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Concanavalin a induced inhibition of 5′-nucleotidase from guinea pig skeletal muscle and bull seminal plasma: A comparative study
Int. J. Biochem. Vol. 18, No. 8, pp. 683-689, 1986 Printed in Great Britain
0020-711X/86 $3.00+ 0.00 Pergamon Journals Ltd
CONCANAVALIN A INDUCED INHIBITION OF 5'-NUCLEOTIDASE FROM GUINEA PIG SKELETAL MUSCLE A N D BULL SEMINAL PLASMA: A COMPARATIVE STUDY CARLO FINIl, MARCELLA CAMICI2, ALBA MINELLIl, ARDESIO FLORIDI1 and PIER LUIGI IPATA2 qstituto Interfacoltfi di Chimica Biologica, Universit/t di Perugia, Via del Giochetto, 06100 Perugia, Italy :Istituto di Biochimica, Biofisica e Genefica, Universit~t di Pisa, Via S. Maria, 55, 56100 Pisa, Italy (Received 16 December 1985)
Abstract--1. Both purified and membrane-bound 5'-nucleotidases (EC 3.1.3.5) from guinea pig skeletal muscle and bull seminal plasma are inhibited by Concanavalin A (Con A). 2. 5'-Nucleotidase purified from skeletal muscle is inhibited by Con A by an apparent uncompetitive process (KI = 160 nM), while the lectin inhibits the particulate enzyme by an apparent non-competitive process (Ki = K~= 50 nM). 3. 5'-Nucleotidase purified from bull seminal plasma is inhibited by Con A by an apparent non-competitive process (K'i= K~= 270 nM), while the membrane-bound enzyme is subjected to a mixed type inhibition by the lectin (K~ > K,; 30 and 14 nM, respectively). 4. The enzyme purified from skeletal muscle exhibits a significant cooperativity in the interaction with Con A. 5. The inhibition of bull seminal plasma particulate 5'-nucleotidase brought about by Con A is not completely reversed by addition of ~-methyl-D-mannoside.
INTRODUCTION Con A, a lectin from Canavalia ensiformis, binds specifically to carbohydrates having the D-arabinoside configuration (Agrawal and Goldstein, 1967; So and Goldstein, 1968). This property allows the binding of Con A to the glycosidic moieties of glycoproteins; this interaction results in an inhibition of 5'-nucleotidase activity in intact cells (Stefanovic et al., 1975; Dornand et al., 1978; Carraway et al., 1979), in membrane preparations (Zachowski and Paraf, 1974; Riordan and Slavik, 1974, 1977; Dornand et al., 1977; Harb et al., 1983), as well as in the purified enzyme (Slavik et al., 1977; Harb et al., 1983). However, a stimulation of 5'-nucleotidase activity of ascites cells and rat liver plasma membranes at low Con A concentrations, has been also reported (Zachowski and Paraf, 1974; Riordan and Slavik, 1974). Moreover, Novogrodsky (1972) and Pommier et al. (1975) showed an activating effect of Con A on ATPase activity of plasma membranes of lymphocytes. It has been shown that the purified 5'-nucleotidase from bovine liver is less sensitive to Con A inhibition than the isolated membranes enzyme (Harb et al., 1983); however, it has been also observed that the enzyme purified from Wistar rat liver plasma membranes was even more sensitive to Con A inhibition than the membrane-bound enzyme (Slavik et al., 1977). Furthermore, the type of Con A inhibition, as well as the cooperativity of the Con A-enzyme interaction depend on either the enzyme source or the degree of enzyme purity, i.e. whether the activity is
measured using a purified enzyme protein or a membrane-bound 5'-nucleotidase preparation (Carraway et al., 1976, 1979; Dornand et al., 1978). For a better understanding of the inhibitory effect of Con A on 5'-nucleotidase, we have undertaken a comparative study on two mammalian 5'-nucleotidases. We have used both the purified and membrane-bound enzymes from guinea pig skeletal muscle and bull seminal plasma. The purification procedures and some kinetic and molecular properties of these enzymes have been previously reported (Camici et al., 1985; Fini et al. 1983). In the present study, we show that Con A inhibits both bull semen and muscle 5'-nucleotidase. However, the enzyme purified from skeletal muscle is inhibited by Con A in a process that is apparently uncompetitive and the inhibition exhibits positive cooperativity, while Con A is a non-competitive inhibitor of the particulate enzyme with no cooperativity. On the other hand, the enzyme purified from bull seminal plasma shows an apparently noncompetitive inhibition that does not exhibit any significant cooperativity; finally, the membranebound seminal enzyme is subjected to a mixed type inhibition with a Hill coefficient close to 1. The purified enzymes from both sources are less sensitive to Con A inhibition than the membrane-bound enzymes. The inhibition exerted by Con A can be completely reversed by addition of a-methyl-Dmannoside to particulate, purified skeletal muscle and purified bull seminal plasma 5'-nucleotidases, while the particulate bull seminal plasma enzyme is only partially reactivated.
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CARLO FIyl et al.
684 MATERIALS AND METHODS
Materials Guinea pig skeletal muscle 5'-nucleotidase was purified according to Camici et al. (1985); bull seminal plasma 5'-nucleotidase was purified as described by Fini et al. (1983). Con A, ~-methyl-o-mannoside, calf intestinal mucosa adenosine deaminase (EC 3.5.4.4) and Y-AMP were purchased from Sigma. All other chemicals were of reagent gradc. Preparation ~)[~membrane-bound 5"-nucleotidases (a) Guinea pig skeletal muscle 5'-nucleotidase: 10 mg of freshly excised tissue were homogenized in 3.5 vol of 50 mM Tris-HCl, pH 7.4. The homogenate was centrifuged at 600 g at 4°C for 30 min. The pellet was discarded. The supernatant was then centrifuged at 15,000g at 4~'C for 20 min and the pellet discarded. Finally, the supernatant was centrifuged at 105,000 g for 1 hr and the pellet, referred to as "microsome fraction" (Kamp and Wirtz, 1974), collected and stored at - 2 f f C . Before use, aliquots of the microsome fraction, containing 0.52 mg of protein, were suspended in 1 ml of 50 mM Tris-HC1, pH 7.4. The 5'-nucleotidase activity associated with this suspension represents the membrane-bound enzyme. (b) Bull semen particulate 5'-nucleotidase: 50 ml of bull semen were centrifuged at 2000g at 4°C for 30 min. The supernatant was withdrawn and centrifuged at 15,000g at 4'C for 30min and the pellet was discarded; the supernatant, referred to as "bull seminal plasma", was centrifuged at 105.000g at 4c'C for 1 hr. The supernatant contained the soluble form of 5'-nucleotidase of bull seminal plasma and was not used in this study. The pellet was suspended in 12ml of 50mM Tris-HCl, pH 7.4 and then centrifuged at 105,000g at 4°C for 90 min. The supernatant was discarded and the pellet was resuspended in 5 ml of 50 mM Tris HCI, pH 7.4 and stored at 0-T'C. This suspension represents the membrane-bound seminal plasma 5'-nucleotidase. Assay The 5'-nucleotidase assay was carried out in 50mM Tris-HCl, pH 7.4, at 37°C using 5'-AMP as substrate in the presence of excess adenosine deaminase, by measuring the decrease in absorbance at 265 nm which accompanies the conversion of adenosine to inosine, according to Ipata (1967). The reaction mixture contained, in a final volume of 1 ml, different amounts of Con A and 5'-nucleotidase preparation, as indicated in the legends to figures. One/~g of adenosine deaminase was also added. We did not observe a time-dependent variation in inhibition of enzyme activity by Con A when the 5'-nucleotidase preparations were preincubated for 5 10 and 20 min in the presence of the lectin. Thus, a preincubation of 10 rain of the enzyme preparations with Con A was carried out before addition of varying amount of 5'-AMP (see legends to figures). One unit of enzyme activity is the amount of enzyme catalyzing the conversion of 1 #mol of substrate per rain in the assay mixture.
