Effects of human fibrinogen and its cleavage products on activation of human plasminogen by streptokinase

The influence of human fibrinogen (Fg) and its terminal plasminolytic digestion products, fragment D and fragment E, on the kinetics of activation of human plasminogen (Pg) by catalytic levels of streptokinase (SK) has been investigated. Both Fg and fragment D enhanced the rates of activation of human Glu1-Pg, Lys77-Pg, and Val442-Pg. Fragment E was refractive in this regard. In the case of Glu1-Pg, the Km for activation by SK, 0.4 microM, was not affected by the presence of Fg or fragment D. The kcat for this same reaction, 0.12 s-1, was elevated to 0.3 s-1 at saturating levels of these effector molecules. On the other hand, the Km for activation of Lys77-Pg, 0.5 microM, was decreased to 0.09 microM, whereas the kcat, 0.33 s-1, was not altered in the presence of saturating concentrations of Fg or fragment D. In the case of Val442-Pg, the Km for this same activation, 2.0 microM, was lowered to 0.4 microM and 0.25 microM in the presence of Fg and fragment D, respectively. The kcat for this process, 1.0 s-1, was unchanged in the presence of these agents. The concentrations of Fg (KFg) and fragment D (KFD) that led to half-maximal stimulation of the activation rates were determined. For Fg with Glu1-Pg, Lys77-Pg, and Val442-Pg, the KFg values were 0.08 microM, 0.14 microM, and 0.17 microM, respectively. The KFD values for these same plasminogens were 0.25 microM, 2.0 microM, and 1.7 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of plasminogen and fibrin in plasminogen activation

Glu1-, Lys77-, miniplasminogens, kringle 1-3, kringle 1-5A, and kringle 1-5R were able to bind with fibrin, while microplasminogen and kringle 4 did not bind significantly. Kringle 1-5A, but not kringle 1-3, effectively inhibited the binding of Glu1-, Lys77-, and miniplasminogens with fibrin. Miniplasminogen also inhibited the binding of Glu1-plasminogen with fibrin. The binding of kringle 1-3 with fibrin was blocked by mini- or Glu1-plasminogen. It is therefore evident that there are two fibrin-binding domains in plasminogen and that the one in kringle 5 is of higher affinity than that in kringle 1-3. CNBr cleavage products of fibrinogen effectively enhanced the activation of Glu1-, Lys77-, or miniplasminogens, but not microplasminogen, by tissue-type plasminogen activator. Kringle 1-5, but not kringle 1-3, dose-dependently inhibited the enhancement by fibrinogen degradation products of Glu1-plasminogen activation by the activator. Lysine and epsilon-aminocaproic acid could inhibit the binding of plasminogens and plasminogen derivatives with fibrin and block the enhancement effect of fibrinogen degradation products on plasminogen activation. The data clearly illustrate that the binding of plasminogen with fibrin, mainly determined by kringle 5, is essential for effective activation by tissue-type plasminogen activator. However, the presence of kringle 1-4 in the plasminogen molecule is required for the full enhancing effect since the kcat/Km of miniplasminogen activation in the presence of fibrinogen degradation products was 8.2 microM-1 min-1 which is significantly less than 52.0 microM-1 min-1 of Glu1-plasminogen.     

The control of the urokinase-catalyzed activation of human glutamic acid 1-plasminogen by positive and negative effectors

The urokinase-catalyzed activation of human Glu1-plasminogen (Glu1Pg) has been found to be inhibited by monovalent anions in the following order of effectiveness: I- greater than SCN- greater than Cl- greater than IO3- greater than HCOO- greater than F- greater than OAc-. The inhibition is reversed by epsilon-aminocaproic acid, with its effectiveness in this capacity generally inversely proportional to the strength of the binding of the anion. The physical basis for the anion inhibition and epsilon-aminocaproic acid stimulation lies in the ability of these effectors to cause measurable opposite alterations in the conformation of Glu1Pg, which are revealed through study of the sedimentation velocity of the protein under various conditions. The kinetic mechanism of the chloride inhibition of Glu1Pg activation has been examined in detail. It has been found that the Glu1Pg.Cl complex serves as an alternate substrate to Glu1Pg for urokinase, with a greatly increased Km (25 +/- 3 and 2.2 +/- 0.3 microM, respectively) for activation. The kcat for the urokinase.Glu1Pg.Cl complex is approximately the same as that for urokinase.Glu1Pg (1.6 +/- 0.2 - 2.0 +/- 0.2/s). Similarly, the stimulation by epsilon-aminocaproic acid also results from effects on the Km of the activation, which is reduced to 1.8 +/- 0.2 microM for the Glu1Pg.Cl.epsilon-aminocaproic acid complex. The kcat for the urokinase.Glu1Pg.Cl.epsilon-aminocaproic acid of 2.4 +/- 0.3/s complex is not greatly different from that for urokinase.Glu1Pg.Cl. Nuclear magnetic resonance studies of the Glu1Pg-induced line broadening of the 35Cl- spectra in the presence and absence of epsilon-aminocaproic acid suggest that Cl- and epsilon-aminocaproic acid simultaneously bind to the protein and that each of these effectors displays its effects through separate binding sites.     

