Tissue-type plasminogen activator and urokinase: differences in the reaction pattern with the active-site titrant 4-methylumbelliferyl-p-guanidinobenzoate hydrochloride

The applicability of 4-methylumbelliferyl-p-guanidinobenzoate hydrochloride (MUGB) as active-site titrant for tissue-type plasminogen activator (t-PA) was studied in comparison to urokinase. Although t-PA was capable of cleaving MUGB, active-site titration of t-PA (one-chain form as well as two-chain form) with MUGB was not possible, whereas MUGB titration of urokinase could be performed. We therefore studied the kinetics of the interaction of these two plasminogen activators with MUGB. The equilibrium dissociation constant, KS, for the interaction between MUGB and urokinase was 2.9 X 10(-6) M, and for the interaction with t-PA 3.13 X 10(-5) M. However, one main requirement for active-site titration, namely a stable acyl enzyme intermediate (ES'), was only fulfilled for MUGB urokinase but not for MUGB t-PA. Whereas for the reaction of MUGB and urokinase the first-order acylation rate constant k2 was found to be about 10(6)-times higher than the first-order deacylation rate constant k3 (k2 = 3.76 X 10(-1) s-1, k3 = 3.7 X 10(-7) s-1), the k2/k3 ratio for the reaction of MUGB and t-PA (one- and two-chain form) was 0.77 to 3.85. Therefore, urokinase and t-PA differ in their reaction with this fluorogenic substrate and MUGB cannot be used for active-site titration of tPA.     

Effect of monovalent cations on the pre-steady-state kinetic parameters of the plasma protease bovine activated protein C

Activated bovine plasma protein C (APC) was not reactive with the substrate p-nitrophenyl p-guanidinobenzoate (NPGB) in the absence of cations. In the presence of increasing concentrations of Na+, the acylation rate constant, k2,app, at 7 degrees C, progressively increased from 0.32 +/- 0.03 s-1 at 12.5 mM Na+ to 1.15 +/- 0.10 s-1 at 62.5 mM Na+. A linear dependence of the reciprocal of k2,app with [Na+]-2 was observed, indicating that at least two monovalent cation sites, or classes of sites, are necessary for the catalytic event to occur. From this latter plot, the k2,max for APC catalysis of NPGB hydrolysis, at saturating [Na+] and [NPGB], was calculated to be 1.21 +/- 0.10 s-1, and the Km for Na+ was found to be 21 +/- 3 mM. The dissociation constant, Ks, for NPGB to APC, at 7 degrees C, was not altered as [Na+] was increased, yielding a range of values of 18.5 X 10(-5) to 19.9 X 10(-5) M as [Na+] was varied from 12.5 to 62.5 mM. The deacylation rate constant, k3, for p-guanidinobenzoyl-APC hydrolysis was also independent of [Na+], with a value of (3.8 +/- 1.0) X 10(-3) s-1 in the absence of Na+ or in the presence of concentrations of Na+ up to 200 mM. Identical kinetic behavior was observed when Cs+ was substituted for Na+ in the above enzymic reaction. The pre-steady-state kinetic parameters were calculated according to the same methodology as described above.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protease nexin. Properties and a modified purification procedure

The present paper describes chemical and functional properties of protease nexin, a serine protease inhibitor released from cultured human fibroblasts. It is shown that protease nexin is actually synthesized by fibroblasts and represents about 1% of their secreted protein. Analysis of the amino acid composition of purified protease nexin indicates that it is evolutionarily related to antithrombin III and heparin cofactor II. Protease nexin contains approximately 6% carbohydrate, with 2.3% amino sugar, 1.1% neutral sugar, and 3.0% sialic acid. The Mr calculated from equilibrium sedimentation analysis is 43,000. Protease nexin is a broad specificity inhibitor of trypsin-like serine proteases. It reacts rapidly with trypsin (kassoc = 4.2 +/- 0.4 X 10(6) M-1 s-1), thrombin (kassoc = 6.0 +/- 1.3 X 10(5) M-1 s-1), urokinase (kassoc = 1.5 +/- 0.1 X 10(5) M-1 s-1), and plasmin (kassoc = 1.3 +/- 0.1 X 10(5) M-1 s-1), and slowly inhibits Factor Xa and the gamma subunit of nerve growth factor but does not inhibit chymotrypsin-like proteases or leukocyte elastase. In the presence of heparin, protease nexin inhibits thrombin at a nearly diffusion-controlled rate. Two heparin affinity classes of protease nexin can be detected. The present characterization pertains to the fraction of protease nexin having the higher affinity for heparin. The low affinity material, which is the minor fraction, is lost during purification.     

