I750
BlOCH EM ICAL SOCl ETY TRANSACTIONS
mammalian smooth muscle (Bass et ul., 1969); the activity is, however, in the range reported by Mitchell et al. (1977) for insect fibrillar flight muscle. CaZ+( I 0.6)-activated ATPase activity of onychophoran smooth-muscle actomyosin is similar to that of mammalian red muscle and some 30 times that of mammalian smooth muscle (Barany, 1967). The rate of, and total Cat+ uptake by onychophoran sarcoplasmic reticulum is similar to that of rabbit red muscle, but is only one-tenth as active as that of rabbit white muscle (Sreter, 1964) or insect flight muscle (Stossel & Zebe, 1968). Cat+uptake by onychophoran sarcoplasmic reticulum is some 6 times that of mammalian smooth muscle (Carsten, 1969). Caz+-dependent ATPase activity of the sarcoplasmic reticulum of onychophoran muscle shows the same comparative position as that of Cat+ uptake. With the exception of succinate dehydrogenase, the relatively high enzyme activities and Cazt uptake of onychophoran muscle suggests that it is a much more rapidly acting muscle than mammalian smooth muscle, thus generally confirming the ultrastructural observations of Lavallard (1966). Although he did not observe fat droplets in onychophoran muscle, the present finding of a moderately high 3-hydroxyacyl-CoA dehydrogenase activity in muscle homogenates suggests that lipids may be stored and utilized also. The significance of an abundant vesicular sarcoplasmic reticulum having a moderately high Ca2+-transportingability in the slow-moving onychophorans remains unclear. Nevertheless, it is apparent that onychophoran smooth muscle has a closer affinity in enzymic terms and some structural features to striated, rather than to smooth mammalian muscle. Bhrhny, M. (1 967) J. Gen. Physiol. 50, 197-2 18 Bass, A., Brdiczka, D., Eyer, P., Hofer, S. & Pette, D. (1969) Eur. J. Biochem. 10, 198-206 Carsten, M. (1969) J . Gen. Physiol. 53,414-426 Heffron, J. J. A., Hepburn, H. R . & Zwi, J. (1976) Naturwissenschafen 63,95 Lavallard, R. (1966) C.R. Hebd. SPances Acad. Sci. Ser. D 263, 148-151 Martonosi, A. & Feretos, R. (1964)J. Biol. Chem. 239,648-658 Mitchell, J. A., Heffron, J. J. A. &Hepburn, H. R. (1977) Comp. Biochem.Physiol. 857,111-1 16 Sreter, F. A. (1964) Fed. Proc. Fed. Am. SOC.Exp. Biol. 23,930-932 Stossel, W. & Zebe, E. (1968) Pflligers Arch. 302, 38-56 Taussky, H. H. & Shorr, E. (1953)J. Biof. Chem. 202,675-685
The Effects of Organic Phosphates and Univalent Cations on the Activity of Adenylate Deaminase from Rabbit Skeletal Muscle D. O’DRISCOLL, N. P. RORING and C . A. ROSS Department of Biochemistry, University College, Cork, Ireland
In the course of our studies on the regulatory properties of adenylate deaminase (EC 3.5.4.6) from rabbit skeletal muscle, we have observed differences in the effects of organic phosphates on enzyme activity from reported effects of these compounds on the enzyme obtained from chicken breast muscle and human skeletal muscle. These differences occur with respect to the relative effectiveness of a series of phosphate esters in inhibiting the enzyme and in the effects of the univalent-cation activator K+ on this inhibition. Suelter and co-workers (Smiley & Suelter, 1967; Smiley et al., 1967) demonstrated the allosteric activation of rabbit muscle adenylate deaminase by K+. They found K+ and Na+ to be equally effective in activating the enzyme, with maximum activation occurring at a concentration of 1 5 0 m ~ Li+, . Rb+ and NH4+ gave 75, 49 and 48% respectively, of the activity obtained with K+. Since then, K+ has been found to be the most effective univalent-cation activator of skeletal-muscle adenylate deaminase of a number of species, including the enzymes from chicken muscle (Sammons et al., 1970) and human muscle (Makarewicz & Stankiewicz, 1974). 1977
571sl MEETING, DUBLIN
1751
In agreement, we have found that the rabbit muscle enzyme, purified as described by Smiley et a / . (1967), is activated by univalent cations, K + being the most effective. Maximum activation was obtained in the presence of 100-150m~KCI, at AMP concentrations of 0 . 0 5 m ~or 0 . 2 m ~Above . ISO~M-KCI, a decrease in activity was observed with increasing KCI concentration. This effect was also reported for the enzyme from rat muscle (Ronca ef al., 1972), and was said to be due to the anionic moiety of the salt. Assaying at 0.05m~-AMPand with a cation (Na+) concentration of 1 5 0 m resulted ~ in 93 % activation relative to that obtained with K+, whereas NH4+ was 50 % effective. The relative activities shown in the presence of Li+ and Rb+, however, 45 % and 18 %respectively, were somewhat lower than those obtained by Suelter’s group (Smiley & Suelter, 1967; Smiley et al., 1967). Askari & Rao (1968) reported that 2,3-bis(phospho)-~-glycerateinhibited adenylate deaminase from human erythrocytes. This was the only organic phosphate of nine examined that was inhibitory, and the effects