Prostromelysin-1 (proMMP-3) stimulates plasminogen activation by tissue-type plasminogen activator.

Matrix metalloproteinase-3 (MMP-3 or stromelysin-1) specifically binds to tissue-type plasminogen activator (t-PA), without however, hydrolyzing the protein. Binding affinity to proMMP-3 is similar to single chain t-PA, two chain t-PA and active site mutagenized t-PA (Ka of 6.3 x 106 to 8.0 x 106 M-1), but is reduced for t-PA lacking the finger and growth factor domains (Ka of 2.0 x 106 M-1). Activation of native Glu-plasminogen by t-PA in the presence of proMMP-3 obeys Michaelis-Menten kinetics; at saturating concentrations of proMMP-3, the catalytic efficiency of two chain t-PA is enhanced 20-fold (kcat/Km of 7.9 x 10-3 vs. 4.1 x 10-4 microM-1.s-1). This is mainly the result of an enhanced affinity of t-PA for its substrate (Km of 1.6 microM vs. 89 microM in the absence of proMMP-3), whereas the kcat is less affected (kcat of 1.3 x 10-2 vs. 3.6 x 10-2 s-1). Activation of Lys-plasminogen by two chain t-PA is stimulated about 13-fold at a saturating concentration of proMMP-3, whereas that of miniplasminogen is virtually unaffected (1.4-fold). Plasminogen activation by single chain t-PA is stimulated about ninefold by proMMP-3, whereas that by the mutant lacking finger and growth factor domains is stimulated only threefold. Biospecific interaction analysis revealed binding of Lys-plasminogen to proMMP-3 with 18-fold higher affinity (Ka of 22 x 106 M-1) and of miniplasminogen with fivefold lower affinity (Ka of 0.26 x 106 M-1) as compared to Glu-plasminogen (Ka of 1.2 x 106 M-1). Plasminogen and t-PA appear to bind to different sites on proMMP-3. These data are compatible with a model in which both plasminogen and t-PA bind to proMMP-3, resulting in a cyclic ternary complex in which t-PA has an enhanced affinity for plasminogen, which may be in a Lys-plasminogen-like conformation. Maximal binding and stimulation require the N-terminal finger and growth factor domains of t-PA and the N-terminal kringle domains of plasminogen.     

Characterization and nucleotide binding properties of a mutant dihydropteridine reductase containing an aspartate 37-isoleucine replacement.

Kinetic constants for the interaction of NADH and NADPH with native rat dihydropteridine reductase (DHPR) and an Escherichia coli expressed mutant (D-37-I) have been determined. Comparison of kcat and Km values measured employing quinonoid 6,7-dimethyldihydropteridine (q-PtH2) as substrate indicate that the native enzyme has a considerable preference for NADH with an optimum kcat/Km of 12 microM-1 s-1 compared with a figure of 0.25 microM-1 s-1 for NADPH. Although the mutant enzyme still displays an apparent preference for NADH (kcat/Km = 1.2 microM-1 s-1) compared with NADPH (kcat/Km = 0.6 microM-1 s-1), kinetic analysis indicates that NADH and NADPH have comparable stickiness in the D-37-I mutant. The dihydropteridine site is less affected, since the Km for q-PtH2 and K(is) for aminopterin are unchanged and the 14-26-fold synergy seen for aminopterin binding to E.NAD(P)H versus free E is decreased by less than 2-fold in the D-37-I mutant. No significant changes in log kcat and log kcat/Km versus pH profiles for NADH and NADPH were seen for the D-37-I mutant enzyme. However, the mutant enzyme is less stable to proteolytic degradation, to elevated temperature, and to increasing concentrations of urea and salt than the wild type. NADPH provides maximal protection against inactivation in all cases for both the native and D-37-I mutant enzymes. Examination of the rat DHPR sequence shows a typical dinucleotide binding fold with Asp-37 located precisely in the position predicted for the acidic residue that participates in hydrogen bond formation with the 2'-hydroxyl moiety of all known NAD-dependent dehydrogenases. This assignment is consistent with x-ray crystallographic results that localize the aspartate 37 carboxyl within ideal hydrogen bonding distance of the 2'- and 3'-hydroxyl moieties of adenosine ribose in the binary E.NADH complex.     