Kinetics of the reactions between streptokinase, plasmin and alpha 2-antiplasmin.

Streptokinase reacts very rapidly with human plasmin (rate constant 5.4 S 10(7) M-1 s-1) forming a 1:1 stoichiometric complex which has a dissociation constant of 5 X 10(-11) M. This plasmin-streptokinase complex is 10(5) times less reactive towards alpha 2-antiplasmin than plasmin, the inhibition rate constant being 1.4 X 10(2) M-1 s-1. The loss of reactivity of the streptokinase-plasmin complex towards alpha 2-antiplasmin is independent of the lysine binding sites in plasmin since low-Mr plasmin, which lacks these sites, and plasmin in which the sites have been blocked by 6-aminohexanoic acid, are both equally unreactive towards alpha 2-antiplasmin on reaction with streptokinase. The plasmin-streptokinase complex binds to Sepharose-lysine and Sepharose-fibrin monomer in the same fashion as free plasmin, showing that the lysine binding sites are fully exposed in the complex. Bovine plasmin is rapidly inhibited by human alpha 2-antiplasmin (k1 = 1.6 X 10(6) M-1 s-1) and similarly loses reactivity towards the inhibitor on complex formation with streptokinase (50% binding at 0.4 microM streptokinase).     

Activation of plasminogen by pro-urokinase. II. Kinetics

The kinetics of the activation of plasminogen by recombinant pro-urokinase obtained by expression of human urokinase cDNA in Escherichia coli was studied. The conversion of pro-urokinase (U) and plasminogen (P) to urokinase (u) and plasmin (p) is represented by a sequence of three reactions which each obey Michaelis-Menten kinetics, i.e. (Formula: see text). In this model, pro-urokinase formally behaves as an enzyme in Reaction I and as a substrate in reaction II. The experimentally measured overall rates of formation of urokinase and plasmin are in good agreement with those calculated from the kinetic parameters and the initial concentrations of pro-urokinase and plasminogen, confirming the validity of the model. It appears that recombinant pro-urokinase is an equally potent activator of plasminogen (k2/Km = 0.05 microM-1 s-1), as in urokinase (k"2/K"m = 0.02 microM-1 s-1). This is due to the fact that the proenzyme, which is virtually inactive toward low Mr substrates for urokinase, forms an intermediate of the Michaelis-Menten type with plasminogen, with a much higher affinity than that of the active enzyme with its substrate. This is an exceptional phenomenon among the serine proteases.     

Purification and characterization of a novel inhibitor of urokinase from human urine. Quantitation and preliminary characterization in plasma

Urokinase-related proteins in human urine occur mainly as a 1:1 complex of urokinase with an inhibitor (Stump, D. C., Thienpont, M., and Collen, D. (1986) J. Biol. Chem. 261, 1267-1273). BALB/c mice were immunized with this urokinase-urokinase inhibitor complex and spleen cells fused with mouse myeloma cells, resulting in hybridomas producing monoclonal antibodies. Three antibodies reacting with the complex but not with urokinase were utilized to develop a sensitive (0.5 ng/ml) enzyme-linked immunosorbent assay for the urokinase inhibitor, which was used for monitoring its purification by chromatography on zinc chelate-Sepharose, concanavalin A-Sepharose, SP-Sephadex C-50, and Sephadex G-100. A homogenous glycoprotein of apparent Mr 50,000 was obtained with a yield of 40 micrograms/liter urine and a purification factor of 320. One mg of the purified protein inhibited 35,000 IU of urokinase within 30 min at 37 degrees C. This protein was immunologically related to both the purified urokinase-urokinase inhibitor complex and to the inhibitor portion dissociated from it by nucleophilic dissociation. It was immunologically distinct from all known protease inhibitors, including the endothelial cell-derived fast-acting inhibitor of tissue-type plasminogen activator, the placental inhibitor of urokinase and protease nexin. In electrophoresis the protein migrated with beta-mobility. Inhibition of urokinase occurred with a second order rate constant (k) of 8 X 10(3) M-1 s-1 in the absence and of 9 X 10(4) M-1 s-1 in the presence of 50 IU of heparin/ml. The urokinase inhibitor was inactive towards single-chain urokinase-type plasminogen activator and plasmin, but it inhibited two-chain tissue-type plasminogen activator with a k below 10(3) M-1 s-1 and thrombin with a k of 4 X 10(4) M-1 s-1 in the absence and 2 X 10(5) M-1 s-1 in the presence of heparin. The concentration of this urokinase inhibitor in plasma from normal subjects determined by immunoassay was 2 +/- 0.7 micrograms/ml (mean +/- S.D., n = 25). The protein purified from plasma by immunoabsorption had the same Mr, amino acid composition, and immunoreactivity as the urinary protein. Furthermore, when urokinase was added to plasma, time-dependent urokinase-urokinase inhibitor complex formation was observed at a rate similar to that observed for the inhibition of urokinase by the purified inhibitor from urine. This urokinase inhibitor, purified from human urine, most probably represents a new plasma protease inhibitor.     

