Eur. J. Biochem. 70, 225-231 (1976)

Studies on the Mechanism of Collagen Glucosyltransferase Reaction Raili MYLLYLA Department of Medical Biochemistry, University of Oulu (Received February 7/August 11, 1976)

The mechanism of collagen glucosyltransferase reaction was studied with enzyme preparations purified about 2500 - 5000-fold from extract of homogenate of whole chick embryos. Data obtained in experiments on initial velocity and inhibition kinetics of the reaction were consistent with an ordered mechanism in which the substrates are bound to the enzyme in the following order: Mn2+, UDP-glucose and collagen substrate, the addition of Mn2+ being at thermodynamic equilibrium and the binding site of the UDP-glucose to the enzyme not being the same as that for Mn2+ and collagen substrate. Only one metal co-factor seems to be involved in the reaction. The collagen substrate can probably also react in some conditions with enzyme-Mn" and with enzyme-Mn2 -UDP, and the UDP with the free enzyme, but in all these instances dead-end complexes are formed. Evidence is presented for an ordered release of the products in the following order: glucosylated collagen, UDP and Mn2+,in which Mn2+ need not leave the enzyme during each catalytic cycle. +

Collagens from interstitial tissues and basement membranes contain hydroxylysine-linked disaccharide units with a structure of 2-O-a-glucosyl-O-~-galactosylhydroxylysine (for review, see [l -41). The linking of glucose to certain galactosylhydroxylysyl residues is catalyzed by collagen glucosyltransferase. This reaction requires Mn2+ as a co-factor and the transfer occurs from UDP-glucose [l- 91. Collagen glucosyltransferase was initially purified at a relatively low level from guinea pig skin [5], rat kidney cortex [6], chick embryo cartilage [7] and bovine arterial tissue [8]. Recently, > 2000-fold purification of the enzyme from whole chick embryos was reported and several properties of the enzyme were examined [9- 111. Essentially nothing is known, however, about the reaction mechanism, except that several observations suggest the requirement of an enzyme-Mn2+ complex for the binding of the gelatin substrate and the UDP-glucose co-substrate [9,11]. In the present study, an attempt was made to examine the mechanism of collagen glucosyltransferase reaction with enzyme preparations purified about 2500 - 5000-fold from extract of homogenate of whole chick embryos. Information about the reaction mechanism was mainly obtained from studies of initial velocity and inhibition kinetics (for recent reviews of the principles of such experiments, see [12- 141). Enzyme. Collagen glucosyltraniferase or UDP-glucose : 5-hydroxylysine-collagen glucosyltransferase (EC 2.4.1.66).

MATERIALS AND METHODS

Materials Calf skin gelatin was prepared as reported earlier [7,9,10]. Glucose was removed from the gelatin by mild acid hydrolysis (0.2 M HCl at 110 "C for 3 h) [15] and the glucose-free gelatin was termed 'collagen substrate'. Glucosylgalactosylhydroxylysine was prepared and purified from sponge collagen [ l l , 161 over 95 % pure and contained no galactosylhydroxylysine when examined with an amino acid analyzer [ll]. Glomerular basement membrane collagen was prepared from human kidney [17,18]. Uridine diphosphate-~-['~C]glucose (227 Ci/mol) was purchased from New England Nuclear and non-radioactive UDPglucose from Sigma. The radioactive UDP-glucose was diluted with the non-labelled compound to a final specific activity of 10 Ci/mol [12]. MnC12 was Merck analytical reagent and was further purified with dithizone [19]. The initial experiments were carried out on enzyme preparations purified from whole chick embryos by the procedure consisting of six conventional protein purification steps [9]. Subsequent studies were performed using enzyme preparations purified either by affinity chromatography on UDP-glucose derivate linked to agarose [ l l ] or collagen linked to agarose [20]. The specific activities of all enzyme preparations used in this study were about 2500- 5000 times higher than those in the original ektract.

