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624 BIOCHEMICAL SOCIETY TRANSACTIONS E-Mg-ATP-ADP(4) E-M%-ADP E-ADP(1) /I \\ E -E-Pyr-P-E-Mg-ADP-Pyr-P I E-Mg- (2) 1 E-Mg-Pyr- P (3) I E-Mg-ATP /\ \ JE E-Mg -ATP-Pyr E-Pyr I E-Pyr-Mg-ADP(5) Scheme 1. Reaction scheme showing five dead-end complexes (1-5) for the pyruvate kinase from the leg muscle of C.maenas Formation of quaternary dead-end complexes is shown only once for convenience. Pyr-P and Pyr represent phosphoenolpyruvate and pyruvate respectively. products to enzyme complexes other than those to which they normally combine in the reverse reaction can change the form of the observed inhibition patterns. Both products, pyruvate and ATP, were non-competitive inhibitors with respect to the substrate ADP. This is the expected pattern if the two dead-end complexes ADP-enzyme-pyruvate and ADP-enzyme-ATP occur in the reaction mechanism. Scheme 1 summarizes a reaction mechanism that is consistent with both the intial rate and product-inhibition data. It is a rapid-equilibrium random Bi Bi mechanism in which five dead-end complexes are present. Ainsworth, S. & MacFarlane, N. (1973) Biochem.J. 131,223-236 Giles, I. G., Poat, P. C. & Munday, K. A. (1975) Biochem. SOC.Trans. 3 , 7 1 4 7 1 6 MacFarlane, N. & Ainsworth, S. (1972) Biochem.J. 129,1035-1047 Mildvan, A. S . & Cohn, M. (1966)J. Biol. Chem. 241,1178-1193 Newton, C. J., Poat, P. C. & Munday, K. A. (1976) Biochem. SOC.Trans. 4,479-481 Reynard, A. M., Has, L. F., Jacobson, D. D. & Boyer, P. D. (1961) J. Biol. Chem. 236,22772283 Equilibrium Isotopic-Exchange Studies of the Reaction Catalysed by Rabbit Muscle Pyruvate Kinase IAN G. GILES, PETER C. POAT and KENNETH A. MUNDAY Department of Physiology and Biochemistry, University of Southampton, Southampton SO9 SNH, U.K. Rabbit muscle pyruvate kinase (EC 2.7.1.40) catalyses a reaction between ADP and phosphoenolpyruvate, resulting in the formation of ATP and pyruvate, which is dependent on the presence of a bivalent cation, usually Mg2+,and a univalent cation, usually K+ (Kachmar & Boyer, 1953). The bivalent cation can bind directly to the enzyme (Mildvan & Cohn, 1965), and it has k e n suggested that it is a true substrate of the reaction in the forward direction (Ainsworth & MacFarlane, 1973). Other workers, however, have suggested that Mg-ADP is the true substrate of the enzyme (Melchior 1965; Cleland, 1967). Notwithstanding the ambiguity in identification of the true substrates, all the product-inhibition experiments of the forward reaction reported are consistent with a rapid-equilibrium random mechanism (Reynard et a f . , 1961 ; Mildvan & Cohn, 1966; Ainsworth & MacFarlane, 1973). 1976 563rd MEETING, LONDON 625 An assay suitable for kinetic studies of the reverse reaction of pyruvate kinase, and based on the transfer of label from [Q2P]ATP to phosphoenolpyruvate, has been reported and used to conduct product-inhibition experiments (Giles et al., 1975). All the inhibitionswerecompetitiveandareconsistentwitharandommechanism. Replots ofthe slopes of the primary double-reciprocal plots (of reaction rate against ATP concentration) as a function of inhibitor concentration were linear. When pyruvate was the variable substrate, however, non-linear-slope replots were obtained. Non-linear-slope effects normally reflect multiple combination of an inhibitor with the enzyme. It is notable therefore that the non-linear-slope replots were seen with both products, but only when pyruvate was the variable substrate. This would appear to rule out formation of a complex involving more than one molecule of the inhibitor in a rapid-equilibrium random mechanism as an explanation of the non-linear-slope effects. As an alternative, it was suggested that the binding (or realease) of pyruvate might not be completely rapidequilibrium and that the reaction possesses some non-rapidequilibriumcharacter (Giles etal., 1975). This possibility has been investigated bymeasuring the initial rates of isotopic exchange at chemical equilibrium. In a truly rapidequilibrium random mechanism the rates of all the isotopic exchanges will be the same in a given set of equilibrium