which forms the glucoprotein required for the glycogen biosynthesis, has an absolute requirement for divalent cations, Mn*+ being more effective (10). The saturation of protein:glucosyltransferase activity by low UDPGlc and protein concentrations (Fig. 3) indicates a high affinity for donor and acceptor substrates and may reflect its critical importance for the glucoprotein biosynthesis. The K, for UDP-Glc reported for glycogen:glucosyltransferase from human platelets is 6.6 mM (4). The biphasic curve obtained with UDP-Glc concentration suggests the formation of a possible glucoprotein intermediate in the initiation of glycogen biosynthesis in platelets as was first reported by Krisman in rat liver system (7). However, a clear connection between the synthesis of the glucoprotein and glycogen was not observed in platelets (Fig. 3). This could be a consequence of the lesser amount of glycogen:glucosyltransferase found in our platelets under our experimental conditions. Also, the characteristic lag period showed in liver (7) and heart (8) for the time course incorporation of glucose into the protein for the formation of the appropriate intermediate involved in the synthesis of glycogen was not observed in platelets. The results obtained with the addition of 1% glycogen to the incubation mixture (Fig. 4) indicate that platelet glucosyltransferases differ from the two glucosyltransferases involved in the biosynthesis of glycogen in other tissues (7-9). While in platelets a significant inhibition was observed in the synthesis of glucoprotein and the polysaccharide, in other systems an inhibition on the glucoprotein synthesis and an increase in radioactivity in the ethanol precipitate of the trichloroacetic acid-soluble fraction have been reported. This effect was shown in platelets, but at very low concentrations of glycogen. Thus in platelets exogenous glycogen in the range 0.002-0.0075% competes with the endogenous acceptor protein and inhibits its own synthesis at higher concentration. Our results suggest that the concentration of glycogen in platelets could be of critical importance in the control of both UDPGlc:glucosyltransferases responsible for the synthesis of the glucoprotein and the polysaccharide. This fact together with the low level of glycogen synthase could explain the low rate of glycogen formation in the cell. In platelets Gear and Schneider (16) have reported glycogen synthase to be in the range of 30% or less of its maximum activity. Platelet glycogen content is about 60 pmole glycogen/lO” platelets (17) and it takes at least 2 hr to reach this glycogen level indicating a very low glycogen turnover. Results from Sephadex G-100 filtration (Fig. 1) indicated that proteinglucosyltransferase was eluted together with the endogenous acceptor. Possible explanations are that the enzyme and the acceptor are two different proteins that copurified after the gel filtration step or that they are a single protein with enzymatic activity and acceptor capacity which could be autoglycosylated. It is well known that purified mammalian glycosyltransferases are glycoproteins and appear to contain up to 15% carbohydrate by weight. Recently two reports have appeared concerning the self-glycosylating enzyme for the formation of the primer of glycogen in rabbit skeletal muscle (10,ll). Our results suggest that the biosynthesis of a glucoprotein takes place in
GLUCOSYLTRANSFERASE
ACTIVITY
157