Mechanism of association of a specific aldehyde inhibitor, leupeptin, with bovine trypsin

Leupeptin (acyl peptidyl-L-argininal) is a potent inhibitor of trypsin and related proteases. We analyzed the association of leupeptim with bovine trypsin kinetically, assuming that it proceeds by a pathway which involves two steps: E + I in equilibrium K1 Complex I k-2 in equilibrium k+2 Complex II. The observed dissociation constant (K1) for the first step was 1.24 X 10(-3) M (at pH 8.2 15 degrees C) and the two first-order rate constants (k+2 and k-2) were 166 s-1 and 1.75 X 10(-3.s-1, respectively (at pH 8.2, 15 degrees C). The dissociation constant (Kd) for the whole process was calculated from these parameters to be 1.34 X 10(-8) M. This value is compatible with that determined directly by an independent static method (2.36 X 10(-8) M). We also measured Kd for the leupeptine complex of anhydrotrypsin, a trypsin derivative in which the active-site hydroxyl group is missing. The observed value was about 5 orders of magnitude larger than Kd and was rather similar to K1 in native trypsin. A elupeptin isomer which contains a D-argininal residue did not show strong affinity towards trypsin. These findings suggest that complex II consists of a covalent hemiacetal adduct formed between the serine hydroxyl group in the enzyme active site and the aldehyde group in the inhibitor. The pH dependencies of the dissociation constant and other parameters show that deprotonation of the charge-relay sustem in the active site is important for the formation and stabilization of complex II.     

On the kinetics of the reaction between human antiplasmin and a low-molecular-weight form of plasmin.

The reaction between antiplasmin (A) and a low-molecular-weight form of plasmin (P) proceeds in at least two steps: a fast reversible second-order reaction followed by a slower irreversible first-order transition, and may be represented by: P +A k1 in equilibrium k-1 PA k2 leads to PA'. The low-Mr plasmin, which is obtained by limited elastase digestion, is composed of an intact B chain and a small A chain lacking the lysine-binding sites. The k1 of the reaction is (6.5 +/- 0.5) x 10(5) M-1 s-1 which is 30--60 times smaller than that for normal plasmin and antiplasmin. The dissociation constant of the first step is 1.9 x 10(-9) M which is 10 times higher than for normal plasmin and antiplasmin. The rate constant of the second step is (4.2 +/- 0.2) x 10(-3) s-1 for both normal and low-Mr plasmin. Low Mr plasmin which has substrate bound to its active site does not react or reacts only very slowly with antiplasmin. The reaction rate, however, is only slightly influenced by 6-aminohexanoic acid in concentrations up to 1 mM which decrease the reaction rate of normal plasmin approximately 50-fold. The findings further indicate that the lysine-binding site(s) of plasmin are of great importance for the rate of its reaction with antiplasmin.     

An elastase-dependent pathway of plasminogen activation

In reaction mixtures containing Glu-plasminogen, alpha 2-antiplasmin, and tissue plasminogen activator or urokinase, either pancreatic or leukocyte elastase enhances the rate of plasminogen activation by 2 or more orders of magnitude. This effect is the consequence of several reactions. (a) In concentrations on the order of 100 nM, elastase degrades plasminogen within 10 min to yield des-kringle1-4-plasminogen (mini-plasminogen), which is 10-fold more efficient than Glu-plasminogen as a substrate for plasminogen activators. Des-kringle1-4-plasminogen is insensitive to cofactor activities of fibrin(ogen) fragments or an endothelial cell cofactor. (b) Des-kringle1-4-plasmin is one-tenth as sensitive as plasmin to inhibition by alpha 2-antiplasmin: k" = 10(6) M-1 s-1 versus 10(7) M-1 s-1. (c) alpha 2-Antiplasmin is disabled efficiently by elastase, with a k" of 20,000 M-1 s-1. The elastase-dependent reactions are not influenced by 6-aminohexanoate. In diluted (10-fold) blood plasma, the capacity of endogenous inhibitors to block plasmin expression is suppressed by 30 microM elastase. It is proposed that elastases provide an alternative pathway for Glu-plasminogen activation and a mechanism for controlling initiation of fibrinolysis by urokinase-type plasminogen activators.     