226

Collagen Glucosyltransferase

Assay of Collagen Glucosyltransferase Activity

The reaction with collagen glucosyltransferase under standard conditions was carried out in a final volume of 100 p1 containing 3-6 pg/ml of enzyme protein, 35 mg/ml collagen substrate, 60 pM UDPglucose (10 Ci/mol), 2 mM MnC12,O. 1 M NaCl, 1 mM dithiothreitol and 50 mM Tris-HC1 buffer, with pH adjusted to 7.4 at 20 "C [7,9,10]. The MnClz concentration is lower than the previously reported 10 mM [7,9, lo], because 2-4 mM was found to be optimal with the highly purified enzyme preparations used here. The collagen substrate was heated to 6O "C for 10 min and rapidly cooled to 0 "C, and immediately after this the substrate was added to the incubation mixture [9, lo]. The samples were incubated at 37 "C for 45 min, and the product assayed as reported previously [7,10]. The radioactivity in the product was converted into molar amount of glucosylgalactosylhydroxylysine on the basis of the specific activity of the UDP-glucose. In experiments in which components of the reaction mixture were varied, the conditions were as described in the legends to corresponding figures or tables. In experiments on metal requirements, cobalt concentration in the reaction mixture was 2.5 mM, magnesium concentration 30 mM and calcium concentration 10mM. These conditions were found to be optimal for these metals [9]. In the inhibition studies the added inhibitors were at the concentrations indicated. RESULTS Initial Velocity Studies

The synthesis of glucosylgalactosylhydroxylysine under the conditions used in this study was linear with time and enzyme concentration, and in all experiments reported below less than 1 % of the galactosylhydroxylysyl residues in the collagen substrate was converted to glucosylgalactosylhydroxylq syl residues. Detailed kinetic studies were carried out by varying the concentration of one substrate in the presence of different fixed concentrations of the second substrate, while the concentration of the third substrate was held constant. UDP-glucose is known to bind Mn2' , the dissociation constant for the Mn2+-UDP-glucose complex being about 19 mM [21]. It can be calculated that less than 10% of the UDP-glucose was in the form of the above complex in all experiments, and thus the data predominantely represents the kinetics of the free UDP-glucose. Reduction of the free Mn2 concentration due to this complex formation was less than 0.5%. The Tris buffer does not bind Mn2+ to any significant extent [22,23], but to make suire that +

binding of some Mn2+ to Tris buffer did not affect the results the experiments reported in Fig.1 and 2 (below) were repeated with 50 mM 3(-N-morpholino-) propanesulphonic acid buffer which likewise does not bind Mn" [24]. The results were identical to those shown in Fig. 1 and 2. The binding of Mn2+ to the collagen substrate was studied by mixing the components of the enzyme incubations with several different ratios of Mn2' to collagen in an Amicon ultrafiltration cell and by ultrafiltering about 10 of the volume through an UM-2 membrane. No collagen was detected in the ultrafiltrate. The Mn2' concentration of the ultrafiltrate measured by atomic absorption spectrophotometry agreed within 20 of that in the ultrafiltration cell with all ratios of Mn2+ to collagen used in this study. It thus seems that although some Mn2+ was bound to collagen, at least 80% was present as the free cation. Preliminary experiments in which the effect of UDP-glucose concentration on the initial velocity of the reaction at various fixed concentrations of Mn2+ and a constant concentration of collagen substrate was studied indicated that double-reciprocal plots intersected left of the ordinate (see Annexes). Because a recent study on kinetics of bovine milk galactosyltransferase [25] indicated that impurities present in commercial MnC12 preparations can affect the results, the same experiment was repeated with MnC12 purified by dithizone. In this experiment the lines intersected on the ordinate (Fig. 1). When the data obtained in these experiments were plotted as velocity-' versus [MnZt 1- at various fixed concentrations of UDP-glucose, the lines intersected to the left of the vertical axis. Slopes of these lines plotted against [UDP-glucose]-' give a line which passes through the origin (Fig. 2). Intersecting lines were obtained when UDP-glucose concentration was varied at different fixed concentrations of collagen substrate and at a constant concentration of Mn2+ (Fig. 3). Similar experiments varying collagen concentration at different fixed concentrations of Mn2+ and at constant concentration of UDP-glucose gave lines likewise intersecting left of the vertical axis (Fig. 4). The apparent and true K, and K, values for the substrates of the enzyme reaction are shown in Table 1. The apparent values were calculated directly from the initial velocity data [12,14,26], and the true values using further corrections given in equations reported elsewhere [26]. A good agreement was found between the apparent and true values.

'

Possible Involvement of Two Metals in the Reaction

Manganese is the most effective metal co-factor for collagen glucosyltransferase [6- 91, but other

R. Myllyla

227

6

12

5

:0

7- 4

1

7- 8

0

0

s -3

E

.