Other methods Protein was determined by the Coomassie Blue binding assay (Bradford. 1976). RESULTS
Effect o f Con A on 5'-nucleotidase The effect o f C o n A was studied on b o t h the particulate a n d the purified 5'-nucleotidase from guinea pig skeletal muscle a n d bull seminal plasma. Figure 1 shows t h a t the p a t t e r n o f inhibition o f the purified enzymes is r e m a r k a b l y different: the C o n A c o n c e n t r a t i o n to o b t a i n 50% inhibition is 400 n M for the skeletal muscle enzyme (Panel A) a n d 100 n M for the bull seminal plasma enzyme (Panel B). O n the o t h e r h a n d , the m e m b r a n e - b o u n d enzymes show almost the same inhibitory p a t t e r n and are more sensitive to C o n A inhibition (50% o f activity is inhibited at approx. 3 0 n M C o n A). The enzyme purified from skeletal muscle exhibits a significant positive cooperative effect in the interaction with C o n A (Fig. 1A; Hill coefficient 2), while the m e m b r a n e b o u n d enzyme does not show any significant cooperativity (Fig. IA; Hill coefficient 0.96). The Hill plot for the bull seminal plasma 5'-nucleotidase inhibition (Fig. I B) exhibits no cooperativity b o t h for the m e m b r a n e - b o u n d a n d the purified enzyme (Hill coefficients 0.95 a n d 1.09, respectively).
Kinetic parameters o f inhibition o f 5'-nucleotidase by Con A Previous studies on the inhibition of 5'-nucleotidase from different sources, by either C o n A or nucleoside di- a n d triphosphates have s h o w n that these c o m p o u n d s exert different inhibitory patterns; thus, b o t h positive a n d negative cooperativity, competitive, n o n - c o m p e t i t i v e a n d mixed types of inhibition have been reported ( C a r r a w a y et al., 1976, 1979; D o r n a n d et al., 1978; R i o r d a n a n d Slavik, 1974; D o r n a n d et al., 1977; H a r b et al., 1983; M u r r a y a n d Friedrichs, 1969; E d w a r d s a n d M c G u i r e , 1970; T a n a k a et al., 1973; Burger a n d Lowenstein, 1975; Stefanovic et al., 1976; N a i t o a n d Lowenstein, 1985). In the present study, we have c o m p a r e d the changes in kinetic p a r a m e t e r s b r o u g h t a b o u t by C o n A inhibition o f two m a m m a l i a n 5'-nucleotidases, varying either the substrate or the inhibitor concentrations. Figures 2 a n d 3 show the plot of s/v vs i a n d sly vs s for skeletal muscle a n d bull semen 5'-nucleotidase, respectively. Ki values were o b t a i n e d graphically: K i represents the dissociation c o n s t a n t for the enzymei n h i b i t o r complex; K'i is the dissociation c o n s t a n t for the e n z y m e - i n h i b i t o r - s u b s t r a t e complex. As shown
Table 1. Inhibition of 5'-nucleotidase activity by Con A Enzyme source
Type of inhibition (/~M) (nM) (nM) Skeletal muscle (a) Purified Uncompetitive 19 _+ 1.1 160 + 8.2 (b) Membrane-bound Non-competitive 25 _+ 1.9 50 _+3.0 50 _+4.1 Bull seminal plasma (a) Purified Non-competitive I 1 + 1.2 270 + 6.9 270 ± 6.9 (b) Membrane-bound Mixed 11 _+ 1.8 14 __+2.5 30 + 2.8 K, and KI values were obtained according to Cornish-Bowden (1974). Experimental conditions are given in the text.
Hill coefficient 2.00_+0.12 0.96 + 0.09 0.95 + 0.05 1.09 + 0.07
Con A inhibition of 5'-nucleotidase (A)
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Fig. I. Effect of Con A on 5'-nucleotidase. Panel (A) shows the inhibitory pattern for guinea pig skeletal muscle 5'-nucleotidase. Left: 35 ng of purified enzyme ( 0 ) or 15.6/~g of microsome fraction (©). The reaction was started by addition of Y-AMP to a final concentration of 62.5/~ M. Right:Hill plot for Con A inactivation of purified ( 0 ) and membrane-bound (©) 5'-nucleotidase. Panel (B) refers to bull seminal plasma 5'-nucleotidase. Left: 20 ng of purified enzyme ( 0 ) or 1 #g of the pellet 105,000g (O). The reaction was started by adding Y-AMP to a final concentration of 50/~M. Right: Hill plot for Con A inactivation of purified ( 0 ) and membrane-bound 5'-nucleotidase (O).
in Fig. 2A, in the plot of s/v vs i, the lines intersect above the i axis giving a KI value of 160 nM; moreover, in the plot of sly vs s, the lines intercept on the vertical axis; these two graphs, together with a Dixon's plot (not shown) which gives rise to parallel lines, suggest that the purified skeletal muscle 5'-nucleotidase is inhibited by Con A in an apparently uncompetitive process. Analysis of the data shown in Fig. 2B, together with a Dixon's plot, here not shown, indicates that the membrane-bound skeletal muscle 5'-nucleotidase is inhibited by an apparent non-competitive process with a KI = Kj value of 50 nM. The same type of analysis was performed on the bull seminal enzyme. The results obtained, shown in Fig. 3, together with the Dixon's plots (not shown), indicate that the purified 5'-nucleotidase (Panel A) is inhibited by Con A by an apparent non-competitive process, with a K I = Ki of 270nM; while the in-
hibition observed for the membrane-bound enzyme (Panel B) is apparently of the mixed type with a K, > K~of 30 nM and 14 nM, respectively. The results are summarized in Table 1.
