Structural and kinetic properties of chymotrypsin from Atlantic cod (Gadus morhua). Comparison with bovine chymotrypsin.

1. Two chymotrypsins with isoelectric points pI 6.2 and 5.8 were purified from the pyloric caeca of Atlantic cod using a phenyl-Sepharose column and chromatofocusing chromatography. The apparent molecular weight was 26,000 as judged by SDS-polyacrylamide gel electrophoresis and gel filtration. 2. The cod enzymes differed from bovine chymotrypsin in having a slightly higher molecular weight and more acidic pI points. N-terminal amino acid sequence analysis of cod chymotrypsin B showed considerable similarity with bovine chymotrypsin. 3. Heat stability and stability towards acidic pH were reduced in the cod enzymes. Generally, the cod and bovine chymotrypsins responded similarly to various protease inhibitors. However, the cod chymotrypsins were less sensitive to aprotinin inhibition but more sensitive towards soybean trypsin inhibitor and cysteine. 4. Kinetic properties were examined and the cod enzymes found to be more active towards both ester (N-benzoyl-tyrosine ethyl ester) and amide (N-benzoyl-tyrosine-p-nitroanilide) substrates. The observed differences in kinetic properties are indicative of an adaptive response towards the low temperature environment in which the cod lives.     

Some kinetic and molecular properties of yeast phosphofructokinase

Yeast PFK had a sedimentation coefficient of 16.7 S both in the absence and in the presence of ATP, and did not dissociate even at very low protein concentrations. Sodium dodecyl-sulphate caused dissociation of the protein to sub-units of 3.2 S.The effects of pH on substrate affinities are described. In the presence of UTP, acting as non-inhibiting phosphate donor, the behaviour of the enzyme towards F-6-P was co-operative, with a Hill coefficient of 2.2.     

Some kinetic properties of gamma-glutamyltransferase from rabbit liver

gamma-Glutamyltransferase ((5-glutamyl)-peptide: amino-acid 5-glutamyltransferase, EC 2.3.2.2) of rabbit liver (detergent form) was purified 1100-fold in order to study its kinetic properties. Kinetic studies were conducted from pH 6.0 to 12.0 in the absence and presence of the acceptor substrate glycylglycine using gamma-glutamyl-3-carboxy-4-nitroanilide as the donor. The existence of more than one binding site for both donor and acceptor is postulated on kinetic evidence such as donor substrate activation, donor substrate inhibition and acceptor substrate activation. Homotropic interaction is also observed, in the form of negative cooperativity, in donor substrate binding, in the absence of acceptor at pH less than 9.0 and positive cooperativity (n = 2), in the absence or presence of acceptor at pH greater than 9.0. Hydrolase reaction reaches a maximum of activity at pH 10 (pK 8.6). Transferase activity under conditions of maximal velocity is maximal at pH 9.0 (pK 7.1). The ratio of transferase activity/hydrolase activity is maximal at pH 7.0-7.5. At low donor substrate concentrations, maximal activity is attained at pH 7.5.     

Some kinetic and chromatographic properties of detergent-dispersed adenylate cyclase

Studies on the reaction kinetics and chromatographic properties of detergent-dispersed adenylate cyclase are described. Detergent-dispersed enzyme was prepared from whole rat cerebellum and from partially purified plasma membranes from rat liver. Data were simulated to fit kinetic models for which an inhibitor is added in constant proportion to the variable substrate. Models were chosen to distinguish whether the adenylate cyclase reaction may be controlled by an inhibitory action of free ATP^?4 (or HATP^?3) or by a stimulatory action of free divalent cations. The various kinetic models were then tested with the dispersed brain adenylate cyclase with both Mg⁺⁺ and Mn⁺⁺ and in two different buffer systems. The experimental data indicate that this enzyme has a distinct cation binding site, but exhibits no significant inhibition by HATP^?3 or ATP^?4. The detergent-dispersed adenylate cyclase both from liver plasma membranes and from brain have been chromatographed on anion exchange material and have been chromatographed on anion exchange material and have been subjected to gel filtration. The presence of detergent was required for elution of cyclase activity from DEAE-Sephadex but was not required when DEAE-agarose was used. Dispersed brain cyclase was also chromatographed on agarose-NH(CH₂)₃ NH(CH₂)₃-NH₂ which exhibits both ionic and hydrophobic properties. Fifty percent of the applied activity was recovered with a fivefold increase in specific activity. The data suggest that the relative effectiveness of a given chromatographic procedure for detergent-dispersed adenylate cyclase may reflect the in fluence of both hydrophobic and ionic factors.     