'-6

. -

1

1

c

2 1

2

0

-0.03 -0.02-6.01

0 0.01 3.02 0.03 0.04 l/[UDP-glucose]( g M - ' )

l/[UDP-glucose] (PM-') Fig.1. The effect of UDP-glucose concentration on the rate of collagen gluco.syltransjerasr reaction at difeerent ji'xed conccwtrations of M n 2 + and a fi'xed concentration of' collagen substrate 135 mgiml). The concentrations of M n Z + were: ( 0 ) 0.25 mM, (m) 0.5 mM, (0) 0.75 mM, (0)1.0 mM. Velocities 1) are expressed as nmol of the product formed in 45 min

0.6

Fig. 3. The eflict of UDP-glucose concentration on the rate of collagen gluco.syltrun$ierase reuction at diferent j h e d conc,entration.s qf collagen suhstrate and n fixed concentration of Mn2+ (2 m M ) . The concentrations of collagen substrate were: ( 0 ) 4.4 mg/ml, (0) 8.8 mg/ml, (m) 13.2 mg/ml, (0) 35 mg/ml. Velocities are expressed as nmol of the product formed in 45 min

-

a

0

0.02 0.04 l/\UDP-glucose] ( FM-')

0.06

Fig. 2. A secondary plot ?fsIqie.s of' lines obtained by replottiiig the data of Fig.1 with Mn2+ as the variable suhstrate and UDPglucose as the fi'xed vuriuhle suhstrate against UDP-glucose coneent ra t ion

divalent metals, such as Co2+,Mg2+ and Ca2+, can partly replace Mn2+ [6,7,9]. Possible involvement in the reaction of two metals (Mn2+and an additional metal), was studied by adding Co", Mg2+ or Ca2+ in the presence of Mn2+ into the incubation mixture. These metals did not cause any inhibition or stimulation of the catalytic activity.

INHIBITION STUDIES

Substrate Inhibition

0 1 2 3 I / [collagen] (rnlirng) Fig. 4. The efJf;ct of collagim suhstiate conc~entrationon thrj rate qf collagen gluco,syltransj~rasereuction at diffivent fixcd conwntrations o f M n Z' andafixed concentration of UDP-glucose 160 @ M i . The concentrations of M n Z t were: ( 0 ) 0.25 mM, (0) 0.75 mM, (A) 1.0 mM. Velocities are expressed as nmol of the product formed in 45 min -1

nexes) and uncompetitive with respect to Mn2+ (Fig. 5). Inhibition by UDP-galoctose or UDP-glucuronicAcid UDP-galactose and UDP-glucuronic acid were found to be inhibitory analogues of UDP-glucose. UDP-galactose showed linear noncompetitive inhibition with respect to collagen substrate, linear competitive inhibition with respect to UDP-glucose (Fig. 6) and uncompetitive inhibition with respect to Mn2 (Fig. 7). The Ki value for UDP-galactose calculated from the secondary plot obtained from Fig.6 was 30 pM. UDP-glucuronic acid gave similar results (not shown), the Ki value being 400 pM. +

Concentrations of collagen in excess of 45 mg/ml were found to inhibit the reaction (not shown). Further studies of this substrate inhibition showed that it was noncompetitive with respect to UDP-glucose (An-

228

Collagen Glucosyltransferase

Table 1. Kinetic constants f o r the substrates of the collagen glucosyltransferase reaction Ki, and Kib represent dissociation constants for the reaction of MnZ+with the free enzyme and the reaction of UDP-glucose with the enzyme-Mn" , respectively; Kb and K, are Michaelis constants for UDP-glucose and collagen substrate, respectively. The apparent values were directly calculated from the initial velocity data in Fig. 1 or 3 [12,14,26], and the true values using further corrections given in equations reported elsewhere [26]. The values were not corrected for possible binding of up to 20% of the Mn2+ to the collagen substrate and up to 10% of the UDP-glucose to Mn2+ (see second paragraph in Results). The values for K , are expressed as molar concentration of galactosylhydroxylysyl residues which corresponds to 13.3 g/l of collagen [lo] Substrate

Kinetic Apparent True constant value value

Figure used for ca.lculation

mM -~

MnZ+(A)

