Chemical and enzymic degradations of nucleoside mono- and diphosphate sugars

203

Biochimica et Biophysica Acta, 584 ( 1 9 7 9 ) 2 0 3 - - 2 1 5 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

BBA 28882

CHEMICAL AND ENZYMIC DEGRADATIONS OF NUCLEOSIDE MONOAND DIPHOSPHATE SUGARS I. DETERMINATION OF THE DEGRADATION RATE DURING THE GLYCOSYLTRANSFERASE ASSAYS

G. S P I K *, P. SIX a n d J. M O N T R E U I L

Laboratoire de Chimie Biologique et Laboratoire Associd au CNRS No. 217, Universit~ des Sciences et Techniques de Lille I. B.P. No. 36, Villeneuve d'Ascq, 59650 (France) (Received July 28th, 1978)

Key words: Nucleoside sugar phosphate degradation; Glycosyltransferase assay

Summary In incorporation experiments used for the determination of glycosyltransferase activities, we demonstrated that the nucleoside diphosphate sugars are decomposed in three different ways: 1, transfer of the monosaccharide to acceptor molecule, catalyzed by glycosyltransferases; 2, degradation of the glycosyl nucleotides by nucleotide pyrophosphatase into monosaccharide 1phosphates which are further hydrolyzed into free monosaccharides by phosphatases; 3, chemical decomposition of UDP-D-[ 14C]Gal; UDP-D-['4C] Glc and UDP-D-['4C] GIcUA into 1,2-cyclic phosphate derivatives of the corresponding monosaccharide. All the breakdown products of the nucleoside mono- and diphosphate sugars which are obtained during the incorporation experiments may be separated by paper chromatography and their amounts may be determined. Galactosyltransferase assays on human and rat serum have shown that the three different ways of decomposition of the nucleoside diphosphate sugars are dependent mostly on the concentration of divalent cations (Mn 2+, Mg2÷). Inhibition of the nucleotide pyrophosphatase activity is obtained with low concen* To whom all correspondence should be addressed. Abbreviations: UDP-D-Gal, uridine diphosphogalactose; UDP-D-Glc, uridine diphosphoglucose; UDP-DGlcUA, uridine diphosphoglucuronic acid; UDP-D-GlcNAc, uridine diphospho-N-acetylglucosamine;GDPD-Man, guanosine diphosphomannose; GDP-L-Fuc, guanosine diphosphofucose; CMP-D-AcNeu, cytosine monophospho-N-acetylneuraminic acid; GaI-1,2-P, galactose 1,2-monophosphate; GIc-I,2-P, glucose 1,2monophosphate; GlcUA-1,2-P, glucuronic acid 1,2-monophosphate.

204

trations of UMP, but increasing concentrations of UMP inhibit also the galactosyltransferase activity and consequently enhance the formation of galactose 1,2-monophosphate. A partial elimination of the nucleotide pyrophosphatase activity was achieved by the addition of increasing concentrations of UDP-D-Gal. These results demonstrate that the determination of glycosyltransferase activities in tissues and in biological fluids is not possible without a concomitant determination of the nucleotide pyrophosphatase activity present in the assay.

Introduction Glycoprotein glycosyltransferase activities are in general determined by measuring the transfer of a labeled monosaccharide from an appropriate labeled nucleoside mono- or diphosphate sugar to an acceptor. When the incubation experiments are performed with crude glycosyltransferases found in tissues or in body fluids, various errors may occur due to the possible modifications of the nucleoside mono- or diphosphate sugars. The various pathways for the destiny of the sugar from the nucleoside diphosphate sugar are summarized in Fig. 1. In addition to the transfer of the sugar by a specific glycosyltransferase, the sugar may be converted by nucleotide epimerases or isomerases, or liberated as sugar phosphate which is subsequently hydrolyzed to free sugar by a phosphatase. Liberation of the labelled sugar transferred to the acceptor may also occur due to the high activity of glycosidases present in the medium. Finally, some of the nucleoside diphosphate sugars may be submitted in vitro to a chemical degradation catalysed by metal ions. This chemical degradation was exhaustively analyzed by Nunez and Barker [ 1 ]. The present studies try to estimate, in classical conditions for the determination of the glycosyltransferase activities, the percentage of enzymic and chemical modifications of the nucleoside mono- or diphosphate sugars. The galactosyltransferase activities present in human and in rat serum were taken as references and the action of various inhibitors of the enzymic and chemical decomposition of the nucleoside diphosphate sugars were determined.

