Biochimica et Biophysica Acta, 1039 (1990) 123-129
123
Elsevier BBAPRO 33655
Mechanism of Na+/K+-ATPase activation by trypsin and kallikrein P. F i n o t t i , A. F a c c h i n e t t i a n d P. P a l a t i n i Department of Pharmacology, University of Padooa, Padova (Italy)
(Received 25 January 1990)
Key words: ATPase,Na+/K+-; Proteinase; Trypsin; Kallikrein The mechanism of the Na+/K+-ATPase activation by trypsin (from bovine pancreas) and kallikrein (from human plasma) was investigated on enzyme preparations from different sources (beef heart and dog kidney) and at different degrees of purification (beef heart). Kallikrein was effective on both beef and dog enzymes, whereas trypsin stimulated only the beef-heart N a ÷ / K +-ATPase. The extent of activation by the proteinases was inversely related to the degree of purification (maximal enzyme activation about 60 and 20% on the partially purified and the more purified enzymes, respectively). Enzyme activation was observed up to 0.5-0.6 pg/ml of proteinase. At higher concentrations the activation decreased and was converted into inhibition at proteinase concentrations above 1.0 p g / m l . N a + / K +-ATPase stimulation was due to an increase in the Vm~x of the enzyme reaction. K m for ATP remained unaffected. The activating effect was favoured by sodium and counteracted by potassium. Accordingly, Na+-ATPase activity was stimulated to a greater extent (up to 350%), whereas K +-dependent p-nitrophenylphosphatase activity proved to he insensitive to the actions of the proteinases. The N a + / K +-ATPase stimulation by both proteinases was antagonized by either ouabain or canrenone, two drugs that bind on the extracellular side of the Na+/K+-ATPase molecule. On the contrary, the enzyme inactivation observed at high proteinase concentrations was not counteracted by these two drugs. The stimulation of either N a + / K ÷- or Na+-ATPase activity was shown to he an irreversible effect without any significant protein degradation detectable by SDS gel electrophoresis. The results obtained suggest that proteinases exert their stimulatory effects by interacting preferentially with the E 2 conformation of N a ÷ / K +-ATPase at site(s) located on the extraceilnlar moiety of the enzyme.
Introduction Several reports have documented the activation by trypsin and trypsin-like proteinases of different membrane enzymes including human platelet GTPase [1], Ca2+-ATPase from human erythrocytes [2,3] and chloroplast ATPase [4]. On the contrary, no proteinase-induced stimulation of Na+/K+-ATPase has been reported so far, although proteinases have been extensively employed to investigate the structure-function relationship of this enzyme [5-7]. Recently, we have shown that an Na+/K÷-ATPase activating substance, purified from plasma of insulin-dependent diabetics,
Abbreviations: TCA, trichloroacetic acid; SDS, sodium dodecyl sulfate; PAGE, polyacrylamidegel electrophoresis; p-NPPase, pnitrophenylphosphatase. Correspondence: P. Finotti, Department of Pharmacology,University of Padova, Largo E. Meneghetti2, 35131 Padova, Italy.
behaves like a serine proteinase [8]. We therefore tested the effect of various proteinases on Na+/K+-ATPase activity and showed that at concentrations lower than those hitherto used on this enzyme, proteinases produced a stimulatory effect [8]. The aim of the present work was to investigate the mechanism whereby two different serine proteinases, trypsin (from bovine pancreas) and kallikrein (from human plasma), stimulate the Na+/K+-ATPase activity. Since it has been shown that the effect of proteinase on Na+/K+-ATPase activity is dependent on the enzyme source [7], N a + / K +ATPase preparations from two different organs (beef heart and dog kidney) were used. Moreover, the beefheart enzyme was obtained at two different degrees of purification in order to evaluate the influence of the purification procedure on the proteinase-induced stimulation. Purification procedures may indeed alter either the lipid composition of the membrane [9], or the protein-lipid interaction which is crucial for determining the type of proteinase-sensitive enzyme conformation [10,111.
0167-4838/90/$03.50 © 1990 Elsevier SciencePublishers B.V. (BiomedicalDivision)
124 Materials and Methods
The Ca 2÷-ATPase activity was calculated by subtracting the activity in the absence of CaC12 (Mg2÷-ATPase) from that obtained in the presence of 0.5 mM CaC12 (Mg2+/Ca2÷-ATPase). The reaction was started by the addition of ATP and terminated after 30 min of incubation by 0.25 ml of 50% TCA. Product formation was linear with time throughout the incubation period. For determination of the Na+-ATPase activity the medium was the same as indicated above for the N a ÷ / K ÷ATPase activity with the exception that NaC1 concentration was 150 mM and KC1 was omitted. Potassium activated p-nitrophenylphosphatase (pNPPase) activity was assayed at 37°C for 10 rain [15] in a medium containing 20 mM KCI, 3 mM MgC12, 18 mM imidazole, 0.1 mM EGTA, 3 mM pnitrophenylphosphate and 36/~g of enzyme proteins in a final volume of 1 ml (pH 7.8). The molarity of the medium was adjusted at 150 mM by the addition of choline chloride. The activity was calculated by subtracting the activity in potassium-free medium from that in the medium containing potassium. SDS-PAGE (7%) was performed according to the method of Laemmli et al. [16].
Beef-heart Na+/K÷-ATPase was prepared according to Matsui and Schwartz [12] either without (partially purified) or with the final step of purification with NaI. The Na+/K÷-ATPase from dog kidney was obtained from Sigma (U.S.A.). Proteins were measured with the method of Lowry et al. [13]. With the exception of the experiments in which the concentration of either MgC12 or ATP was varied, a typical incubation medium contained in a final volume of 1 ml: 100 mM NaC1, 20 mM KC1, 3.5 mM MgC12, 3 mM Na2ATP (pH 7.1), imidazole-histidine buffer (18 mM each, pH 7.1), 0.1 mM EGTA, 30-40 #g of proteins of the partially purified or 20 /~g of the more purified enzyme preparations. The reaction was started by the addition of ATP and terminated after 30 min of incubation at 37 °C by the addition of 0.25 ml of 50% (w/v) trichloroacetic acid (TCA). Pi released in the supernatant was measured according to the method of Fiske and SubbaRow [14]. The Na÷/K+-ATPase activity was calculated by subtracting the activity in the presence of 0.2 mM ouabain (Mg2÷-ATPase) from that in the absence of ouabain (total ATPase) and correcting it for appropriate blank. Product formation was always linear with time for the duration of the assays (30 min for beef-heart enzymes and 10 min for dog-kidney enzyme) either in the presence or absence of proteinases. When the Ca2+-ATPase activity was tested, the incubation medium was the same as reported above with the exception that NaC1 was omitted, EGTA was 0.5 mM and enzyme protein concentration was 50 t~g/ml.