occurred at physiological K + concentration. Sammons et a / . (1970) carried out a similar study, with 11 phosphate esters, on adenylate deaminase from chicken breast muscle. They found 2,3-bis(phospho)-~glycerate to be the most effective inhibitor, with the remaining phosphate esters producing various but smaller decreases in activity. Inhibition was considerably greater in the presence of K+ than in its absence. Makarewicz & Stankiewicz (1974) demonstrated that 3-phospho-~-glyceratewas the most effective inhibitor of three esters examined on human muscle adenylate deaminase, and showed that inhibition was independent of K+. Our studies on the effects of organic phosphates on rabbit skeletal muscle adenylate deaminase involved seven phosphate esters, namely2,3-bis(phospho)-~-glycerate,ribose Sphosphate, 3-phospho-~-glycerate,fructose I $-bisphosphate, fructose 6-phosphate, glucose 6-phosphate and 2-phosphoenolpyruvate, all of which were used by Sammons et al. (1970). The last five phosphate esters, being glycolytic intermediates, are of particular interest in view of the reported association of high adenylate deaminase activity with muscles of high potential for glycolytic activity (Conway & Cooke, 1939; Raggi et a/.,1969). Our initial experiments showed no inhibition by any of these esters at con-
c
loo
I I 0
I .o
I
2.0
2
3.0
[Phosphateester] (mM) Fig. 1 . Inhihition of’ adettylatr dearnitme by phosphate esters Activity was measured as described by Kalckar (1945), by monitoring the decrease in A265that accompanies the deamination of AMP. Assays were performed with 0 . 2 m ~ AMP in O.OSM-imidazole/HCI buffer, pH 6.5, at 3 0 T , in the presence of: A, 2-phosB,3-phosphoglycerate. A value of phoenolpyruvate; e, 2,3-bis(phospho)-~-glycerate; 100 was taken as the activity in the absence of phosphate esters. VOl. 5
1752
BIOCHEMICAL SOCIETY TRANSACTIONS
Table I . Summary of the effects ofphosphate esters on aderiylate deaminase activity Activity was assayed as detailed in the legend to Fig. 1 , except for the presence of the indicated cation-activator concentration. A value of 100 is taken as the activity in the absence of phosphate esters at each activator concentration. -, Not tested. Relative activity Phosphate ester (3 mM) 3-Phospho-~-glycerate 2,3-Bis(phospho)-~-glycerate Fructose 1,6-bisphosphate Ribose 5-phosphate Fructose 6-phosphate Glucose 6-phosphate 2-Phosphoenolpyruvate
[Cation-activator] (mM)
.._
0 7 43 -
42
50 48 64 56 90
99 73 73
150 100 97 98 95 99 97 100
centrations up to 3mM in the presence of ~ S O ~ M - K Cwhich I , was surprising in view of previous reports. However, in the absence of K+ or at decreased cation-activator concentrations, considerable inhibition was obtained with some of these esters. This is illustrated in Fig. 1, where the effects on enzyme activity of increasing concentrations of those esters that were available as tris or cyclohexylammonium salts, are shown in the absence of KCI. Table 1 summarizes the effects of all seven phosphate esters on enzyme activity at cation-activator concentrations up to that giving optimal activity. In the case of phosphate esters used as sodium salts, KCI was added to the assay medium so that the sum of K+ and the Na+ added as ester (Na+ having virtually the same effect on activity as K+) gave the indicated cation-activator concentration. Overall, 3-phospho-~-glycerate had the greatest inhibitory effect, with 2,3-bis(phospho)-D-glycerate and 2-phosphoenolpyruvate, in the absence of cations, causing somewhat lower inhibition. Fructose 6-phosphate did not inhibit, and only a slight decrease of activity was observed with ribose 5-phosphate. These findings differ from the results obtained with the chicken enzyme, where 2,3-bis(phospho)-~-glyceratewas the most effective inhibitor, with 3-phospho-~-glyceratethe next most inhibitory. These results also differ from both of the previous studies (Sammons et a/., 1970; Makerewicz & Stankiewicz, 1974) on the protection from inhibition which was afforded by the presence of K+, the most effective cation activator of all three enzymes. In the previous reports, no such protective effects were apparent. That 2,3-bis(phospho)-~-glycerateinhibition of the chicken muscle enzyme might be of physiological significance was suggested by Sammons e t at. (1970), since inhibition of this enzyme was found to take place at phosphate ester and K+ concentrations that are believed to be within the physiological range. However, the present results do not support this suggestion, either for 2,3-bis(phospho)-~-glycerate or for any of the other phosphate esters, because of the opposing effect of K+. This is such that no inhibition occurs at cation concentrations within the physiological range. D. O’D. and N. P. R. acknowledge receipt of research-maintenancegrants from An Roinn Oideachais. The Wellcome Foundation is thanked for a gift of a recording spectrophotometer.