1,2-cyclic phosphate derivative of sugar

~ XDP-Sugar (2) c Sugar-l-P d Sugar

1 , 2 - c y c l i c p h o s p h a t e ~f t[a d XDP-Sugar (1) c Sugar-l-P ~ Sugar derivative of sugar 15 Acceptor-Sugar

e

Fig. 1. Possible p a t h w a y s for the destiny of the sugar from glycosyl nucleotides during incorporation experiments for the determination of glycosyltransferase activity, a, XDP-epimerase; XDP-isomerase; UDP-dehydrogenase; b, glycosyltransferase; e, nucleotide pyrophosphatase; d, phosphatase; e, glyeosidase; f, chemical degradation.

205 Materials and Methods

Materials Chemicals. The following nucleoside mono- and diphosphate sugars: UDPD-[14C]glucose (296Ci/mol), UDP-D-[~4C]galactose {290Ci/mol), UDP-Nacetyl-D-[14C]glucosamine (300 Ci/mol), UDP-D-[14C]glucuronic acid (321 Ci/ mol), GDP-L-[14C]fucose (140 Ci/mol), GDP-D-[~4C]mannose (154 Ci/mol) and CMP-D-[X4C]sialic acid (214 Ci/mol) were purchased from Radiochemical Centre (Amersham, U.K.). Unlabeled UDP-D-galactose, UDP-D-glucose, UDP-Nacetyl-D-glucosamine, UDP-D-glucuronic acid, GDP-D-mannose, and a-D-galactose 1-phosphate (Gal-l-P), a-D-glucose 1-phosphate (Glc-l-P), a-D-glucuronic acid 1-phosphate (GlcUA-1-P) and a-D-mannose 1-phosphate (Man-l-P) were obtained from Sigma. Free monosaccharides and the tris-(hydroxymethyl)aminomethane were from Fluka AG (Buchs, Switzerland). All other reagents were products of the highest purity available. Ovomucoid [2] and the glycopeptides from ovomucoid containing five residues of N-acetylglucosamine at terminal nonreducing positions [ 3] were used as acceptors for [ ~4C]galactose. Serum. Blood samples were obtained from normal adult human donors and, after decapitation, from normal male Wistar rats. The serum from the clotted blood samples was separated at 4°C and centrifuged at 4°C for 20 min at 1500 × g. The serum samples were used immediately or stored at --20°C for periods of up to 3 months. Methods Analytical methods. Protein was estimated by the method of Lowry et al. [4] or by the fluorescamine method of Udenfried et al. [5] using crystalline bovine serum albumin {Sigma) as standard. Monosaccharides were assayed by classical colorimetric methods [6] and total phosphate was analyzed according to Allen [7]. Identification of the breakdown products of nucleoside diphosphate sugars was performed by subjecting the incubating mixtures to descending paper chromatography on Whatman No. 3 paper using ethyl acetate/pyridine/glacial acetic acid/water (5 : 5 : 1 : 3) as solvent [8]. After chromatography the papers were dried and cut at 1-cm intervals. The strips were counted in an Intertechnique liquid scintillation spectrometer. Nonradioactive standards were chromatographed simultaneously and were visualized by the silver nitrate [ 9] or triphenyltetrazolium reagents [ 10]. The phosphoryl compounds were stained by the reagent of Hanes and Isherwood [ 11]. Identification of 1,2-cyclic phosphate derivatives of the different sugars was confirmed by the procedures used by Paladini or Leloir [12] and Nunez and Barker [ 1]. Chemical and enzymic degradation of the nucleoside diphosphate sugars. The test system {total volume 100 ~l) was 150 mM in Tris/HC1 buffer (pH 7.3) and 0--40 mM in MnSO4 or MgCI~ and also contained 30 pl of human or rat serum and 0.03--0.1 nmol of [14C]glycosyl nucleotides. The controls were prepared by omission of the serum. The solutions were mixed and incubated at 37°C for 1 h and the reaction was stopped by the addition of 30 gl of 0.3 M EDTA.