Results
The effects of increasing proteinase concentrations on the N a + / K +- and Mg2+-ATPase activities of the beef-heart enzyme preparations are shown in Fig. 1. Both trypsin and kallikrein produced a significant stimulation (up to about 60%) of the Na+/K+-ATPase activity of the partially purified preparation (panel A).
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Fig. 1. Effect of proteinases on beef-heart Mg 2+- and N a ÷ / K + - A T P a s e activities. (A) N a + / K ÷ - A T P a s e activity of partially purified beef-heart enzyme in the presence of increasing concentrations of trypsin (e) or kallikrein (zx). ATPase assay was as specified in Materials and Methods. Incubation time was 30 min and enzyme proteins 29 ~g. Each point represents the mean ( + S.E.) of at least four different experiments. Basal enzyme activity was 3.1 + 0.21 # m o l / m g per h (mean + S.E. of six experiments). (B) N a + / K +-ATPase (continuous line) and Mg 2 +-ATPase (dotted lines) activities of purified-beef-heart enzyme in the presence of increasing concentrations of trypsin (O©) or kallikrein (&t,). Each point is the mean of three different experiments with S.E. equal or less than 3.2%. Mean basal values of N a + / K + - A T P a s e and MgZ+-ATPase activities were 12.65 + 0.12 and 0.69 + 0.02 ~ m o l / m g per h (n = 3), respectively.
125
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Fig. 2. Percent stimulation of dog-kidney Na+/K+-ATPase (left ordinate) and Mg2+-ATPase activities (right ordinate) by scalar concentrations of kallikrein. ATPase assay was as specified in Materials and Methods, incubation time was 10 min and enzyme proteins 23/~g. Each point is the mean (+ S.E.) of three different experiments. Basal Na+/K+-ATPase and Mg2+-ATPase activities were 62.5+2.25 and 4.78 + 1.23/~mol/mg per h, respectively(mean + S.E. of three experiments).
At concentrations greater than 0.5-0.6 vg/ml the stimulatory effect decreased and was converted into inhibition above 1 # g / m l . N o significant stimulation of the Mg2+-ATPase activity was observed (results not shown). Smaller activating effects (10 and 20% with kallikrein and trypsin, respectively) were observed on the N a + / K + - A T P a s e activity of the more purified preparation (panel B).
Only kallikrein gave a significant stimulatory effect (about 150%) of the Mg2+-ATPase activity. Similar effects were elicited by kallikrein on the purified-dogkidney preparation (Fig. 2), i.e., modest activation of N a + / K + - A T P a s e but considerable stimulation of Mg2÷-ATPase. Dog-kidney enzyme activities were not stimulated by trypsin. A comparison of Figs. 1B and 2 with Fig. 1A reveals that the more purified the enzyme preparation, the smaller is the activating effect on N a ÷ / K + - A T P a s e and the narrower the range of effective proteinase concentrations. Virtually identical resuits were obtained when beef-heart enzyme was purified according to the method of Akera et al. [17]. Fig. 3 shows that regardless of the enzyme source, N a + / K +- and Mg2÷-ATPase stimulation occurred without any apparent lag-phase. With the partially purified beef-heart enzyme (panel A) the stimulation of N a + / K ÷ - A T P a s e by trypsin increased up to 30 min and then decreased. On the contrary, a constant stimulation of the Mg2÷-ATPase activity was observed up to 60 rain. Identical stimulatory patterns were observed with kallikrein (data not shown). On dog-kidney ATPases (panel B) kallikrein exerted a stimulatory effect only within the first 15 rain of incubation. F r o m this time on, no further activation was observed. The effect of proteinases on the kinetics of the N a + / K + - A T P a s e reaction is shown in Fig. 4. It is apparent that both proteinases increased the Vmax of the reaction (40 and 100% by 0.3/,g of trypsin and 0.5/~g of kallikrein, respectively, as determined from a direct linear plot [18]). On the contrary, no major change was observed in the Michaelis constant for ATP. Quite similar results were obtained with the more purified beef-heart preparation (data not shown).
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Fig. 3. Time-course of Mg2÷- and Na+/K+-ATPase activities of beef-heart and dog-kidney enzymes: influence of trypsin and kallikrein. (A) Na+/K+-dependent (continuous lines) and Mg2+-dependent (dotted lines) ATPase activities of partially purified beef-heart enzyme in the absence (control: Oo) or presence of 0.5/,g/ml trypsin (Azx).Enzyme proteins were 29/,g. Other conditions as specified in the Materials and Methods. (B) Na +/K+-dependent (continuous lines) and Mg2+-dependent (dotted lines) ATPase activities from dog-kidney enzyme in the absence (control: o o) or presence of 0.2/,g/ml kallikrein (*zx). Enzyme proteins were 23/*g.
