Purification and some properties of glyoxylate reductase (NADP+) and its functional location in mitochondria in Euglena gracilis z

Abstract

Euglena mitochondria contain both glyoxylate reductase (NADP+) and glycollate dehydrogenase to constitute the glycollate-glyoxylate cycle [Yokota & Kitaoka (1979) Biochem. J. 184, 189-192]. Euglena glyoxylate reductase (NADP+) was purified and its submitochondrial location was determined in order to elucidate the cycle. The purified glyoxylate reductase was homogeneous on polyacrylamide-gel electrophoresis. Difference spectra of the purified enzyme revealed that the enzyme was a flavin enzyme. The Mr of the enzyme was 82 000. The enzyme was specific for NADPH, with an apparent Km of 3.9 microM, and for glyoxylate, with an apparent Km of 45 microM. It was 30% as active with oxaloacetate as with glyoxylate. NADH and hydroxypyruvate did not support the activity at all. The optimum pH was 6.45. Submitochondrial fractionation of purified mitochondria showed that the enzyme was located in the intermembrane space and loosely bound to the outer surface of the inner membrane. These properties and the submitochondrial localization of NADPH-glyoxylate reductase facilitate the operation of the glycollate-glyoxylate cycle in combination with glycollate dehydrogenase, which is tightly bound to the inner membrane of Euglena mitochondria.