K,,

0.375

0.375

1

UDP-glucose (B)

Kb Kb

0.033 0.038 0.012

3

K,b

0.028 0.045 0.014

K,

0.150

0.150

3

Collagen (C)

l/[UDP-glucose] (pM-')

Fig. 6. Inhibition of collagen glucosyltransferase reaction by UDPgalactose with respect to UDP-glucose. The concentrations of UDPgalactose were: (W) none, (A) 50 pM, (A) 100 pM, (0) 200 pM. Velocities are expressed as nmol of the product formed in 45 min

1 3

l2

-

t

4

i

t 3

Fig. 7. Inhibition of collngen glucosyltransferuse reaction by U D P galactose with respect to M n 2 + . The concentrations of UDP-galactose were: (m) none, (A) 50pM, (A) 100pM and (0) 200pM. Velocities are expressed as nmol of product formed in 45 min. Similar parallel lines were obtained in four additional experiment

l / [ M n 2 + ] (rnM-')

Fig. 5. Inhibition of collagen glucosyltransferuse reaction by collagen substrate with Mn2+ as the variable component. The concentrations of collagen substrate were: (0)49 mg/ml, (0)80 mg/ml, (0)109 mg/ ml. Velocities are expressed as nmol of the product formed in . 45 min. Similar parallel lines were obtained in four additional experiments with slightly different collagen concentratioins

Product Inhibition

Inhibition studies could not be carried out on free UDP since it has a strong affinity for Mn2+ (see [24]). It would have been difficult to examine the relative inhibitory properties of free UDP and the Mn2+-UDP complex separately and it was decided to study the effect of the Mn2+-UDP complex only. Tlherefore, the conditions were selected so that the rati'o of free UDP to Mn2+-complexed UDP will be small and the effect of free UDP may be considered to be nlegligible. The Mn2+-UDP complex was found to be a linear

noncompetitive inhibitor with respect to Mn2+ (Annexes), a linear noncompetitive inhibitor with respect to collagen substrate (not shown), and a linear competitive inhibitor with respect to UDP-glucose (Fig. 8) giving a Ki value about 4 pM. A possible product inhibition of the glucosylgalactosylhydroxylysyl residues was studied by using both free glucosylgalactosylhydroxylysine and glomerular basement membrane collagen which contains a high number of hydroxylysine-linked carbohydrate units, almost exclusively in the form of the disaccharide [27-291. Both substances were found to cause an inhibition (Table 2). When the values were expressed as molar concentration of the product sites, protein-bound glucosylgalactosylhydroxylsine was clearly a more effective inhibitor than the free compound (Table 2). This finding is in agreement with previous data, indicating that higher-molecular-weight

R. Myllyla

4t

229 Table 3. Inhibition of collagen glucosyftransjerase by various compounds All compounds which were found to be inhibitory were linear noncompetitive inhibitors in relation to the collagen substrate. The apparent Ki values were calculated from the secondary plots. Ki, is the Ki calculated from the slopes and Kii the Ki calculated from the intercepts

t

Kis

Inhibitor

Kii

mM ~~

UTP UMP Uridine ATP ADP AMP CDP GDP Glucose Lactose

1 / [UDP-glucose] ((IM-')

Fig. 8. Inhibition of collugen glucosyltransferase reaction by UDP with respect to UDP-glucose. The UDP concentrations used were: (W) none, (0)20 pM, ( 0 )30 pM, (A) 40 pM, (0)50 pM. Velocities are expressed as nmol of the product formed in 45 min

Table 2. The effect of glueosylgalactosylhydroxylysineor glomerulur basement membrane collagen on the rate of collagen glucosyltransferase reaction The reaction was carried out as described in Methods with 35 mg/ml collagen substrate and with an addition of glucosylgalactosylhydroxylysine or glomerular basement membrane collagen in the concentration indicated. Values for glucosyltransferase activity are given as amount of product formed in 45 min with relative activity in parentheses Inhibitor

Glucosylgalactosylhydroxylysine

Glomerular basement membrane collagen

Amount Concen- Glucosyltranstration ferase activity of product site mg/ml

pmol/ml

nmol (%)

0 8 16 32

-

16.9 33.8 67.6

0.108 0.090 0.072 0.064

0 10

-

1.5

(100) ( 83) ( 67) ( 59)