platelets, which may be involved in the biosynthesis of glycogen. Both molecules are synthetized by two different enzymes. The role of platelet glucoprotein in other physiological processes remains to be established. SUMMARY Human platelets exhibited significant glucosyltransferase activity, that transfer [‘4C]glucose from UDP-Glc to an endogenous protein acceptor. The enzyme protein:glucosyltransferase responsible for the catalysis was characterized and compared with glycogen:glucosyltransferase. We describe a partial separation of both activities, the ratio of protein:glucosyltransferase/glycogen:glucosyltransferase varied from 7 : 1 in a crude homogenate of platelets to 36: 1 in the Sephadex G-100 column, This procedure failed to separate the protein:glucosyltransferase from its endogenous acceptor. Glucosylation of protein demonstrated dependence with respect to time and both protein and UDP-Glc concentration, and was saturated by very low concentration of donor and acceptor substrates. It was inhibited 76% by 5 mM Mn2+ concentration and was activated 23 and 11% by 5 mM concentrations of Ca*+ and MgZf , respectively. With respect to glycogen:glucosyltransferase, when the effect of time, protein, and substrate concentration were determined under identical conditions, it did not show the same dependence. At 5 mM concentration, Mn2+, Ca2’, and Mg2+ were activators of the enzyme 43, 80, and 200%, respectively. On the basis of these characteristics, we conclude that the synthesis of glucoprotein and glycogen are catalyzed by two distinct enzymes. Addition of exogenous glycogen (range 0.002-l%) inhibited the protein: glucosyltransferase, whereas at O.OOl-0.007% concentration it was acceptor substrate for glycogen:glucosyltransferase activity. At higher concentrations this activity was strongly inhibited. The concentration of glycogen in platelets could play a regulatory role in forming the glucoprotein and the glycogen molecules. ACKNOWLEDGMENTS We thank Dr. Francisco Herrera for his review of the manuscript, Dra. Graciela Le6n from Municipal Blood Bank for supplying the platelet concentrates, Mr. Alfonso Tablante for his technical assistance, Miss Elizabeth Gonztiez for secretarial work, and Mrs. Dhuwya Otero for drawing the figures. This work was supported in part by Project Sl-1986, CONICIT.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Barber, A. .I., and Jamieson, G. A., Biochim. Biophys. Acta 252, 533 (1971). Bosmann, H. B., Biochem. Biophys. Res. Commun. 43, 1118 (1971). Vainer, H., and Wattiaux, R., Nature(London) 217, 951 (1968). Karpatkin, S., Charmatz, A., and Langer, R. M., J. Clin. Invest. 19, 140 (1970). Greenberg, J. H., Fletcher, A. P., and Jamieson, G. A., Thromb. Diath. Haemorrh. 30, 307 (1973). Rodriguez, P., Bello, 0.. Tablante, A., and Apitz-Castro, R., Biochem. Med. Metab. Biol. 40, 151 (1988). Krisman, C. R., Ann. N. Y. Acad. Sci. 210, 81 (1973). Blumenfeld, M. L., Whelan, W. J., and Krisman, C. R., Eur. J. Biochem. 135, 175 (1983). Blumenfeld, M. L., and Krisman, C. R., Eur. J. Biochem. 156, 163 (1986). Pitcher, J., Smythe, C., and Cohen, P., Eur. J. Biochem. 176, 391 (1988).
158 11. 12. 13. 14. 15. 16. 17.
RODRIGUEZ
AND DURANTE
Lomako, J., Lomako, W. M., and Whelan, W. J., FASEB J. 2, 3097 (1988). Peterson, G. L., Anal. Biochem. 83, 346 (1977). Rodriguez, P., Bello, 0.. and Apitz-Castro, R., Biochem. Med. Mefab. Biol. 37, 197 (1987) Banvois, B., Montreuil, J., and Verbert, A., Biochim. Biophys. Acta 788, 234 (1984). Aguilar, J., and Rosell-PCrez, M., Rev. Espagfi. Fysiol. 31, 151 (1975). Gear, A. R. L., and Schneider, W., Biochim. Biophys. Acta 392, 111 (1975). Akkerman, J. W. N., Thromb. Haemostasis 39, 712 (1978).