Reversal of Con A inhibition of 5"-nucleotidase by ct-methyl-o-mannoside The K~ values obtained indicate that Con A is a powerful inhibitor of 5'-nucleotidase from both guinea pig skeletal muscle and bull seminal plasma. The addition of ct-methyl-D-mannoside, which binds specifically to Con A, completely reversed the inhibition induced by the lectin of both purified and membrane-bound skeletal muscle 5'-nucleotidase (Fig. 4A), as well as of purified bull seminal plasma 5'-nucleotidase (Fig. 4B). However, under the same conditions, the sugar failed in fully reactivating the bull semen particulate enzyme, and no more than
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Fig. 2. Kinetics of Con A inhibition of 5'-nucleotidase from guinea pig skeletal muscle. Panel (A). Purified 5'-nucleotidase. Left: 35 ng of enzyme were incubated in the presence of the indicated amount o f Con A; the reaction was started by adding 5'-AMP to a final concentration of 40 # M (Q), 80/aM (ll), and 120 # M (A). Right: 35 ng of enzyme were incubated in the absence ( O ) or in the presence o f 85 nM (11) or 170 nM ( A ) C o n A. Panel (B). Membrane-bound enzyme. Lqft: the microsome fraction (15.6/ag o f protein) was incubated in the presence of Con A; the reaction was started by adding 5 ' - A M P to a final concentration of 3 7 . 5 # M (O), 62.5/aM (m), and 125/aM (A). Right: the microsome fraction was incubated in the absence ( O ) or in the presence of Con A to a final concentration of 17 nM (11) or 68 n M (A).
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Fig. 3. Kinetics of Con A inactivation of 5'-nucleotidase from bull seminal plasma. Pane/ (A). Purified 5'-nucleotidase. Left: 20 ng of enzyme were incubated in the presence of the indicated a m o u n t of Con A; the reaction was started by adding Y - A M P to a final concentration of 12.5/aM (O), 50/aM (m), and 100/aM (A). Right: 20 ng of enzyme were incubated in the absence (O) or in the presence of 136.5 nM (11) or 455 nM ( A ) Con A. Panel (B) m e m b r a n e - b o u n d enzyme. Le/?: 1 /ag of protein was incubated in the presence of Con A: the reaction was started by adding 5'-AMP to a final concentration of 12.5 t~M (OI. 50/aM (11), and 1001tM (A). Right: 1 /ag of protein was incubated in the absence ( O ) or in the presence of 13.65 nM ( D ) or 45.5 nM ( A ) Con A.
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Con A inhibition of 5'-nucleotidase
687
1975; Zachowski et al., 1975; Akedo et al., 1976; Young et al., 1976). Being ecto 5'-nucleotidase a glycoprotein acting as Con A receptor (Carraway et 80 al., 1979), the study of the lectin-enzyme interaction might be of great help for understanding cell surface biochemical phenomena as well as relationships be60 tween transmembrane cell surface proteins and the cytoskeleton system. 40 >. In the present study, we have compared the inhibitory effect of Con A on 5'-nucleotidase from two i i i i i ~ zo mammalian sources: guinea pig skeletal muscle and "6 1oo bull seminal plasma. Experiments were carried out on both the purified and the particulate enzymes. The data obtained show that the purified enzymes are less ~ 80 g: sensitive to Con A inhibition than the membranebound enzymes: the concentration of inhibitor re60 quired for half maximal inactivation was 3-10 times higher for the purified 5'-nucleotidase than for the 40 particulate enzymes. Moreover, the comparison between the purified enzymes shows that the bull semi20 nal plasma 5'-nucleotidase is more sensitive to Con A inhibition than the guinea pig skeletal muscle enI i i zyme; on the other hand, almost the same inhibitory 2 4 6 ~ ~b pattern was obtained for the membrane-bound en[ = - M e t h y l - D - m a n n o s i d e ] (raM) zymes. Analysis of the types of inhibition of the different Fig. 4. Reversal of Con A inhibition of 5'-nucleotidase by enzyme preparations brought about by Con A allows ~-methyl-o-mannoside. Panel (A). Skeletal muscle 5'-nucleotidase. 35 ng of purified enzyme (O) were incu- some speculations. Thus, it appears that the interbated 10 min at 37°C in the presence of 680 nM Con A. action of Con A with purified 5'-nucleotidase of Then increasing amounts of ~-methyl-o-mannoside were skeletal muscle (uncompetitive type inhibition) affects added and the activity was assayed after addition of Y-AMP the breakdown of the enzyme-substrate complex, to a final concentration of 62.5/~M. The microsome fraction with proportional changes in Km and V (a 1.5 time (15.6 #g of protein) (O) was incubated l0 min at 37°C in the decrease in Km and V in the presence of 170 nM Con presence of 170 nM Con A, before addition of increasing A); on the other hand, the interaction of purified bull amounts of ~t-methyl-o-mannoside, then the activity was seminal plasma 5'-nucleotidase with Con A (nonassayed as described before. Panel (B) bull seminal plasma competitive type inhibition) results in a reduction of 5'-nucleotidase. 20 ng of the purified enzyme (11) were incubated 10 min at 37°C in the presence of 455 nM Con A. V (from 2.080mU, in the absence of Con A, to Then, increasing amounts of ~t-methyl-o-mannoside were 0.943 mU, in the presence of 455 nM Con A) and no added and the activity was assayed after addition of Y-AMP effect on Km is observed. Differences were also obto a final concentration of 50/~M. The particulate enzyme served for the membrane-bound enzymes. Thus, the (1/~g of protein) (I-q) was incubated 10 nm at 37°C in the interaction Con A-particulate skeletal muscle presence of 45.5 nM Con A, before addition of increasing 5'-nucleotidase (non-competitive type inhibition) reamounts of ~-methyl-D-mannoside, then the activity was duces V (from 2.041 to 1.504 mU in the absence and assayed as described above. presence of 17nM Con A, respectively), with no effect on Kin; while the seminal particulate enzyme is 60% of the initial activity was restored by an subjected to a mixed type inhibition in which the Km ~t-methyl-D-mannoside concentration of 10.4nM, a is shifted from ! 1 #M (in the absence of the lectin) to 18 # M (in the presence of 45.5 nM Con A) and V is 228 x 103 excess over Con A concentration. shifted from 2.080 mU to i.0 mU in the presence of 45.5 nM Con A. It appears then, that Con A interDISCUSSION action does not affect the combination of 5'-AMP with both the membrane-bound skeletal muscle and Con A from Canavalia ensiformis is a tetrameric metalloprotein which agglutinates erythrocytes, lym- the purified bull seminal plasma enzymes, while the inhibitory lectin can affect both the substrate combiphocytes and transforms cells. This lectin binds nation and the breakdown of the enzyme-substrate specifically to carbohydrates such as Orb-glucose and complex in the case of the membrane-bound bull ct-o-mannose and their derivatives, as well as to seminal plasma enzyme. The differences in the mechglycoproteins containing such glycosidic moieties. anism of inhibition, observed for the two purified Since glycoproteins both in intact cells and plasma 5'-nucleotidases, may be related to differences in the membranes are exposed on the external surface, they may be regarded as Con A receptors. The interaction topology of the active centre, as well as to the between Con A and these receptors has been widely particular localization of the glycosidic moiety (which allows the binding of Con A) with respect to the studied to obtain information on biological processes substrate binding site. We have already observed occurring at the cell surface (Carraway et al., 1975, some differences in substrate specificity between the 1979; Riordan and Slavik, 1974; Novogrodsky, 1972; two enzymes (Camici et al., 1985; Fini et al., 1983) Jarret and Smith, 1974; Pommier et al., 1975; Podoland more recently (experiment in progress) a minor sky and Weiser, 1975; Gahmberg and Hakomori, 100
