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.     

Sodium binding sites of gramicidin A: sodium-23 nuclear magnetic resonance study.

In ethanol-water mixtures (90:10), the gramicidin dimer binds Na+ cations at well-defined sites, with a binding constant K = 4 M-1. Partial desolvation of Na+ occurs upon binding, as judged from the magnitude of the quadrupolar coupling constant (1.7 MHz) for bound sodium. The binding sites are identified with the outer sites flanking the channel entrances. The rate constants for binding and release are k+ less than or equal to 2.2 X 10(9) M-1 s-1 and k- less than or equal to 5.5 X 10(8) s-1, respectively.     

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+.     

Mechanism of reaction of myeloperoxidase with hydrogen peroxide and chloride ion.

The reaction of myeloperoxidase compound I (MPO-I) with chloride ion is widely assumed to produce the bacterial killing agent after phagocytosis. Two values of the rate constant for this important reaction have been published previously: 4.7 x 106 M-1.s-1 measured at 25 degrees C [Marquez, L.A. and Dunford, H.B. (1995) J. Biol. Chem. 270, 30434-30440], and 2.5 x 104 M-1.s-1 at 15 degrees C [Furtmüller, P.G., Burner, U. & Obinger, C. (1998) Biochemistry 37, 17923-17930]. The present paper is the result of a collaboration of the two groups to resolve the discrepancy in the rate constants. It was found that the rate constant for the reaction of compound I, generated from myeloperoxidase (MPO) and excess hydrogen peroxide with chloride, decreased with increasing chloride concentration. The rate constant published in 1995 was measured over a lower chloride concentration range; the 1998 rate constant at a higher range. Therefore the observed conversion of compound I to native enzyme in the presence of hydrogen peroxide and chloride ion cannot be attributed solely to the single elementary reaction MPO-I + Cl- --> MPO + HOCl. The simplest mechanism for the overall reaction which fit the experimental data is the following: MPO+H2O2 ⇄k-1k1 MPO-I+H2O MPO-I+Cl- ⇄k-2k2 MPO-I-Cl- MPO-I-Cl- -->k3 MPO+HOCl where MPO-I-Cl- is a chlorinating intermediate. We can now say that the 1995 rate constant is k2 and the corresponding reaction is rate-controlling at low [Cl-]. At high [Cl-], the reaction with rate constant k3 is rate controlling. The 1998 rate constant for high [Cl-] is a composite rate constant, approximated by k2k3/k-2. Values of k1 and k-1 are known from the literature. Results of this study yielded k2 = 2.2 x 106 M-1.s-1, k-2 = 1.9 x 105 s-1 and k3 = 5.2 x 104 s-1. Essentially identical results were obtained using human myeloperoxidase and beef spleen myeloperoxidase.     

Fructose 1,6-bisphosphate aldolase from rabbit muscle. The isomerization of the enzyme-dihydroxyacetone phosphate complex.

The formation and dissociation of the aldolase-dihydroxyacetone phosphate complex were studied by following changes in A240 [Topper, Mehler & Bloom (1957), Science 126, 1287-1289]. It was shown that the enzyme-substrate complex (ES) slowly isomerizes according to the following reaction: (formula: see text) the two first-order rate constants for the isomerization step being k+2 = 1.3s-1 and k-2 = 0.7s-1 at 20 degrees C and pH 7.5. The dissociation of the ES complex was provoked by the addition of the competitive inhibitor hexitol 1,6-bisphosphate. At 20 degrees C and pH 7.5, k+1 was 4.7 X 10(6)M-1-S-1 and k-1 was 30s-1. Both the ES and the ES* complexes react rapidly with 1.7 mM-glyceraldehyde 3-phosphate, the reaction being practically complete in 40 ms. This shows that the ES* complex is not a dead-end complex. Evidence was also provided that aldolase binds and utilizes only the keto form of dihydroxyacetone phosphate.     