Photoinduced electron-transfer kinetics of singly labeled ruthenium bis(bipyridine) dicarboxybipyridine cytochrome c derivatives

Cytochrome c derivatives labeled at specific lysine amino groups with ruthenium bis(bipyridine) dicarboxybipyridine [RuII(bpy)2(dcbpy)] were prepared by using the procedure described previously [Pan, L. P., Durham, B., Wolinska, J., & Millett, F. (1988) Biochemistry 27, 7180-7184]. Four additional singly labeled derivatives were purified, bringing the total number to 10. These derivatives have a strong luminescence emission centered at 662 nm arising from the excited state, RuII*. Transient absorption spectroscopy was used to directly measure the rate constants for the photoinduced electron-transfer reaction from RuII* to the ferric heme group (k1) and for the thermal back-reaction from the ferrous heme group to RuIII (k2). The rate constants were found to be k1 = 14 X 10(6) s-1 and k2 = 24 X 10(6) s-1 for the derivative modified at lysine 72, which has a distance of 8-16 A between the ruthenium and heme groups. Similar rate constants were found for the derivatives modified at lysines 13 and 27, which have distances of 6-12 A separating the ruthenium and heme groups. The rate constants were significantly slower for the derivatives modified at lysine 25 (k1 = 1 X 10(6) s-1, k2 = 1.5 X 10(6) s-1) and lysine 7 (k1 = 0.3 X 10(6) s-1, k2 = 0.5 X 10(6) s-1), which have distances of 9-16 A. Transients due to photoinduced electron transfer could not be detected for the remaining derivatives, which have larger distances between the ruthenium and heme groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduction of laccase type 1 copper by 3,4-dihydroxyphenylalanine and other catechol derivatives

3,4-Dihydroxyphenylalanine (DOPA) is not a preferred substrate of Rhus vernicifera laccase, as rate constants for the anaerobic reduction of the type 1 cupric atom by L-DOPA (6.3 X 10(1) M-1 s-1), D-DOPA (2.6 X 10(1) M-1 s-1), and L-DOPA methyl ester (2.6 X 10(1) M-1 s-1) are considerably smaller than k1 (catechol) (7 X 10(2) M-1 s-1) and rate constants characteristic of numerous other nonphysiological organic substrates (25 degrees C, pH 7.0, I = 0.5 M). The reactions of DOPA derivatives with laccase are unique, however, in that a two-term rate law pertains: kobsd = k0 + k1[phenol]; k0(L-DOPA) = 7 X 10(-2) s-1. The reactivities of other catechol derivatives (pyrogallol, gallic acid, and methyl gallate) with laccase type 1 copper were also examined.     

Binding of Ca2+ to the calcium adenosinetriphosphatase of sarcoplasmic reticulum

The binding of Ca2+ and the resulting change in catalytic specificity that allows phosphorylation of the calcium ATPase of sarcoplasmic reticulum by ATP were examined by measuring the amount of phosphoenzyme formation from [32P]ATP, or 45Ca incorporation into vesicles, after the simultaneous addition of ATP and EGTA at different times after mixing enzyme and Ca2+ (25 degrees C, pH 7.0, 5 mM MgSO4, 0.1 M KCl). A "burst" of calcium binding in the presence of high [Ca2+] gives approximately 12% phosphorylation and internalization of two Ca2+ at very short times after the addition of Ca2+ with this assay. This shows that calcium binding sites are available on the cytoplasmic-facing side of the free enzyme. Calcium binding to these sites induces the formation of cE.Ca2, the stable high-affinity form of the enzyme, with k = 40 s-1 at saturating [Ca2+] and a half-maximal rate at approximately 20 microM Ca2+ (from Kdiss = 7.4 X 10(-7) M for Ca.EGTA). The formation of cE.Ca2 through a "high-affinity" pathway can be described by the scheme E 1 in equilibrium cE.Ca1 2 in equilibrium cE.Ca2, with k1 = 3 X 10(6) M-1 s-1, k2 = 4.3 X 10(7) M-1 s-1, k-1 = 30 s-1, k-2 = 60 s-1, K1 = 9 X 10(-6) M, and K2 = 1.4 X 10(-6) M. The approach to equilibrium from E and 3.2 microM Ca2+ follows kobsd = kf + kr = 18 s-1 and gives kf = kr = 9 s-1. The rate of exchange of 45Ca into the inner position of cE.Ca2 shows an induction period and is not faster than the approach to equilibrium starting with E and 45Ca. The dissociation of 45Ca from the inner position of cE.45Ca.Ca in the presence of 3.2 microM Ca2+ occurs with a rate constant of 7 s-1. These results are inconsistent with a slow conformational change of free E to give cE, followed by rapid binding-dissociation of Ca2+.     