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sensitivity of the skeletal muscle enzyme to inhibition by antibodies against bull seminal plasma 5'-nucleotidase, compared to the purified bull seminal plasma enzyme itself. It can be argued that the differences in Con A-enzyme interaction might originate from dissimilarities in the polypeptide chains of the two 5'-nucleotidases; further information will be obtained from amino acid analysis that is underway in our laboratory. It has been longly debated (Carraway et al., 1976, 1979; Snow et al., 1980) whether the change in cooperativity in the Con A inhibition observed between the purified and particulate 5'-nucleotidase was due to an involvement of cytoskeleton elements, such as actin, to which 5'-nucleotidase might be anchored (Dieckhoff and Mannherz, 1985; Rohr and Mannherz, 1979; Mesher et al., 1981). Recently, Craik et al. (1985) have shown that cooperativity changes probably result from modification of the nucleotidase or a small, closely associated membrane moiety, rather than from involvement of actinmicrofilaments. In our experimental conditions, we have found that the purified skeletal muscle 5'-nucleotidase shows a positive cooperativity in the interaction with Con A, while the particulate enzymelectin interaction does not show any cooperativity. Owing to the methods used by us to prepare the "microsome fraction", actin filaments are not left intact; therefore, our results suggest that some other factor, other than the detachment of 5'-nucleotidase from the cell cytoskeleton, must be involved to explain the observed increased cooperativity in the interaction of the purified enzyme with Con A. Membrane-bound skeletal muscle and particulate bull semen 5'-nucleotidases are inhibited by Con A by distinct inhibitory mechanisms. This finding, together with the lack of complete reactivation of the bull semen enzyme, as compared to the skeletal muscle 5'-nucleotidase, suggest that interactions involving components of the membrane other than the enzyme itself might play an important role. SUMMARY
Concanavalin A inactivation of 5'-nucleotidase (EC 3.1.3.5) has been studied using both the purified and membrane-bound enzyme, obtained from two different mammalian sources: guinea pig skeletal muscle and bull seminal plasma. The purified enzymes from both sources are less sensitive to inhibition by the lectin than the membrane-bound enzymes, the concentration of inhibitor required for half maximal inactivation being 3-10 times higher for the purified 5'-nucleotidases than for the particulate enzymes. The enzyme purified from skeletal muscle is inhibited by the lectin by an apparent uncompetitive process, while Con A inhibits the particulate skeletal muscle 5'-nucleotidase by an apparent noncompetitive process. The Hill coefficient for the inactivation of purified skeletal muscle 5'-nucleotidase is shifted to 2, indicating significant cooperativity in the lectin-enzyme interaction. On the other hand, 5'-nucleotidase purified from bull seminal plasma is inhibited by Con A by an apparent non-competitive process, while the membrane-bound enzyme is subjected to a mixed type inhibition by the lectin. The
significance of such differences in the inhibition by Con A of the 5'-nucleotidase preparations studied is discussed. REFERENCES
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Con A inhibition of 5'-nucleotidase Jarret L. and Smith R. M. (1974) The stimulation of adipocyte plasma membrane magnesium ion-stimulated triphosphatase by insulin and Concanavalin A. J. biol. Chem. 249, 5195-5199. Kamp H. H. and Wirtz K. W. (1974) Phosphatidylcboline exchange protein from beef liver. In Methods in Enzymology (Edited by Fleisher S. and Packer L.), Vol. XXXII, pp. 140-142. Academic Press, New York. Mesher M. F., Jose M. J. L. and Balk S. P. (1981) Actin-containing matrix associated with the plasma membrane of murine tumour and lymphoid cells. Nature, Lond. 289, 139-144. Murray A. W. and Friedrichs B. (1969) Inhibition of 5'-nucleotidase from Ehrlich ascites-tumor cells by nucleoside triphosphates. Biochem. J. 111, 83-89. Naito Y. and Lowenstein J. M. (1985) 5'-Nucleotidase from rat heart membranes. Inhibition by adenine nucleotides and related compounds. Biochem. J. 226, 645~51. Novogrodsky A. (1972) Concanavalin A stimulation of rat lymphocyte ATPase. Biochim. biophys. Acta 266, 343-349. Podolsky D. K. and Weiser M. M. (1975) Role of cell membrane galactosyltransferase in Concanavalin A agglutination of erythrocytes. Biochem. J. 146, 213-221. Pommier G., Pipert G., Azoulay E. and Depieds R. (1975) Effect of Concanavalin A on membrane-bound enzymes from mouse lymphocytes. Biochim. biophys. Acta 389, 483-494. Riordan J. R. and Slavik M. (1974) Interactions of lectin with membrane glycoproteins. Effects of Concanavalin A on 5'-nucleotidase. Biochim. biophys. Acta 373, 356-360. Rohr G. and Mannherz H. G. (1979) The activation of actin: Dnase I complex with rat liver plasma membranes. The possible role of 5'-nucleotidase. FEBS Lett. 99, 351-356. Slavik M., Kartner N. and Riordan J. C. (1977) Lectininduced inhibition of plasma membrane 5'-nucleotidase:
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sensitivity of purified enzyme. Biochem. biophys. Res. Commun. 75, 342-349. Snow L. D., Doss R. C. and Carraway K. L. (1980) Cooperativity of the concanavalin A inhibition of bovine milk fat globule membrane 5'-nucleotidase. Response to extraction of nucleotidase and putative cycloplasmic surface coat components. Biochim. biophys. Acta 611, 333-341. So L. L. and Goldstein I. J. (1968) Protein-carbohydrate interaction. XX. On the number of combining sites on concanavalin A, the phytohemagglutinin of the jack bean. Biochim. biophys. Acta 165, 398-404. Stefanovic V., Mandel P. and Rosenberg A. (1975) Concanavalin A inhibition of ecto-5'-nucleotidase of intact cultured C6 glioma cells. J. biol. Chem. 250, 7081-7083. Stefanovic V., Mandel P. and Rosenberg A. (1976) Ecto 5'-nucleotidase of intact cultured C6 glioma cells. J. biol. Chem. 251, 3900-3905. Tanaka R., Teruya A. and Morita H. (1973) Effects of divalen cations on 5'-mononucleotidase of bovine cerebral cortex. Can. J. Biochem. 51, 841-848. Young M. E. M., Moscarello M. A. and Riordan J. R. (1976) Concanavalin A binding to membranes of Golgi apparatus and resultant modification of galactosyltransferase activity. J. biol. Chem. 251, 5860-5865. Zachowski A., Evans W. H. and Paraf A. (1981) Immunological evidence that plasma membrane 5'-nucleotidase is a transmembrane protein. Biochim. biophys. Acta 644, 121-126. Zachowski A. and Paraf A. (1974) Use of Concanavalin A polymer to isolate right side-out vesicles of purified plasma membranes from eukariotic cells. Biochem. biophys. Res. Commun. 57, 787-792. Zachowski A., Migliore-Samour D., Paraf A. and Jolles P. (1975) Non specific effector-induced enzyme modulation in isolated plasma membranes. FEBS Lett. 52, 57-61.
