This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Hampton, M. L. Articles by Freese, E. Search for Related Content PubMed PubMed Citation Articles by Hampton, M. L. Articles by Freese, E.

Explanation for the Apparent Inefficiency of Reduced Nicotinamide Adenine Dinucleotide in Energizing Amino Acid Transport in Membrane Vesicles

¹ Laboratory of Molecular Biology, National Institute of Neurological Diseases and Stroke, Bethesda, Maryland 20014

ABSTRACT

Lineweaver-Burk plots of reduced nicotinamide adenine dinucleotide (NADH) oxidation by membrane preparations from Bacillus subtilis are biphasic, with two K_m values for NADH. The higher K_m corresponds to the only K_m observed for NADH oxidation by whole cells, whereas the lower K_m corresponds to that observed with open cell envelopes. Membrane preparations apparently contain a small fraction of open or inverted vesicles which is responsible for the low K_m reaction, whereas entry of NADH into the larger portion of closed, normally oriented vesicles is rate limiting and responsible for the high K_m reaction. In contrast, the oxidation of L--glycerol-phosphate (glycerol-P) by membrane preparations shows only one K_m that corresponds to that of glycerol-P oxidation by whole cells or lysates. Since glycerol-P dehydrogenase (NAD independent) has the same K_m, this enzyme reaction rather than entry of glycerol-P into vesicles represents the rate-limiting step for glycerol-phosphate oxidation. The K_m for amino acid uptake by vesicles in the presence of NADH corresponds to the high K_m for NADH oxidation, indicating that NADH energizes transport only if it enters closed, normally oriented vesicles. Studies with rotenone and proteolytic enzymes support this interpretation. The apparent efficiency of NADH in energizing uptake seems to be lower than that of glycerol-P because, under the experimental conditions usually employed, open or inverted vesicles that do not participate in amino acid uptake are responsible for the major portion of NADH oxidation. When the results are corrected for this effect, the efficiency of NADH is essentially the same as that of L--glycerol-P.