Rate of ATP synthesis by dynein

The rates of ATP synthesis and release by the dynein ATPase were determined in order to estimate thermodynamic parameters according to the pathway: (Formula: see text). Dynein was incubated with high concentrations of ADP and Pi to drive the net synthesis of ATP, and the rate of ATP production was monitored fluorometrically by production of NADPH through a coupled assay using hexokinase and glucose-6-phosphate dehydrogenase. The turnover number for the rate of release of ATP from 22S dynein was 0.01 s-1 per site at pH 7.0, 28 degrees C, assuming a molecular weight of 750 000 per site. The same method gave a rate of ATP synthesis by myosin subfragment 1 of 3.4 X 10(-4) s-1 at pH 7.0, 28 degrees C. The rate of ATP synthesis at the active site was estimated from the time dependence of medium phosphate-water oxygen exchange. Dynein was incubated with ADP and [18O] Pi, and the rate of loss of the labeled oxygen to water was monitored by 31P NMR. A partition coefficient of 0.31 was determined, which is equal to k-2/(k-2 + k3). Assuming k3 = 8 s-1 [Johnson, K.A. (1983) J. Biol. Chem. 258, 13825-13832], k-2 = 3.5 s-1. From the rates of ATP binding and hydrolysis measured previously (Johnson, 1983), the equilibrium constants for ATP binding and hydrolysis could be calculated: K1 = 5 X 10(7) M-1 and K2 = 14.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nerve growth factor receptors. Characterization of two distinct classes of binding sites on chick embryo sensory ganglia cells

Steady state and kinetic studies on the binding of 125I-beta nerve growth factor (NGF) to single cells from sensory ganglia of 8-day-old chick embryos show two distinct, saturable binding sites with dissociation constants of Kd(I) = 2.3 X 10(-11) M and Kd(II) = 1.7 X 10(-9) M. The difference in the affinities is due to different rate constants of dissociation (k-1(I) = 10(-3) s-1, k-1(II) = 2 X 10(-1 s-1). The association to both sites is apparently diffusion controlled (k+1(I) = 4.8 X 10(7) M-1s-1, k+2(II) = 10(7) to 10(8) M-1s-1). The binding of betaNGF to both sites is specific, since none of a number of hormones or proteins tested compete for the binding of 125I-betaNGF to either of those two sites. The heterogeneity of the binding of 125I-betaNGF is not due to heterogeneity of the 125I-betaNGF preparation nor to a negatively cooperative binding. In experiments where the dissociation of 125I-betaNGF is induced by the addition of saturating amounts of unlabeled betaNGF, the ratio of the 125I-betaNGF released with either of the two dissociation rate constants is solely dependent on the occupancy of the two sites before dissociation is started and is independent of the total occupancy of the sites during dissociation. The rate of dissociation of 125I-betaNGF from the higher affinity binding site I is accelerated by unlabeled betaNGF under conditions where the occupancy is both increased and decreased. Although the dissociation characteristics of 125I-beta NGF change with increasing times of exposure of the cells to the ligand, and 125I-beta NGF is degraded after it binds to the cells, these secondary processes do not interfere with the analysis of the binding data. At the lowest concentration of 125I-beta NGF used for the analysis less than 10% of the 125I-beta NGF is degraded. Both kinetic and steady state binding data reveal the two NGF binding sites at 2 degrees C as well as at 37 degrees C.     

Kinetics of slow, tight-binding inhibitors of angiotensin converting enzyme

Five phosphorus-containing inhibitors of angiotensin converting enzyme were found to exhibit slow, tight-binding kinetics by using furanacryloyl-L-phenylalanylglycylglycine as substrate at pH 7.50 and T = 25 degrees C. Two of the inhibitors, (O-ethylphospho)-Ala-Pro (2) and (O-isopropylphospho)-Ala-Pro (3), are found to follow at minimum a two-step mechanism of binding (mechanism B) to the enzyme. This mechanism consists of an initial fast formation of a weaker enzyme-inhibitor complex (Ki = 130 nM for 2 and 180 nM for 3) followed by a slow reversible isomerization to a tighter complex with measurable forward (K3) and reverse (k4) rate constants (k3 = 4.5 X 10(-2) s-1 for 2 and 5.4 X 10(-2) s-1 for 3; k4 = 9.2 X 10(-3) s-1 for 2 and 3.5 X 10(-3) s-1 for 3). For the remaining three inhibitors, phospho-Ala-Pro (1), (O-benzyl-phospho)-Ala-Pro (4), and (P-phenethylphosphono)-Ala-Pro (5), a one-step binding mechanism (mechanism A) is observed under the conditions of the experiment. The second-order rate constants k1 (M-1 s-1) for the binding of these inhibitors to converting enzyme are found to have values more than 3 orders of magnitude lower than the diffusion-controlled limit for a bimolecular reaction involving the enzyme, viz., 3.9 X 10(5) for 1, 2.2 X 10(5) for 4, and 4.8 X 10(5) for 5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics and mechanism of bilirubin binding to human serum albumin