MEDICINE
AND
METABOLIC
Characterization
BIOLOGY
43, 147-158 (1990)
of a Protein:Glucosyltransferase in Human Platelets
Activity
PROVIDENCIA RODRIGUEZ AND PAULA DURANTE Laboratorio
de Trombosis Experimental, Centro de Biojikica y Bioquimica, Institute Venezolano de Investigaciones CientQicas, Apartado 21827, Caracas 1020-A, Venezuela Received July 10, 1989, and in revised form November 27, 1989
The presence of glucosyltransferase in human blood platelets has been established by several authors (l-6). Collagen:glucosyltransferase was found in the plasma membrane and has been implicated in the mechanism of platelet:collagen adhesion (1,2). Several reports have described the enzyme glycogen synthase (glycogen:glucosyltransferase) responsible for the synthesis of the polysaccharide in platelets (3-5). With the help of this enzyme, the platelets are able to build up the required glycogen stores necessary as an energy source. The first evidence of an enzymatic activity that transfers glucose from uridine diphosphate glucose (UDP-Glc) to an endogenous protein acceptor in platelets was reported by Greenberg et al. (5). They did not characterize the enzyme and concluded from hydrolytic experiments with P-amylase, trypsin, and collagenase that the glycogen present in their platelet preparation may be complexed to protein, causing it to precipitate with trichloroacetic acid together with the protein. Previous results from our laboratory showed the existence of a protein:glucosyltransferase in platelets and demonstrated that this enzyme was present in a higher amount than the protein:galactosyltransferase reported (6). On the other hand, from its properties it was concluded that the protein:glucosyltransferase and protein:galactosyltransferase were two distinct enzymes in terms of their substrate specificity and solubilization with Triton X-100. On the formation of glycogen, it has been suggested that a glucoprotein is synthesized as a precursor of the polysaccharide for several tissues, and two distinct enzymes are required for this (7-l 1). Since platelets contain glycogen and protein:glucosyltransferases, we have tried to characterize the enzyme responsible for the formation of the glucoprotein and at the same time determine the synthesis of glycogen under such conditions. In this report, we present evidence that these glucosylation reactions in platelets are carried out by two different enzymes. In addition, a partial separation of both activities is shown. 147 0885-45OY90 $3.00 Copyright 0 1990 by Academic Press. Inc. All rights of reproduction in any form reserved.
148
RODRiGUEZ
MATERIALS
AND
DURANTE
AND METHODS
Concentrated platelets were provided by the Municipal Blood Bank. UDP-[U“C]glucose (sp act 223 mCi/mmole) was obtained from New England Nuclear Corp. (Boston, MA). Sephadex G-25 and G-100 were obtained from Pharmacia Fine Chemical (Upsala). Bovine liver glycogen, sodium dodecyl sulfate (SDS), and all other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Preparation of Platelets and Platelet Homogenates Platelets from two concentrates of human platelets were isolated and homogenized according to the procedure described previously (6). The homogenate was centrifuged at 105,000g for 60 min at 4”C, and the pellet was resuspended in 30 mM Tris-HCl buffer, pH 7.5. The demonstration of glucosylation of platelet proteins and the characterization of the enzyme reported here were conducted with this fraction. The 105,OOOg supernatant was chromatographed on Sephadex G-100. Protein was measured by the procedure described by Peterson (12) using bovine serum albumin as the standard. Glucosylation of Endogenous Platelet Proteins and Enzyme Assays The standard incubation mixture contained in 100 ~1 total volume, 15 PM UDP[‘4ClGlc (20,000 dpm), 50 mM Tris-HCl, pH 7.5 (standard buffer), and 20 pg of protein. The incubation was carried out at 37°C for 20 min and stopped by addition of 0.5 ml 10% chilled trichloroacetic acid. The reaction mixture was cooled in ice for 30 min and centrifuged at 10,OOOg. The precipitate was considered as the trichloroacetic acid-insoluble protein (fraction I). The resulting supernatant (fraction II) was retained in order to assay the trichloroacetic acidsoluble glycogen. Controls with denatured enzyme were run with each incubation and the values obtained were subtracted from those of the test samples. Chromatography
of Reaction
Product
Fraction I was washed with 10% trichloroacetic acid, and the pellet was dissolved with 1% SDS in 0.1 N acetic acid. This sample was then run through a Sephadex G-25 column (0.7 x 5 cm) equilibrated with standard buffer. The column was eluted with the same buffer, and aliquots of the fractions were counted in 3 ml of Aquasol. Untreated fraction II was also chromatographed on the same column. Endogenous Protein: Glucosyltransferase Activity Fraction I was washed free of acid-soluble radioactivity with 0.5 ml portions of 10% trichloroacetic acid and rinsed twice with 95% ethanol. The pellet was digested in 200 ~1 of Soluene-350. The radioactivity was measured in 3 ml of Aquasol containing 200 ,ul of 0.5 N HCl. Enzyme activity was expressed as dpm of [‘4C]glucose transferred to endogenous acceptor protein.