0.161 (100) 0.093 ( 58)

acceptors constitute better substrate for the enzyme than low-molecular-weight acceptors [6, lo]. Because it seemed possible that glucosylgalactosylhydroxylysine, due to its low molecular weight, might act differently compared to normal products of the reaction, and because purified glomerular basement membrane collagen was not available in large quantities, kinetics of inhibition with these products were not analyzed. Other Inhibition Studies A series of other compounds was also tested as inhibitors : UMP, UTP, AMP, ADP, ATP, CDP, GDF:

0.040 0.8

~

a

-

-

2.5 1.5 1.o 0.15

2.5 1.6 1.2 0.16

-

-

-b

-b -b

-b

-

0.030 0.75

~

No inhibition at 1 mM concentration. Only slight inhibition at 1 mM concentration.

uridine, glucose and lactose (Table 3). Uridine and GDP did not give any inhibitions at 1 mM concentration and glucose or lactose were only slightly inhibitory at this concentration. CDP was an effective inhibitor at 0.2 mM concentration: it was a linear competitive inhibitor with respect to UDP-glucose and a linear noncompetitive inhibitor with respect to collagen substrate or Mn2+ (not shown). DISCUSSION Kinetic mechanisms for enzyme reactions fall into two major groups. Those in which all reactants must combine with the enzyme before reaction can take place and any products can be released are called sequential. Mechanisms in which one or more products are released before all substrates are bound are called substitution mechanisms. Sequential mechanism are called ordered if reactants combine with the enzyme and dissociate in an obligatory order, or random if alternate pathways exist and the order of combination or release is not obligatory (see reviews 112- 141). In this study, the intersecting pattern of lines in double-reciprocal plots given by every pair of the three substrates, Mn2+, UDP-glucose and collagen substrate, indicates that collagen glucosyltransferase reaction occurs by a sequential mechanism [12-141, either ordered or random. Experiments in which the effects of UDP-giucose concentration at various fixed concentrations of purified MnClz and a fixed concentration of collagen substrate were studied, gave lines intersecting on the vertical axis. When the data were plotted as velocity-' versus [Mn2'1 at various fixed concentrations of

Collagen Glucosyltransferase

230

UDP-glucose, the lines intersected to the left of the vertical axis, and slopes of these lines plotted against [UDP-glucose]- passed through the origin. These data indicate an ordered mechanism for the aiddition of Mn2+ and UDP-glucose, with the addition of Mn2+ being at thermodynamic equilibrium [12- 141. It thus follows that the addition of Mn2+ takes place before addition of UDP-glucose, Mn2 cannot dissociate once UDP-glucose has been added, and Mn2 need not leave the enzyme during each catalytic cycle but may remain bound to the enzyme [12- 141. Lines intersecting left of the vertical axis were obtained when substrate concentration was varied at different fixed concentrations of purified MnC12 and a constant concentration of UDP-glucose. These results suggest that the enzyme operates through an entirely ordered mechanism rather than a mechanism in which randomorder equilibrium binding of UDP-glucose and collagen substrate follows the binding of Mn2+. Some divalent cations, such as Co2+, Me?+ and Ca2+, can partly replace Mn2+ as a co-factor for collagen glucosyltransferase [6,7,9]. In the present experiments, no stimulation of the reaction was found after addition of Co2+, Mg2+ or Ca2+ together with Mn2 into the incubation mixture, which suggests that only one metal is required in the reaction. Neither did these metals cause inhibition, suggesting an inability of Co2+, Mg2+ or Ca2+ to act as an effective metal co-factor in the presence of M n 2 + . Inhibition studies with analogues of UDP-glucose, such as UDP-galactose, indicated an uncornpetitive inhibition with respect to Mn2+, but competitive inhibition with respect to UDP-glucose. Accordingly, the analogues apparently reacted with an enzymeMn2+ complex competing with the addition of UDPglucose to this complex, and the experiments, in accordance with the initial velocity studies (above), suggest the addition of Mn2+ to the enzyme prior to the addition of UDP-glucose. These studies, further suggest that the binding site of the co-substrate is not the same as that for the metal co-factor or the collagen substrate. Previous studies on the binding of collagen glucosyltransferase to an affinity column, in which UDPglucuronic acid is coupled from its carboxyl group to amino group in AH-Sepharose 4B, indicated that the binding was markedly enhanced by the presence of manganous ions [ l l ] . This suggests the requirement of an enzyme-Mn2 complex for the binding of UDP-glucose. In another study the protection of collagen glucosyltransferase by the co-substrate against inhibition with sulphydryl reagents was examined [9]. The results likewise suggest the requirement of an enzyme-Mn2+ complex for the binding of UDP-glucose. Collagen glucosyltransferase has in the presence of Mn2+ a high affinity to a column prepared by +