Some spectral and steady-state kinetic properties of Pseudomonas cytochrome oxidase.

Some spectra of Pseudomonas cytochrome oxidase are reported, both for comparison with those of other workers and to illustrate the differences between the ascorbate- and dithionite-reduced forms of the enzyme. A spectrum of the reduced enzyme-CO complex, prepared in the absence of added reductants by incubation under CO, is also included. Ultracentrifugation studies yielded a value for the sedimentation coefficient (s20,w) of 7.5S, and an isoelectric point of pH6.9 was determined by isoelectric focusing. Steady-state kinetic constants of the electron donors, quinol, sodium ascorbate, reduced Pseudomonas azurin and Pseudomonas ferrocytochrome c551 were investigated giving Km values of 30mM, 4mM, 49muM and 5.6muM respectively. The two protein substrates were observed to be subject to product inhibition and the Ki for oxidized Pseudomonas azurin was evaluated at 4.9muM. Steady-state kinetics were also used to investigate the effects of the oxidation products of dithionite on the oxidase and nitrite reductase activities of Pseudomonas cytochrome oxidase. These experiments showed that whereas the oxidase activity was inhibited, the nitrite reductase activity was slightly enhanced.     

Some kinetic and steady-state properties of sodium channels after removal of inactivation

To study the kinetic and steady-state properties of voltage-dependent sodium conductance activation, squid giant axons were perfused internally with either pronase or N-bromoacetamide and voltage clamped. Parameters of activation, tau m and gNa(V), and deactivation, tau Na, were measured and compared with those obtained from control axons under the assumption that gNa oc m3h of the Hodgkin-Huxley scheme. tau m(V) values obtained from the turn-on of INa agree well with control axons and previous determinations by others. tau Na(V) values derived from Na tail currents were also unchanged by pronase treatment and matched fairly well previously published values. tau m(V) obtained from 3 x tau Na(V) were much larger than tau m(V) obtained from INa turn-on at the same potentials, resulting in a discontinuous distribution. Steady-state In (gNa/gNa max - gNa) vs. voltage was not linear and had a limiting logarithmic slope of 5.3 mV/e-fold gNa. Voltage step procedures that induce a second turn-on of INa during various stages of the deactivation (Na tail current) process reveal quasiexponential activation at early stages that becomes increasingly sigmoid as deactivation progresses. For moderate depolarizations, primary and secondary activation kinetics are superimposable. These data suggest that, although m3 can describe the shape of INa turn-on, it cannot quantitatively account for the kinetics of gNa after repolarization. Kinetic schemes for gNa in which substantial deactivation occurs by a unique pathway between conducting and resting states are shown to be unlikely. It appears that the rate-limiting step in linear kinetic models of activation may be between a terminal conducting state and the adjacent nonconducting intermediate.     

Some kinetic and regulatory properties of the pea mitochondrial pyruvate dehydrogenase complex

The pyruvate dehydrogenase complex was isolated, partially purified, and characterized from green pea (Pisum sativum L., cv Little Marvel) leaf mitochondria. The pH optimum for the overall reaction was 7.6. The divalent cation requirement was best satisfied by Mg²⁺. Reaction velocity was maximal at 40°C. Pyruvate was a better substrate than 2-oxo-butyrate; other 2-oxo-acids were not substrates. Michaelis constants for substrates were; pyruvate, 57 micromolar; NAD, 122 micromolar; Coenzyme-A, 5 micromolar; Mg²⁺, 0.36 millimolar; Mg-thiamine pyrophosphate, 80 nanomolar. The products, NADH and acetyl-Coenzyme-A, were linear competitive inhibitors with respect to NAD and Coenzyme A. Inhibition constants were 18 and 10 micromolar, respectively. Glyoxylate inhibited complex activity only in the absence of thiol reagents. Glyoxylate inhibition was competitive with respect to pyruvate with an inhibition constant of 51 micromolar. Among mitochondrial metabolites examined as potential effectors, only ADP with an inhibition constant of 0.57 millimolar could be of physiological significance.