GLUCOSYLTRANSFERASE
Glycogen:
Glucosyltransferase
ACTIVITY
149
Activity
Fraction II was precipitated with 95% ethanol (1.5 vol) in the presence of 1.2 mg of glycogen carrier. The tube was heated in a bath of boiling water for 1 min and then stored for 16 hr at 4°C. To separate the glycogen the sample was centrifuged at 12,OOOg; the pellet was washed free of ethanol-soluble radioactivity with 0.5 ml portions of 95% ethanol and recentrifuged. The precipitate was digested and the radioactivity counted as described above. Enzyme activity was expressed as dpm of [‘4C]glucose incorporated into glycogen. Sephadex
G-100 Chromatography
The 105,OOOg supernatant was applied to a Sephadex G-100 column (1.5 x 25 cm) which had been equilibrated and eluted with the standard buffer. Enzymatic activities and absorbance at 280 nm were determined in the fractions. RESULTS
A representative partial purification of protein and glycogen:glucosyltransferase from human platelets is shown in the Table 1. Both enzymes were present in the crude homogenate in an approximate ratio of 7: 1 and after Sephadex G-100 this ratio varied to 36: 1. The protein:glucosyltransferase was recovered with an approximate 50% yield after an 1l-fold purification and glycogen:glucosyltransferase was obtained in this procedure with a purification factor of 5 and a yield of 10%. In every preparation the activities for both glucosyltransferases in the 105,OOOg pellet were significantly higher than in the homogenate, and in consequence the aggregate yield of activities of supernatant and pellet obtained from the homogenate was always greater than 100%. When the 105,OOOg supernatant fraction was applied to a Sephadex column, the enzymatic activities were eluted as shown in Fig. 1. A major protein peak I (fractions 25-55) showed protein:glucosyltransferase activity. Contamination with glycogen:glucosyltransferase activity was less than 3% in this step of fractionation and did not increase when the enzyme was assayed in the presence of exogenous glycogen. A minor peak II (fractions 83-100) contained protein:glucosyltransferase only, but at a very low concentration (Fig. 1). To test if peak II behaved as an endogenous acceptor for the protein:glucosyltransferase, the enzymatic activity of the first peak was assayed with aliquots of the second one as substrate. No stimulation of glucosyltransferase was observed. Incubation of UDP-[14C]Glc with a platelet preparation which sediments at 105,OOOg led to incorporation of radioactivity into a fraction precipitable by 10% trichloroacetic acid (fraction I) and solubilized by SDS. The elution profile obtained on Sephadex G-25 gel filtration (Fig. 2) of the product formed under these conditions showed that one peak of radioactivity eluted with the V,,; 1.2 of the total radioactivity was incorporated into protein. When the supernatant (fraction II) was filtered on the same column, the peak eluting with VT (Fig. 2) corresponded to unreacted UDP[‘4C]Glc added to the assay mixture or radioactive sugar liberated from the sugar nucleotide. In a parallel incubation we determined
52 15.6
24.9 2.2
0.9
6 2.3
1
Protein (mg)
6.5 5.2
Volume (ml)
5
1217 234
468 393
5.5
48.9 106.4
9 25.2
Specific activity (KU/mg protein)
1
260 50
100 84
Yield (%)
a One unit is the amount of enzyme which transfer 1 dpm of [‘4C]glucose per 20 min at 37°C. b No detectable enzymatic activity was found.