The kinetics of bilirubin binding to human serum albumin at pH 7.40, 4 degrees C, was studied by monitoring changes in bilirubin absorbance. The time course of the absorbance change at 380 nm was complex: at least three kinetic events were detected including the bimolecular association (k1 = 3.8 +/- 2.0 X 10(7) M-1 S-1) and two relaxation steps (52 = 40.2 +/- 9.4 s-1 and k3 = 3.8 +/- 0.5 s-1). The presence of the two slow relaxations was confirmed under pseudo-first order conditions with excess albumin. Curve-fitting procedures allowed the assignment of absorption coefficients to the intermediate species. When the bilirubin-albumin binding kinetics was observed at 420 nm, only the two relaxations were seen; apparently the second order association step was isosbestic at this wavelength. The rate of albumin-bound bilirubin dissociation was measured by mixing the pre-equilibrated human albumin-bilirubin complex with bovine albumin. The rate constant for bilirubin dissociation measured at 485 nm was k-3 = 0.01 s-1 at 4 degrees C. A minimum value of the equilibrium constant for bilirubin binding to human albumin determined from the ratio k1/k-3 is therefore approximately 4 X 10(9) M-1.     

Rapid spectral scan and stopped-flow studies of carbon monoxide binding to bovine adrenocortical cytochrome P-450scc

Carbon monoxide binding with both cholesterol-free (low-spin) and cholesterol-bound (high-spin) reduced forms of purified cytochrome P-450scc has been investigated by rapid-scan and stopped-flow spectrometry. CO binding occurs within 150 ms at 25 degrees C for both forms of P-450scc, with a typical absorption maximum at 450 nm. Isosbestic points occur at the following wavelengths: between reduced-CO and reduced cholesterol-free P-450scc at 434 and 471 nm; between reduced-CO and reduced cholesterol-bound P-450scc at 433 and 469 nm. Both the 'on' (k1) and 'off' rate constants (k-1) are found to be independent of pH between pH 5 and 9. The mean values of k1 for cholesterol-free (1.8 +/- 0.2) X 10(5) M-1 X s-1) and cholesterol-bound [1.9 +/- 0.1) X 10(5) M-1 X s-1) P-450scc are almost identical, while the mean value of k-1 for the former [2.3 +/- 0.3) X 10 s-1) is about double that of the latter [1.2 +/- 0.1) X 10 s-1). This suggests the instability of the reduced-CO complex in the absence of cholesterol.     

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.     

The reaction between cytochrome c1 and cytochrome c

The kinetics of electron transfer between the isolated enzymes of cytochrome c1 and cytochrome c have been investigated using the stopped-flow technique. The reaction between ferrocytochrome c1 and ferricytochrome c is fast; the second-order rate constant (k1) is 3.0 . 10(7) M-1 . s-1 at low ionic strength (I = 223 mM, 10 degrees C). The value of this rate constant decreases to 1.8 . 10(5) M-1 . s-1 upon increasing the ionic strength to 1.13 M. The ionic strength dependence of the electron transfer between cytochrome c1 and cytochrome c implies the involvement of electrostatic interactions in the reaction between both cytochromes. In addition to a general influence of ionic strength, specific anion effects are found for phosphate, chloride and morpholinosulphonate. These anions appear to inhibit the reaction between cytochrome c1 and cytochrome c by binding of these anions to the cytochrome c molecule. Such a phenomenon is not observed for cacodylate. At an ionic strength of 1.02 M, the second-order rate constants for the reaction between ferrocytochrome c1 and ferricytochrome c and the reverse reaction are k1 = 2.4 . 10(5) M-1 . s-1 and k-1 = 3.3 . 10(5) M-1 . s-1, respectively (450 mM potassium phosphate, pH 7.0, 1% Tween 20, 10 degrees C). The 'equilibrium' constant calculated from the rate constants (0.73) is equal to the constant determined from equilibrium studies. Moreover, it is shown that at this ionic strength, the concentrations of intermediary complexes are very low and that the value of the equilibrium constant is independent of ionic strength. These data can be fitted into the following simple reaction scheme: cytochrome c2+1 + cytochrome c3+ in equilibrium or formed from cytochrome c3+1 + cytochrome c2+.     