Catalytic residues and substrate specificity of scytalidoglutamic peptidase, the first member of the eqolisin in family (G1) of peptidases

Scytalidoglutamic peptidase (SGP) is the first-discovered member of the eqolisin family of peptidases with a unique structure and a presumed novel catalytic dyad (E136 and Q53) [Fujinaga et al., PNAS 101 (2004) 3364-3369]. Mutants of SGP, E136A, Q53A, and Q53E lost both the autoprocessing and enzymatic activities of the wild-type enzyme. Coupled with the results from the structural analysis of SGP, Glu136 and Gln53 were identified as the catalytic residues. The substrate specificity of SGP is unique, particularly, in the preference at the P3 (basic amino acid), P1' (small a.a.), and P3' (basic a.a.) positions. Superior substrates and inhibitors have been synthesized for kinetic studies based on the results reported here. kcat, Km, and kcat/Km of SGP for D-Dap(MeNHBz)-GFKFF*ALRK(Dnp)-D-R-D-R were 34.8 s-1, 0.065 microM, and 535 microM-1 s-1, respectively. Ki of Ac-FKF-(3S,4S)-phenylstatinyl-LR-NH2 for SGP was 1.2x10(-10) M. Taken together, we can conclude that SGP has not only structural and catalytic novelties but also a unique subsite structure.     

The pH dependence of pre-steady-state and steady-state kinetics for the papain-catalyzed hydrolysis of N-alpha-carbobenzoxyglycine p-nitrophenyl ester

Pre-steady-state and steady-state kinetics of the papain (EC 3.4.22.2)-catalyzed hydrolysis of N-alpha-carbobenzoxyglycine p-nitrophenyl ester (ZGlyONp) have been determined between pH 3.0 and 9.5 (I = 0.1 M) at 21 +/- 0.5 degrees C. The results are consistent with the minimum three-step mechanism involving the acyl X enzyme intermediate E X P: (Formula: see text). The formation of the E X S complex may be regarded as a rapid pseudoequilibrium process; the minimum values for k+1 are 8.0 microM-1 s-1 (pH less than or equal to 3.5) and 0.40 microM-1 s-1 (pH greater than 6.0), and that for k-1 is 600 s-1 (pH independent). The pH profile of k+2/Ks (= kcat/Km; Ks = k-1/k+1) reflects the ionization of two groups with pK' values of 4.5 +/- 0.1 and 8.80 +/- 0.15 in the free enzyme. The pH dependence of k+2 and k+3 (measured only at pH values below neutrality) implicates one ionizing group in the acylation and deacylation step with pK' values of 5.80 +/- 0.15 and 3.10 +/- 0.15, respectively. As expected from the pH dependences of k+2/Ks (= kcat/Km) and k+2, the value of Ks changes with pH from 7.5 X 10(1) microM (pH less than or equal to 3.5) to 1.5 X 10(3) microM (pH greater than 6.0). Values of k-2 and k-3 are close to zero over the whole pH range explored (3.0 to 9.5). The pH dependence of kinetic parameters indicates that at acid pH values (less than or equal to 3.5), the k+2 step is rate limiting in catalysis, whereas for pH values higher than 3.5, k+3 becomes rate limiting. The observed ionizations probably reflect the acid-base equilibria of residues involved in the catalytic diad of papain, His159-Cys25. Comparison with catalytic properties of ficins and bromelains suggests that the results reported here may be of general significance for cysteine proteinase catalyzed reactions.     

Importance of lysine-286 at the NADP site of glutamate dehydrogenase from Salmonella typhimurium.