126
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was observed for the stimulation of dog-kidney ATPase by kallikrein. As sodium and potassium appeared to favour and counteract, respectively, the proteinase stimulation of N a + / K + - A T P a s e activity, we also tested the proteinase effect on the Na÷-ATPase and p-NPPase activities, i.e., the enzyme activities in the absence of potassium or sodium, respectively. Fig. 5 shows the effects of trypsin and kaUikrein on the Na+-ATPase activity of the dog-kidney enzyme as a function of substrate concentration (the Na+-ATPase activity of the beef-heart enzyme was too low to be assayed). Trypsin, which was devoid of any stimulatory activity on dog-kidney Na+/K+-ATPase, showed a modest (20%) but constant stimulatory effect on Na +ATPase activity (panel A). A dramatic stimulatory effect was observed with kallikrein (panel B), which yielded a maximal stimulation of about 350% at 1.5 mM ATP. It can be noted that, apart from an increase in Vmax, the two proteinases did not produce any other major changes in the kinetics of the enzyme reaction. The p-NPPase activity from dog kidney remained unaltered in the presence of different kallikrein concentrations (data not shown), confirming the antagonistic effect of potassium on the proteinase stimulation of N a * / K + - A T P a s e . As the' effects of Na + and K ÷ on the stimulatory action of the proteinases are similar to those exerted by these cations on ouabain inhibition, the possibility was considered that these ligands possess identical or at least overlapping binding sites. To test this hypothesis, we studied the influence of ouabain and canrenone on the proteinase stimulating activity. Canrenone is a partial inhibitor which binds reversibly to the digitalis receptor site of N a ÷ / K + - A T P a s e [20]. It was used to
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Fig. 4. N a + / K + - A T P a s e activity of partially purified beef-heart enzyme as a function of A T P concentration in the absence (e) and presence of either 0.3 # g / m l trypsin (z~) or 0.5 # g / m l kallikrein (o). Time of incubation was 30 rain, enzyme proteins 29 #g. Mg 2+ concentrations were 0.5 m M in excess of each A T P concentration. This Mg 2÷ excess was determined to be optimal for enzyme activity at the lowest and highest A T P concentration used.
Since Na ÷ has been shown to influence the proteolytic effect of trypsin on N a + / K ÷ - A T P a s e [19], we tested the effect of this cation on the proteinase-induced stimulation by varying the N a + / K + concentration ratio from 1 to 11, while maintaining the total Na + plus K + concentration constant at 120 mM. Activation of the beef-heart enzyme by the two proteinases became apparent only at a N a + / K + concentration ratio greater than 5 and increased upon further increase of the concentration of sodium over that of potassium. The same
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Fig. 5. Dog-kidney Na+-ATPase activity in the absence (e) and in the presence of either 0.1 # g / m l trypsin (&) or 0.2 # g / m l kallikrein (o). Na+-ATPase activity was measured as specified in the Materials and Methods. Enzyme proteins were 22.5 #g, time of incubation 10 min. For Mg 2+ concentration, see legend to Fig. 4.
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[CANRENONE1, pM Fig. 6. Effect of canrenone on the stimulation of beef heart N a + / K +ATPase activity by trypsin and kallikrein. Control experiments in which the proteolytic activity of trypsin and kallikrein was assayed on tosyl-sulfonyl-L-arginine methyl ester (TAME) [8] showed that canrenone had no effect on either proteinase. O, control; (3, 0.5 / t g / m l trypsin; and /,, 0.17/~g/ml kallikrein. Enzyme proteins were 39 /~g, time of incubation 30 min. Basal enzyme activity was 3.4 # m o l / m g per h.
this purpose, since, as a reversible ligand, it is theoretically more suitable than ouabain for demonstrating a possible competition with other ligands. Unlike ouabain, it can also be employed at saturating concentrations, since, as a partial inhibitor, it does not drive the enzyme velocity to zero. Fig. 6 shows that the activating effect of trypsin on Na+/K÷-ATPase was counteracted in a dose-dependent manner by canrenone and virtually abolished at the maximal drug concentration used, suggesting competitive antagonism between the two ligands. The
TABLE I
Irreversibility of dog-kidney Na +/ K +-,,4 TPase stimulation by kallikrein Dog-kidney enzyme was incubated in a typical assay medium for determination of the N a + / K + - A T P a s e activity in either the absence (control) or presence of kallikrein. After 10 rain incubation, an aliquot was taken up and Pi (liberated) was determined [14]. The remaining portions were centrifuged for 60 min at 194000× g in a Beckman L7-55 ultracentrifuge and the pellet was resuspended in 180 mM imidazole-histidine buffer (pH 7.1). After protein determination [13], aliquots of these suspensions were added to an Na+/K+-ATPase medium and enzyme activity was again determined after 10 min incubation. The values are the mean ( + S.E.) of five different experiments. Enzyme activity (/~mol/mg per h)
Before centrifugation Aftercentrifugation
~
control
kallikrein
stimulation
(0.15 ~g/ml)
(~)
58.35 ( + 2.05) 19.80(+3.30)
72.05 ( + 2.56) 25.45(+2.67)
24.3 ( + 0.5) 29.0(+1.2)
11)0 200 300 [CANRENONE], JJM
400
Fig. 7. The effect of cartrenone on the stimulation of dog-kidney Na+-ATPase activity by kallikrein. Kallikrein concentraton was 0.2 # g / m l , proteins were 22.5 /~g and time of incubation 10 min. ATP and Mg 2÷ concentration were 1.4 and 1.9 raM, respectively.