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.     

Kinetic study of the reaction between vitamin E radical and alkyl hydroperoxides in solution

Kinetic study of the reaction between vitamin E radical and alkyl hydroperoxides has been performed, as a model for the reactions of lipid hydroperoxides with vitamin E radical in biological systems. The rates of reaction of hydroperoxides (n-butyl hydroperoxide 1, sec-butyl hydroperoxide 2, and tert-butyl hydroperoxide 3) with vitamin E radical (5,7-diisopropyl-tocopheroxyl 4) in benzene solution have been determined spectrophotometrically. The second-order rate constants, k-1, obtained are 1.34 x 10(-1) M-1s-1 for 1, 2.42 x 10(-1) M-1s-1 for 2, and 3.65 x 10(-1) M-1s-1 for 3 at 25.0 degrees C. The result indicates that the rate constants increase as the total electron donating capacity of the alkyl substituents at alpha-carbon atom of hydroperoxides increases. The above rates, k-1, are about seven order of magnitude lower than those, k1, for the reaction of vitamin E with peroxyl radical.     

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.     

CAMP-dissociation kinetics in hormone-dependent and -independent rat mammary tumor cytosols

Cytosols from 7, 12-dimethylbenz (alpha) anthracene-induced rat mammary tumors which exhibit different hormone-responsiveness were compared with respect to their cAMP-dissociation kinetics. At 22 degree C, pH 4.5, 1 micrometer cAMP, hormone-dependent mammary tumors exhibited monophasic dissociation rates with a rate constant of k-1 = 0.06 min-1. In contrast, hormone-independent mammary tumors exhibited biphasic dissociation curves with rate constants of k-1 = 0.47 and k-2 = 0.06 min-1. The binding of cAMP was completely reversible; radio-labeled ligand was completely dissociated by 1mM nonradioactive cAMP; the binding protein could be reassociated to its original binding level after dextran-coated charcoal adsorption. The mammary cytosols exhibited specific binding for cAMP which could be displaced partially by cGMP but not by ATP, ADP, AMP, or adenosine. Receptor inactivation during the course of incubation was negligible. Both mammary tissue cytosols exhibited similar association rates at 22 degree C, pH 4.5, 1 micrometer cAMP (k+1 = 5-7 x 10(5)M-1 min-1). These data indicate that mammary tissues exhibit 2 cAMP dissociation rates. Hormone-dependent mammary tumors exhibit a dissociation constant of a high affinity binding site (k-1/k+1 = 0.07 micrometer) whereas hormone-independent mammary tumors exhibit dissociation constants of one high affinity (k-1/k+1 = 0.07 micrometer) and a second low affinity site (k-1/k+1 = 0.05 micrometer).     

Studies on the effect of serine protease inhibitors on activated contact factors. Application in amidolytic assays for factor XIIa, plasma kallikrein and factor XIa

Amidolytic assays have been developed to determine factor XIIa, factor XIa and plasma kallikrein in mixtures containing variable amounts of each enzyme. The commercially available chromogenic p-nitroanilide substrates Pro-Phe-Arg-NH-Np (S2302 or chromozym PK), Glp-Pro-Arg-NH-Np (S2366), Ile-Glu-(piperidyl)-Gly-Arg-NH-Np (S2337), and Ile-Glu-Gly-Arg-NH-Np (S2222) were tested for their suitability as substrates in these assays. The kinetic parameters for the conversion of S2302, S2222, S2337 and S2366 by beta factor XIIa, factor XIa and plasma kallikrein indicate that each active enzyme exhibits considerable activity towards a number of these substrates. This precludes direct quantification of the individual enzymes when large amounts of other activated contact factors are present. Several serine protease inhibitors have been tested for their ability to inhibit those contact factors selectively that may interfere with the factor tested for. Soybean trypsin inhibitor very efficiently inhibited kallikrein, inhibited factor XIa at moderate concentrations, but did not affect the amidolytic activity of factor XIIa. Therefore, this inhibitor can be used to abolish a kallikrein and factor XIa contribution in a factor XIIa assay. We also report the rate constants of inhibition of contact activation factors by three different chloromethyl ketones. D-Phe-Pro-Arg-CH2Cl was moderately active against contact factors (k = 2.2 X 10(3) M-1 s-1 at pH 8.3) but showed no differences in specifity. D-Phe-Phe-Arg-CH2Cl was a very efficient inhibitor of plasma kallikrein (k = 1.2 X 10(5) M-1 s-1 at pH 8.3) whereas it slowly inhibited factor XIIa (k = 1.4 X 10(3) M-1 s-1) and factor XIa (k = 0.11 X 10(3) M-1 s-1). Also Dns-Glu-Gly-Arg-CH2Cl was more reactive towards kallikrein (k = 1.6 X 10(4) M-1 s-1) than towards factor XIIa (k = 4.6 X 10(2) M-1 s-1) and factor XIa (k = 0.6 X 10(2) M-1 s-1). Since Phe-Phe-Arg-CH2Cl is highly specific for plasma kallikrein it can be used in a factor XIa assay selectively to inhibit kallikrein. Based on the catalytic efficiencies of chromogenic substrate conversion and the inhibition characteristics of serine protease inhibitors and chloromethyl ketones we were able to develop quantitative assays for factor XIIa, factor XIa and kallikrein in mixtures of contact activation factors.     