206

Galactosyltransferase activity. Galactosyltransferase activity of human and rat serum was assayed by the transfer of [~4C]galactose from UDP[~4C]galacrose to 500 ~g of ovomucoid or to 250/~g of the glycopeptide of ovomucoid. The conditions used were identical to those described for the study of the degradation of the glycosyl nucleotides. Glycosidase activity of the serum. After the transfer reaction of [14C]galactose onto ovomucoid, the labeled glycopeptide was eluted from the paper chromatograph and used as the substrate for the serum galactosidases activity. 500 ~g of this [~4C]glycopeptide was added to the test system containing Tris/ HC1 buffer, pH 7.3, 20 mM of MnSO4 and 30 gl of human or rat serum. The tubes were incubated for 1 h at 37°C and the liberation o f free [14C]galactose was analyzed b y paper chromatography. Effects of UMP and inorganic pyrophosphate. The effects of UMP and inorganic pyrophosphate on the enzymic and chemical decomposition of UDPGal was analyzed in the following way. Concentrations of UMP or inorganic pyrophosphate, PPi, with a final range 0.2--10 mM were added to the test system contained in a total volume of 100 pl: 150 mM Tris/HC1 (pH 7.3), 20 mM MnSO4, 500 pg ovomucoid, 30 pl human or rat serum and 0.1 nmol of UDPD-[~4C]galactose. The solutions were mixed and incubated at 37°C for 1 h. The reaction was stopped and the products were determined as described previously. Effects of increasing amounts of UDP-D-galactose. Increasing concentrations of UDP-D-galactose (1 pM--1 mM) were added to the test system containing ovomucoid as acceptor. The transfer of galactose to the acceptor and the amounts of the breakdown products were determined in the presence of human or rat serum. Results

Identification of the breakdown products present in the standards of [14C]nucleoside diphosphate sugars When the purity of the tested [t4C]nucleoside diphosphate sugars was analyzed by paper chromatography [6], in addition to the [14C]nucleosidediphosphate sugars, the samples of UDP-D-[ ~4C]Gal; UDP-D-[ ~4C]Glc and UDPD-[~4C]GlcUA were contaminated b y minor components which possessed the migrations of Gal-I,2-P; Glc-I,2-P and GIcUA-1,2-P reported in Table I. These components were identified as the 1,2-cyclic phosphate derivatives of [14C]Gal; [ 14C] Glc and [ ~4C] GlcUA on the basis of the following results: TABLE I

Migration o f t h e 1 , 2 - c y c l i c p h o s p h a t e d e r i v a t i v e s o f galactose0 g l u c o s e a n d g l u c u r o n i c a c i d o n paper chromatography using ethyl acetate/pyridine/glacial acetic acid/water (5:5:1:3)as s o l v e n t after 20 h o f m i g r a t i o n for U D P G a l and U D P G l c and 4 0 h f o r UDPGlcUA. T h e m i g r a t i o n o f t h e f r e e m o n o s a c c h a r i d e s w a s t a k e n as r e f e r e n c e . UDP-D-Gal Gal-l-P Gal-1,2-P Gal

0.26 0.35 0.50 1

UDP-D-GIc Gle-I-P Glc-l,2-P Glc

0.26 0.37 0.50 1

UDP-D-GlcUA GIeUA-1-P GteUA-1,2-P GlcUA

0.19 0.29 0.48 1

207 (a) The 1,2-cyclic phosphate derivatives obtained from unlabeled UDP-DGal; UDP-D-Glc and UDP-D-GIcUA were hydrolyzed (2 M HCI 1 h at 100°C) into free monosaccharides and phosphoric acid in a molar ratio of I to 1. They did not possess reducing power and were not stained by the triphenyltetrazolium reagent which is known to give no coloration with 2-substituted sugars [10]. (b) Partial hydrolysis of the 1,2-cyclic phosphate derivatives, in the conditions described by Paladini and Leloir [12] leads to the identification of monosaccharide-2-phosphates. These derivatives possessed reducing power, contained monosaccharide and phosphoric acid in a ratio of 1 : 1 and were not stained by the triphenyltetrazolium reagent. It may be noticed that the amount of these ~4C-labeled 1,2-cyclic phosphate derivatives increased in the presence of increasing concentration of Mn 2÷, at pH 7.3, after 1 h incubation at 37°C (Table II) and by repeating freezing and thawing of the ~4C-labeled nucleoside diphosphate sugars. The identification of other contaminant products found in some samples of UDP-D-[14C]Gal; UDP-D-[14C]Glc; UDP-D-[14C]GlcNAc and UDP-D-[~4C]GlcUA which migrated on paper chromatography just behind the ~4C-labeled nucleoside diphosphate sugars was not pursued as the amounts of these products were very low. Chemical and enzymic decompositions o f the 14C-labeled nucleoside diphosphate sugars in the presence o f human and rat serum The results of the chemical and enzymic decompositions of the 14C-labeled nucleoside diphosphate sugars by human serum are summarized in Table III. From these results it appears that under the conditions commonly used for the assay of the glycosyltransferase activities all the ~4C-labeled nucleoside diphosphate sugars analyzed were decomposed essentially into [~4C]sugar 1-phosphate. The amounts of free [ 14C]sugars released by the hydrolysis of the mono-

