29] Microbial phosphorylating preparations: Azotobacter

Abstract

Publisher Summary This chapter describes the microbial phosphorylating preparations of Azotobacter . A cell-free extract of Azotobacter vinelandii is prepared, which contains particles capable of catalyzing the oxidation of NADH and other substrates, and a soluble factor, which is required for coupled phosphorylation. The factor is bound to the particles in the presence of a suitably high concentration of salt, and released when the salt concentration is decreased. This release is more complete in the presence of low concentrations (1-2 mM) of EDTA or phosphate. Cell-free extracts may also be prepared by grinding with Alumina or glass powder; however, the yield of phosphorylating particles in these preparations is much lower. Both phosphorylating and nonphosphorylating particles catalyze the oxidation of suecinate, malate, lactate, NADPH, and NADH; the phosphorylating particles can catalyze phosphorylation of ADP to ATP coupled to these oxidations. The P:NADH ratios obtained with phosphorylating particles made from fresh extract vary between 0.25 and 0.5. Compared with mammalian preparations, the oxidation and phosphorylation in Azotobacter is less sensitive toward most inhibitors or uncouplers.