+

+

+

coupling rat-skin citrate-soluble collagen to agarose [20]. This finding indicates that the enzyme-Mn2+ complex can in certain conditions become bound to the citrate-soluble collagen prior to the addition of the UDP-glucose, thus forming an enzyme-Mn2+collagen complex. The studies of protection against inhibition with sulphydryl reagents likewise suggest the formation of the above-mentioned complex [9]. However, the data obtained in substrate inhibition experiments suggest that the enzyme-Mn2+-collagen is a dead-end complex, because the inhibition was uncompetitive with respect to Mn2+ and noncompetitive with respect to UDP-glucose [12- 141. These inhibition studies further suggest the formation of an additional dead-end complex, namely enzyme-Mn2 UDP-collagen [12- 141. Product inhibition studies with Mn2+-UDP complex gave noncompetitive, competitive and noncompetitive inhibitions with respect to Mn2+, UDPglucose and collagen substrate, respectively. These results would be consistent with an ordered ter-ter mechanism, in which the UDP combines with the same enzyme complex as the UDP-glucose, except that in such a situation the inhibition with respect to Mn2+ should be uncompetitive and not noncompetitive [24]. However, it is possible that, in the presence of a high UDP concentration, a dead-end complex enzymeUDP is also formed and in such a situation the inhibition with respect to Mn2+ should be noncompetitive, as consistent with the present data. The results further suggest that the dead-end complex enzyme-Mn2+-collagen was not formed to a large extent under the conditions of these product inhibition experiments, because in such a situation the inhibition with respect to UDP-glucose should be noncompetitive [24]. Allowing for these reservations it seems probable that UDP leaves the enzyme after the glucosylated collagen. On the basis of the data discussed above, it seems likely that collagen glucosyltransferase from chick embryos operates predominantly through an ordered mechanism as shown in Fig.9. The substrates are bound to the enzyme in the following order: Mn2+, UDP-glucose and collagen, the addition of Mn2+ being at thermodynamic equilibrium and UDP-glucose attaching at a separate site from Mn2+ and collagen substrate. Evidently only one metal is involved in the reaction. The collagen substrate can probably also react in some conditions with enzyme-Mn2+and with enzyme-Mn2'-UDP, and the UDP with the free enzyme, but in all these instances dead-end complexes are formed. Evidence was presented for an ordered release of the products in the following order: glucosylated collagen, U D P and Mn2+, in which Mn2+ need not leave the enzyme during each catalytic cycle. It should be noted that kinetic analysis of enzyme reactions has in many instances proved rather + -

ur :-23 1

R. Myllyll

T

Gluc.Coll Mn2' UDP-Glc E E.M&

3

E.Mn2: UDPGlc E~Mn2'.UDPGlcColl E.M&UDP

.1

E.Mn**.Col I

E.M8

E

E.Mn2: UDPColl .1

Fig.9. A schematic presentation of the proposed mechunisni for collugeri glu~~osyltrun~~/t,ruIse rcwtioiz. UDP-Glc, Coll and Gluc.Coll represent UDP-glucose, collagen substrate and glucosylated collagen, respectively. The dashed line indicates that Mn2 need not leave the enzyme during each catalytic cycle +

complicated [12- 141, and the present analysis is certainly not complete; for instance, studies on product inhibition were complicated by the binding of UDP to M n 2 + , and kinetics of inhibition with glucosylated collagen were not studied. Thus the scheme is only a tentative one which seems to be consistent with the data presently available. It is of interest that this scheme is very similar to that suggested for N-acetyllactosamine synthetase from human or bovine milk [24-26, 30-321.