T A B L E II C h e m i c a l d e c o m p o s i t i o n o f 1 4 C - l a b e l e d n u c l e o s i d e d i p h o s p h a t e sugars dissolved in Tris/HC1 b u f f e r , p H 7 . 3 , in the presence o f increasing c o n c e n t r a t i o n s o f M n S O 4 a f t e r 1 h i n c u b a t i o n at 3 7 ° C . Nucleoside d i p h o s p h a t e sugars

Concentration of MnSO 4 (raM)

Residual UDP[ 14C]sugar

[ 14 C ] S u g a r 1-phosphate

14 C-labeled 1,2-cyclic p h o s p h a t e sugar

U D P . D . [ 14 C] G a l

0 10 20 40

94 91 75 52

2 2 2 2

4 7 23 46

U D P . D . [ 1 4 C] Glc

0 10 20 40

98 90 79 62

0 0 0 0

2 10 21 38

U D P - D - [ 14 C ] Glc U A

0 10

95 84

0 0

5 16

20 40

74 57

0 0

26 43

208 TABLE

III

Enzymic and of increasing buffer, after In e a c h c a s e ,

chemical decomposition of [ 14C] nucleoside mono-and diphosphate sugars in the presence concentrations o f M n S O 4 a n d MgC12 a n d h u m a n s e r u m a d j u s t e d t o p H 7 , 3 w i t h T r i s / H C l 1 h incubation at 3 7 ° C . T h e r e s u l t s are g i v e n as t h e p e r c e n t a g e s o f t h e t o t a l r a d i o a c t i v i t y . A refers to addition of MnSO4, B to addition of MgCl 2 .

Nucleoside

MnSO 4

Residual

[14C]Sugar-

[14C]Sugar-

Free [14C]-

monosugars

or MgCl 2 (raM)

nucleoside mono or diphosphate sugars

1-phosphate

1,2 cyclic phosphate

sugar

A

B

A

B

A

B

A

0 10 20 40

49 54 45 43

47 22 13 8

44 36 40 29

45 64 65 65

3 7 11 25

4 4 5 7

0

48

48

47

44

2

2

3

6

10 20 40

68 60 42

19 19 22

27 29 32

57 58 54

2 7 17

2 2 4

3 4 9

22 21 20

or diphosphate

UDP-D-[14C]Gal

UDP-D-[14C]Glc

UDP-D-[14C]GIcUA

UDP-D-[14C]GlcNAc

GDP-D-[14C]Man

GDP-L-[14C]Fuc

CMP-D-[14C]AcNeu

[14C]