Affinity labeling studies of NADP(+)-glutamate dehydrogenase from Salmonella typhimurium have shown that the peptide Leu-282-Lys-286 is located near the coenzyme site [Haeffner-Gormley et al. (1991) J. Biol. Chem. 266, 5388-5394]. The present study was undertaken to evaluate the role of lysine-286. The mutant enzymes K286R, K286Q, and K286E were prepared by site-directed mutagenesis, expressed in Escherichia coli, and purified. The Vmax values (micromoles of NADPH per minute per milligram of protein) were similar for WT (270), K286R (529), K296Q (409), and K286E (382) enzymes. As measured at pH 7.9, the Km value for NADPH was much greater for K286E (280 microM) than for WT (9.8 microM), K286R (30 microM), or K286Q (66 microM) enzymes. The efficiencies (kcat/Km) of the WT and K286R mutant were similar (1.2 x 10(3) min-1 microM-1 and 1.0 x 10(3) min-1 microM-1, respectively) while those of K286Q (0.30 x 10(3) min-1 microM-1) and K286E (0.07 x 10(3) min-1 microM-1) were greatly reduced. The decreased efficiency of the K286E mutant results from the increase in Km-NADPH, consistent with a role for a basic residue at position 286 which enhances the binding of NADPH. Plots of Vmax vs pH showed the pH optima to be 8.1-8.3 for all enzymes at saturating NADPH concentrations. A 40-fold increase in Km-NADPH for K286E was observed as the pH increased from 5.98 to 8.08, from which a unique pKe of 6.5 was calculated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of a fluorescence plate reader for measuring kinetic parameters with inner filter effect correction

A general method is presented here for the determination of the Km, kcat, and kcat/Km of fluorescence resonance energy transfer (FRET) substrates using a fluorescence plate reader. A simple empirical method for correcting for the inner filter effect is shown to enable accurate and undistorted measurements of these very important kinetic parameters. Inner filter effect corrected rates of hydrolysis of a FRET peptide substrate by hepatitis C virus (HCV) NS3 protease at various substrate concentrations enabled measurement of a Km value of 4.4 +/- 0.3 microM and kcat/Km value of 96,500 +/- 5800 M-1 s-1. These values are very close to the HPLC-determined Km value of 4.6 +/- 0.7 microM and kcat/Km value of 92,600 +/- 14,000 M-1 s-1. We demonstrate that the inner filter effect correction of microtiter plate reader velocities enables rapid measurement of Ki and Ki' values and kinetic inhibition mechanisms for HCV NS3 protease inhibitors.     

Kinetic studies of three different molecular forms of urokinase for the activation of native human plasminogen

The kinetic parameters of three different molecular forms of urokinase (UK) for the activation of native Glu-plasminogen were compared. The apparent Michaelis constant (Km. app.) of each UK was almost of the same order of magnitude (31-38 microM), but the catalytic constants (kc) were observed to be different: UKh (high molecular weight form, molecular weight 53,000), 2.4 +/- 0.2 s-1; UK+ (low molecular weight form, molecular weight 33,000), 0.83 +/- o.10 s-1, and UKl (trypsin-digested form, molecular weight 36,000), 0.91 +/- 0.18 s-1. The overall second order rate constant, kc/Km calculated for UKh was 7.7 X 10(4) M-1 s-1, higher than for UKl (2.2 X 10(4) M-1 s-1) or UKt (2.4 X 10(4) M-1 s-1), indicating the possibility of a much higher degree of enzymatic specificity and efficiency.     

Definition of the structural elements in plasminogen required for high-affinity binding to apolipoprotein(a): a study utilizing surface plasmon resonance

Lipoprotein(a) [Lp(a)] is suggested to link atherosclerosis and thrombosis owing to the similarity between the apolipoprotein(a) [apo(a)] moiety of Lp(a) and plasminogen. Lp(a) may interfere with tPA-mediated plasminogen activation in fibrinolysis, thereby generating a hypercoaguable state in vivo. The present study employed surface plasmon resonance (SPR) to examine the binding interaction between plasminogen and a physiologically relevant, 17-kringle recombinant apo(a) species [17K r-apo(a)] in real time. Native, intact Glu(1)-plasminogen bound to apo(a) with substantially higher affinity (K(D) approximately 0.3 microM) compared to a series of plasminogen fragments (K1-5, K1-3, K4, K5P, and tail domain) that interacted weakly with apo(a) (K(D) > 50 microM). Treatment of Glu(1)-plasminogen with citraconic anhydride (a lysine modification reagent) completely abolished binding to wild-type 17K r-apo(a), whereas citraconylated 17K r-apo(a) decreased binding to wild-type Glu(1)-plasminogen by approximately 50%; inhibition of binding was also observed using the lysine analogue epsilon-aminocaproic acid. Whereas native Glu(1)-plasminogen exhibited monophasic binding to 17K r-apo(a), truncated Lys(78)-plasminogen exhibited biphasic binding. Altering Glu(1)-plasminogen from its native, closed conformation (in chloride buffer) to an open conformation (in acetate buffer) also yielded biphasic isotherms. These SPR data are consistent with a two-state kinetic model in which a conformational change in the plasminogen-apo(a) complex may occur following the initial binding event. Differential binding kinetics between Glu(1)-/Lys(78)-plasminogen and apo(a) may explain why Lp(a) is a stronger inhibitor of tPA-mediated Glu(1)-plasminogen activation compared to Lys(78)-plasminogen activation.     

