Re-Activation of the Expression of Glyoxysomal Genes in Green Plant Tissue

Biochemie, Fachbereich Chemie, Universität Marburg, Hans-Meerwein-Straße, D-3550 Marburg, Bundesrepublik Deutschland Z. Naturforsch. 45c, Isocitrate Lyase c DNA, Malate Synthase c DNA, Glyoxysome, Leaf Peroxisome, Transition of Organelles Glyoxysomes are being replaced by leaf-type peroxisom es during the greening of dark-grown cucumber cotyledons. Light functions in this process as negative modulator of the gene expression of glyoxylate cycle enzymes but as positive regulator for the activation of glycollate oxidase formation. The differential gene expression was investigated at the level of m RNA amounts using c DNA probes hybridizing with malate synthase m RNA, isocitrate lyase m RNA, and glycollate oxidase m RNA. Hybrid ization probes were obtained from a c DNA library complementary to the germinationspecific m RNA s of cucumber cotyledons. The process of replacem ent of glyoxysomal proteins by leaf peroxisom al proteins was reversed to a certain extend when greened cotyledons were brought back in the dark. Hybridization on Northern b lots provided evidences that in greened cotyledons the amount of malate synthase m RNA and isocitratelyase m RNA starts to increase up on dark treatment.

A luminometric assay for peroxisomal β-oxidation. Effects of fasting and streptozotocin-diabetes on peroxisomal β-oxidation

1. A luminometric assay for acyl-CoA oxidase activity is described. The assay uses the luminol/microperoxidase system to monitor continuously acyl-CoA-dependent generation of H2O2. The assay is rapid, convenient, and lends itself to automation with an LKB 1251 luminometer. The assay is extremely sensitive, requiring at the most 10 micrograms of liver-homogenate protein per assay. 2. The assay can also be used to measure other oxidases, e.g. glycollate oxidase (EC 1.1.3.15), D-aspartate oxidase (EC 1.4.3.1) and urate oxidase (EC 1.7.3.3), the only modification being substitution of substrates to appropriate concentration. 3. With rat liver homogenates, spectrophotometrically measured rates of palmitoyl-CoA-dependent NAD+ reduction and acyl-CoA oxidase activity [Hryb & Hogg (1979) Biochem. Biophys. Res. Commun. 87, 1200-1206] was generally found in good agreement with luminometrically measured acyl-CoA oxidase activity. 4. With liver homogenates from streptozotocin-diabetic rats, however, rates of palmitoyl-CoA-dependent NAD+ reduction were consistently lower than the corresponding acyl-CoA oxidase activity. This difference was most marked with respect to luminometrically assayed acyl-CoA oxidase activity.

Inhibition of endogenous oxalate production: biochemical considerations of the roles of glycollate oxidase and lactate dehydrogenase

1. Both the peroxisomal, flavin-linked glycollate oxidase [(S)-2-hydroxy-acid oxidase; EC 1.1.3.15] and the cytosolic, nicotinamide–adenine dinucleotide (NAD)-linked lactate dehydrogenase (l-lactate dehydrogenase; EC 1.1.1.27) are thought to contribute to the formation of oxalate from its immediate precursors, glycollate and glyoxylate, but the relative contributions of each enzyme to endogenous oxalate production is not known. 2. In rat liver homogenates, [14C]oxalate production from labelled glycollate is halved and that from labelled glyoxylate is increased fourfold by the addition of either NAD or NADH. 3. In isolated rat hepatocytes, the 3-hydroxy-1H-pyrrole-2,5-dione derivatives of glycollate, which are specific inhibitors of glycollate oxidase, have a greater effect on glycollate metabolism than on glyoxylate metabolism. 4. These findings are consistent with an important role for lactate dehydrogenase in oxalate formation from glyoxylate. 5. With human and rat liver homogenates and with purified human liver glycollate oxidase and rabbit muscle lactate dehydrogenase, dl-phenyl-lactate (2 mmol/l) completely inhibits glycollate oxidase but has no effect on lactate dehydrogenase. On the other hand, the reduced form of a chemically synthesized, NAD–pyruvate adduct (1 mmol/l) almost completely inhibited lactate dehydrogenase but had no effect on glycollate oxidase. 6. Either alone or in combination, dl-phenyl-lactate and reduced NAD–pyruvate adduct reduce oxalate production from glycollate and glyoxylate in isolated rat hepatocytes, but do not abolish it completely. 7. These findings support a role for another enzyme, probably glycollate dehydrogenase (EC 1.1.99.14), in oxalate production in integrated cell metabolism. 8. In relation to renal oxalate stone disease, these results suggest that the therapeutic inhibition of glycollate oxidase or lactate dehydrogenase would not completely prevent the endogenous formation of oxalate.