4 3 4 4

B 4 10 17 20

0

13

17

72

68

3

3

12

12

10 20 40

26 18 17

3 3 0

59 62 54

64 56 56

5 10 20

4 4 4

10 10 9

29 37 40

0 10 20 40

63 66 63 57

60 51 32 23

33 30 34 35

36 44 60 68

0 0 0 0

0 0 0 0

4 4 3 8

4 5 8 9

0

44

45

52

49

0

0

4

6

10 20 40

52 55 60

35 17 10

45 39 35

55 64 61

0 0 0

0 0 0

3 6 5

10 19 29

0

46

40

40

40

0

0

14

20

10 20 40

59 57 60

26 17 10

26 30 28

41 24 29

0 0 0

0 0 0

15 13 12

33 59 61

0

95

93

0

0

0

0

5

7

10 20 40

95 90 90

90 89 85

0 0 0

0 0 0

0 0 0

0 0 0

5 10 10

10 11 15

saccharide 1-phosphates by the alkaline phosphatase were very low. These results indicate that the nucleotide pyrophosphatase activity of human serum is effective towards various nucleoside diphosphate sugars, and does not require the addition of Mn 2÷ or Mg 2+. UDP-D-[ 14C] GlcUA appears to be the most easily hydrolysed of the nucleoside diphosphate sugars. Increasing the concentration (10--40 mM) of Mn 2÷ in the incubation mixture increases the formation of the 1,2-cyclic phosphate derivatives of galactose, glucose and glucuronic acid and leads to a decrease in the amount of [14C]sugar 1-phosphate. The amounts of residual 14C-labeled nucleoside diphosphate sugars remained relatively stable. Increasing the concentration (10--40 mM) of Mg :÷ in the serum increases the level of [14C]sugar 1-phosphate in the case of UDP-D-[14C]Gal; UDP-D-[14C]Glc; UDP-D-[14C]GlcNAc; GDP-D-[14C]Man and the level of free [14C]sugars:

209

glucose; glucuronic acid; mannose and fucose. Addition of MgC12 has no effect on the formation of 1,2-cyclic phosphate derivatives of the three nucleoside diphosphate sugars. In the presence of rat serum and in the presence of 20 mM MnSO4, CMP-D[~4C]AcNeu and all the other labeled nucleoside diphosphate sugars tested are rapidly decomposed principally into 14C-labeled free sugars (Table IV) and there is no formation of 1,2-cyclic phosphate derivative of galactose, glucose or glucuronic acid. These results indicate that rat serum possesses a nucleotide pyrophosphatase activity which is effective towards all the nucleoside mono- and diphosphate sugars analyzed and that this activity as well as the activity of the alkaline phosphatase is much higher than that present in the human serum. As the decomposition of the substrates b y the nucleotide pyrophosphatase is very fast, there is no formation of 1,2-cyclic phosphate derivatives of the three monosaccharides. In contrast with the results previously obtained, addition of increasing concentrations of Mn 2÷ did not induce the formation of the 1,2-cyclic phosphate derivatives. Competition between nucleotide pyrophosphatase and galactosyltransferase activities in human and rat serum To determine the competition between the nucleotide pyrophosphatase and the galactosyl transferase activities in human and rat serum, ovomucoid was used as acceptor for galactose and the amounts of residual UDP-D-[14C]Gal; [14C]Gal-l-P; [14C]Gal-l,2-P; [14C]Gal, and labeled glycoprotein were determined. In Fig. 2, are summarized the results obtained after 1 h of incorporation in the presence of increasing concentrations of Mn 2+. The maximum activity of the human serum galactosyltransferase was obtained for a concentration of 40 mM in Mn 2÷. The level of [14C]Gal-1-P decreases when the concentration of Mn 2÷ increases from 0 to 15 mM and remains stable between 15 mM and 40 mM. The amount of Gal-I,2-P remains relatively stable when the concentration of Mn 2÷ increases from 25 to 40 mM.

T A B L E IV E n z y m i c a n d c h e m i c a l d e c o m p o s i t i o n s o f 14C.labele d nucleoside m o n o - and d i p h o s p h a t e sugars in t h e p r e s e n c e o f 20 mM MnSO4 and rat s e r u m a d j u s t e d t o p H 7.3 a f t e r 1 h i n c u b a t i o n at 3 7 ° C . T h e results are given as t h e p e r c e n t a g e s o f the t o t a l radioactix~.ty.

Nucleoside m o n o - or diphosphate sugars

Residual 14C-labeled nucleoside monoor diphosphate sugars

[14C]Sugar-l-p

UDP-D-[I 4 C ] G a l UDP.D.[I 4 C] Glc UDP.D.[I 4 C] GIcUA UDP-D-[I 4 C] GIcNAc G D P . D . [ 14 C ] M a n GDP-L-[ 1 4 C ] F u c CMP.D.[I 4 C ] A c N e u

2 4 0 4 3 1 7

31 26 3 75 29 5 0

* The 1 , 2 - p h o s p h o c y c l i c derivative o f t h e c o r r e s p o n d i n g m o n o s a c c h a r i d e .