conditions. The experiments were performed at pH7.4 and 25°C in the presence of 25m~-Tris/ HCI, 1mM-EGTA [ethanedioxybis(ethylamine)tetra-acetate], 100m~-K~,,.,and 0.5 rnM-Mg:;.. In these conditions the apparent equilibrium constant of the reaction was measured as 23500 when expressed in terms of total substrate concentrations. In all the experiments, except the pyruvate-into-phosphoenolpyruvateexchange, the equilibrium mixture of reactants was incubated at 25°C for 5min, after which sufficient enzyme to give a linear exchange rate was added. A further lOmin incubation was carried out in the presence of theenzyme to ensure chemical equilibrium. Control experiments showed that no loss of enzyme activity occurred in this incubation. The exchange reaction was initiated by addition of a small volume (less than 1 % of the volume of the incubation mixture) of a labelled substrate. In three cases ([2-3H]ATP, [2-3H]ADP and [p3'P]ATP) addition of the isotope resulted in less than a 0.01 % increase in the concentration of that Time (min) Fig. 1. Linear appearance of IabeI from [I-'4C]pyruvate into phosphoenolpyruvate as a frrnction of time The exchange was measured at pH7.4 and 25°C in the presence of 25m~-Tris/HCI, 1 mM-EGTA, 100rnM-K:,,,,, 0.5mM-Mg?Ae, 1m ~ - A T P ,12.9m~-pyruvate,2 5 f l ~ - A D P and 22.1 ,u~-phosphoenolpyruvate.The pyruvate contained 0.16pCi of [I -14C]pyruvate/,umol. The exchange reaction was initiated by addition of 0.6pg of rabbit muscle pyruvate kinase per ml of reaction medium. VOl. 4 626 Exchange measured BIOCHEMICAL SOCIETY TRANSACTIONS (I) Fig. 2. Histogram showing the relative exchange rates for the rabbit niuscle pyruoate kinase reaction The exchanges measured were: (1) ATP into phosphoenolpyruvate; ( 2 ) ATP into ADP; (3) ADP into ATP; (4) pyruvate into phosphoenolpyruvate; (5) phosphoenolpyruvate into pyruvate. The equilibrium conditions of the unhatched parts are as in Fig. 1. Those for the hatched parts are the same as in Fig. 1 except that the concentrations of ATP, ADP, pyruvate and phosphoenolpyruvate were 7 . 6 5 m ~ ,5 0 p ~ 7, . 6 5 m ~and 5 0 p ~ respectively. The results are expressed as a ratio to the slowest exchange (phosphoenolpyruvate into pyruvate). The rate of this slowest exchange in the second (hatched) condition was 66 % of that in the first equilibrium set of conditions. substrate. In the fourth case (phosph~enol[l-~~C]pyruvate) the increase was 5 %. Samples of the reaction mixture were taken at various times over 6min and added to trichloroacetic acid and placed on ice to stop further exchange. The pyruvate-intophosphoenolpyruvate exchange was initiated by addition of enzyme to a mixture at 25°C already containing isotope. The acid-quenched samples were neutralized with Tris base and the labelled product resolved by chromatography on Dowex 1 (C1- form). The product was counted for radioactivity by using conventional liquid-scintillation counting techniques. The exchange rate was determined from the linear slope of the d.p.m. appearing in the product as a function of time, and from the specific radioactivity of the labelled substrate. Five of the six possible exchanges were measured and all gave a linear exchange over the time-course studied, even though the zero time value may be high relative to the slope of the line. This is illustrated in Fig. 1 for the unfavourable pyruvate-into-phosphoenolpyruvate exchange. Measuring the rate of exchange between a substrate/product pair in either direction gave values that were within experimental error of each other, indicating that the system was at chemical equilibrium. Further, it was found that extending the preincubation period with the enzyme for up to 1h caused no change in the exchange rate, again indicating the equilibrium condition. Control experiments showed that exchange was dependent on the presence of enzyme and all reactants. This is evidence that a direct phosphate transfer occurs. The initial rates of isotopic exchange were determined in two sets of equilibrium conditions (Fig. 2). The substrate/product ratios used were limited, unfortunately, by the unfavourable equilibrium constant of the reaction and by the difficulty in removing ADP inpurity from ATP. The data obtained, however, indicate that the phosphoenolpyruvateinto-pyruvate exchange was slower than the other exchanges, including the ATP-intophosphoenolpyruvate exchange, which has phosphoenolpyruvate as a common partner. 