Homogenate 105,OOOg supematant 105,OOOgpellet Sephadex G-100 fractions 33-37 Fractions 96-97
Step
Total activity (KU)
4.9 6.7 -
NDh
1.6 1.3
Specific activity (KU/mg protein)
Glycogen:glucosyltransferase
122 6.5
25 68
Total activity (KU)”
from Human Platelet
Protein:glucosyltransferase
TABLE 1 Protein and GlycogenXilucosyltransferase
-
179 10
37 loo
Yield @)
z $ 3 m
:
E, ? i-t
g
GLUCOSYLTRANSFERASE
Fraction
ACTIVITY
Number
FIG. 1. Gel filtration on Sephadex G-100. Supematant (105,OOOg) was applied to the Sephadex G-100 column (1.5 x 25 cm) equilibrated in the standard buffer. Fractions (0.5 ml) were eluted with the same buffer. Aliquots were assayed for protein:glucosyltransferase activity (0); glycogen:glucosyltransferase activity (0); and protein absorbance at 280 nm (A). Enzymatic activities are plotted per fraction.
that 0.1% of the total radioactivity was incorporated into glycogen (fraction II) synthetized by the 105,OOOg pellet. The denatured protein routinely yielded blank values of less than 0.05% of the radioactivity incubated. The effect of time, UDP-Glc, and protein concentration on the incorporation of [‘4C]glucose into fractions I and II were determined (Fig. 3). Glucosylation of the protein was linear with time for 20 min (Fig. 3A), at 60 min of incubation the activity was 83% of the maximum observed. The incorporation of glucose into glycogen was not linear with time and was completed in approximately 15 min (Fig. 3A). The formation of glucoprotein was dependent on the protein concentration and reached a maximum between 0.3 and 1.0 pg/ml (Fig. 3B). At protein concentrations above this level the radioactivity decreased slightly and the activity remained constant between 2.0 and 200 pg/ml. Figure 2 also shows that no significant variation of radioactivity in fraction II was obtained with increasing protein concentration. The curve obtained for the glucosylation of the protein with various UDP-Glc concentrations showed a biphasic pattern (Fig. 3C). As can be seen, a first plateau was reached between a substrate level of 7 and 10 PM and a second one at a substrate concentration of 15 PM. This maximal
I-
152
4.c
T 0 .-g t .I-e ‘;
vo 4
RODRIGUEZ I
AND DURANTE I
I VT i
)-
2.c
0.1
,”
!
I
IO Fraction
20 Number
30
40
FIG. 2. Chromatography on Sephadex G-25 of fractions I and II. Incubation of the lO5,OOOg pellet was performed under standard assay conditions, and fractions I and II were separated as described under Materials and Methods. 14C-fraction I (trichloroacetic acid-insoluble) dissolved in 0.1% SDS and 14C-fraction II (trichloroacetic acid-soluble) were subject to gel filtration on Sephadex G-25. The column (0.7 x 5 cm) was equilibrated with the standard buffer. Fractions (0.5 ml) were collected, and radioactivity was measured for each fraction.
incorporation of radioactivity into fraction I was maintained even with lOOO-fold higher UDP-Glc concentration. The synthesis of glycogen was very low and invariable to 10 PM UDP-Glc. Concentrations of substrate greater than 10 PM progressively increased the radioactivity and became constant between 15 PM and 15 mM (Fig. 3C). As indicated in Table 2 the enzyme responsible for the formation of the glucoprotein was found to be inhibited 76% by Mn2+ (5 mM) whereas at lower concentrations Mn2+ had no effect on the activity. Ca2’ and Mg2+ (5 mM) ions increased the activity 23 and 1l%, respectively. Glycogen:glucosyltransferase activity was stimulated 43, 80, and 200% with Mn2+, Ca2+ , and Mg2+, respectively. The effect of Triton X-100 on protein and glycogen:glucosyltransferase was studied between 0 and 0.75% v/v concentration (data not shown). The activities
GLUCOSYLTRANSFERASE
Time
Protein
(min)
60
I
c
UDP-Glc
concentmtion
153
ACTIVITY
concentration
@g/ml)
4
(PM)
FIG. 3. Glucosyltransferase activity as a function of (A) time, (B) protein concentration, and (C) UDP-Glc concentration. Incubations were carried out under the conditions described under Material and Methods, except with the variations indicated. Protein:glucosyltransferase (0) and glycogen:glucosyltransferase (0) activities were measured with the 105,OOOgpellet.