activating effect of kallikrein was also antagonized, although not totally abolished. Fig. 7 shows that when tested on Na+-ATPase, canrenone was able to prevent completely the activating effect of kallikrein. This result is in accord with the previously reported observation that canrenone has a greater affinity for the Na+-conformation of the enzyme [20]. A decrease in the percent activation of either Na+/K+-ATPase or Na+-ATPase activity by proteinases was also observed in the presence of ouabain at concentrations producing limited enzyme inhibition. At variance with the stimulatory effect, the inactivation of Na+/K+-ATPase produced by high proteinase concentrations was not affected by either ouabain or canrenone (experiments not shown). Table I shows that the kallikrein-induced activation of Na+/K+-ATPase is an irreversible effect, since enzyme stimulation persisted after removal of the proteinase by centrifugation as well. However, the electrophoretic patterns of kallikrein-treated and untreated enzymes were overlapping (results not presented), indicating that no extensive proteolysis had taken place. Identical results, i.e., irreversibility of the activating effects of kallikrein and trypsin and no detectable proteolysis were observed when the proteinase effect was tested on the dog-kidney Na+-ATPase activity. Discussion
The foregoing results have shown that the proteinaseinduced stimulation of the Na+/K+-ATPase occurs without lag-phase, is constant over a long period of time and is competitively antagonized by drugs, such as ouabain and canrenone, which interact with a specific
128 receptor on the enzyme [20]. These data suggest that the activating effect of trypsin and kallikrein on N a + / K +ATPase is mediated by a receptorial mechanism. The irreversibility of the stimulatory effect and the fact that no proteolysis is detectable by gel electrophoresis, indicate that the stimulation is a consequence of a minimal proteolytic effect at the receptor site. It is indeed known that only an extensive enzyme digestion can cause significant changes in the electrophoretic pattern of N a + / K + - A T P a s e [21]. Analogous indications of a receptorial mechanism have been obtained by Jakobs and Aktories [1] and by Martin et al. [22] for the stimulation by serine proteinases of G T P hydrolysis and Ca 2+ and A T P secretion from h u m a n platelets, respectively. Several lines of evidence indicate that N a + / K ÷ATPase activation is consequent to proteinase interaction with the E 2 conformation of the enzyme at a site located on the extracellular side: (a) activation is favoured by sodium and antagonized by potassium. Accordingly, the proteinase activation is maximal on the Na+-ATPase activity, whereas no activating effect is observed on the p-NPPase activity. Sodium and potassium are known to increase and decrease, respectively, the steady-state concentration of the E 2 conformation of the enzyme [23]; (b) the same dependence on sodium and potassium is observed for the stimulatory effect of the proteinases and the inhibitory effect of either ouabain or canrenone which interact with the E 2 conformation of N a + / K + - A T P a s e [20,23]; (c) ouabain and canrenone, that bind to the extracellular moiety of the enzyme, can prevent the activation by trypsin and kallikrein; (d) both proteinases increase only the Vm~, of the enzyme reaction while leaving unaffected the g m for ATP, whose binding site is located on the cytoplasmic side of the enzyme [24]; (e) neither trypsin nor kallikrein activated the Ca2+-ATPase from beef heart. If the activation of N a + / K + - A T P a s e were due to an interaction of the proteinases with the cytoplasmic moiety of the enzyme, then Ca2+-ATPase would be expected to be activated as well, since these two enzymes have the major areas of homology on the cytoplasmic side of the membrane [251. The enzyme inactivation produced by high concentrations of proteinases appears consequent to interaction with sites distinct from those mediating the activating effect, since: (a) the enzyme stimulation can be prevented by drugs that have no effect on the inactivation observed at high proteinase concentration; (b) inactivation of N a + / K + - A T P a s e by trypsin has been shown to be the result of trypsin interaction with a cytoplasmic binding site [5,7], whereas the site(s) responsible for activation appear(s) located on the external side. N o unequivocal explanation can be given for the observation that the extent of N a + / K + - A T P a s e activation is inversely related to the degree of enzyme purifi-
cation. Considering the importance of the type of protein-lipid interaction in determining the susceptibility of m e m b r a n e - b o u n d enzymes to the action of proteinases [7,9,26], it is likely that an alteration in the lipid-protein interaction induced by the purification procedure [9] is responsible for the observed effect. The phospholipid depletion that ensues the purification procedure has been shown to favour the interaction of the proteinases with the cytoplasmic sites responsible for the N a + / K + - A T P a s e inactivation [27]. In purified enzyme preparations, the stimulation may, therefore, be masked by the enhanced inactivating effect. These last observations, at any rate, indicate that the enzyme must be in its native state in order to be activated by the proteinases, in accordance with the physiopathological role previously proposed for this proteinase effect [8]. The fact that only Kallikrein was active on dog-kidney N a + / K + - A T P a s e may be related to the well-known organ-specific function played by this proteinase in the renal tissue [28,29].
References 1 Jakobs, K.M. and Aktories, K. (1988) Biochem. J. 249, 639-643. 2 Rossi, J.P.F.C., Garrahan, P.J. and Rega, A.F. (1986) Biochim. Biophys. Acta 858, 21-30. 3 Ranies, C.A. and Hipkins, H.F. (1988) Biochim. Biophys. Acta 933, 172-178. 4 Wang, K.K.W., Villalobo, A. and Roufogalis, B.D. (1988) Arch. Biochem. Biophys. 260, 696-704. 5 Jorgensen, P.L. and Farley, R.A. (1988) Methods Enzymol. 156, 291-301. 6 Anner, B.M., Ting-Beall, H.P. and Robertson, J.D. (1984) Biochim. Biophys. Acta 773, 262-270. 7 Chin, G. and Forgac, M. (1983) Biochemistry22, 3405-3410. 8 Finotti, P. and Verbaro, R. (1987) Clin. Chim. Acta 170, 121-134. 9 Roelofsen, B. (1981) Life Sci. 29, 2235-2247. 10 London, Y. and Vossenberg, F.G.A. (1973) Biochim. Biophys. Acta 306, 478-490. 11 Sarkadi, B., Enyedi, A. and Gardos, G. (1987) Biochim. Biophys. Acta 899, 129-133. 12 Matsui, M. and Schwartz, A. (1966) Biochim. Biophys. Acta 128, 380-390. 13 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 14 Fiske, C.H. and SubbaRow, Y. (1925) J. Biol. Chem. 66, 375-400. 15 Robinson, J.D. (1969) Biochemistry 8, 3348-3355. 16 Laemmli, U.K. (1970) Nature 227, 680-685. 17 Akera, T., Larsen, F.S. and Brody, T.M. (1970) J. Pharmacol. Exp. Ther. 170, 17-26. 18 Fromm, H.J. (1975) Initial Rate Enzyme Kinetics, pp. 42-82, Springer, Berlin. 19 Jorgensen, P.L. (1977) Biochim. Biophys. Acta 466, 97-108. 20 Finotti, P. and Palatini, P. (1981) J. Pharmacol. Exp. Ther. 217, 784-790. 21 Jorgensen, P.L. (1975) Biochim. Biophys. Aeta 401, 399-415. 22 Martin, B.M., Feinman, R.D. and Detwiler, T.C. (1975) Biochemistry 14, 1308-1314. 23 Hootman, S.R. and Ernst, S.A. (1988) Methods Enzymol. 156, 213-229. 24 Jorgensen, P.L. and Klodos, I. (1978) Biochim. Biophys. Acta 507, 8-16.
129 25 Shull, G.E., Schwartz, A. and Lingrel, J.B. (1985) Nature 316, 691-695. 26 Steck, T.L., Fairbanks, G. and Wallach, D.F.H. (1971) Biochemistry 10, 2617-2624. 27 London, Y. and Vossemberg, F.G.A. (1973) Biochim. Biophys. Acta 307, 478-490.