0020-711X/86 $3.00+ 0.00 Pergamon Journals Ltd
CONCANAVALIN A INDUCED INHIBITION OF 5'-NUCLEOTIDASE FROM GUINEA PIG SKELETAL MUSCLE A N D BULL SEMINAL PLASMA: A COMPARATIVE STUDY CARLO FINIl, MARCELLA CAMICI2, ALBA MINELLIl, ARDESIO FLORIDI1 and PIER LUIGI IPATA2 qstituto Interfacoltfi di Chimica Biologica, Universit/t di Perugia, Via del Giochetto, 06100 Perugia, Italy :Istituto di Biochimica, Biofisica e Genefica, Universit~t di Pisa, Via S. Maria, 55, 56100 Pisa, Italy (Received 16 December 1985)
Abstract--1. Both purified and membrane-bound 5'-nucleotidases (EC 3.1.3.5) from guinea pig skeletal muscle and bull seminal plasma are inhibited by Concanavalin A (Con A). 2. 5'-Nucleotidase purified from skeletal muscle is inhibited by Con A by an apparent uncompetitive process (KI = 160 nM), while the lectin inhibits the particulate enzyme by an apparent non-competitive process (Ki = K~= 50 nM). 3. 5'-Nucleotidase purified from bull seminal plasma is inhibited by Con A by an apparent non-competitive process (K'i= K~= 270 nM), while the membrane-bound enzyme is subjected to a mixed type inhibition by the lectin (K~ > K,; 30 and 14 nM, respectively). 4. The enzyme purified from skeletal muscle exhibits a significant cooperativity in the interaction with Con A. 5. The inhibition of bull seminal plasma particulate 5'-nucleotidase brought about by Con A is not completely reversed by addition of ~-methyl-D-mannoside.
INTRODUCTION Con A, a lectin from Canavalia ensiformis, binds specifically to carbohydrates having the D-arabinoside configuration (Agrawal and Goldstein, 1967; So and Goldstein, 1968). This property allows the binding of Con A to the glycosidic moieties of glycoproteins; this interaction results in an inhibition of 5'-nucleotidase activity in intact cells (Stefanovic et al., 1975; Dornand et al., 1978; Carraway et al., 1979), in membrane preparations (Zachowski and Paraf, 1974; Riordan and Slavik, 1974, 1977; Dornand et al., 1977; Harb et al., 1983), as well as in the purified enzyme (Slavik et al., 1977; Harb et al., 1983). However, a stimulation of 5'-nucleotidase activity of ascites cells and rat liver plasma membranes at low Con A concentrations, has been also reported (Zachowski and Paraf, 1974; Riordan and Slavik, 1974). Moreover, Novogrodsky (1972) and Pommier et al. (1975) showed an activating effect of Con A on ATPase activity of plasma membranes of lymphocytes. It has been shown that the purified 5'-nucleotidase from bovine liver is less sensitive to Con A inhibition than the isolated membranes enzyme (Harb et al., 1983); however, it has been also observed that the enzyme purified from Wistar rat liver plasma membranes was even more sensitive to Con A inhibition than the membrane-bound enzyme (Slavik et al., 1977). Furthermore, the type of Con A inhibition, as well as the cooperativity of the Con A-enzyme interaction depend on either the enzyme source or the degree of enzyme purity, i.e. whether the activity is
measured using a purified enzyme protein or a membrane-bound 5'-nucleotidase preparation (Carraway et al., 1976, 1979; Dornand et al., 1978). For a better understanding of the inhibitory effect of Con A on 5'-nucleotidase, we have undertaken a comparative study on two mammalian 5'-nucleotidases. We have used both the purified and membrane-bound enzymes from guinea pig skeletal muscle and bull seminal plasma. The purification procedures and some kinetic and molecular properties of these enzymes have been previously reported (Camici et al., 1985; Fini et al. 1983). In the present study, we show that Con A inhibits both bull semen and muscle 5'-nucleotidase. However, the enzyme purified from skeletal muscle is inhibited by Con A in a process that is apparently uncompetitive and the inhibition exhibits positive cooperativity, while Con A is a non-competitive inhibitor of the particulate enzyme with no cooperativity. On the other hand, the enzyme purified from bull seminal plasma shows an apparently noncompetitive inhibition that does not exhibit any significant cooperativity; finally, the membranebound seminal enzyme is subjected to a mixed type inhibition with a Hill coefficient close to 1. The purified enzymes from both sources are less sensitive to Con A inhibition than the membrane-bound enzymes. The inhibition exerted by Con A can be completely reversed by addition of a-methyl-Dmannoside to particulate, purified skeletal muscle and purified bull seminal plasma 5'-nucleotidases, while the particulate bull seminal plasma enzyme is only partially reactivated.
