Proline Dehydrogenase from Escherichia coli K12
- 1 July 1983
- journal article
- research article
- Published by Wiley in European Journal of Biochemistry
- Vol. 134 (1), 77-82
- https://doi.org/10.1111/j.1432-1033.1983.tb07533.x
Abstract
The oxidative activities of inverted cytoplasmic membrane preparations from E. coli bearing proline dehydrogenase were examined. The measurements include direct substrate:2,6-dichloroindophenol and substrate:O2 oxidoreductase assays and the 9-aminoacridine fluorescence assay for proton translocation, employing succinate and NADH dehydrogenases as comparative standards. Membranes prepared in a new buffer system bear proline dehydrogenase that is stable in activity and membrane association. This membrane-associated enzyme shows an apparent Km for proline 20-fold lower than that estimated from the solubilized and purified enzyme. Electrons are transferred from proline to O2 via the respiratory chain since proline utilization requires porphyrin synthesis and it is coupled to trans-membrane proton translocation. Patterns of inhibition by 5-ethyl-5-isopentyl barbituric acid (Amytal) and 2-heptyl-4-hydroxyquinoline-N-oxide (HpHOQnO) suggest that parallel pathways of electron flux from NADH and proline converge at a cyanide-sensitive terminal oxidase. Succinate:O2 and succinate:DCIP [2,6-dichloroindolphenol] oxidoreductase activities are stimulated by HpHOQnO and Amytal, and the former is inhibited by cyanide in this system. Amytal is a noncompetitive inhibitor of proline dehydrogenase. Analysis of the fluorescence data suggests that Amytal and HpHOQnO dissipate .DELTA.pH at concentrations as low as 5 and 8.5 .mu.M, respectively, in this system.This publication has 28 references indexed in Scilit:
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