Microsomal electron transport with NADH as donor.

Macaluso, Peter Michael (1971) Microsomal electron transport with NADH as donor. Masters thesis, Memorial University of Newfoundland.

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Abstract

α-NADH is neither active as an electron donor when tested with several β-NADH-linked dehydrogenases, nor as a competitive inhibitor of the β-isomer in these enzyme systems. Recently, however, an α-NADH-oxidoreductase activity was found in hepatic microsomes and the soluble fraction of rat liver. -- The enzymatic oxidation of α-NADH by the hepatic microsomal electron transport system has been investigated. A comparative study of the factors responsible for the microsomal catalyzed oxidation of α- and β-NADH has implicated that the enzymatic activity of α-NADH is most likely a manifestation of isoenzyme reductases associated with the microsomal electron transport system, rather than a manifestation of a broad electron acceptor specificity of the β-NADH-linked microsomal reductases. -- Substrate specificity studies revealed that the most active electron acceptors for the -e-NADH-linked reductases are completely inactive as substrates in the α-NADH linked reductase system. -- Using cytochrome c and DCPIP as final electron acceptors it was observed that: The enzymatic oxidation of α-NADH is optimal at a more acidic pH than is β-NADH oxidation; the apparent Km values for α-NADH are somewhat higher than those for β-NADH; α-NADH oxidation is more readily inhibited by sulfhydryl group reagents and X-irradiation than is the corresponding β-NADH oxidation; after acetone extraction the enzymatic oxidation of α- and β-NADH respond differently to the addition of phospholipid micelles; TTFA readily inhibits α-NADH oxidation whereas β-NADH oxidation is relatively resistant; trypsin digestion results in a dramatic increase in the Km for α-NADH with little effect on the rate of α-NADH oxidation. In contrast, the effect on the rate of β-NADH oxidation is inactivated by trypsin with the Km for β-NADH remaining unaltered. -- DOC solubilization of microsomes followed by DEAE-cellulose chromatography results in the resolution of the α- and β-NADH-DCPIP reductases. -- Evidence inconsistant with the existence of microsomal isoenzyme reductases has been described and discussed. It was observed that: α-NADH may serve as a competitive inhibitor of the β-isomer with the β-NADH-DCPIP and cytochrome c reductases; β-NAD⁺ may protect both the α- and β-NADH-DCPIP and cytochrome c reductase activities against pCMB inhibition; the lysosomal solubilization of microsomes followed by DEAE-cellulose chromatography and isoelectric focusing revealed that α- and β-NADH-DCPIP reductases and β-NADH-Fe(CN)₆⁻³ reductase possess identical isoelectric points. -- Hypothetical model systems illustrating the possible heterogeneity of the microsomal electron transport system has also been presented.

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