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CARLO FIyl et al.
684 MATERIALS AND METHODS
Materials Guinea pig skeletal muscle 5'-nucleotidase was purified according to Camici et al. (1985); bull seminal plasma 5'-nucleotidase was purified as described by Fini et al. (1983). Con A, ~-methyl-o-mannoside, calf intestinal mucosa adenosine deaminase (EC 3.5.4.4) and Y-AMP were purchased from Sigma. All other chemicals were of reagent gradc. Preparation ~)[~membrane-bound 5"-nucleotidases (a) Guinea pig skeletal muscle 5'-nucleotidase: 10 mg of freshly excised tissue were homogenized in 3.5 vol of 50 mM Tris-HCl, pH 7.4. The homogenate was centrifuged at 600 g at 4°C for 30 min. The pellet was discarded. The supernatant was then centrifuged at 15,000g at 4~'C for 20 min and the pellet discarded. Finally, the supernatant was centrifuged at 105,000 g for 1 hr and the pellet, referred to as "microsome fraction" (Kamp and Wirtz, 1974), collected and stored at - 2 f f C . Before use, aliquots of the microsome fraction, containing 0.52 mg of protein, were suspended in 1 ml of 50 mM Tris-HC1, pH 7.4. The 5'-nucleotidase activity associated with this suspension represents the membrane-bound enzyme. (b) Bull semen particulate 5'-nucleotidase: 50 ml of bull semen were centrifuged at 2000g at 4°C for 30 min. The supernatant was withdrawn and centrifuged at 15,000g at 4'C for 30min and the pellet was discarded; the supernatant, referred to as "bull seminal plasma", was centrifuged at 105.000g at 4c'C for 1 hr. The supernatant contained the soluble form of 5'-nucleotidase of bull seminal plasma and was not used in this study. The pellet was suspended in 12ml of 50mM Tris-HCl, pH 7.4 and then centrifuged at 105,000g at 4°C for 90 min. The supernatant was discarded and the pellet was resuspended in 5 ml of 50 mM Tris HCI, pH 7.4 and stored at 0-T'C. This suspension represents the membrane-bound seminal plasma 5'-nucleotidase. Assay The 5'-nucleotidase assay was carried out in 50mM Tris-HCl, pH 7.4, at 37°C using 5'-AMP as substrate in the presence of excess adenosine deaminase, by measuring the decrease in absorbance at 265 nm which accompanies the conversion of adenosine to inosine, according to Ipata (1967). The reaction mixture contained, in a final volume of 1 ml, different amounts of Con A and 5'-nucleotidase preparation, as indicated in the legends to figures. One/~g of adenosine deaminase was also added. We did not observe a time-dependent variation in inhibition of enzyme activity by Con A when the 5'-nucleotidase preparations were preincubated for 5 10 and 20 min in the presence of the lectin. Thus, a preincubation of 10 rain of the enzyme preparations with Con A was carried out before addition of varying amount of 5'-AMP (see legends to figures). One unit of enzyme activity is the amount of enzyme catalyzing the conversion of 1 #mol of substrate per rain in the assay mixture.
Other methods Protein was determined by the Coomassie Blue binding assay (Bradford. 1976). RESULTS
Effect o f Con A on 5'-nucleotidase The effect o f C o n A was studied on b o t h the particulate a n d the purified 5'-nucleotidase from guinea pig skeletal muscle a n d bull seminal plasma. Figure 1 shows t h a t the p a t t e r n o f inhibition o f the purified enzymes is r e m a r k a b l y different: the C o n A c o n c e n t r a t i o n to o b t a i n 50% inhibition is 400 n M for the skeletal muscle enzyme (Panel A) a n d 100 n M for the bull seminal plasma enzyme (Panel B). O n the o t h e r h a n d , the m e m b r a n e - b o u n d enzymes show almost the same inhibitory p a t t e r n and are more sensitive to C o n A inhibition (50% o f activity is inhibited at approx. 3 0 n M C o n A). The enzyme purified from skeletal muscle exhibits a significant positive cooperative effect in the interaction with C o n A (Fig. 1A; Hill coefficient 2), while the m e m b r a n e b o u n d enzyme does not show any significant cooperativity (Fig. IA; Hill coefficient 0.96). The Hill plot for the bull seminal plasma 5'-nucleotidase inhibition (Fig. I B) exhibits no cooperativity b o t h for the m e m b r a n e - b o u n d a n d the purified enzyme (Hill coefficients 0.95 a n d 1.09, respectively).
Kinetic parameters o f inhibition o f 5'-nucleotidase by Con A Previous studies on the inhibition of 5'-nucleotidase from different sources, by either C o n A or nucleoside di- a n d triphosphates have s h o w n that these c o m p o u n d s exert different inhibitory patterns; thus, b o t h positive a n d negative cooperativity, competitive, n o n - c o m p e t i t i v e a n d mixed types of inhibition have been reported ( C a r r a w a y et al., 1976, 1979; D o r n a n d et al., 1978; R i o r d a n a n d Slavik, 1974; D o r n a n d et al., 1977; H a r b et al., 1983; M u r r a y a n d Friedrichs, 1969; E d w a r d s a n d M c G u i r e , 1970; T a n a k a et al., 1973; Burger a n d Lowenstein, 1975; Stefanovic et al., 1976; N a i t o a n d Lowenstein, 1985). In the present study, we have c o m p a r e d the changes in kinetic p a r a m e t e r s b r o u g h t a b o u t by C o n A inhibition o f two m a m m a l i a n 5'-nucleotidases, varying either the substrate or the inhibitor concentrations. Figures 2 a n d 3 show the plot of s/v vs i a n d sly vs s for skeletal muscle a n d bull semen 5'-nucleotidase, respectively. Ki values were o b t a i n e d graphically: K i represents the dissociation c o n s t a n t for the enzymei n h i b i t o r complex; K'i is the dissociation c o n s t a n t for the e n z y m e - i n h i b i t o r - s u b s t r a t e complex. As shown
Table 1. Inhibition of 5'-nucleotidase activity by Con A Enzyme source
Type of inhibition (/~M) (nM) (nM) Skeletal muscle (a) Purified Uncompetitive 19 _+ 1.1 160 + 8.2 (b) Membrane-bound Non-competitive 25 _+ 1.9 50 _+3.0 50 _+4.1 Bull seminal plasma (a) Purified Non-competitive I 1 + 1.2 270 + 6.9 270 ± 6.9 (b) Membrane-bound Mixed 11 _+ 1.8 14 __+2.5 30 + 2.8 K, and KI values were obtained according to Cornish-Bowden (1974). Experimental conditions are given in the text.