Askari, A. & Rao, S. N. (1968) Biochim. Biophys. Acfa 151,198-203 Conway, E. J. & Cooke, R. (1939) Biochem. J. 37,695-726 Kalckar, H. M. (1945) J. Biol. Chem. 167,445-459 Makarewicz, W. & Stankiewicz, A. (1974) Biochem. Med. 10,180-197 Raggi, A,, Ronca-Testoni,S . & Ronca, G. (1969) Biochim. Biophys. Acta 178,619-622 Ronca, G., Raggi, A. & Ronca-Testoni,S. (1972) Zral. J . Biochem. 21,305-321 Sammons, D. W., Henry, H. & Chilson, 0. P. (1970) J. Biol. Chem. 245, 2109-2113 Smiley, K. L., Jr. &Suelter, C. H. (1967)J. Biol. Chem. 242,1980-1981 Smiley, K. L., Jr., Berry, A. J. & Suelter, C. H. (1967) J. Biol. Chern. 242,2502-2506 1977
BlOCH EM ICAL SOCl ETY TRANSACTIONS
mammalian smooth muscle (Bass et ul., 1969); the activity is, however, in the range reported by Mitchell et al. (1977) for insect fibrillar flight muscle. CaZ+( I 0.6)-activated ATPase activity of onychophoran smooth-muscle actomyosin is similar to that of mammalian red muscle and some 30 times that of mammalian smooth muscle (Barany, 1967). The rate of, and total Cat+ uptake by onychophoran sarcoplasmic reticulum is similar to that of rabbit red muscle, but is only one-tenth as active as that of rabbit white muscle (Sreter, 1964) or insect flight muscle (Stossel & Zebe, 1968). Cat+uptake by onychophoran sarcoplasmic reticulum is some 6 times that of mammalian smooth muscle (Carsten, 1969). Caz+-dependent ATPase activity of the sarcoplasmic reticulum of onychophoran muscle shows the same comparative position as that of Cat+ uptake. With the exception of succinate dehydrogenase, the relatively high enzyme activities and Cazt uptake of onychophoran muscle suggests that it is a much more rapidly acting muscle than mammalian smooth muscle, thus generally confirming the ultrastructural observations of Lavallard (1966). Although he did not observe fat droplets in onychophoran muscle, the present finding of a moderately high 3-hydroxyacyl-CoA dehydrogenase activity in muscle homogenates suggests that lipids may be stored and utilized also. The significance of an abundant vesicular sarcoplasmic reticulum having a moderately high Ca2+-transportingability in the slow-moving onychophorans remains unclear. Nevertheless, it is apparent that onychophoran smooth muscle has a closer affinity in enzymic terms and some structural features to striated, rather than to smooth mammalian muscle. Bhrhny, M. (1 967) J. Gen. Physiol. 50, 197-2 18 Bass, A., Brdiczka, D., Eyer, P., Hofer, S. & Pette, D. (1969) Eur. J. Biochem. 10, 198-206 Carsten, M. (1969) J . Gen. Physiol. 53,414-426 Heffron, J. J. A., Hepburn, H. R . & Zwi, J. (1976) Naturwissenschafen 63,95 Lavallard, R. (1966) C.R. Hebd. SPances Acad. Sci. Ser. D 263, 148-151 Martonosi, A. & Feretos, R. (1964)J. Biol. Chem. 239,648-658 Mitchell, J. A., Heffron, J. J. A. &Hepburn, H. R. (1977) Comp. Biochem.Physiol. 857,111-1 16 Sreter, F. A. (1964) Fed. Proc. Fed. Am. SOC.Exp. Biol. 23,930-932 Stossel, W. & Zebe, E. (1968) Pflligers Arch. 302, 38-56 Taussky, H. H. & Shorr, E. (1953)J. Biof. Chem. 202,675-685
The Effects of Organic Phosphates and Univalent Cations on the Activity of Adenylate Deaminase from Rabbit Skeletal Muscle D. O’DRISCOLL, N. P. RORING and C . A. ROSS Department of Biochemistry, University College, Cork, Ireland