Interaction of thrombin with PAR1 and PAR4 at the thrombin cleavage site

Investigations determined the critical amino acids for alpha-thrombin's interaction with protease-activated receptors 1 and 4 (PAR1 and PAR4, respectively) at the thrombin cleavage site. Recombinant PAR1 wild-type (wt) exodomain was cleaved by alpha-thrombin with a Km of 28 microM, a kcat of 340 s-1, and a kcat/Km of 1.2 x 10(7). When the P4 or P2 position was mutated to alanine, PAR1-L38A or PAR1-P40A, respectively, the Km was unchanged, 29 or 23 microM, respectively; however, the kcat and kcat/Km were reduced in each case. In contrast, when Asp39 at P3 was mutated to alanine, PAR1-D39A, Km and kcat were both reduced approximately 3-fold, making the kcat/Km the same as that of PAR1-wt exodomain. Recombinant PAR4-wt exodomain was cleaved by alpha-thrombin with a Km of 61 microM, a kcat of 17 s-1, and a kcat/Km of 2.8 x 10(5). When the P5 or P4 position was mutated to alanine, PAR4-L43A or PAR4-P44A, respectively, there was no change in the Km (69 or 56 microM, respectively); however, the kcat was lowered in each case (9.7 or 7.7 s-1, respectively). Mutation of the P2 position (PAR4-P46A) also had no effect on the Km but markedly lowered the kcat and kcat/Km approximately 35-fold. PAR1-wt exodomain and P4 and P3 mutants were noncompetitive inhibitors of alpha-thrombin hydrolyzing Sar-Pro-Arg-pNA. However, PAR1-P40A displayed a mixed type of inhibition. Mutation of P4, P3, or P2 had no effect on the Ki. All PAR4 exodomains were competitive inhibitors of alpha-thrombin. Mutation of P5, P4, or P2 had no effect on the Ki. These investigations show that Leu at P4 in PAR1 or P5 in PAR4 critically influences the kinetics of alpha-thrombin binding and cleavage of PAR1 and PAR4 exodomains. It also implies that factors other than the hirudin-like binding region on PAR1 exodomain predominate in influencing PAR1 cleavage on cells.     

Barnase has subsites that give rise to large rate enhancements.

Barnase is found to have a series of subsites for binding its substrates that confers large rate enhancements. Ribonucleotide substrates of the type Zp0Gp1Xp2Y have been synthesized, where p is phosphate, X, Y, and Z are nucleosides, and G is guanosine. G occupies the primary specificity site. The most important subsite is for p2, followed by that for Y. There appears to be no subsite for the Z or p0 positions. Occupation of the subsite for p2 gives rise to a 1000-fold increase in kcat/Km, composed of a 100-fold increase in kcat and a 10-fold decrease in Km. The Y subsite gives rise to further 20-fold increase in kcat/Km. Rates approaching diffusion control for kcat/Km are observed. kcat for the dinucleotide monophosphate GpU = 0.55 s-1, and Km = 240 microM; this compares with 53 s-1 and 20 microM for GpUp, and 3.3 x 10(3) s-1 and 17 microM for GpApA (the best substrate tested). Cleavage occurs at the 3'-phosphate of guanosine in all cases. There are differences in base specificity at the two subsites for X and Y downstream of the scissile bond. The binding energies of different substrates have been analyzed using thermodynamic cycles. These show that the contributions of the X and Y sites are nonadditive.     

Activation of human Hageman factor by Pseudomonas aeruginosa elastase in the presence or absence of negatively charged substance in vitro.

