Pressure relaxation of the equilibrium of the pig heart lactate dehydrogenase system

Abstract

The relaxation behaviour of a system of reactants equilibrated in the presence of pig heart lactate dehydrogenase was studied after pressure perturbation. Two relaxations were observed when protein fluorescence was recorded, but only the slower relaxation was apparent in observations ofA340. The faster relaxation therefore involves transfer between free and enzyme-bound NADH, whereas the slower relaxation represents the reduction of NAD+. Both relaxations were observed in Tris buffer, where there is little effect of pressure on pH, and in phosphate buffer, where pH changes are significant; however, the amplitudes depended on the buffer used. The slower reciprocal relaxation time increases with increasing total enzyme concentration and decreases slightly with increasing NAD+ concentration. Computer simulations, based on a proposed mechanism, were compared with the experimentally determined amplitudes and relaxation times as a test of the mechanism.Preliminary observations (Davis & Gutfreund, 1976) of the pressure perturbation of a system of reactants equilibrated in the presence of pig heart lactate dehydrogenase showed the existence of relaxation phenomena. Transient kinetic analysis of stopped-flow experiments in this laboratory (Whitaker et al., 1974; Boland & Gutfreund, 1975;Holbrook et al., 1976) has resulted in considerable elucidation of our understanding of the reaction of lactate dehydrogenase with its substrates and allows pressure-relaxation effects to be analysed in * Present address:

Relationship between the mechanisms of the esterase and dehydrogenase activities of the cytoplasmic aldehyde dehydrogenase from sheep liver. An alternative view

“…Stopped-Flow Experiments. These were carried out on a Durrum-Gibson D110 stopped-flow spectrophotometer as described by Hardman et al (1977) in either the fluorescence or absorbance modes. Proton release experiments were performed with enzyme which was dialyzed against 0.5 mM, pH 7.6, phosphate buffer and made up in solutions containing 0.1 M Na2S04, 0.1 M NaN03, and phenol red (10-30 µ ) as we have described previously (Agnew et al, 1981;Bennett et al, 1982).…”

Protein-bound ATP: properties of a key intermediate of the magnesium-dependent subfragment 1 ATPase from rabbit skeletal muscle

“…Signals were stored on disc or tape with a PDP 1103 computer with facilities for computer averaging and calculation of the rate constants of exponential processes. Kinetic simulations were done with the system described by Hardman et al (1978). Steady-state fluorescence measurements were made on a photon counting SLM spectrofluorometer.…”

Magnesium ion dependent rabbit skeletal muscle myosin guanosine and thioguanosine triphosphatase mechanism and a novel guanosine diphosphatase reaction

“…The quenched-flow apparatus used has been described by Gutfreund (1969) and the stopped-flow apparatus for measuring absorption or fluorescence changes by Gutfreund (1972) and Bagshaw et al (1972), respectively. Data from stopped-flow experiments were either recorded photographically or collected and processed as described by Hardman et al (1978) as is illustrated in Figure 4.…”