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Pyruvate synthesis by a partially purified enzyme from Clostridiumacidi-urici

1965; Elsevier BV; Volume: 18; Issue: 3 Linguagem: Inglês

10.1016/0006-291x(65)90703-5

1090-2104

Samuel Raeburn, Jesse C. Rabinowitz,

Biochemical and Molecular Research

This chapter describes the assay method, purification procedure, and properties of pyruvate-ferredoxin oxidoreductase from Clostridium acidi-urici. The enzyme activity can be determined by measuring the radioactivity of pyruvate formed in the exchange reaction between pyruvate and 14CO2 or by the decrease in absorption at 450 nm caused by the reduction of FAD in the presence of pyruvate. All purification steps are carried out at 0–4° unless otherwise noted. Buffer I contains 50 mM potassium phosphate buffer, pH 7.4, 40 mM EDTA, 0.1 mM ferrous ammonium sulfate, and 0.5 mg of reduced glutathione per milliliter. It is freshly prepared for use. Buffer II has the same composition as buffer I except that the concentration of potassium phosphate is reduced to 20 mM and the ferrous ammonium sulfate concentration was increased to 1 mM. The enzyme has a molecular weight of approximately 240,000 based on sedimentation studies. It contains nonheme iron and inorganic sulfide and thiamine. The enzyme catalyzes (a) a pyruvate-CO2 exchange reaction, (b) oxidation of pyruvate with reduction of a variety of electron acceptors, (c) acetoin formation from pyruvate and acetalde hyde, and (d) the synthesis of pyruvate from acetyl-CoA and bicarbonate in the presence of reduced ferredoxin. FAD, FMN, clostridial ferredoxin, rubredoxin, and various viologen and tetrazolium derivatives can function as electron acceptors in the enzymic oxidation of pyruvate catalyzed by the enzyme. Other properties are also discussed.