Human Hageman factor, a plasma proteinase zymogen, was activated in vitro under a near physiological condition (pH 7.8, ionic strength I = 0.14, 37 degrees C) by Pseudomonas aeruginosa elastase, which is a zinc-dependent tissue destructive neutral proteinase. This activation was completely inhibited by a specific inhibitor of the elastase, HONHCOCH(CH2C6H5)CO-Ala-Gly-NH2, at a concentration as low as 10 microM. In this activation Hagemen factor was cleaved, in a limited fashion, liberating two fragments with apparent molecular masses of 40 and 30 kDa, respectively. The appearance of the latter seemed to correspond chronologically to the generation of activated Hageman factor. Kinetic parameters of the enzymatic activation were kcat = 5.8 x 10(-3) s-1, Km = 4.3 x 10(-7) M and kcat/Km = 1.4 x 10(4) M-1 x s-1. This Km value is close to the plasma concentration of Hageman factor. Another zinc-dependent proteinase, P. aeruginosa alkaline proteinase, showed a negligible Hageman factor activation. In the presence of a negatively charged soluble substance, dextran sulfate (0.3-3 micrograms/ml), the activation rate by the elastase increased several fold, with the kinetic parameters of kcat = 13.9 x 10(-3) s-1, Km = 1.6 x 10(-7) M and kcat/Km = 8.5 x 10(4) M-1 x s-1. These results suggested a participation of the Hageman factor-dependent system in the inflammatory response to pseudomonal infections, due to the initiation of the system by the bacterial elastase.     

Zymogen-activation kinetics. Modulatory effects of trans-4-(aminomethyl)cyclohexane-1-carboxylic acid and poly-D-lysine on plasminogen activation.

The kinetics of plasminogen activation catalysed by urokinase and tissue-type plasminogen activator were investigated. Kinetic measurements are performed by means of a specific chromogenic peptide substrate for plasmin, D-valyl-L-leucyl-L-lysine 4-nitroanilide. Two methods are proposed for the analysis of the resulting progress curve of nitroaniline formation in terms of zymogen-activation kinetics: a graphical transformation of the parabolic curve and transformation of the curve for nitroaniline production into a linear progress curve by the addition of a specific inhibitor of plasmin, bovine pancreatic trypsin inhibitor. The two methods give similar results, suggesting that the reaction between activator and plasminogen is a simple second-order reaction at least at plasminogen concentrations up to about 10 microM. The kinetics of both Glu1-plasminogen (residues 1-790) and Lys77-plasminogen (residues 77-790) activation were investigated. The results confirm previous observations showing that trans-4-(aminomethyl)cyclohexane-1-carboxylic acid at relatively low concentrations enhances the activation rate of Glu1-plasminogen but not that of Lys77-plasminogen. At higher concentrations both Glu1- and Lys77-plasminogen activation are inhibited. The concentration interval for the inhibition of urokinase-catalysed reactions is shown to be very different from that of the tissue-plasminogen activator system. Evidence is presented indicating that binding to the active site of urokinase (KD = 2.0 mM) is responsible for the inhibition of the urokinase system, binding to the active site of tissue-plasminogen activator is approx. 100-fold weaker, and inhibition of the tissue-plasminogen activator system, when monitored by plasmin activity, is mainly due to plasmin inhibition. Poly-D-lysine (Mr 160 000) causes a marked enhancement of plasminogen activation catalysed by tissue-plasminogen activator but not by urokinase. Bell-shaped curves of enhancement as a function of the logarithm of poly-D-lysine concentration are obtained for both Glu1- and Lys77-plasminogen activation, with a maximal effect at about 10 mg/litre. The enhancement of Glu1-plasminogen activation exerted by trans-4-(aminomethyl)cyclohexane-1-carboxylic acid is additive to that of poly-D-lysine, whereas poly-D-lysine-induced enhancement of Lys77-plasminogen activation is abolished by trans-4-(aminomethyl)cyclohexane-1-carboxylic acid. Analogies are drawn up between the effector functions of poly-D-lysine and fibrin on the catalytic activity of tissue-plasminogen activator.     