In the course of our studies on the regulatory properties of adenylate deaminase (EC 3.5.4.6) from rabbit skeletal muscle, we have observed differences in the effects of organic phosphates on enzyme activity from reported effects of these compounds on the enzyme obtained from chicken breast muscle and human skeletal muscle. These differences occur with respect to the relative effectiveness of a series of phosphate esters in inhibiting the enzyme and in the effects of the univalent-cation activator K+ on this inhibition. Suelter and co-workers (Smiley & Suelter, 1967; Smiley et al., 1967) demonstrated the allosteric activation of rabbit muscle adenylate deaminase by K+. They found K+ and Na+ to be equally effective in activating the enzyme, with maximum activation occurring at a concentration of 1 5 0 m ~ Li+, . Rb+ and NH4+ gave 75, 49 and 48% respectively, of the activity obtained with K+. Since then, K+ has been found to be the most effective univalent-cation activator of skeletal-muscle adenylate deaminase of a number of species, including the enzymes from chicken muscle (Sammons et al., 1970) and human muscle (Makarewicz & Stankiewicz, 1974). 1977
571sl MEETING, DUBLIN
1751
In agreement, we have found that the rabbit muscle enzyme, purified as described by Smiley et a / . (1967), is activated by univalent cations, K + being the most effective. Maximum activation was obtained in the presence of 100-150m~KCI, at AMP concentrations of 0 . 0 5 m ~or 0 . 2 m ~Above . ISO~M-KCI, a decrease in activity was observed with increasing KCI concentration. This effect was also reported for the enzyme from rat muscle (Ronca ef al., 1972), and was said to be due to the anionic moiety of the salt. Assaying at 0.05m~-AMPand with a cation (Na+) concentration of 1 5 0 m resulted ~ in 93 % activation relative to that obtained with K+, whereas NH4+ was 50 % effective. The relative activities shown in the presence of Li+ and Rb+, however, 45 % and 18 %respectively, were somewhat lower than those obtained by Suelter’s group (Smiley & Suelter, 1967; Smiley et al., 1967). Askari & Rao (1968) reported that 2,3-bis(phospho)-~-glycerateinhibited adenylate deaminase from human erythrocytes. This was the only organic phosphate of nine examined that was inhibitory, and the effects occurred at physiological K + concentration. Sammons et a / . (1970) carried out a similar study, with 11 phosphate esters, on adenylate deaminase from chicken breast muscle. They found 2,3-bis(phospho)-~glycerate to be the most effective inhibitor, with the remaining phosphate esters producing various but smaller decreases in activity. Inhibition was considerably greater in the presence of K+ than in its absence. Makarewicz & Stankiewicz (1974) demonstrated that 3-phospho-~-glyceratewas the most effective inhibitor of three esters examined on human muscle adenylate deaminase, and showed that inhibition was independent of K+. Our studies on the effects of organic phosphates on rabbit skeletal muscle adenylate deaminase involved seven phosphate esters, namely2,3-bis(phospho)-~-glycerate,ribose Sphosphate, 3-phospho-~-glycerate,fructose I $-bisphosphate, fructose 6-phosphate, glucose 6-phosphate and 2-phosphoenolpyruvate, all of which were used by Sammons et al. (1970). The last five phosphate esters, being glycolytic intermediates, are of particular interest in view of the reported association of high adenylate deaminase activity with muscles of high potential for glycolytic activity (Conway & Cooke, 1939; Raggi et a/.,1969). Our initial experiments showed no inhibition by any of these esters at con-
c
loo
I I 0
I .o
I
2.0
2
3.0
[Phosphateester] (mM) Fig. 1 . Inhihition of’ adettylatr dearnitme by phosphate esters Activity was measured as described by Kalckar (1945), by monitoring the decrease in A265that accompanies the deamination of AMP. Assays were performed with 0 . 2 m ~ AMP in O.OSM-imidazole/HCI buffer, pH 6.5, at 3 0 T , in the presence of: A, 2-phosB,3-phosphoglycerate. A value of phoenolpyruvate; e, 2,3-bis(phospho)-~-glycerate; 100 was taken as the activity in the absence of phosphate esters. VOl. 5
1752
