EVIDENCE FOR AND CHARACTERISTICS OF A TRANSMEMBRANE REDOX SYSTEM IN THE PLASMA MEMBRANE OF DAUCUS CAROTA L. (NADH DEHYDROGENASE, ELECTRON TRANSPORT, RESPIRATION, IRON)

THEODORE A CRAIG, Purdue University

Abstract

The plasma membrane of Daucus carota (carrot) cells has an electron transport system reducing extracellular acceptors. The electron acceptor ferricyanide was reduced by cells without entering the cytosol. Release of reductants was not responsible for the major portion of reduction as indicated by (i) lack of reductants in the extracellular medium, (ii) low cell breakage, (iii) low cytochrome c reduction, and (iv) low catechol release. The K(,M) for ferricyanide was 13 (+OR-) 4 (mu)M. The increase in reduction with increasing ferricyanide displayed a pattern expected for an enzyme catalyzed reaction. Electron transfer by carrot cells was insensitive to the mitochondrial inhibitors cyanide and antimycin a indicating a mitochondria from broken cells were not reducing ferricyanide. A unique redox system was also indicated from the sensitivity to azide and HOQNO. Failure of membrane impermeant sulfhydryl reagent PCMBS to inhibit reduction indicated thiols were neither excreted nor available on the plasma membrane surface. The permeant thiol reagent NEM inhibited 78% of reduction showing that an internal thiol is involved in the redox function. Ethanol stimulation of reduction indicated NADH as a cytosolic electron donor, suggesting the redox enzyme in the plasma membrane may be an NADH dehydrogenase. Consistent with lack of a rapidly oxidized, excreted reductant, anaerobiosis inhibited ferricyanide reduction. The electron transfer to ferricyanide was shown to be pH dependent, increasing above pH 5, reaching a maximum at pH 7.5-8.0. Membrane impermeable, protein reactive reagents indicate tyrosine or histidine is present in the redox protein since diazonium benzene sulfonate inhibited reduction but trinitrobenzene sulfonate failed to do so. From the above, a model is presented in which a transmembrane redox protein transports H('+), and electrons, normally returning to reduce cytosolic oxygen, are short circuited to reduce external ferricyanide. The ferricyanide increased H('+) release from cells with increased H('+) / e-transferred to ferricyanide of 1 supports this model. Hormone control was indicated from 2,4-D inhibition of increased ferricyanide reduction after cell washing and fusicoccin stimulation.

Degree

Ph.D.

Subject Area

Biochemistry

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