Studies of the role of factor Va in the factor Xa-catalyzed activation of prothrombin, fragment 1.2-prethrombin-2, and dansyl-L-glutamyl-glycyl-L-arginine-meizothrombin in the absence of phospholipid

In order to specifically evaluate the role of Factor Va in the prothrombinase complex, studies of the activation of prothrombin, Fragment 1.2-prethrombin-2, and active-site-blocked meizothrombin were carried out, both in the absence of phospholipid and at concentrations of substrates and Factor Va sufficient to approach saturation in all components. Km values were independent of Factor Va concentrations, whereas kcat (apparent) values approached saturation with respect to Factor Va concentrations. The three respective substrates exhibited the following parameters of kinetics (Km, microM; kcat, s-1 at saturating [Factor Va]): prothrombin (9.0 +/- 0.4; 31 +/- 1); Fragment 1.2-prethrombin-2 (5.4 +/- 0.4; 13 +/- 2); and meizothrombin (3.6 +/- 0.3; 51 +/- 5). Models of kinetics were constructed to interpret the results, and two of these were formally consistent with experimental results. Both models indicated that the variation of kcat(app) with concentrations of Factor Va reflects the formation of a Factor Va-Factor Xa binary complex. Analysis of kinetics indicated Kd values for this interaction of 1.3 +/- 0.1, 3.0 +/- 0.5, and 1.0 +/- 0.1 microM for the three respective substrates. The models differed in the interpretation of Km. One indicated that Km reflects a binary interaction between Factor Xa and prothrombin, whereas the other indicated a binary interaction between Factor Va and prothrombin. Both indicated that two of the three possible binary interactions between the three components would be reflected in Km and kcat values but not the third. To distinguish these models, the binary interactions were studied by extrinsic fluorescence (Va.Xa), light-scattering (Factor Va.prothrombin), and competition kinetics (Xa.II). The first two interactions were detected and were characterized by Kd values of 2.7 +/- 0.1 microM (Va.Xa) and 8.8 +/- 0.8 microM (Factor Va.prothrombin). No active-site-dependent interaction between prothrombin and Factor Xa could be detected in the absence of Factor Va. The results of these studies suggest that Factor Va interacts with both Factor Xa and prothrombin and effectively presents one to the other in the formation of a ternary enzyme-substrate-cofactor complex. In addition, a comparison of the parameters of kinetics of conversion of prothrombin and its intermediates indicates that meizothrombin is the major intermediate of prothrombin activation in the absence, as well as in the presence of phospholipid.     

Enzymatic properties of single-chain and two-chain forms of a Lys158----Glu158 mutant of urokinase-type plasminogen activator

Recombinant single-chain urokinase-type plasminogen activator (rscu-PA), in which the plasmin-sensitive peptide bond Lys158-Ile159 is destroyed by site-specific mutagenesis of Lys158 to Glu (rscu-PA-Glu158), is quantitatively converted to two-chain urokinase-type plasminogen activator (rtcu-PA-Glu158) by treatment with endoproteinase Glu-C (Staphylococcus aureus V8 proteinase). The catalytic efficiency (k2/Km) of rscu-PA-Glu158 for the activation of plasminogen is 20 times lower (0.0001 microM-1 s-1) than that of rscu-PA (0.002 microM-1 s-1). In contrast, rtcu-PA-Glu158 has very similar properties to rtcu-PA obtained by digestion of rscu-PA with plasmin, including binding to benzamidine-Sepharose, catalytic efficiency for the activation of plasminogen (0.035 microM-1 s-1 versus 0.046 microM-1 s-1) and fibrinolytic activity in an in vitro plasma clot lysis system. It is concluded that the amino acid in position 158 is a main determinant of the functional properties of single-chain urokinase-type plasminogen activator but not of the two-chain form.     

Distinction between the two basic mechanisms of cation transport in the cardiac Na(+)-Ca2+ exchange system

In order to distinguish between the Ping-Pong and sequential mechanisms of cation transport in the cardiac Na(+)-Ca2+ exchange system, the initial rates of the Nai-dependent 45Ca uptake (t = 1 s) were measured in reconstituted proteoliposomes, loaded with a Ca chelator. Under "zero-trans" conditions ([Na]o = [Ca]i = 0) at a fixed [Na]i = 10-160 mM with varying [45Ca]o = 2.5-122 microM for each [Na]i, the Km and Vmax values increased from 7.7 to 33.5 microM and from 2.3 to 9.0 nmol.mg-1.s-1, respectively. The Vmax/Km values show a +/- 2-10% deviation from the average value of 0.274 nmol.mg-1.s-1.microM-1 over the whole range of [Na]i. These deviations are within the standard error of Vmax (+/- 3-7%), Km (+/- 11-17%), and Vmax/Km (+/- 11-19%). This suggests that, under conditions in which Vmax and Km are [Na]i dependent and vary 4-5-fold, the Vmax/Km values are constant within the experimental error. In the presence of K(+)-valinomycin the Vmax/Km values are 0.85 +/- 0.17 and 1.08 +/- 0.18 nmol.mg-1.s-1.microM-1 at [Na]i = 20 and 160 mM, respectively, suggesting that under conditions of "short circuit" of the membrane potential the Vmax/Km values still exhibit the [Na]i independence. At a very low fixed [45Ca]o = 1.1 microM with varying [Na]i = 10-160 mM, the initial rates were found to be [Na]i independent. At a high fixed [45Ca]o = 92 microM the initial rates show a sigmoidal dependence on the [Na]i with Vmax = 13.8 nmol.mg-1.s-1, KmNa = 21 mM, and Hill coefficient nH = 1.5. The presented data support a Ping-Pong (consecutive) mechanism of cation transport in the Na(+)-Ca2+ exchanger.     