28 Margolius, H.M. Horwitz, D., Pisano, J.J. and Keiser, H.R. (1976) Fed. Proc. 35, 203-206. 29 Ovedack, A., Backer-Kreutz, E., Ressel, C., Muller, H.M., Kolloch, R. and Sturape K.O. (1986) Clin. Sci. 70, 13-17.
123
Elsevier BBAPRO 33655
Mechanism of Na+/K+-ATPase activation by trypsin and kallikrein P. F i n o t t i , A. F a c c h i n e t t i a n d P. P a l a t i n i Department of Pharmacology, University of Padooa, Padova (Italy)
(Received 25 January 1990)
Key words: ATPase,Na+/K+-; Proteinase; Trypsin; Kallikrein The mechanism of the Na+/K+-ATPase activation by trypsin (from bovine pancreas) and kallikrein (from human plasma) was investigated on enzyme preparations from different sources (beef heart and dog kidney) and at different degrees of purification (beef heart). Kallikrein was effective on both beef and dog enzymes, whereas trypsin stimulated only the beef-heart N a ÷ / K +-ATPase. The extent of activation by the proteinases was inversely related to the degree of purification (maximal enzyme activation about 60 and 20% on the partially purified and the more purified enzymes, respectively). Enzyme activation was observed up to 0.5-0.6 pg/ml of proteinase. At higher concentrations the activation decreased and was converted into inhibition at proteinase concentrations above 1.0 p g / m l . N a + / K +-ATPase stimulation was due to an increase in the Vm~x of the enzyme reaction. K m for ATP remained unaffected. The activating effect was favoured by sodium and counteracted by potassium. Accordingly, Na+-ATPase activity was stimulated to a greater extent (up to 350%), whereas K +-dependent p-nitrophenylphosphatase activity proved to he insensitive to the actions of the proteinases. The N a + / K +-ATPase stimulation by both proteinases was antagonized by either ouabain or canrenone, two drugs that bind on the extracellular side of the Na+/K+-ATPase molecule. On the contrary, the enzyme inactivation observed at high proteinase concentrations was not counteracted by these two drugs. The stimulation of either N a + / K ÷- or Na+-ATPase activity was shown to he an irreversible effect without any significant protein degradation detectable by SDS gel electrophoresis. The results obtained suggest that proteinases exert their stimulatory effects by interacting preferentially with the E 2 conformation of N a ÷ / K +-ATPase at site(s) located on the extraceilnlar moiety of the enzyme.
Introduction Several reports have documented the activation by trypsin and trypsin-like proteinases of different membrane enzymes including human platelet GTPase [1], Ca2+-ATPase from human erythrocytes [2,3] and chloroplast ATPase [4]. On the contrary, no proteinase-induced stimulation of Na+/K+-ATPase has been reported so far, although proteinases have been extensively employed to investigate the structure-function relationship of this enzyme [5-7]. Recently, we have shown that an Na+/K÷-ATPase activating substance, purified from plasma of insulin-dependent diabetics,
Abbreviations: TCA, trichloroacetic acid; SDS, sodium dodecyl sulfate; PAGE, polyacrylamidegel electrophoresis; p-NPPase, pnitrophenylphosphatase. Correspondence: P. Finotti, Department of Pharmacology,University of Padova, Largo E. Meneghetti2, 35131 Padova, Italy.
behaves like a serine proteinase [8]. We therefore tested the effect of various proteinases on Na+/K+-ATPase activity and showed that at concentrations lower than those hitherto used on this enzyme, proteinases produced a stimulatory effect [8]. The aim of the present work was to investigate the mechanism whereby two different serine proteinases, trypsin (from bovine pancreas) and kallikrein (from human plasma), stimulate the Na+/K+-ATPase activity. Since it has been shown that the effect of proteinase on Na+/K+-ATPase activity is dependent on the enzyme source [7], N a + / K +ATPase preparations from two different organs (beef heart and dog kidney) were used. Moreover, the beefheart enzyme was obtained at two different degrees of purification in order to evaluate the influence of the purification procedure on the proteinase-induced stimulation. Purification procedures may indeed alter either the lipid composition of the membrane [9], or the protein-lipid interaction which is crucial for determining the type of proteinase-sensitive enzyme conformation [10,111.
0167-4838/90/$03.50 © 1990 Elsevier SciencePublishers B.V. (BiomedicalDivision)
124 Materials and Methods
The Ca 2÷-ATPase activity was calculated by subtracting the activity in the absence of CaC12 (Mg2÷-ATPase) from that obtained in the presence of 0.5 mM CaC12 (Mg2+/Ca2÷-ATPase). The reaction was started by the addition of ATP and terminated after 30 min of incubation by 0.25 ml of 50% TCA. Product formation was linear with time throughout the incubation period. For determination of the Na+-ATPase activity the medium was the same as indicated above for the N a ÷ / K ÷ATPase activity with the exception that NaC1 concentration was 150 mM and KC1 was omitted. Potassium activated p-nitrophenylphosphatase (pNPPase) activity was assayed at 37°C for 10 rain [15] in a medium containing 20 mM KCI, 3 mM MgC12, 18 mM imidazole, 0.1 mM EGTA, 3 mM pnitrophenylphosphate and 36/~g of enzyme proteins in a final volume of 1 ml (pH 7.8). The molarity of the medium was adjusted at 150 mM by the addition of choline chloride. The activity was calculated by subtracting the activity in potassium-free medium from that in the medium containing potassium. SDS-PAGE (7%) was performed according to the method of Laemmli et al. [16].