[14C]Sugar1,2-P *

sugar

Free [14C]

4 2 4 0 0 0 0

63 68 93 21 68 94 93

210

A

lO(3

75 o ~5

e

50

g E ~ 25

0

A

10

&

A

A

X

X- ~ - - - - X ~

20 30 Mn 2. concentration (mM)

40

10C

>~ 7~ .>_ o o

"5 ¢1

B E 25 n

0

10 20 30 Mn 2÷ concentration (mM)

40

Fig. 2. D e t e r m i n a t i o n o f t h e g a l a c t o s y l t r a n s f e r a s e a c t i v i t y o f h u m a n s e r u m (A) a n d r a t s e r u m (B) adj u s t e d t o p H 7.3 a n d d e t e r m i n a t i o n o f t h e e n z y m i c a n d c h e m i c a l d e g r a d a t i o n s of t h e UDP-D-[ 1 4 C ] g a l a c . tose a f t e r 1 h i n c u b a t i o n at 3 7 ° C in t h e p r e s e n c e of i n c r e a s i n g c o n c e n t r a t i o n s o f Mn 2+. o • UDPGal; A ~, G a I - I - P ; • A Gal; X - - X , Gal-l,2-P; • i Ovomueoid[14C]Gal.

If t h e p e r c e n t a g e s o f t h e b r e a k d o w n p r o d u c t s o f U D P - D - [ 1 4 C ] G a l o b t a i n e d w i t h and w i t h o u t t h e substrate for t h e g a l a c t o s y l t r a n s f e r a s e are c o m p a r e d (Fig. 2 and Table III), it is n o t i c e a b l e t h a t for a c o n c e n t r a t i o n o f 40 m M in Mn :÷ t h e a m o u n t o f [ 14C]Gal-1-P is relatively similar in b o t h cases, b u t t h a t t h e a m o u n t

211 of residual UDP-D-[14C]Gal decreased from 42% in the experiment without ovomucoid to 0% in the presence of this acceptor. The formation of [l*C]Gal1,2-P is lower when an acceptor of galactose is added to the mixture. All these results indicate that the activity of nucleotide pyrophosphatase is not greatly modified by the addition of a substrate for the galactosyltransferase activity and that the transfer of [~*C]galactose to ovomucoid is effected with the residual UDP-D-[ ~4C]Gal as substrate. In the presence of rat serum, the UDP-D-[14C]Gal is decomposed to a great extent into [ 14C] Gal-l-P and after that into free [ 14C]Gal; the transfer reaction of galactose onto ovomucoid is very low and is not considerably enhanced by addition of increasing concentration of Mn ~÷. From these results it can be concluded that the determination of glycosyl transferase activity cannot by dissociated from the determination of nucleotide pyrophosphatase activity and that the nucleotide pyrophosphatase activity is much higher than that of the glycosyl transferase activity.

Competition between fl-D-galactosidase and galactosyltmnsferase activities In order to determine whether the [14C]Gal transferred onto the glycopeptide of ovomucoid can be hydrolyzed b y the galactosidases present in human and rat serum, hydrolysis was performed on the labeled glycopeptide isolated after the transfer of [ 14C] Gal. After 2 h incubation at 37°C, galactosidases from human and rat serum were not capable of liberating free [I*C]Gal from the labeled substrate. Similar results were obtained by Kim et al. [13] who have studied the endogenous glycosidase activities present in rat small intestinal mucosal homogenates during the determination of glycosyltransferase activities and have coneluded that the hydrolysis of the glycosidic bond of the terminal radioactive sugar is minimal. These results indicate that competition between galactosyltransferase and galactosidase, if it exists, is very low.

