Biomedical subjects

K J Laidler

Publications and source records attributed to K J Laidler.

At least 37 records · Page 2Linked to original sources

PubMed1975

Immobilized electric eel acetylcholinesterase. I. Kinetics of acetylcholinesterase trapped in polyacrylamide membranes.

Techniques are described for the trapping of electric eel acetylcholinesterase in polyacrylamide gel. The activity of the trapped enzyme was substantially reduced, the effect being due to inhibition by acrylamide, but the emzyme immobilized in polyacrylamide was considerable more stable than that in free solutionma kinetic study was made of the hydrolysis of acetylthiocholine, covering a range of membrane thicknesses, enzyme concentrations, substrate concentrations and temperatures. The results were interpreted with reference to the theoretical treatment of Sundaram, Tweedale and Laidler, and of Kobayaski and Laidler, and provided support for those treatments; Clear evidence was obtained for diffusion control with the thicker membranes. An activation energy was obtained for the diffusion of the substrate within the membrane, by combining the temperature results for thick and thin membranes at low substrate concentrations. The results lead to the conclusion that the in vivo kinetics of acetylcholinesterase are largely diffusion-free in muscle filaments, but are substantially diffusion-controlled in fibrils and fibers.

Acetylcholinesterase↗

PubMed1975

Immobilized electric eel acetylcholinesterasemii. II. Flow kinetics of acetylcholinesterase chemically attached to nylon tubing.

Acetylcholinesterase has been attached covalently to the inner surface of nylon tubing. An experimental study has been carried out on the flow kinetics; solutions of acetylthiocholine at various concentrations were passed through tubing at various flow rates, and measurements made of the rates of formation of product. The results were analyzed in the light of the theoretical treatment of Kobayashi and Laidler, four different methods of analysis being employed. It is found that at lower substrate concentrations and flow rates the reactions are largely diffusion controlled. The Km(app) values are substantially higher than the Km value for diffusion-free conditions, but approach it as the flow rate is increased, when the diffusion layer becomes less important. The results are entirely consistent with the Kobayaski-Laidler theory, and provide guidelines for the design of open tubular heterogeneous enzyme reactors, both for industrial and analytic purposes.

Acetylcholinesterase↗

PubMed1975

A flow method for determination of the kinetic parameters for immobilized enzymes.

A flow method is described for determination of the kinetics parameters (V-m and K-m) for enzymes that are bound to particles, to membranes, and to the interior surfaces of tubes. Substrate solution is pumped through Tygon tubing to a microvolume flow cell and back into the reaction mixture, the flow rate being adjusted to be faster than the rate of formation of product. To illustrate the technique, it is applied to the determination of the parameters for electric-eel acetylcholinesterase attached to particles, to membranes, and to the inner surface of nylon tubing.

Acetylcholinesterase↗

PubMed1975

Transient kinetics of the acetylcholinesterase catalyzed hydrolysis of N-methylindoxyl acetate.

An experimental study has been made of the kinetics of the hydrolysis of N-methylindoxyl acetate catalyzed by electric-eel acetylcholinesterase, both in the steady state and the pre-steady statemstopped-flow and temperature-jump experiments revealed a fast transient and a slow one. The fast transient is correlated with the conventional mechanism E+A in equilibrium EA yields X-yieldsEA' YIELDS E+Y. The slow transient is attributed to conformational changes involving E or EA. Analysis of it revealed two exponential terms of the form e- minus lambda t, and the two lambda values were obtained over the temperature range 5Yand 25Ydegrees C. The results are interpreted in terms of two alternative mechanisms; in one, the enzyme undergoes a conformational change before it adds on the substrate molecule; in the other, the conformational change occurs after the substrate addition. Both mechanisms may be involved, but the results exclude a concerted mechanism in whivh the conformational change occurs concurrently with the addition of substrate. Kinetic parameters (delta S not equal to and E) are obtained for this conformational change and for the conversion of EA into EA'+X.

Acetates↗

PubMed1975

Flow kinetics of beta-galactosidase chemically attached to nylon tubing.

Beta-Galactosidase (EC 3.2.1.23) has been attached covalently to the inner surface of nylon tubing. An experimental study has been made of the flow kinetics for the hydrolysis of o-nitrophenylgalactose, the substrate concentration and flow rate being varied. The results were analyzed in the light of the theoretical treatment of Kobayashi and Laidler, three different methods of analysis being employed. It is concluded that at the lower substrate concentrations and flow rates employed, the reactions are largely diffusion controlled; with increase in flow rate and substrate concentration the width of the Nernst diffusion layer decreases, and there is found to be less diffusion control. The values of Km(app) vary with flow rate VF, being linear in VF-1/3, and the value extrapolated to very high flow rate agrees well with the Km value for beta-galactosidase in free solution. The theory and results are shown to provide guidelines for the design of open tubular heterogeneous enzyme reactors for industrial, biomedical, and analytical applications.

Diffusion↗

PubMed1975

Kinetics and thermodynamics of lactate dehydrogenases from beef heart, beef muscle, and flounder muscle.

The eight rate constants for a four-step ordered ternary-complex mechanism have been compared for lactate dehydrogenases (EC1.1.1.27) from three sources, beef heart, beef muscle, and flounder muscle. The rate constants were determined at temperatures ranging from 5 degrees C to 50 degrees C, and the corresponding activation parameters deltaG not equal to, deltaH not equal to, and deltaS not equal to were calculated. Significant differences are noted for the values for the three types of enzyme. The relative heights of the activation barriers are much the same in all three cases, differences in kinetic behavior resulting mainly from differences in the stable binary and ternary enzyme-substrate complexes. These complexes are, in general, at lower free-energy and enthalpy levels of the beef-heart and beef-muscle enzymes than for the flounder-muscle enzyme. A high degree of compensation is found between the enthalpies and entropies of activation, resulting in relatively small differences between the free energies (and rates) for homologous steps with different enzymes. Analysis of the results, on the assumption that the compensation effect is due to weak-bonding effects, suggests that there are fewer weak bonds in the stable complexes of the muscle enzymes.

Animals↗

PubMed1974