were enhanced 37 and 30% respectively in the presence of O.Ol-0.04% Triton X-100. Concentrations above this level inhibited the enzymes, and when O.l0.75% of detergent was added 75 and 86% inhibition was observed. Effect of glycogen on [‘4Clglucose incorporation into protein and glycogen: The addition of 0.005-1.0% glycogen progressively inhibited the incorporation of [‘4C]glucose into glucoprotein from 65 to 95% (Fig. 4). However, 0.002, 0.005,
154
RODRiGUEZ
AND DURANTE
TABLE 2 Effect of Various Ions on Protein and GlycogenGlucosyltransferase Activity (% of control) Ion added
Protein: glucosyltransferase
None (control) Mn*’ Ca2+ MgZf
Glycogen: glucosyltransferase
100 24 123 111
100 143 180 300
Note. Protein and glycogen:glucosyltransferases were assayed as described under Material and Methods, with the fraction 105,OOOg pellet. The ions were added at 5 mM concentrations.
and 0.007% polysaccharide stimulated 2, 3, and 2.5fold respectively the incorporation into ethanol precipitate of the trichloroacetic acid-soluble fraction. Concentrations of glycogen greater than 0.007% severely inhibited the radioactivity incorporated into glycogen, which returned close to basal values observed in the absence of glycogen (Fig. 4).
40 i
I
0.025
: 0-
I
0.05 Percent Glycogen (W/VI
FIG. 4. The effect of glycogen on the protein and g1ycogen:glucosyltransferase activities. The enzymes were assayed as indicated under Material and Methods, except that the concentrations of glycogen added to the reaction mixture were increased as shown. Determinations of protein (0) and glycogen:glucosyltransferase (0) activities were made with the 105,OOOg pellet.
GLUCOSYLTRANSFERASE
ACTIVITY
155
When the effect of glycogen was assayed with the more purified Sephadex column (fractions 33-37) the protein:glucosyltransferase showed a response identical to that described earlier. However, the glycogen:glucosyltransferase activity did not exhibit any variation with added glycogen in the range studied (data not shown). DISCUSSION The results summarized in the preceding section demonstrate that human platelets can glycosylate endogenous protein acceptor. The synthesis of a glucoprotein is judged from the insolubility in 10% trichloroacetic acid, solubility in 1% SDS, and the gel filtration. These data are consistent with our earlier results (6,13). It is possible that the assay incubation used by Greenberg et al. (5) in the presence of the Mn*’ ion (0.22 M) inhibited substantially the formation of the glucoprotein. The partial purification of the enzyme (Table 1) has shown it to be distinct from that of the glycogen:glucosyltransferase. The change of the ratio from 7: 1 (protein:glucosyltransferase/glycogen:glucosyltransferase) in the homogenate to 36: 1 in peak I from the Sephadex column supports this conclusion. The fact that the yield and purification factor observed for protein:glucosyltransferase activity are greater than those shown by glycogen:glucosyltransferase (Table 1) could be explained by the fact that they are two different enzymes and are separable. A complete separation of both activities from rabbit muscle has been reported (11). The authors showed an increase in the specific activity of protein:glucosyltransferase and a decrease in glycogen:glucosyltransferase activity. Similar results were reported for two separable sialyltransferase activities in human platelets (14). Our results of the higher yield obtained by adding the activities in the supernatant and particulate fractions (Table 1) could reflect that the extent of glycosylation has no quantitative relation to the real transferase activities present in the homogenate. This could suggest the presence of an unknown inhibitor in the homogenate that produces an apparent lower activity for both enzymes in that fraction. With respect to protein:glucosyltransferase activity, it is also possible that the amount of acceptor present in the homogenate is limiting; the manipulation could provoke the appearance of more efficient endogenous substrate for the enzyme. In the case of glycogen:glucosyltransferase the higher yield after the centrifugation