Abstract
The time-resolved and steady state fluorescence properties were measured for pig heart cytoplasmic malate dehydrogenase at pH 6.0 and 8.0. The fluorescence decay can be described by two rate processes, according to the functions: I(t) = 0.7e(-t/1.0) + 0.3e(-t/4.4) for the free enzyme and I(t) = 0.7e(-t/0.8) + 0.3e(-t/2.0 for the enzyme . NADH complex. Quenching by NADH of the tryptophan fluorescence is linear. The only effect of pH is to change the association constant for NADH binding; the fluorescence of the free enzyme and the fluorescence quenching by NADH, I-, and acrylamide are unaffected by pH. Thus there are no changes in conformation of the free enzyme or of the NADH complex over the range of pH 6 to 8.
Highlights
The studies reported in this work define the time-resolved fluorescence properties of subunits in cytoplasmic malate dehydrogenase (s-MDH), in order to examine the extent to which structural and enzymological homology is reflected in fluorescence properties
A principal conclusion from this work is that the fluorescence emissions of lactate dehydrogenase (LDH) and s-MDH are similar, and that, in particular, both the linear NADH quenching of s-MDH and the nonlinear NADH quenching of LDH emission are due to energy transfer
Evident are the structural similarities between the subunits in cytoplasmic malate dehydrogenase (s-MDH)’ and lactate dehydrogenase (LDH)
Summary
Was prepared by the method of Glatthaar et al (7). but the acid precipitation and CM-cellulose steps were omitted, and chromatography on hydroxylapatite (8) was added as a final step. The first two of the three peaks of activity eluted from the hydroxylapatite column were used. MDH was stored as a precipitate in 90% saturated (NHd)zSOd solution of pH 7, containing 1 mM Na2EDTA and 1 mM P-mercaptoethanol. MDH concentrations, given as moles of monomer (active site)/liter, were determined using ~280 = 7.2 x lo[4] M-’ cm-‘, measured in this laboratory. Purified s-MDH had a specific activity of 740 units/mg and gave a single band in disc-gel electrophoresis. Pure Grade) was obtained from P-L Biochemicals and was purified by the method of Mueggler et al (4), using a linear gradient from 0.05. NaCl. The purified NADH stock solutions had an absorbance ratio (AZM/A340) of 2.25 to 2.28. Concentrations of NADH were determined using ~340 = 6320 Mm’ cm-’ (9)
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