Effects of UMP and PPi on glycosyltransferase and nucleotide pyrophosphatase activities Monophosphonucleoside or diphosphonucleosides have been used to inhibit the nucleotide pyrophosphatase activity [14--18]. In order to determine the action o f UMP which is a final product of the galactosyltransferase activity on serum galactosyltransferase and nucleotide pyrophosphatase activities, increasing concentrations o f UMP from 0 to 10 mM were added to the human and rat serum containing 20 mM Mn 2÷ and 500 pg ovomucoid. The percentages of galactose transfer and of the different breakdown products were determined and the results are reported in Fig. 3. Addition of increasing concentrations of UMP to human serum inhibits the activity of the nucleotide pyrophosphatase and also the galactosyltransferase activity. In these conditions, the formation of Gal-l,2-P is considerably increased. Maximum transfer of galactose onto ovomucoid was obtained for a concentration of UMP of 0.1 mM. Addition of increasing concentrations of 'UMP to rat serum as in human serum inhibits the galactosyltransferase and the nucleotide pyrOphosphatase activities, and enhances the formation of Gal-I,2-P. However, for a concentra-

212

A

10C

7E

._>

sc !

"6 2E

9 u fl_ AA

&

2

a

A

g

A

g

~o

UMP concentration (raM) i

100

7E

____--@ .>_

f

8 5c

9 "5

_ _

x

x

2E

g a_ 0

2

4 6 8 UMP concentration (mM)

10

F i g . 3. D e t e r m i n a t i o n o f t h e g a l a c t o s y l t r a n s f e r a s e a c t i v i t y of h u m a n s e r u m ( A ) a n d rat S e r u m (B) adj u s t e d t o p H 7.3 a n d d e t e r m i n a t i o n o f t h e e n z y m i c a n d c h e m i c a l d e g r a d a t i o n s o f t h e UDP-D-[ 14 C] galactose a f t e r 1 h i n c u b a t i o n a t 37~C in t h e p r e s e n c e o f 2 0 m M Mn 2+ a n d i n c r e a s i n g c o n c e n t r a t i o n s o f UMP. • •, UDPGal; ~ '~, G a I - I - P ; • A Gall X ×, G a l - l , 2 - P ; • a Ovomucoid[ 14C]Gal.

t i o n o f 0.3 m M U M P t h e transfer o f [14C]galactose o n t o o v o m u c o i d w a s for rat s e r u m increased 3 times. T h e s e results i n d i c a t e t h a t t h e c o n c e n t r a t i o n o f t h e i n h i b i t o r s m u s t be d e t e r m i n e d e x a c t l y in order t o use a c o n c e n t r a t i o n w h i c h d o e s n o t a f f e c t t h e g l y c o s y l t r a n s f e r a s e activity. A d d i t i o n o f UMP did n o t c o n -

213 TABLE V E f f e c t o f i n c r e a s i n g c o n c e n t r a t i o n s of U D P - D - G a l o n t h e i n c o r p o r a t i o n of galactose o n t o o v o m u c o i d in t h e p r e s e n c e of h u m a n a n d r a t s e r u m . T h e i n c u b a t i o n m i x t u r e c o n t a i n s : 5 0 0 Dg o f o v o m u c o i d ; 2 0 m M M n S O 4 ; 0.1 n m o l UDP-D-[ 1 4 C ] G a l ; 3 0 #l o f h u m a n o r r a t s e r u m , i n c r e a s i n g c o n c e n t r a t i o n s of UDP-DGal in a t o t a l v o l u m e of 1 0 0 ~1. C o n c e n t r a t i o n of UDP-D-Gal a d d e d in pM

0 10 100 1000

T r a n s f e r o f galactose in t h e p r e s e n c e of Human serum

Rat serum

A*

B**

A*

B**

37.4 37.2 18.3 1.9

15.6 350 1654 1711

6.6 7.9 S.1 1.9

2.8 74 732 1710

* T h e results are e x p r e s s e d as t h e p e r c e n t a g e s o f t h e t o t a l r a d i o a c t i v i t y p r e s e n t in t h e a s s a y . * * T h e results are e x p r e s s e d as t h e t o t a l galactose ( p m o l ) i n c o r p o r a t e d o n t o o v o m u c o i d .

siderably increase the glycosyltransferase activity as the level of nucleoside diphosphate sugar chemically decomposed into 1,2-cyclic phosphate derivative of the [ 14C]monosaccharide increases. Inorganic pyrophosphate was also tested as inhibitor of the nucleotide pyrophosphatase. Addition of increasing concentrations of PPi (0--10 mM) to human and rat serum did not cause any modification of the nucleotide pyrophosphatase or the galactosyltransferase activities. The absence of effects of the pyrophosphate m a y be explained if it is considered that the pyrophosphate may be destroyed b y the inorganic pyrophosphatase (EC 3.6.1.1) present in human [19] and in the rat serum. Another explanation is that the serum galactosyltransferase and nucleotide pyrophosphatase of human and rat serum are not inhibited b y pyrophosphate.