Hill coefficient 2.00_+0.12 0.96 + 0.09 0.95 + 0.05 1.09 + 0.07
Con A inhibition of 5'-nucleotidase (A)
m
,ooZe. ilI\ 0
I 200
I 300
I 400
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.
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Fig. I. Effect of Con A on 5'-nucleotidase. Panel (A) shows the inhibitory pattern for guinea pig skeletal muscle 5'-nucleotidase. Left: 35 ng of purified enzyme ( 0 ) or 15.6/~g of microsome fraction (©). The reaction was started by addition of Y-AMP to a final concentration of 62.5/~ M. Right:Hill plot for Con A inactivation of purified ( 0 ) and membrane-bound (©) 5'-nucleotidase. Panel (B) refers to bull seminal plasma 5'-nucleotidase. Left: 20 ng of purified enzyme ( 0 ) or 1 #g of the pellet 105,000g (O). The reaction was started by adding Y-AMP to a final concentration of 50/~M. Right: Hill plot for Con A inactivation of purified ( 0 ) and membrane-bound 5'-nucleotidase (O).
in Fig. 2A, in the plot of s/v vs i, the lines intersect above the i axis giving a KI value of 160 nM; moreover, in the plot of sly vs s, the lines intercept on the vertical axis; these two graphs, together with a Dixon's plot (not shown) which gives rise to parallel lines, suggest that the purified skeletal muscle 5'-nucleotidase is inhibited by Con A in an apparently uncompetitive process. Analysis of the data shown in Fig. 2B, together with a Dixon's plot, here not shown, indicates that the membrane-bound skeletal muscle 5'-nucleotidase is inhibited by an apparent non-competitive process with a KI = Kj value of 50 nM. The same type of analysis was performed on the bull seminal enzyme. The results obtained, shown in Fig. 3, together with the Dixon's plots (not shown), indicate that the purified 5'-nucleotidase (Panel A) is inhibited by Con A by an apparent non-competitive process, with a K I = Ki of 270nM; while the in-
hibition observed for the membrane-bound enzyme (Panel B) is apparently of the mixed type with a K, > K~of 30 nM and 14 nM, respectively. The results are summarized in Table 1.
Reversal of Con A inhibition of 5"-nucleotidase by ct-methyl-o-mannoside The K~ values obtained indicate that Con A is a powerful inhibitor of 5'-nucleotidase from both guinea pig skeletal muscle and bull seminal plasma. The addition of ct-methyl-D-mannoside, which binds specifically to Con A, completely reversed the inhibition induced by the lectin of both purified and membrane-bound skeletal muscle 5'-nucleotidase (Fig. 4A), as well as of purified bull seminal plasma 5'-nucleotidase (Fig. 4B). However, under the same conditions, the sugar failed in fully reactivating the bull semen particulate enzyme, and no more than
(B)
80
0
/
500
50 100 [ConA] (nM)
j
250
/
J
40
80
120
80
0
120
I
I
50 lOO [AMP] (/..¢M)
,
Fig. 2. Kinetics of Con A inhibition of 5'-nucleotidase from guinea pig skeletal muscle. Panel (A). Purified 5'-nucleotidase. Left: 35 ng of enzyme were incubated in the presence of the indicated amount o f Con A; the reaction was started by adding 5'-AMP to a final concentration of 40 # M (Q), 80/aM (ll), and 120 # M (A). Right: 35 ng of enzyme were incubated in the absence ( O ) or in the presence o f 85 nM (11) or 170 nM ( A ) C o n A. Panel (B). Membrane-bound enzyme. Lqft: the microsome fraction (15.6/ag o f protein) was incubated in the presence of Con A; the reaction was started by adding 5 ' - A M P to a final concentration of 3 7 . 5 # M (O), 62.5/aM (m), and 125/aM (A). Right: the microsome fraction was incubated in the absence ( O ) or in the presence of Con A to a final concentration of 17 nM (11) or 68 n M (A).
0
~o x 100
b
120
(A)
0
25
250
EConA] (nM)
100 ~
(B)
0
~2o/
, 50
500
40
0
60
io
4O
80
120
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I 50
I
50
I
100
I
100
Fig. 3. Kinetics of Con A inactivation of 5'-nucleotidase from bull seminal plasma. Pane/ (A). Purified 5'-nucleotidase. Left: 20 ng of enzyme were incubated in the presence of the indicated a m o u n t of Con A; the reaction was started by adding Y - A M P to a final concentration of 12.5/aM (O), 50/aM (m), and 100/aM (A). Right: 20 ng of enzyme were incubated in the absence (O) or in the presence of 136.5 nM (11) or 455 nM ( A ) Con A. Panel (B) m e m b r a n e - b o u n d enzyme. Le/?: 1 /ag of protein was incubated in the presence of Con A: the reaction was started by adding 5'-AMP to a final concentration of 12.5 t~M (OI. 50/aM (11), and 1001tM (A). Right: 1 /ag of protein was incubated in the absence ( O ) or in the presence of 13.65 nM ( D ) or 45.5 nM ( A ) Con A.