ANNEXES Additional kinetic data have been deposited at the Archives originuks du cenfre de documen/u/ion du C.N.R.S., F-75971 Paris Cedex-20, France, where they may be ordered as microfiche or photocopies. Reference no. : A.O.-554. a) The effect of UDP-glucose concentration on the rate of collagen glucosyltransferase reaction at different fixed concentrations of analytical grade M n Z +and a fixed concentration of collagen substrate (35 mgiml) (Annex Fig. 1). b) Inhibition of collagen glucosyltransferase reaction by collagen substrate with UDP-glucose as the variable component (Annex Fig. 2). c) Inhibition of collagen glucosyltransferase reaction by UDP with respect to Mn2' (Annex Fig. 3). The present work was supported in part by a grant from the Medical Research Council of the Academy of Finland. The author gratefully acknowledges the valuable suggestions and comments of Professor Kari I. Kivirikko and Professor Martti Koivusalo and the expert technical assistance of Miss Raija Leinonen and Mrs Raija Harju.

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6. Spiro, R. G. & Spiro, M. J. (1971) J . B i d . C'hem. 246, 48'194909. 7. Myllyla, R.. Risteli, L. & Kivirikko. K. 1. (1975) Eur. J . Biochem. 52,401 -410. 8. Henkel, W. & Buddecke, E. (1975) Hoppt>-Seykr's Z. Phjxiol. Chem. 356, 921 928. 9. Myllyla, R., Risteli, L. & Kivirikko, K. 1. (1975) Eur. J . Biochem. 61, 59-67. 10. Myllyll. R., Risteli, L. & Kivirikko, K. I. (1975) Eur. J . Biochem. S X , 517- 521. 11. Anttinen, H. & Kivirikko, K. I. (1976) Biochim. B k ~ h j ~ .Acfu. \. 429, 750-758. 12. Cleland, W. W. (1970) in The Enn_ymcJ (Boyer, P. D., ed.) vol. TI, pp. 1-66, Academic Press, New York. 13. Cleland, W. W. (1967) Annu. Rev. Biochem. 36. 77-112. 14. Plowman, K. M. (1972) Enzyme Kinetics, pp. 171, McGrawHill Book Company, New York. , 375-383. 15. Askenasi, R. (1973) Biochim. Biophys. A i ~ f u304, 16. Katzman, R. L., Halford, M . H., Reinhold, V. M. & Jeanloz, R. W. (1972) Biochrmistry, 11. 1161-1167. 17. Kel'alides, N. (1971) J . Clin. h v e s t . 53, 403-407. 18. Kefalides, N . (1 974) Biochem. Biophy.'. Ri's. ('omniim. 45. 226 234. 19. Morrison, J. F. & Uhr, M. L. (1966) Biochim. Biuph1.s. A c / u , 122, 57 - 74. 20. Risteli, L., Myllyll, R . & Kivirikko, K. I . (1976) Eur. J . Biochem. 67,197-202. 21. Berliner, L. J. & Wong, S. S. (1975) Biochemisrr.v, 14. 49774982. 22. Hanlon, D. P., Watt, D. S. & Westhead, E. W. (1966) Anul. Biochem. 16, 225-233. 23. Good, N. E. & Izawa, S. (1972) Methods Enzynzol. 24B, 53-68. 24. Khatra, B. S., Herries. D. G. & Brew, K . (1974) EuI.. J . Biochem. 44, 537- 560. 25. Geren, C . R., Geren, L. M. & Ebner, K. I . (1975) Biochem. Biophvs. Res. Commun. 66, 139- 143. 26. Morrison, J. F. & Ebner, K. E. (1971) J . Biol. Chtw. 246. 3977 3984. 27. Spiro, R . G . (1969) J . Biol. Chem. 244. 602-612. 28. Spiro, R. G. (1967) J . Bid. Chrm. 242, 4813-4823. 29. Spiro, R . G . (1972) in G1yroprotein.s. Their Compo.sition,Structure undFunction (Gottschalk, A , , ed.) pp. 964-999. Elsevier Publishing Company, Amsterdam. 30. Powel, J. T. & Brew, K. (1974) Eur. J . BiochtJm.48, 217-228. 31. Morrison, J. F. & Ebner, K. E. (1971) J . Biol. Chun. 246, 3985- 3991. 32. Morrison, J . I+'. & Ebner, K. E. (1971) J . B i d . Chem. 246. 3992- 3998. -

-

-

R. Myllyll, Oulun yliopiston IHlketieteellisen kemian laitos, Kajaanintie 52A, SF-90220 Ouln 22, Finland