Effects of increasing concentrations of UDP-D-Gal on glycosyltransferase and nucleotide pyrophosphatase activities Similar incorporation of galactose onto ovomucoid may be obtained as is shown in Table V by the galactosyltransferases of human and rat serum when 1 mM UDP-D-Gal is added to the incubation. Under these conditions the amount of UDP-D-Gal is sufficient to obtain a saturation of the nucleotide pyrophosphatase and the a m o u n t of UDP-D-Gal which is left by the nucleotide pyrophosphatase may be used as substrate for the galactosyltransferases of human and rat serum. Discussion

The comparative determination of the galactosyltransferase activities in human and rat serum which possess different nucleotide pyrophosphatase activities shows clearly that the determination of galactosyltransferase is dependent on the nucleotide pyrophosphatase activity of the medium. In b o d y fluids and in different membranes of cells it has been shown by various authors [20--26] that the presence of nucleotide pyrophosphatase activity is associated with the presence of glycosyltransferase activities.

214 The presence of nucleotide pyrophosphatase and glycosyltransferase as well as the phosphatase activity in the b o d y fluids and on the membranes may play an important role in the regulation of the biosynthesis of the glycan moiety of the glycoproteins. Elimination of the nucleotide pyrophosphate activity may be achieved by various methods. Purification of the glycosyltransferase activities or utilization of transformed cells in which the nucleotide pyrophosphatase activity is blocked [27] may be considered as the best methods for precise determination of the glycosyltransferase activities. Various nucleoside mono- or diphosphates have been used in the literature to inhibit the activity of the nucleotide pyrophosphatase. The present study demonstrates that inhibition of the nucleotide pyrophosphatase activity may be also accompanied by inhibition of the glycosyltransferase activities, and in the case of UDPgalactose, UDPglucose and UDP-N-acetylglucosamine, with a significant increase of the 1,2-cyclic phosphate derivative of the corresponding monosaccharide. The appearance of a c o m p o n e n t called 'spot X' during the hydrolysis of UDP[3H]Gal by BHK cells or during the hydrolysis of UDPgalactose by the pyrophosphatase of norreal rat endometrium was noticed respectively by Deppert et al. [28] and by Jato-Rodrigues et al. [29]. We can confirm from this study and from the results previously obtained by Paladini and Leloir [12] and by Nunez and Barker [1] that the unknown c o m p o u n d corresponds to the 1,2-cyclic phosphate derivative of galactose. The cyclic derivative of monosaccharide exists in the commercial labeled nucleoside diphosphate galactose, glucose and glucuronic acid, its amount is increased in the presence of increasing concentrations of Mn 2+ at neutral pH. In rat serum where the nucleotide pyrophosphatase activity is very high, the formation of the 1,2-cyclic phosphate derivative is inhibited. As a considerable increase of this derivative was noticed after inhibition of the nucleotide pyrophosphatase activity, it is difficult in the cases of determination of the galactosyl, glucosyl and glucuronyltransferases to obtain an exact value for these glycosyltransferase activities. During the study of effectors of glycosyltransferase activities it must be kept in mind that these effectors also may modify the activity of the nucleotide pyrophosphatase. If the effects on both enzymes are not determined, erroneous interpretations may be made. For example, Debeire et al. [30] using the method of determination of the breakdown products of nucleoside diphosphate sugars that we described in this paper, have demonstrated that the cyclic nucleotides did not affect the glycosyltransferase activities by direct stimulation of the glycosyltransferases but by inhibiting the nucleotide pyrophosphatases. In another case, Geren and Ebner [31] have demonstrated that the stimulatory effect of folic acid and 5'-AMP on the galactosyltransferase activity is obtained by substrate protection due to the inhibition of the nucleotide pyrophosphatase activity. Misleading interpretation of data on the inhibition or stimulation of the glycosyltransferase activities present in normal and pathological b o d y fluids or on cell membranes may be made if, in the pathological case, there is protection or disappearance of the substrate due to the modification of the nucleotide pyrophosphatase activity. Since in each of human and rat serum the tested nucleoside diphosphate

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