could be the result of the existence of two interconvertible forms of the enzyme in platelets, which change its activity. Concerning this point there have been considerable discussion and evidence for less active forms of the enzyme (15-17). The characterization of both enzymes (Fig. 3) indicated that protein:glucosyltransferase exhibits a definite dependence on the time of incubation, protein, and UDP-Glc concentration. This is in striking contrast to glycogen:glucosyltransferase, which did not significantly increase its activity, in response to those variables. This fact appears to reflect a lesser abundance of this enzyme in the mature platelet, or that our conditions are not optimal for glycogen synthesis. There are also differences between the two activities with respect to the metal ion requirements (Table 2). Mn*+ shows opposite effects on both activities, and Ca*+ and Mg*+ ions exerted a greater
156
RODRiGUEZ
AND
DURANTE
stimulation on glycogen:glucosyltransferase than that on protein:glucosyltransferase. It has been reported that in rabbit skeletal muscle the protein:glucosyltransferase, which forms the glucoprotein required for the glycogen biosynthesis, has an absolute requirement for divalent cations, Mn*+ being more effective (10). The saturation of protein:glucosyltransferase activity by low UDPGlc and protein concentrations (Fig. 3) indicates a high affinity for donor and acceptor substrates and may reflect its critical importance for the glucoprotein biosynthesis. The K, for UDP-Glc reported for glycogen:glucosyltransferase from human platelets is 6.6 mM (4). The biphasic curve obtained with UDP-Glc concentration suggests the formation of a possible glucoprotein intermediate in the initiation of glycogen biosynthesis in platelets as was first reported by Krisman in rat liver system (7). However, a clear connection between the synthesis of the glucoprotein and glycogen was not observed in platelets (Fig. 3). This could be a consequence of the lesser amount of glycogen:glucosyltransferase found in our platelets under our experimental conditions. Also, the characteristic lag period showed in liver (7) and heart (8) for the time course incorporation of glucose into the protein for the formation of the appropriate intermediate involved in the synthesis of glycogen was not observed in platelets. The results obtained with the addition of 1% glycogen to the incubation mixture (Fig. 4) indicate that platelet glucosyltransferases differ from the two glucosyltransferases involved in the biosynthesis of glycogen in other tissues (7-9). While in platelets a significant inhibition was observed in the synthesis of glucoprotein and the polysaccharide, in other systems an inhibition on the glucoprotein synthesis and an increase in radioactivity in the ethanol precipitate of the trichloroacetic acid-soluble fraction have been reported. This effect was shown in platelets, but at very low concentrations of glycogen. Thus in platelets exogenous glycogen in the range 0.002-0.0075% competes with the endogenous acceptor protein and inhibits its own synthesis at higher concentration. Our results suggest that the concentration of glycogen in platelets could be of critical importance in the control of both UDPGlc:glucosyltransferases responsible for the synthesis of the glucoprotein and the polysaccharide. This fact together with the low level of glycogen synthase could explain the low rate of glycogen formation in the cell. In platelets Gear and Schneider (16) have reported glycogen synthase to be in the range of 30% or less of its maximum activity. Platelet glycogen content is about 60 pmole glycogen/lO” platelets (17) and it takes at least 2 hr to reach this glycogen level indicating a very low glycogen turnover. Results from Sephadex G-100 filtration (Fig. 1) indicated that proteinglucosyltransferase was eluted together with the endogenous acceptor. Possible explanations are that the enzyme and the acceptor are two different proteins that copurified after the gel filtration step or that they are a single protein with enzymatic activity and acceptor capacity which could be autoglycosylated. It is well known that purified mammalian glycosyltransferases are glycoproteins and appear to contain up to 15% carbohydrate by weight. Recently two reports have appeared concerning the self-glycosylating enzyme for the formation of the primer of glycogen in rabbit skeletal muscle (10,ll). Our results suggest that the biosynthesis of a glucoprotein takes place in