Peptide models of protein metastable binding sites: competitive kinetics of isomerization and hydrolysis

alpha 2-Macroglobulin and the complement components C3 and C4 each contain a metastable binding site that is essential for covalent attachment. Two cyclic peptides are useful models of these unusual protein sites. Five-membered lactam 1 (CH3CO-Gly-Cys-Gly-Glu-Glp-Asn-NH2) contains an internal residue of pyroglutamic acid (Glp). Fifteen-membered thiolactone 2 (CH3CO-Gly-Cys-Gly-Glu-Glu-Asn-NH2 15-thiolactone) contains a thiol ester bond between Cys-2 and Glu-5. These isomeric hexapeptides are spontaneously interconverted in water. Competing with the two isomerization reactions are three reactions involving hydrolysis of 1 and 2. These five processes were found to occur simultaneously under physiologic conditions (phosphate-buffered saline, pH 7.3, 37 degrees C). Best estimates of the five rate constants for these apparent first-order reactions were obtained by comparing the observed molar percentages of peptides 1-4 with those calculated from a set of exponential equations. Both isomerization reactions (ring expansion of 1 to 2, k1 = 6.4 X 10(-5) s-1; ring contraction of 2 to 1, k-1 = 69 X 10(-5) s-1) proceeded faster than any of the hydrolysis reactions: alpha-cleavage of 1 with fragmentation to form dipeptide 3 (k2 = 3.3 X 10(-5) s-1), gamma-cleavage of 1 with ring opening to yield mercapto acid 4 (k3 = 0.35 X 10(-5) s-1), and hydrolysis of 2 with ring opening to give 4 (k4 = 1.9 X 10(-5) s-1). The isomerization rate ratio (k1/k-1 = 10.9) agreed with the isomer ratio at equilibrium (1:2 = 11 starting from 1 and 10 starting from 2). The alpha/gamma regioselectivity ratio (k2/k3 = 9.7) for hydrolysis of the internal Glp residue of 1 was consistent with results for model tripeptides. Part of the chemistry of the protein metastable binding sites can be explained by similar isomerization and hydrolysis reactions.     

Serine proteinase inhibition by the active site titrant N alpha-(N, N-dimethylcarbamoyl)-alpha-azaornithine p-nitrophenyl ester. A comparative study.

Kinetics for the hydrolysis of the chromogenic active-site titrant N alpha-(N,N-dimethylcarbamoyl)-alpha-azaornithine p-nitrophenyl ester (Dmc-azaOrn-ONp) catalysed by bovine beta-trypsin, bovine alpha-thrombin, bovine Factor Xa, human alpha-thrombin, human Factor Xa, human Lys77-plasmin, human urinary kallikrein, Mr 33 000 and Mr 54 000 species of human urokinase, porcine pancreatic beta-kallikrein-A and -B and Ancrod (the coagulating serine proteinase from the Malayan pit viper Agkistrodon rhodostoma venom) have been obtained between pH 6.0 and 8.0, at 21.0 degrees C, and analysed in parallel with those for the enzymatic cleavage of N alpha-(N,N-dimethylcarbamoyl)-alpha-azalysine p-nitrophenyl ester (Dmc-azaLys-ONp). The enzyme kinetics are consistent with the minimum three-step catalytic mechanism of serine proteinases, the rate-limiting step being represented by the deacylation process. Bovine beta-trypsin kinetics are modulated by the acid-base equilibrium of the His57 catalytic residue (pKa approximately 6.9). Dmc-azaOrn-ONp and Dmc-azaLys-ONp bind stoichiometrically to the serine proteinase active site, and allow the reliable determination of the active enzyme concentration between 1.0 x 10-6 M and 3.0 x 10-4 M. The affinity and the reactivity for Dmc-azaOrn-ONp (expressed by Ks and k+2/Ks, respectively) of the serine proteinases considered are much lower than those for Dmc-azaLys-ONp. The very different affinity and reactivity properties for Dmc-azaOrn-ONp and Dmc-azaLys-ONp have been related to the different size of the ornithine/lysine side chains, and to the ensuing different positioning of the active-site titrants upon binding to the enzyme catalytic centre (i.e. to P1-S1 recognition). These data represent the first detailed comparative investigation on the catalytic properties of serine proteinases towards an ornithine derivative (i. e. Dmc-azaOrn-ONp).     

