Dermal toxicity of paraquat

Abstract

This chapter describes the assay method, purification procedure, and properties of glyoxylate reductase. Two enzymes that catalyze the reduction of glyoxylate to glycolate may be obtained from Pseudomonas. Reduced pyridine nucleotides act as hydrogen donors for the enzymes. The NADH-linked glyoxylate reductase is entirely specific for its coenzyme, but the NADPH-linked reductase shows some affinity toward NADH. The enzymes are assayed by measuring the rate of oxidation of either NADH or NADPH in the presence of glyoxylate. The oxidation of the coenzyme is followed spectrophotometrically at 340 nm. The enzymes are obtained from a strain of Pseudomonas fluorescens isolated from soil, which has the ability to grow on butane-2,3-diol as sole carbon source. All subsequent operations of purification are conducted at 2°. The chapter discusses the purification of the NADPH-linked glyoxylate reductase, and the purification of the NADH-linked glyoxylate reductase. The NADH-linked enzyme is entirely specific for its coenzyme, but the NADPH-linked reductase shows some affinity toward NADH. Both enzymes convert hydroxypyruvate to glycerate. The preparation containing the NADH-linked enzyme is contaminated with malate and lactate dehydrogenases; a gradual but not total removal of the contaminating enzymes is achieved during the purification procedure. Both reductases show maximal activity between pH 6.0 and 6.8, but the NADPH-linked enzyme retains its activity over a wider range of pH values. Oxo acids inhibit enzyme activity by combining with the active centers. Both enzymes are strongly dependant on free thiol groups for activity, as shown by inhibition with p-chloromercuribenzoate. The reduction of glyoxylate and hydroxypyruvate is not stimulated by anions.