RESPIRATION AND PROTEIN SYNTHESIS IN ESCHERICHIA COLI MEMBRANE-ENVELOPE FRAGMENTS

Abstract

This paper describes experiments conducted with membranous and soluble fractions obtained from Escherichia coli that had been grown on succinate, malate, or enriched glucose media. Oxidase and dehydrogenase activities were studied with the following substrates : nicotinamide adenine dinucleotide, reduced form (NADH), nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), succinate, malate, isocitrate, glutamate, pyruvate, and a-ketoglutarate . Respiration was virtually insensitive to poisons that are commonly used to inhibit mitochondrial systems, namely, rotenone, antimycin, and azide . Succinate dehydrogenase and NADH, NADPH, and succinate oxidases were primarily membrane-bound whereas malate, isocitrate, and NADH dehydrogenases were predominantly soluble . It was observed that E. coli malate dehydrogenase could be assayed with the dye 2,6-dichlorophenol indophenol, but that porcine malate dehydrogenase activity could not be assayed, even in the presence of E . coli extracts. The characteristics of E. coli NADH dehydrogenase were shown to be markedly different from those of a mammalian enzyme . The enzyme activities for oxidation of Krebs cycle intermediates (malate, succinate, isocitrate) did not appear to be under coordinate genetic control .

RESPIRATION AND PROTEIN SYNTHESIS IN ESCHERICHIA COLI MEMBRANE-ENVELOPE FRAGMENTS

“…E . coli W-6, a proline auxotroph (4), was grown on proline-supplemented Difco Antibiotic medium #3, and spheroplasts were produced by growth in the presence of penicillin (1) . The spheroplasts were recovered by centrifugation, washed, stored overnight in liquid nitrogen, and thawed as previously described (1) .…”

“…coli W-6, a proline auxotroph (4), was grown on proline-supplemented Difco Antibiotic medium #3, and spheroplasts were produced by growth in the presence of penicillin (1) . The spheroplasts were recovered by centrifugation, washed, stored overnight in liquid nitrogen, and thawed as previously described (1) . The suspension was then subjected to two 20-sec bursts of ultrasonic irradiation with the Branson sonifier and centrifuged for 10 min at 3500 g (1) .…”

“…The spheroplasts were recovered by centrifugation, washed, stored overnight in liquid nitrogen, and thawed as previously described (1) . The suspension was then subjected to two 20-sec bursts of ultrasonic irradiation with the Branson sonifier and centrifuged for 10 min at 3500 g (1) . The resulting preparation is referred to as GS1 (glucose supernatant 1 [1]) .…”

“…The suspension was then subjected to two 20-sec bursts of ultrasonic irradiation with the Branson sonifier and centrifuged for 10 min at 3500 g (1) . The resulting preparation is referred to as GS1 (glucose supernatant 1 [1]) . The oxidative activities of GS1 prepared at many different times during the course of I Y 2 yr have been highly reproducible for the substrates listed in the introduction to this paper .…”

“…The first paper described techniques for preparing membrane-376 envelope fragments from E . coli, and presented data for their dehydrogenase and oxidase activities with use of the following substrates : NADH, NADPH, succinate, malate, isocitrate, glutamate, pyruvate, and a-ketoglutarate (1) . In the current work we have tried to determine whether these preparations are capable of using fatty acids as an energy source .…”