Enzymatic hydrolysis of p-nitroacetanilide: mechanistic studies of the aryl acylamidase from Pseudomonas fluorescens.

Aryl acylamidase (EC 3.1.5.13; AAA) catalyzes the hydrolysis of p-nitroacetanilide (PNAA) via the standard three-step mechanism of serine hydrolases: binding of substrate (K(s)), acylation of active-site serine (k(acyl)), and hydrolytic deacylation (k(deacyl)). Key mechanistic findings that emerged from this study include that (1) AAA requires a deprotonated base with a pK(a) of 8.3 for expression of full activity toward PNAA. Limiting values of kinetic parameters at high pH are k(c) = 7 s(-1), K(m) = 20 microM, and k(c)/K(m) = 340 000 M(-1) s(-1). (2) At pH 10, where all the isotope effects were conducted, k(c) is equally rate-limited by k(acyl) and k(deacyl). (3) The following isotope effects were determined: (D)()2(O)(k(c)/K(m)) = 1.7 +/- 0.2, (D)()2(O)k(c) = 3.5 +/- 0.3, and (beta)(D)(k(c)/K(m)) = 0.83 +/- 0.04, (beta)(D)k(c) = 0.96 +/- 0.01. These values, together with proton inventories for k(c)/K(m) and k(c), suggest the following mechanism: (i) The initial binding of substrate to enzyme to form the Michaelis complex is accompanied by solvation changes that generate solvent deuterium isotope effects originating from hydrogen ion fractionation at multiple sites on the enzyme surface. (ii) From within the Michaelis complex, the active site serine attacks the carbonyl carbon of PNAA with general-base catalysis to form a substantially tetrahedral transition state enroute to the acyl-enzyme. (iii) Finally, deacylation occurs through a process involving a rate-limiting solvent isotope effect, generating conformational change of the acyl-enzyme that positions the carbonyl bond in a polarizing environment that is optimal for attack by water.     

Kinetic studies on the interaction of eglin c with human leukocyte elastase and cathepsin G

We have investigated the inhibition of human leukocyte elastase and cathepsin G by recombinant Eglin c under near physiological conditions. The association rate constants k on of Eglin c for elastase and cathepsin G were 1.3 X 10(7) M-1 s-1 and 2 X 10(6) M-1 s-1, respectively. Under identical conditions, the k on for the association of human plasma alpha 1-proteinase inhibitor with the two leukocproteinases were 2.4 X 10(7) M-1 s-1 and 10(6) M-1 s-1, respectively. The consistency of these data could be verified using a set of competition experiments. The elastase-Eglin c interaction was studied in greater detail. The dissociation rate constant k off was determined by trapping of free elastase from an equilibrium mixture of elastase and Eglin c with alpha 1-proteinase inhibitor or alpha 2-macroglobulin. The rate of dissociation was very low (k off = 3.5 X 10(-5) s-1). The calculated equilibrium dissociation constant of the complex, Ki(calc) = k off/k on, was found to be 2.7 X 10(-12) M. Ki was also measured by adding elastase to mixtures of Eglin c and substrate and determining the steady-state rates of substrate hydrolysis. The Ki determined from these experiments (7.5 X 10(-11) M) was significantly higher than Ki(calc). This discrepancy might be explained by assuming that the interaction of Eglin c with elastase involves two steps: a fast binding reaction followed by a slow isomerization step. From the above kinetic constants it may be inferred that at a therapeutic concentration of 5 X 10(-7) M, Eglin c will inhibit leukocyte elastase in one second and will bind this enzyme in a "pseudo-irreversible" manner.     