Beef-heart Na+/K÷-ATPase was prepared according to Matsui and Schwartz [12] either without (partially purified) or with the final step of purification with NaI. The Na+/K÷-ATPase from dog kidney was obtained from Sigma (U.S.A.). Proteins were measured with the method of Lowry et al. [13]. With the exception of the experiments in which the concentration of either MgC12 or ATP was varied, a typical incubation medium contained in a final volume of 1 ml: 100 mM NaC1, 20 mM KC1, 3.5 mM MgC12, 3 mM Na2ATP (pH 7.1), imidazole-histidine buffer (18 mM each, pH 7.1), 0.1 mM EGTA, 30-40 #g of proteins of the partially purified or 20 /~g of the more purified enzyme preparations. The reaction was started by the addition of ATP and terminated after 30 min of incubation at 37 °C by the addition of 0.25 ml of 50% (w/v) trichloroacetic acid (TCA). Pi released in the supernatant was measured according to the method of Fiske and SubbaRow [14]. The Na÷/K+-ATPase activity was calculated by subtracting the activity in the presence of 0.2 mM ouabain (Mg2÷-ATPase) from that in the absence of ouabain (total ATPase) and correcting it for appropriate blank. Product formation was always linear with time for the duration of the assays (30 min for beef-heart enzymes and 10 min for dog-kidney enzyme) either in the presence or absence of proteinases. When the Ca2+-ATPase activity was tested, the incubation medium was the same as reported above with the exception that NaC1 was omitted, EGTA was 0.5 mM and enzyme protein concentration was 50 t~g/ml.
Results
The effects of increasing proteinase concentrations on the N a + / K +- and Mg2+-ATPase activities of the beef-heart enzyme preparations are shown in Fig. 1. Both trypsin and kallikrein produced a significant stimulation (up to about 60%) of the Na+/K+-ATPase activity of the partially purified preparation (panel A).
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Fig. 1. Effect of proteinases on beef-heart Mg 2+- and N a ÷ / K + - A T P a s e activities. (A) N a + / K ÷ - A T P a s e activity of partially purified beef-heart enzyme in the presence of increasing concentrations of trypsin (e) or kallikrein (zx). ATPase assay was as specified in Materials and Methods. Incubation time was 30 min and enzyme proteins 29 ~g. Each point represents the mean ( + S.E.) of at least four different experiments. Basal enzyme activity was 3.1 + 0.21 # m o l / m g per h (mean + S.E. of six experiments). (B) N a + / K +-ATPase (continuous line) and Mg 2 +-ATPase (dotted lines) activities of purified-beef-heart enzyme in the presence of increasing concentrations of trypsin (O©) or kallikrein (&t,). Each point is the mean of three different experiments with S.E. equal or less than 3.2%. Mean basal values of N a + / K + - A T P a s e and MgZ+-ATPase activities were 12.65 + 0.12 and 0.69 + 0.02 ~ m o l / m g per h (n = 3), respectively.
125
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0.2 0.3 0.4 KALLIKREIN (JJg/roll
0.5
Fig. 2. Percent stimulation of dog-kidney Na+/K+-ATPase (left ordinate) and Mg2+-ATPase activities (right ordinate) by scalar concentrations of kallikrein. ATPase assay was as specified in Materials and Methods, incubation time was 10 min and enzyme proteins 23/~g. Each point is the mean (+ S.E.) of three different experiments. Basal Na+/K+-ATPase and Mg2+-ATPase activities were 62.5+2.25 and 4.78 + 1.23/~mol/mg per h, respectively(mean + S.E. of three experiments).
At concentrations greater than 0.5-0.6 vg/ml the stimulatory effect decreased and was converted into inhibition above 1 # g / m l . N o significant stimulation of the Mg2+-ATPase activity was observed (results not shown). Smaller activating effects (10 and 20% with kallikrein and trypsin, respectively) were observed on the N a + / K + - A T P a s e activity of the more purified preparation (panel B).
Only kallikrein gave a significant stimulatory effect (about 150%) of the Mg2+-ATPase activity. Similar effects were elicited by kallikrein on the purified-dogkidney preparation (Fig. 2), i.e., modest activation of N a + / K + - A T P a s e but considerable stimulation of Mg2÷-ATPase. Dog-kidney enzyme activities were not stimulated by trypsin. A comparison of Figs. 1B and 2 with Fig. 1A reveals that the more purified the enzyme preparation, the smaller is the activating effect on N a ÷ / K + - A T P a s e and the narrower the range of effective proteinase concentrations. Virtually identical resuits were obtained when beef-heart enzyme was purified according to the method of Akera et al. [17]. Fig. 3 shows that regardless of the enzyme source, N a + / K +- and Mg2÷-ATPase stimulation occurred without any apparent lag-phase. With the partially purified beef-heart enzyme (panel A) the stimulation of N a + / K ÷ - A T P a s e by trypsin increased up to 30 min and then decreased. On the contrary, a constant stimulation of the Mg2÷-ATPase activity was observed up to 60 rain. Identical stimulatory patterns were observed with kallikrein (data not shown). On dog-kidney ATPases (panel B) kallikrein exerted a stimulatory effect only within the first 15 rain of incubation. F r o m this time on, no further activation was observed. The effect of proteinases on the kinetics of the N a + / K + - A T P a s e reaction is shown in Fig. 4. It is apparent that both proteinases increased the Vmax of the reaction (40 and 100% by 0.3/,g of trypsin and 0.5/~g of kallikrein, respectively, as determined from a direct linear plot [18]). On the contrary, no major change was observed in the Michaelis constant for ATP. Quite similar results were obtained with the more purified beef-heart preparation (data not shown).
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Fig. 3. Time-course of Mg2÷- and Na+/K+-ATPase activities of beef-heart and dog-kidney enzymes: influence of trypsin and kallikrein. (A) Na+/K+-dependent (continuous lines) and Mg2+-dependent (dotted lines) ATPase activities of partially purified beef-heart enzyme in the absence (control: Oo) or presence of 0.5/,g/ml trypsin (Azx).Enzyme proteins were 29/,g. Other conditions as specified in the Materials and Methods. (B) Na +/K+-dependent (continuous lines) and Mg2+-dependent (dotted lines) ATPase activities from dog-kidney enzyme in the absence (control: o o) or presence of 0.2/,g/ml kallikrein (*zx). Enzyme proteins were 23/*g.
