Purification of glycollate oxidase from greening cucumber cotyledons

Abstract

Glycollate oxidase (glycollate: oxygen oxidoreductase, EC 1.1.3.1) was purified to apparent homogeneity from crude extracts of greening cucumber cotyledons (Cucumis sat vus). Molecular sieving and chromatofocusing resulted in 700-fold purification and specific activity of 1 μkat mg(-1) protein. The enzyme exhibited a Mr of 180,000, or 700,000, respectively, and is a tetramer or 16-mer made of identical subunits of Mr 43,000. Monospecific antibodies were raised against the homogeneous protein.

Characterization of a cDNA Encoding Lens culinaris Glycolate Oxidase and Developmental Expression of Glycolate Oxidase mRNA in Cotyledons and Leaves

“…Poly(A+) RNA was prepared from the green leaves and used as template for preparation of cDNA (9,10). The presence of translatable glycolate oxidase mRNA was evidenced by translation in vitro and subsequent immunoprecipitation with anti-glycolate oxidase antiserum (4). cDNA was obtained using the cDNA synthesis kit and the procedure of Pharmacia (Uppsala, Sweden) for purification by phenol extraction and chromatography on Sephacryl S-200.…”

“…The filters were washed twice for 10 min each time in 2x SSC and 0.1% SDS. Positive clones were examined both by rescreening with insert of pE56 and by screening with antibodies against lentil glycolate oxidase (4).…”

“…Addition of light leads to a dramatic increase in enzyme activities reponsible for the peroxisomal part of photorespiration (16,27,29). Among the light induced peroxisomal enzymes, glycolate oxidase has been studied at the level of protein (4,8). mRNA expression was demonstrated by protein synthesis (8) and translation in vitro (9).…”

Oxidation of Ethylene Glycol and Glycolic Acid by Glycerol Oxidase

“…This oxidase also oxidized glycolic acid to glyoxylic acid. The oxidative reaction of glycolic acid has been studied in detail using some glycolate oxidases (glycolate: oxygen oxidoreductase, Ee 1.1.3.1) from green plants such as spinach,4) pea, 5) pumpkin,6) tabacco,7) and cucumber, 8) and lactate oxidase 9 ) (L-Iactate: oxygen 2-oxidoreductase (decarboxylating), Ee 1.13.12.4) from Mycobacterium smegma tis, but not with fungal enzymes. This paper describes some characterizations of the oxidative reaction of ethylene glycol and glycolic acid by glycerol oxidase and provides an evaluation of glycerol oxidase for the production of glyoxal and glyoxylic acid.…”

Purification and some characteristics of chicken liver L‐2‐hydroxyacid oxidase A

“…Its absorbance at 280 nm was rather low and in fair agreement with Trp content, and its molecular mass (37 760 Da), as derived from Table II, was consistent with that obtained by SDS-polyacrylamide gel electrophoresis. Since a value of 169 kDa was obtained under nondenaturing conditions (not shown), it was concluded that chicken liver L-2-HAOX A is composed of 4 apparently identical subunits as all the enzymes of the A-type so far isolated from mammals [3,7] and plants [13,20].…”