Molecular mechanisms of olfactory reception. VI. Kinetic characteristics of camphor interaction with binding sites of rat olfactory epithelium

Camphor binding to a possible receptor of rat olfactory epithelium has been studied within the ligand concentration range 10(-11)-10(-6) M. At these concentrations camphor is bound by a set of receptors. They are distinguished by both the affinity to the ligand (K1 = 5 X 10(-10) M, K2 = 3.5 X 10(-8) M, K3 approximately equal to 10(-6) M) and their amount in the epithelium. The differences in the affinities are due to different values of the association rate constant of camphor (k1), which varies from 10(6) M-1 X s-1 for the receptors with high affinity up to 2 X 10(2) M-1 X s-1 for those with low affinity. These data are discussed in terms of equilibrium and kinetic models of the receptor-stimulus interaction.     

Urokinase: a chemotactic factor for polymorphonuclear leukocytes in vivo

The effects of injecting urokinase into subdermal air sacs on the back of mice was studied. Urokinase was leukotactic in the concentration range of 2 X 10(-13) to 2 X 10(-15) M. This response was absolutely dependent on the enzyme activity of the serine esterase, but was found to be independent of generation of the chemotactic complement split product C5a. At high doses of urokinase (greater than 2 X 10(-12) M), no cellular infiltration was observed. Injection of 2 X 10(-10) M urokinase i.p. led to the systemic desensitization of mice when challenged in the skin with a lower dose (2 X 10(-14) M) of urokinase. Urokinase desensitization did not alter the ability of mice to respond to the chemical chemotactic factor f-met-leu-phe or to respond to C5a-dependent chemotactic stimuli. Urokinase desensitized mice failed to demonstrate a chemotactic response to nerve growth factor, thrombin, plasmin, or factor X activating enzyme, all of which were chemotactic in non-urokinase pre-treated animals. The results of these studies indicate the presence of three physiologically independent inflammatory pathways in mice: independent of C5 and not influenced by pretreatment with urokinase, independent of C5 and inhibited by pretreatment with urokinase, and dependent on C5 and not influenced by pretreatment with urokinase.     

Reversible, slow, tight-binding inhibition of human leukocyte elastase

CBz-Ala-Ala-Pro-ambo-Val-CF3 (1) was synthesized. The compound inhibits human Leucocyte elastase with Ki = 1.0 x 10(-9) M. This inhibitor is reversible, slow, tight-binding inhibitor with k on = 2 x 10(4) M-1 s-1 k off = 1.9 x 10(-5) s-1. For the solubilization of elastin by HLE by 1 I.C. 50 = 110 nM. This inhibitor is the most effective aldehyde or ketone inhibitor of a serine proteinase yet described.     

A kinetic study of protein-protein interactions.

Kinetic studies have been carried out of the monomer-dimer interaction of insulin, beta-lactoglobulin, and alpha-chymotrypsin using stopped-flow and temperature-jump techniques. The pH indicators bromothymol blue, bromophenol blue, and phenol red were used to monitor pH changes associated with the monomer-dimer interaction. In all three cases a kinetic process was observed which could be attributed to a simple monomer-dimer equilibrium, and association (k1) and dissociation (k-1) rate constants were determined. The results obtained are as follows: for insulin at 23 degrees C, pH 6.8, 0.125 M KNO3, k1 = 1.14 X 10(8) M-1 s-1, k-1 - 1.48 X 10(4)s(-1); for beta-lactoglobulin AB at 35 degrees C, pH 3.7, 0.025 M KNO3, d1 = 4.7 X 10(4) M-1 s-1, k-1 = 2.1 s-1; for alpha-chymotrypsin at 25 degreesC, pH 4.3, 0.05 M KNO3 k1 - 3.7 X 10(3) M-1 s-1, k-1 - 0.68 s-1. The kinetic behavior of the separated beta-lactoglobulin A and B was similar to that of the mixture. In the case of chymotrypsin, bromophenol blue was found to activate the enzyme catalyzed hydrolysis of p-nitrophenyl acetate, and a rate process was observed with the temperature jump which could be attributed to a conformational change of the indicator-protein complex. The association rate constant for dimer formation of insulin approaches the value expected for a diffusion-controlled process, while the values obtained for the other two proteins are below those expected for a diffusion-controlled reaction unless unusally large steric and electrostatic effects are present.