When 32P-glycolate and phosphoglycolate phosphatase from spinach are mixed, 32P is incorporated into acid precipitated protein. Properties that relate the phosphorylation of the enzyme to the phosphatase are: the Km value for P-glycolate is similar for protein phosphorylation and substrate hydrolysis; the 32P in the phosphoenzyme is diluted by unlabeled P-glycolate or the specific alternative substrate, ethyl-P; the activator Cl- enhances the effectiveness of ethyl-P as a substrate and as an inhibitor of the formation of 32P-enzyme; and 32P is lost from the enzyme when 32P-glycolate is consumed. The phosphorylated protein has a molecular weight of 34,000, which is half that of the native protein and is similar in size to the labeled band that is seen on sodium dodecyl sulfate-polyacrylamide gels. The enzyme-bound phosphoryl group appears to be an acylphosphate from its pH stability, being quite stable at pH 1, less stable at pH 5, and very unstable above pH 5. The bond is readily hydrolyzed in acid molybdate and it is sensitive to cleavage by hydroxylamine at pH 6.8. The demonstration of enzyme phosphorylation by 32P-glycolate resolves the dilemma presented by initial rate studies in which alternative substrates appeared to have different mechanisms (Rose, Z. B., Grove, D. S., and Seal, S. N. (1986) J. Biol. Chem. 261, 10996-11002).
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Photosynthetica
February 2024
Department of Agricultural Biotechnology, Akdeniz University, 07058 Antalya, Turkey.
Animals (Basel)
August 2024
College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China.
Ram sperm undergo a sequence of physiological and biochemical changes collectively termed as capacitation to perform oocyte fertilization. However, the protein changes induced by capacitation remain in need of further exploration. Thus, the present study investigated the comparative proteomic profiling in ram spermatozoa under non-capacitating (NC) and capacitating (CAP) conditions in vitro using a liquid chromatography-tandem mass spectrometry combined with tandem mass tag labeling strategy.
View Article and Find Full Text PDFMethods Mol Biol
June 2024
Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad, India.
Photorespiration, an essential metabolic component, is a classic example of interactions between the intracellular compartments of a plant cell: the chloroplast, peroxisome, mitochondria, and cytoplasm. The photorespiratory pathway is often modulated by abiotic stress and is considered an adaptive response. Monitoring the patterns of key enzymes located in different subcellular components would be an ideal approach to assessing the modulation of the photorespiratory metabolism under abiotic stress.
View Article and Find Full Text PDFMethods Mol Biol
June 2024
Université Paris-Saclay, CNRS, INRAe, Université Paris Cité, Université d'Evry, Institute of Plant Sciences Paris-Saclay (IPS2), Gif-sur-Yvette, France.
To measure the kinetic properties of photorespiratory enzymes, it is necessary to work with purified proteins. Protocols to purify photorespiratory enzymes from leaves of various plant species require several time-consuming steps. It is now possible to produce large quantities of recombinant proteins in bacterial cells.
View Article and Find Full Text PDFMethods Mol Biol
June 2024
Université Paris-Saclay, CNRS, INRAe, Université Paris Cité, Université d'Evry, Institute of Plant Sciences Paris-Saclay (IPS2), Gif-sur-Yvette, France.
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 O and releases HO. The oxidation of o-dianisidine by HO 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.
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