Effects of substrate and inhibitor binding on thermal and proteolytic inactivation of rat liver transhydrogenase

Abstract

The thermostability and proteolytic inactivation of rat liver submitochondrial particle transhydrogenase was studied in the presence of pyridine dinucleotide substrates and a variety of divalent metal and nucleotide inhibitors. Relative to the unliganded enzyme, the NADPH-enzyme complex was more thermostable and showed a twofold greater rate of tryptic inactivation, while the NADP+-enzyme complex was more thermolabile and only slightly more susceptible to tryptic inactivation. Neither NAD+ nor NADH significantly affected thermostability or proteolysis. Similar effects of these ligands were observed for the non-energy-linked and energy-linked transhydrogenase reactions, indicating that both activities are catalyzed by the same enzyme. In thermal experiments, acetyl-CoA, 2'-AMP, and NMNH stabilized, palmitoyl-CoAlabilized, and dephospho-CoA, CoA, NMN+, and 5'-AMP had little effect on enzyme stability. Tryptic inactivation was inhibited by 2'-AMP and NMN+ but was not influenced by the other nucleotide inhibitors. Divalent metal ion inhibitors (Mg2+, Ca2+, Mn2+, Ba2+, and Sr2+) stabilized transhydrogenase against thermal inactivation and promoted tryptic inactivation.

Inhibition of nicotinamide nucleotide transhydrogenase in rat liver submitochondrial particles by dicyclohexylcarbodi-imide and butanedione

“…Nicotinamide nucleotide transhydrogenase (EC 1.6.1.1), an integral protein of the mitochondrial inner membrane, catalyses the reversible transfer of protons across the membrane according to the equation: NADPH + NAD+ + nHm = NADP+ + NADH + nHtc where H+ ahd H+ represent matrix and cytosolic protons respectively (Mitchell, 1966;Blazyk et al, 1976).…”

Reconstitution of bovine heart mitochondrial transhydrogenase: a reversible proton pump

“…The reverse reaction was assayed by the reduction of Ac-PyAD+ by NADPH as described (Blazyk et al, 1976). For the assay of the reconstituted enzyme in either direction, an initial coupled rate was obtained, followed by determination of the rate in the presence of 1.3 µ FCCP.…”

Evidence that reconstituted bovine heart mitochondrial transhydrogenase functions as a proton pump

“…The forward transhydrogenase reaction was assayed by the reduction of thio-NADP+ by NADH [lo] ; reaction mixtures (3 ml) at 25°C contained 80 mM potassium phosphate, pH 6.8,0.5 I.IM rotenone, 175 I.IM NADH and 117 PM thio-NADP'. The reverse reaction was assayed by the reduction of 3-acetylpyridine adenine dinucleotide by NADPH as in [7].…”