Kinetic studies of calcium binding to parvalbumins from bullfrog skeletal muscle

In addition to steady-state properties of calcium binding to parvalbumins, kinetic studies are required for adequate evaluation of the physiological roles of parvalbumins. By using a dual-wavelength spectrophotometer equipped with a stopped-flow accessory, the transient kinetics of calcium binding to parvalbumins (PA-1 and 2) from bullfrog skeletal muscle was examined at 20 degrees C in medium containing 20 mM MOPS-KOH, pH 6.80, 0.13 mM tetramethylmurexide, 25 microM CaCl2, metal-deprived PA-1 or PA-2, various concentrations of Mg2+, and KCl to adjust the ionic strength of the medium to 0.106. The results can be explained in terms of the following rate constants under the conditions mentioned above when a second-order kinetic scheme is assumed. For PA-1, the association and apparent dissociation rate constants for Ca2+ are 1.5 X 10(7) M-1 X s-1 and 1.5 s-1, respectively, or more. The rate constants for Mg2+ are 7,500 M-1 X s-1 and 5-6 s-1, respectively. For PA-2, the rate constants for Ca2+ are 7 X 10(6) M-1 X s-1 and 1.16 s-1, respectively, and those for Mg2+ are 3,500 M-1 X s-1 and 3.5-4 s-1, respectively. Increased affinities for Ca2+ and Mg2+ at 10 degrees C are largely due to decreased apparent dissociation rate constants for these divalent cations, because no significant change in the association rate constants was found.     

Nitrogenase of Klebsiella pneumoniae. Kinetic studies on the Fe protein involving reduction by sodium dithionite, the binding of MgADP and a conformation change that alters the reactivity of the 4Fe-4S centre.

The kinetics of reduction of indigocarmine-dye-oxidized Fe protein of nitrogenase from Klebsiella pneumoniae (Kp2ox) by sodium dithionite in the presence and absence of MgADP were studied by stopped-flow spectrophotometry at 23 degrees C and at pH 7.4. Highly co-operative binding of 2MgADP (composite K greater than 4 X 10(10) M-2) to Kp2ox induced a rapid conformation change which caused the redox-active 4Fe-4S centre to be reduced by SO2-.(formed by the predissociation of dithionite ion) with k = 3 X 10(6) M-1.s-1. This rate constant is at least 30 times lower than that for the reduction of free Kp2ox (k greater than 10(8) M-1.s-1). Two mechanisms have been considered and limits obtained for the rate constants for MgADP binding/dissociation and a protein conformation change. Both mechanisms give rate constants (e.g. MgADP binding 3 X 10(5) less than k less than 3 X 10(6) M-1.s-1 and protein conformation change 6 X 10(2) less than k less than 6 X 10(3) s-1) that are similar to those reported for creatine kinase (EC 2.7.3.2). The kinetics also show that in the catalytic cycle of nitrogenase with sodium dithionite as reductant replacement of 2MgADP by 2MgATP occurs on reduced and not oxidized Kp2. Although the Kp2ox was reduced stoichiometrically by SO2-. and bound two equivalents of MgADP with complete conversion into the less-reactive conformation, it was only 45% active with respect to its ability to effect MgATP-dependent electron transfer to the MoFe protein.