Acetyl phosphate as a substrate for the calcium ATPase of sarcoplasmic reticulum

Acetyl phosphate is hydrolyzed by the calcium ATPase of leaky sarcoplasmic reticulum vesicles from rabbit skeletal muscle with Km = 6.5 mM and kcat = 7.9 s-1 in the presence of 100 microM calcium (180 mM K+, 5 mM MgSO4, pH 7.0, 25 degrees C). In the absence of calcium, hydrolysis is 6% of the calcium-dependent rate at low and 24% at saturating concentrations of acetyl phosphate. Values of K0.5 for calcium are 3.5 and 2.2 microM (n = 1.6) in the presence of 1 and 50 mM acetyl phosphate, respectively; inhibition by calcium follows K0.5 = 1.6 mM (n approximately 1.1) with 50 mM acetyl phosphate and K0.5 = 0.5 mM (n approximately 1.3) with 1.5 mM ATP. The calcium-dependent rate of phosphoenzyme formation from acetyl phosphate is consistent with Km = 43 mM and kf = 32 s-1 at saturation; decomposition of the phosphoenzyme occurs with kt = 16 s-1. The maximum fraction of phosphoenzyme formed in the steady state at saturating acetyl phosphate concentrations is 43-46%. These results are consistent with kc congruent to 30 s-1 for binding of Ca2+ to E at saturating [Ca2+], to give cE.Ca2, in the absence of activation by ATP. Phosphoenzyme formed from ATP and from acetyl phosphate shows the same biphasic reaction with ADP, rate constants for decomposition that are the same within experimental error, and similar or identical activation of decomposition by ATP. It is concluded that the reaction pathways for acetyl phosphate and ATP in the presence of Ca2+ are the same, with the exception of calcium binding and phosphorylation; an alternative, faster route that avoids the kc step is available in the presence of ATP. The existence of three different regions of dependence on ATP concentration for steady state turnover is confirmed; activation of hydrolysis at high ATP concentrations involves an ATP-induced increase in kt.     

Mammalian brain phosphoproteins as substrates for calcineurin

Calcineurin, a Ca2+/calmodulin-dependent phosphoprotein phosphatase found in several tissues, is highly concentrated in mammalian brain. In an attempt to identify endogenous brain substrates for calcineurin, kinetic analyses of the dephosphorylation of several well-characterized phosphoproteins purified from brain were performed. The proteins studied were: G-substrate, a substrate for cyclic GMP-dependent protein kinase; DARPP-32, a substrate for cyclic AMP-dependent protein kinase; Protein K.-F., a substrate for a cyclic nucleotide- and Ca2+-independent protein kinase; and synapsin I, a substrate for cyclic AMP-dependent (site I) and a Ca2+/calmodulin-dependent protein kinase (site II). Calcineurin dephosphorylated each of these proteins in a Ca2+/calmodulin-dependent manner. Similar Km values were obtained for each substrate: G-substrate, 3.8 microM; DARPP-32, 1.6 microM; Protein K.-F., approximately 3 microM (S0.5); synapsin I (site I), 7.0 microM; synapsin I (site II), 4.4 microM. However, significant differences were obtained for the maximal rates of dephosphorylation. The kcat values were: G-substrate, 0.41 s-1; DARPP-32, 0.20 s-1; Protein K.-F., 0.7 s-1; synapsin I (site I), 0.053 s-1; synapsin I (site II), 0.040 s-1. Comparisons of the catalytic efficiency (kcat/Km) for each substrate indicated that DARPP-32, G-substrate, and Protein K.-F. are all potential substrates for calcineurin in vivo.