Determination of Phosphoglycolate Phosphatase Activity via a Coupled Reaction Using Recombinant Glycolate Oxidase

Methods Mol Biol. 2024:2792:29-39. doi: 10.1007/978-1-0716-3802-6_3.

Abstract

Phosphoglycolate phosphatase (PGLP) dephosphorylates 2-phosphoglycolate to glycolate that can be further metabolized to glyoxylate by glycolate oxidase (GOX) via an oxidative reaction that uses O2 and releases H2O2. The oxidation of o-dianisidine by H2O2 catalyzed by a peroxidase can be followed in real time by an absorbance change at 440 nm. Based on these reactions, a spectrophotometric method for measuring PGLP activity using a coupled reaction with recombinant Arabidopsis thaliana GOX is described. This protocol has been used successfully with either purified PGLP or total soluble proteins extracted from Arabidopsis rosette leaves.

Keywords: Coupled reaction; Enzyme activity; Glycolate oxidase; Phosphoglycolate phosphatase; Photorespiration; Spectrophotometry.

MeSH terms

  • Alcohol Oxidoreductases* / chemistry
  • Alcohol Oxidoreductases* / genetics
  • Alcohol Oxidoreductases* / metabolism
  • Arabidopsis Proteins / genetics
  • Arabidopsis Proteins / metabolism
  • Arabidopsis* / enzymology
  • Arabidopsis* / genetics
  • Arabidopsis* / metabolism
  • Enzyme Assays / methods
  • Glycolates / metabolism
  • Hydrogen Peroxide / metabolism
  • Oxidation-Reduction
  • Phosphoric Monoester Hydrolases* / genetics
  • Phosphoric Monoester Hydrolases* / metabolism
  • Plant Leaves / enzymology
  • Plant Leaves / metabolism
  • Recombinant Proteins* / genetics
  • Recombinant Proteins* / metabolism
  • Spectrophotometry / methods

Substances

  • glycollate oxidase
  • Recombinant Proteins
  • Alcohol Oxidoreductases
  • Phosphoric Monoester Hydrolases
  • phosphoglycolate phosphatase
  • Glycolates
  • Hydrogen Peroxide
  • glycolic acid
  • Arabidopsis Proteins
  • phosphoglycolate