BIOCHEMICAL SOCIETY TRANSACTIONS
Table I . Summary of the effects ofphosphate esters on aderiylate deaminase activity Activity was assayed as detailed in the legend to Fig. 1 , except for the presence of the indicated cation-activator concentration. A value of 100 is taken as the activity in the absence of phosphate esters at each activator concentration. -, Not tested. Relative activity Phosphate ester (3 mM) 3-Phospho-~-glycerate 2,3-Bis(phospho)-~-glycerate Fructose 1,6-bisphosphate Ribose 5-phosphate Fructose 6-phosphate Glucose 6-phosphate 2-Phosphoenolpyruvate
[Cation-activator] (mM)
.._
0 7 43 -
42
50 48 64 56 90
99 73 73
150 100 97 98 95 99 97 100
centrations up to 3mM in the presence of ~ S O ~ M - K Cwhich I , was surprising in view of previous reports. However, in the absence of K+ or at decreased cation-activator concentrations, considerable inhibition was obtained with some of these esters. This is illustrated in Fig. 1, where the effects on enzyme activity of increasing concentrations of those esters that were available as tris or cyclohexylammonium salts, are shown in the absence of KCI. Table 1 summarizes the effects of all seven phosphate esters on enzyme activity at cation-activator concentrations up to that giving optimal activity. In the case of phosphate esters used as sodium salts, KCI was added to the assay medium so that the sum of K+ and the Na+ added as ester (Na+ having virtually the same effect on activity as K+) gave the indicated cation-activator concentration. Overall, 3-phospho-~-glycerate had the greatest inhibitory effect, with 2,3-bis(phospho)-D-glycerate and 2-phosphoenolpyruvate, in the absence of cations, causing somewhat lower inhibition. Fructose 6-phosphate did not inhibit, and only a slight decrease of activity was observed with ribose 5-phosphate. These findings differ from the results obtained with the chicken enzyme, where 2,3-bis(phospho)-~-glyceratewas the most effective inhibitor, with 3-phospho-~-glyceratethe next most inhibitory. These results also differ from both of the previous studies (Sammons et a/., 1970; Makerewicz & Stankiewicz, 1974) on the protection from inhibition which was afforded by the presence of K+, the most effective cation activator of all three enzymes. In the previous reports, no such protective effects were apparent. That 2,3-bis(phospho)-~-glycerateinhibition of the chicken muscle enzyme might be of physiological significance was suggested by Sammons e t at. (1970), since inhibition of this enzyme was found to take place at phosphate ester and K+ concentrations that are believed to be within the physiological range. However, the present results do not support this suggestion, either for 2,3-bis(phospho)-~-glycerate or for any of the other phosphate esters, because of the opposing effect of K+. This is such that no inhibition occurs at cation concentrations within the physiological range. D. O’D. and N. P. R. acknowledge receipt of research-maintenancegrants from An Roinn Oideachais. The Wellcome Foundation is thanked for a gift of a recording spectrophotometer.
Askari, A. & Rao, S. N. (1968) Biochim. Biophys. Acfa 151,198-203 Conway, E. J. & Cooke, R. (1939) Biochem. J. 37,695-726 Kalckar, H. M. (1945) J. Biol. Chem. 167,445-459 Makarewicz, W. & Stankiewicz, A. (1974) Biochem. Med. 10,180-197 Raggi, A,, Ronca-Testoni,S . & Ronca, G. (1969) Biochim. Biophys. Acta 178,619-622 Ronca, G., Raggi, A. & Ronca-Testoni,S. (1972) Zral. J . Biochem. 21,305-321 Sammons, D. W., Henry, H. & Chilson, 0. P. (1970) J. Biol. Chem. 245, 2109-2113 Smiley, K. L., Jr. &Suelter, C. H. (1967)J. Biol. Chem. 242,1980-1981 Smiley, K. L., Jr., Berry, A. J. & Suelter, C. H. (1967) J. Biol. Chern. 242,2502-2506 1977