126
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was observed for the stimulation of dog-kidney ATPase by kallikrein. As sodium and potassium appeared to favour and counteract, respectively, the proteinase stimulation of N a + / K + - A T P a s e activity, we also tested the proteinase effect on the Na÷-ATPase and p-NPPase activities, i.e., the enzyme activities in the absence of potassium or sodium, respectively. Fig. 5 shows the effects of trypsin and kaUikrein on the Na+-ATPase activity of the dog-kidney enzyme as a function of substrate concentration (the Na+-ATPase activity of the beef-heart enzyme was too low to be assayed). Trypsin, which was devoid of any stimulatory activity on dog-kidney Na+/K+-ATPase, showed a modest (20%) but constant stimulatory effect on Na +ATPase activity (panel A). A dramatic stimulatory effect was observed with kallikrein (panel B), which yielded a maximal stimulation of about 350% at 1.5 mM ATP. It can be noted that, apart from an increase in Vmax, the two proteinases did not produce any other major changes in the kinetics of the enzyme reaction. The p-NPPase activity from dog kidney remained unaltered in the presence of different kallikrein concentrations (data not shown), confirming the antagonistic effect of potassium on the proteinase stimulation of N a * / K + - A T P a s e . As the' effects of Na + and K ÷ on the stimulatory action of the proteinases are similar to those exerted by these cations on ouabain inhibition, the possibility was considered that these ligands possess identical or at least overlapping binding sites. To test this hypothesis, we studied the influence of ouabain and canrenone on the proteinase stimulating activity. Canrenone is a partial inhibitor which binds reversibly to the digitalis receptor site of N a ÷ / K + - A T P a s e [20]. It was used to
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Fig. 4. N a + / K + - A T P a s e activity of partially purified beef-heart enzyme as a function of A T P concentration in the absence (e) and presence of either 0.3 # g / m l trypsin (z~) or 0.5 # g / m l kallikrein (o). Time of incubation was 30 rain, enzyme proteins 29 #g. Mg 2+ concentrations were 0.5 m M in excess of each A T P concentration. This Mg 2÷ excess was determined to be optimal for enzyme activity at the lowest and highest A T P concentration used.
Since Na ÷ has been shown to influence the proteolytic effect of trypsin on N a + / K ÷ - A T P a s e [19], we tested the effect of this cation on the proteinase-induced stimulation by varying the N a + / K + concentration ratio from 1 to 11, while maintaining the total Na + plus K + concentration constant at 120 mM. Activation of the beef-heart enzyme by the two proteinases became apparent only at a N a + / K + concentration ratio greater than 5 and increased upon further increase of the concentration of sodium over that of potassium. The same
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Fig. 5. Dog-kidney Na+-ATPase activity in the absence (e) and in the presence of either 0.1 # g / m l trypsin (&) or 0.2 # g / m l kallikrein (o). Na+-ATPase activity was measured as specified in the Materials and Methods. Enzyme proteins were 22.5 #g, time of incubation 10 min. For Mg 2+ concentration, see legend to Fig. 4.
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this purpose, since, as a reversible ligand, it is theoretically more suitable than ouabain for demonstrating a possible competition with other ligands. Unlike ouabain, it can also be employed at saturating concentrations, since, as a partial inhibitor, it does not drive the enzyme velocity to zero. Fig. 6 shows that the activating effect of trypsin on Na+/K÷-ATPase was counteracted in a dose-dependent manner by canrenone and virtually abolished at the maximal drug concentration used, suggesting competitive antagonism between the two ligands. The
TABLE I
Irreversibility of dog-kidney Na +/ K +-,,4 TPase stimulation by kallikrein Dog-kidney enzyme was incubated in a typical assay medium for determination of the N a + / K + - A T P a s e activity in either the absence (control) or presence of kallikrein. After 10 rain incubation, an aliquot was taken up and Pi (liberated) was determined [14]. The remaining portions were centrifuged for 60 min at 194000× g in a Beckman L7-55 ultracentrifuge and the pellet was resuspended in 180 mM imidazole-histidine buffer (pH 7.1). After protein determination [13], aliquots of these suspensions were added to an Na+/K+-ATPase medium and enzyme activity was again determined after 10 min incubation. The values are the mean ( + S.E.) of five different experiments. Enzyme activity (/~mol/mg per h)
Before centrifugation Aftercentrifugation
~
control
kallikrein
stimulation
(0.15 ~g/ml)
(~)
58.35 ( + 2.05) 19.80(+3.30)
72.05 ( + 2.56) 25.45(+2.67)
24.3 ( + 0.5) 29.0(+1.2)
11)0 200 300 [CANRENONE], JJM
400
Fig. 7. The effect of cartrenone on the stimulation of dog-kidney Na+-ATPase activity by kallikrein. Kallikrein concentraton was 0.2 # g / m l , proteins were 22.5 /~g and time of incubation 10 min. ATP and Mg 2÷ concentration were 1.4 and 1.9 raM, respectively.
activating effect of kallikrein was also antagonized, although not totally abolished. Fig. 7 shows that when tested on Na+-ATPase, canrenone was able to prevent completely the activating effect of kallikrein. This result is in accord with the previously reported observation that canrenone has a greater affinity for the Na+-conformation of the enzyme [20]. A decrease in the percent activation of either Na+/K+-ATPase or Na+-ATPase activity by proteinases was also observed in the presence of ouabain at concentrations producing limited enzyme inhibition. At variance with the stimulatory effect, the inactivation of Na+/K+-ATPase produced by high proteinase concentrations was not affected by either ouabain or canrenone (experiments not shown). Table I shows that the kallikrein-induced activation of Na+/K+-ATPase is an irreversible effect, since enzyme stimulation persisted after removal of the proteinase by centrifugation as well. However, the electrophoretic patterns of kallikrein-treated and untreated enzymes were overlapping (results not presented), indicating that no extensive proteolysis had taken place. Identical results, i.e., irreversibility of the activating effects of kallikrein and trypsin and no detectable proteolysis were observed when the proteinase effect was tested on the dog-kidney Na+-ATPase activity. Discussion