7
(A)
7---
r~
C
('3
o~
Con A inhibition of 5'-nucleotidase
687
1975; Zachowski et al., 1975; Akedo et al., 1976; Young et al., 1976). Being ecto 5'-nucleotidase a glycoprotein acting as Con A receptor (Carraway et 80 al., 1979), the study of the lectin-enzyme interaction might be of great help for understanding cell surface biochemical phenomena as well as relationships be60 tween transmembrane cell surface proteins and the cytoskeleton system. 40 >. In the present study, we have compared the inhibitory effect of Con A on 5'-nucleotidase from two i i i i i ~ zo mammalian sources: guinea pig skeletal muscle and "6 1oo bull seminal plasma. Experiments were carried out on both the purified and the particulate enzymes. The data obtained show that the purified enzymes are less ~ 80 g: sensitive to Con A inhibition than the membranebound enzymes: the concentration of inhibitor re60 quired for half maximal inactivation was 3-10 times higher for the purified 5'-nucleotidase than for the 40 particulate enzymes. Moreover, the comparison between the purified enzymes shows that the bull semi20 nal plasma 5'-nucleotidase is more sensitive to Con A inhibition than the guinea pig skeletal muscle enI i i zyme; on the other hand, almost the same inhibitory 2 4 6 ~ ~b pattern was obtained for the membrane-bound en[ = - M e t h y l - D - m a n n o s i d e ] (raM) zymes. Analysis of the types of inhibition of the different Fig. 4. Reversal of Con A inhibition of 5'-nucleotidase by enzyme preparations brought about by Con A allows ~-methyl-o-mannoside. Panel (A). Skeletal muscle 5'-nucleotidase. 35 ng of purified enzyme (O) were incu- some speculations. Thus, it appears that the interbated 10 min at 37°C in the presence of 680 nM Con A. action of Con A with purified 5'-nucleotidase of Then increasing amounts of ~-methyl-o-mannoside were skeletal muscle (uncompetitive type inhibition) affects added and the activity was assayed after addition of Y-AMP the breakdown of the enzyme-substrate complex, to a final concentration of 62.5/~M. The microsome fraction with proportional changes in Km and V (a 1.5 time (15.6 #g of protein) (O) was incubated l0 min at 37°C in the decrease in Km and V in the presence of 170 nM Con presence of 170 nM Con A, before addition of increasing A); on the other hand, the interaction of purified bull amounts of ~t-methyl-o-mannoside, then the activity was seminal plasma 5'-nucleotidase with Con A (nonassayed as described before. Panel (B) bull seminal plasma competitive type inhibition) results in a reduction of 5'-nucleotidase. 20 ng of the purified enzyme (11) were incubated 10 min at 37°C in the presence of 455 nM Con A. V (from 2.080mU, in the absence of Con A, to Then, increasing amounts of ~t-methyl-o-mannoside were 0.943 mU, in the presence of 455 nM Con A) and no added and the activity was assayed after addition of Y-AMP effect on Km is observed. Differences were also obto a final concentration of 50/~M. The particulate enzyme served for the membrane-bound enzymes. Thus, the (1/~g of protein) (I-q) was incubated 10 nm at 37°C in the interaction Con A-particulate skeletal muscle presence of 45.5 nM Con A, before addition of increasing 5'-nucleotidase (non-competitive type inhibition) reamounts of ~-methyl-D-mannoside, then the activity was duces V (from 2.041 to 1.504 mU in the absence and assayed as described above. presence of 17nM Con A, respectively), with no effect on Kin; while the seminal particulate enzyme is 60% of the initial activity was restored by an subjected to a mixed type inhibition in which the Km ~t-methyl-D-mannoside concentration of 10.4nM, a is shifted from ! 1 #M (in the absence of the lectin) to 18 # M (in the presence of 45.5 nM Con A) and V is 228 x 103 excess over Con A concentration. shifted from 2.080 mU to i.0 mU in the presence of 45.5 nM Con A. It appears then, that Con A interDISCUSSION action does not affect the combination of 5'-AMP with both the membrane-bound skeletal muscle and Con A from Canavalia ensiformis is a tetrameric metalloprotein which agglutinates erythrocytes, lym- the purified bull seminal plasma enzymes, while the inhibitory lectin can affect both the substrate combiphocytes and transforms cells. This lectin binds nation and the breakdown of the enzyme-substrate specifically to carbohydrates such as Orb-glucose and complex in the case of the membrane-bound bull ct-o-mannose and their derivatives, as well as to seminal plasma enzyme. The differences in the mechglycoproteins containing such glycosidic moieties. anism of inhibition, observed for the two purified Since glycoproteins both in intact cells and plasma 5'-nucleotidases, may be related to differences in the membranes are exposed on the external surface, they may be regarded as Con A receptors. The interaction topology of the active centre, as well as to the between Con A and these receptors has been widely particular localization of the glycosidic moiety (which allows the binding of Con A) with respect to the studied to obtain information on biological processes substrate binding site. We have already observed occurring at the cell surface (Carraway et al., 1975, some differences in substrate specificity between the 1979; Riordan and Slavik, 1974; Novogrodsky, 1972; two enzymes (Camici et al., 1985; Fini et al., 1983) Jarret and Smith, 1974; Pommier et al., 1975; Podoland more recently (experiment in progress) a minor sky and Weiser, 1975; Gahmberg and Hakomori, 100
CARLO FINI et al.
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sensitivity of the skeletal muscle enzyme to inhibition by antibodies against bull seminal plasma 5'-nucleotidase, compared to the purified bull seminal plasma enzyme itself. It can be argued that the differences in Con A-enzyme interaction might originate from dissimilarities in the polypeptide chains of the two 5'-nucleotidases; further information will be obtained from amino acid analysis that is underway in our laboratory. It has been longly debated (Carraway et al., 1976, 1979; Snow et al., 1980) whether the change in cooperativity in the Con A inhibition observed between the purified and particulate 5'-nucleotidase was due to an involvement of cytoskeleton elements, such as actin, to which 5'-nucleotidase might be anchored (Dieckhoff and Mannherz, 1985; Rohr and Mannherz, 1979; Mesher et al., 1981). Recently, Craik et al. (1985) have shown that cooperativity changes probably result from modification of the nucleotidase or a small, closely associated membrane moiety, rather than from involvement of actinmicrofilaments. In our experimental conditions, we have found that the purified skeletal muscle 5'-nucleotidase shows a positive cooperativity in the interaction with Con A, while the particulate enzymelectin interaction does not show any cooperativity. Owing to the methods used by us to prepare the "microsome fraction", actin filaments are not left intact; therefore, our results suggest that some other factor, other than the detachment of 5'-nucleotidase from the cell cytoskeleton, must be involved to explain the observed increased cooperativity in the interaction of the purified enzyme with Con A. Membrane-bound skeletal muscle and particulate bull semen 5'-nucleotidases are inhibited by Con A by distinct inhibitory mechanisms. This finding, together with the lack of complete reactivation of the bull semen enzyme, as compared to the skeletal muscle 5'-nucleotidase, suggest that interactions involving components of the membrane other than the enzyme itself might play an important role. SUMMARY
Concanavalin A inactivation of 5'-nucleotidase (EC 3.1.3.5) has been studied using both the purified and membrane-bound enzyme, obtained from two different mammalian sources: guinea pig skeletal muscle and bull seminal plasma. The purified enzymes from both sources are less sensitive to inhibition by the lectin than the membrane-bound enzymes, the concentration of inhibitor required for half maximal inactivation being 3-10 times higher for the purified 5'-nucleotidases than for the particulate enzymes. The enzyme purified from skeletal muscle is inhibited by the lectin by an apparent uncompetitive process, while Con A inhibits the particulate skeletal muscle 5'-nucleotidase by an apparent noncompetitive process. The Hill coefficient for the inactivation of purified skeletal muscle 5'-nucleotidase is shifted to 2, indicating significant cooperativity in the lectin-enzyme interaction. On the other hand, 5'-nucleotidase purified from bull seminal plasma is inhibited by Con A by an apparent non-competitive process, while the membrane-bound enzyme is subjected to a mixed type inhibition by the lectin. The
significance of such differences in the inhibition by Con A of the 5'-nucleotidase preparations studied is discussed. REFERENCES
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