GLUCOSYLTRANSFERASE
ACTIVITY
157
platelets, which may be involved in the biosynthesis of glycogen. Both molecules are synthetized by two different enzymes. The role of platelet glucoprotein in other physiological processes remains to be established. SUMMARY Human platelets exhibited significant glucosyltransferase activity, that transfer [‘4C]glucose from UDP-Glc to an endogenous protein acceptor. The enzyme protein:glucosyltransferase responsible for the catalysis was characterized and compared with glycogen:glucosyltransferase. We describe a partial separation of both activities, the ratio of protein:glucosyltransferase/glycogen:glucosyltransferase varied from 7 : 1 in a crude homogenate of platelets to 36: 1 in the Sephadex G-100 column, This procedure failed to separate the protein:glucosyltransferase from its endogenous acceptor. Glucosylation of protein demonstrated dependence with respect to time and both protein and UDP-Glc concentration, and was saturated by very low concentration of donor and acceptor substrates. It was inhibited 76% by 5 mM Mn2+ concentration and was activated 23 and 11% by 5 mM concentrations of Ca*+ and MgZf , respectively. With respect to glycogen:glucosyltransferase, when the effect of time, protein, and substrate concentration were determined under identical conditions, it did not show the same dependence. At 5 mM concentration, Mn2+, Ca2’, and Mg2+ were activators of the enzyme 43, 80, and 200%, respectively. On the basis of these characteristics, we conclude that the synthesis of glucoprotein and glycogen are catalyzed by two distinct enzymes. Addition of exogenous glycogen (range 0.002-l%) inhibited the protein: glucosyltransferase, whereas at O.OOl-0.007% concentration it was acceptor substrate for glycogen:glucosyltransferase activity. At higher concentrations this activity was strongly inhibited. The concentration of glycogen in platelets could play a regulatory role in forming the glucoprotein and the glycogen molecules. ACKNOWLEDGMENTS We thank Dr. Francisco Herrera for his review of the manuscript, Dra. Graciela Le6n from Municipal Blood Bank for supplying the platelet concentrates, Mr. Alfonso Tablante for his technical assistance, Miss Elizabeth Gonztiez for secretarial work, and Mrs. Dhuwya Otero for drawing the figures. This work was supported in part by Project Sl-1986, CONICIT.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Barber, A. .I., and Jamieson, G. A., Biochim. Biophys. Acta 252, 533 (1971). Bosmann, H. B., Biochem. Biophys. Res. Commun. 43, 1118 (1971). Vainer, H., and Wattiaux, R., Nature(London) 217, 951 (1968). Karpatkin, S., Charmatz, A., and Langer, R. M., J. Clin. Invest. 19, 140 (1970). Greenberg, J. H., Fletcher, A. P., and Jamieson, G. A., Thromb. Diath. Haemorrh. 30, 307 (1973). Rodriguez, P., Bello, 0.. Tablante, A., and Apitz-Castro, R., Biochem. Med. Metab. Biol. 40, 151 (1988). Krisman, C. R., Ann. N. Y. Acad. Sci. 210, 81 (1973). Blumenfeld, M. L., Whelan, W. J., and Krisman, C. R., Eur. J. Biochem. 135, 175 (1983). Blumenfeld, M. L., and Krisman, C. R., Eur. J. Biochem. 156, 163 (1986). Pitcher, J., Smythe, C., and Cohen, P., Eur. J. Biochem. 176, 391 (1988).
158 11. 12. 13. 14. 15. 16. 17.
RODRIGUEZ
AND DURANTE
Lomako, J., Lomako, W. M., and Whelan, W. J., FASEB J. 2, 3097 (1988). Peterson, G. L., Anal. Biochem. 83, 346 (1977). Rodriguez, P., Bello, 0.. and Apitz-Castro, R., Biochem. Med. Mefab. Biol. 37, 197 (1987) Banvois, B., Montreuil, J., and Verbert, A., Biochim. Biophys. Acta 788, 234 (1984). Aguilar, J., and Rosell-PCrez, M., Rev. Espagfi. Fysiol. 31, 151 (1975). Gear, A. R. L., and Schneider, W., Biochim. Biophys. Acta 392, 111 (1975). Akkerman, J. W. N., Thromb. Haemostasis 39, 712 (1978).