1976 563rd MEETING, LONDON 627 This suggests that the steps involving the binding (or release) of pyruvate may contribute towards the overall rate of the reaction. If this is true, it follows that, strictly, it is invalid to use the rapidequilibrium assumption to describe the rabbit muscle pyruvate kinase reaction, although in the forward reaction all the steady-state kinetic data available are consistent with this assumption. Of the other kinases studied, creatine kinase is truly rapid-equilibrium (Morrison & Cleland, 1966), whereas the sugar-sugar phosphate exchange of galactokinase (Gulbinsky & Cleland, 1968) and hexokinase (Fromm et af., 1964) is slower than the other exchanges by a factor of 1.5 to 2.5. Ainsworth, S. & MacFarlane, N. (1973) Biochem. J. 131,223-236 Cleland, W. W. (1967) Annu. Rev. Biochem. 36,77-112 Frornrn, H . J., Silverstein, E. & Boyer, P. D. (1964)J. Biol. Chem. 239,3645-3652 Giles, I. G., Poat, P. C. & Munday, K. A. (1975) Biochem. SOC.Trans. 3,312-314 Gulbinsky, J. S. & Cleland, W. W. (1968) Biochemistry 7 , 566-575 Kachmar, J. F. & Boyer, P. D. (1953) J. Biol. Chem. 200,669-682 Melchior, J. (1965) Biochemistry 4, 1518-1525 Mildvan, A. S . & Cohn, M. (1965)J. Biol. Chem. 240,238-246 Mildvan, A. S. & Cohn, N. (1966) J. Biol. Chem. 241, 1178-1193 Morrison, J. F. & Cleland, W. W. (1966)J. Biol. Chem. 241,673-683 Reynard,A. M.,Hass, L. F., Jacobsen,D. D. &Boyer P. D. (1961)J.Biol.Chem. 236,2277-2283 Collagenolytic Cathepsin Activity in Rabbit Peritoneal Polymorphonuclear Leucocytes WALTER T. GIBSON,* DAVID W. MILSOM,* FRANK S. STEVEN* and JOHNS. LOWEt *Department of Medical Biochemistry, University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, U.K., and t Department of Biochemistry, I.C.I. Pharmaceuticals Division, Alderley Park, Macclesfield, Cheshire, U.K. The discovery of collagenolytic cathepsins in rat and human liver (Milsom ef al., 1972), rat leucocyte granules (Anderson, 1971), post-partum rat uterus (Etherington, 1973) and bovine spleen (Etherington, 1976), as well as the demonstration of the collagenolytic activity of purified cathepsin B1 (Burleigh eta!., 1974; Etherington, 1974), suggest that acid proteinases play an important role in collagen degradation. Although collagenase has been found in rabbit polymorphonuclear leucocytes (Robertson er al., 1972), there has been no report of an acid proteinase with collagenolytic activity in these cells, although Cochrane & Aikin (1966) showed that extracts of rabbit polymorphonuclear leucocytes will release peptides from purified glomerular basement membrane when incubated at an acid pH. This activity was attributed to cathepsins D and E, which, in addition to cathepsin A, were detected in rabbit polymorphonuclear-leucocyte granules by Wasi e f al. (1966). No cathepsin B or C activitywas observed. In this study, we report that rabbit polymorphonuclear leucocytes contain an enzyme capable of degrading polymeric collagen at an acid pH. Rabbit peritoneal exudatepolymorphonuclear leucocytes were obtained by themethod of Cohn & Hirsch (1960). Lysates of these cells were capable of solubilizing polymeric collagen, isolated by the method of Steven (1967), at pH4.0 and 37°C. The collagen preparations showed a small but variable susceptibility to degradation by trypsin, sometimes exceeding 10%. Activity in cell lysates and isolated granules usually exceeded that of trypsin on the same preparation of collagen, and, as further indication of its ability to degrade native collagen, the enzyme was shown to be able to solubilize polymeric collagen that had been pretreated with trypsin. Optimal activity of the collagenolytic cathepsin was found at around pH3, but assays were generally performed at pH4.0 to minimize the risk of denaturation during incubation at 37°C. There was no increase in the trypsin susceptibility of polymeric collagen Vol. 4