The foregoing results have shown that the proteinaseinduced stimulation of the Na+/K+-ATPase occurs without lag-phase, is constant over a long period of time and is competitively antagonized by drugs, such as ouabain and canrenone, which interact with a specific
128 receptor on the enzyme [20]. These data suggest that the activating effect of trypsin and kallikrein on N a + / K +ATPase is mediated by a receptorial mechanism. The irreversibility of the stimulatory effect and the fact that no proteolysis is detectable by gel electrophoresis, indicate that the stimulation is a consequence of a minimal proteolytic effect at the receptor site. It is indeed known that only an extensive enzyme digestion can cause significant changes in the electrophoretic pattern of N a + / K + - A T P a s e [21]. Analogous indications of a receptorial mechanism have been obtained by Jakobs and Aktories [1] and by Martin et al. [22] for the stimulation by serine proteinases of G T P hydrolysis and Ca 2+ and A T P secretion from h u m a n platelets, respectively. Several lines of evidence indicate that N a + / K ÷ATPase activation is consequent to proteinase interaction with the E 2 conformation of the enzyme at a site located on the extracellular side: (a) activation is favoured by sodium and antagonized by potassium. Accordingly, the proteinase activation is maximal on the Na+-ATPase activity, whereas no activating effect is observed on the p-NPPase activity. Sodium and potassium are known to increase and decrease, respectively, the steady-state concentration of the E 2 conformation of the enzyme [23]; (b) the same dependence on sodium and potassium is observed for the stimulatory effect of the proteinases and the inhibitory effect of either ouabain or canrenone which interact with the E 2 conformation of N a + / K + - A T P a s e [20,23]; (c) ouabain and canrenone, that bind to the extracellular moiety of the enzyme, can prevent the activation by trypsin and kallikrein; (d) both proteinases increase only the Vm~, of the enzyme reaction while leaving unaffected the g m for ATP, whose binding site is located on the cytoplasmic side of the enzyme [24]; (e) neither trypsin nor kallikrein activated the Ca2+-ATPase from beef heart. If the activation of N a + / K + - A T P a s e were due to an interaction of the proteinases with the cytoplasmic moiety of the enzyme, then Ca2+-ATPase would be expected to be activated as well, since these two enzymes have the major areas of homology on the cytoplasmic side of the membrane [251. The enzyme inactivation produced by high concentrations of proteinases appears consequent to interaction with sites distinct from those mediating the activating effect, since: (a) the enzyme stimulation can be prevented by drugs that have no effect on the inactivation observed at high proteinase concentration; (b) inactivation of N a + / K + - A T P a s e by trypsin has been shown to be the result of trypsin interaction with a cytoplasmic binding site [5,7], whereas the site(s) responsible for activation appear(s) located on the external side. N o unequivocal explanation can be given for the observation that the extent of N a + / K + - A T P a s e activation is inversely related to the degree of enzyme purifi-
cation. Considering the importance of the type of protein-lipid interaction in determining the susceptibility of m e m b r a n e - b o u n d enzymes to the action of proteinases [7,9,26], it is likely that an alteration in the lipid-protein interaction induced by the purification procedure [9] is responsible for the observed effect. The phospholipid depletion that ensues the purification procedure has been shown to favour the interaction of the proteinases with the cytoplasmic sites responsible for the N a + / K + - A T P a s e inactivation [27]. In purified enzyme preparations, the stimulation may, therefore, be masked by the enhanced inactivating effect. These last observations, at any rate, indicate that the enzyme must be in its native state in order to be activated by the proteinases, in accordance with the physiopathological role previously proposed for this proteinase effect [8]. The fact that only Kallikrein was active on dog-kidney N a + / K + - A T P a s e may be related to the well-known organ-specific function played by this proteinase in the renal tissue [28,29].
References 1 Jakobs, K.M. and Aktories, K. (1988) Biochem. J. 249, 639-643. 2 Rossi, J.P.F.C., Garrahan, P.J. and Rega, A.F. (1986) Biochim. Biophys. Acta 858, 21-30. 3 Ranies, C.A. and Hipkins, H.F. (1988) Biochim. Biophys. Acta 933, 172-178. 4 Wang, K.K.W., Villalobo, A. and Roufogalis, B.D. (1988) Arch. Biochem. Biophys. 260, 696-704. 5 Jorgensen, P.L. and Farley, R.A. (1988) Methods Enzymol. 156, 291-301. 6 Anner, B.M., Ting-Beall, H.P. and Robertson, J.D. (1984) Biochim. Biophys. Acta 773, 262-270. 7 Chin, G. and Forgac, M. (1983) Biochemistry22, 3405-3410. 8 Finotti, P. and Verbaro, R. (1987) Clin. Chim. Acta 170, 121-134. 9 Roelofsen, B. (1981) Life Sci. 29, 2235-2247. 10 London, Y. and Vossenberg, F.G.A. (1973) Biochim. Biophys. Acta 306, 478-490. 11 Sarkadi, B., Enyedi, A. and Gardos, G. (1987) Biochim. Biophys. Acta 899, 129-133. 12 Matsui, M. and Schwartz, A. (1966) Biochim. Biophys. Acta 128, 380-390. 13 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 14 Fiske, C.H. and SubbaRow, Y. (1925) J. Biol. Chem. 66, 375-400. 15 Robinson, J.D. (1969) Biochemistry 8, 3348-3355. 16 Laemmli, U.K. (1970) Nature 227, 680-685. 17 Akera, T., Larsen, F.S. and Brody, T.M. (1970) J. Pharmacol. Exp. Ther. 170, 17-26. 18 Fromm, H.J. (1975) Initial Rate Enzyme Kinetics, pp. 42-82, Springer, Berlin. 19 Jorgensen, P.L. (1977) Biochim. Biophys. Acta 466, 97-108. 20 Finotti, P. and Palatini, P. (1981) J. Pharmacol. Exp. Ther. 217, 784-790. 21 Jorgensen, P.L. (1975) Biochim. Biophys. Aeta 401, 399-415. 22 Martin, B.M., Feinman, R.D. and Detwiler, T.C. (1975) Biochemistry 14, 1308-1314. 23 Hootman, S.R. and Ernst, S.A. (1988) Methods Enzymol. 156, 213-229. 24 Jorgensen, P.L. and Klodos, I. (1978) Biochim. Biophys. Acta 507, 8-16.
129 25 Shull, G.E., Schwartz, A. and Lingrel, J.B. (1985) Nature 316, 691-695. 26 Steck, T.L., Fairbanks, G. and Wallach, D.F.H. (1971) Biochemistry 10, 2617-2624. 27 London, Y. and Vossemberg, F.G.A. (1973) Biochim. Biophys. Acta 307, 478-490.
28 Margolius, H.M. Horwitz, D., Pisano, J.J. and Keiser, H.R. (1976) Fed. Proc. 35, 203-206. 29 Ovedack, A., Backer-Kreutz, E., Ressel, C., Muller, H.M., Kolloch, R. and Sturape K.O. (1986) Clin. Sci. 70, 13-17.
