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Identification of a 64-kDa protein phosphorylated with glucose in human polymorphonuclear leukocytes in a cell-free
1Department of Antibiotics, National Institute of Health, Tokyo, Japan.
Abstract:
We previously reported that a 64-kDa protein (p64) in human polymorphonuclear leukocytes (PMN) was phosphorylated with [gamma-32P]ATP under a micromolar concentration of glucose in a cell-free system. The present paper presents the results of analysis of phosphorylation reaction and the identification of phosphoprotein. The findings that p64 was also phosphorylated with glucose-6-[32P]phosphate and that phosphorylation was inhibited with mannoheptulose suggested that the reaction was mediated by hexokinase. In fact, it was found that [32P]phosphate in glucose-6-[32P]phosphate was incorporated into either p64 or rabbit muscle phosphoglucomutase and that glucose-6-phosphate formation from glucose and ATP was detected in over 100-kDa fraction of PMN cytosol. These results showed that p64 was phosphoglucomutase in PMN and that phosphate incorporation into p64 was a conversion of a phosphate group in glucose-6-phosphate produced by hexokinase. It was further demonstrated by analysis of two-dimensional electrophoresis that p64 phosphorylated with glucose induction was different from another 64-kDa protein phosphorylated by stimulation with formyl-methionyl-leucyl-phenylalanine in vivo.
Insights
Human polymorphonuclear leukocytes (PMN) contain a 64-kDa protein identified as phosphoglucomutase. This protein is phosphorylated via glucose-6-phosphate produced by hexokinase, distinct from other phosphorylated proteins.
Area of Science:
- Biochemistry
- Cell Biology
- Immunology
Background:
- A 64-kDa protein (p64) in human polymorphonuclear leukocytes (PMN) was previously shown to be phosphorylated by [gamma-32P]ATP in a cell-free system with glucose.
- The current study investigates the phosphorylation reaction mechanism and identifies the specific phosphoprotein involved.
Purpose of the Study:
- To identify the 64-kDa phosphoprotein in human PMN.
- To elucidate the mechanism of p64 phosphorylation in response to glucose.
- To differentiate glucose-induced p64 phosphorylation from other phosphorylation events in PMN.
Main Methods:
- Cell-free phosphorylation assays using [gamma-32P]ATP and glucose-6-[32P]phosphate.
- Enzyme inhibition studies using mannoheptulose.
- Analysis of [32P]phosphate incorporation into proteins and glucose-6-phosphate formation.
- Two-dimensional electrophoresis to compare phosphorylated proteins.
Main Results:
- p64 phosphorylation was observed with glucose-6-[32P]phosphate and inhibited by mannoheptulose, suggesting hexokinase involvement.
- Evidence indicated that p64 is phosphoglucomutase and phosphate incorporation involves glucose-6-phosphate produced by hexokinase.
- Two-dimensional electrophoresis revealed that glucose-induced p64 phosphorylation differs from in vivo phosphorylation stimulated by formyl-methionyl-leucyl-phenylalanine.
Conclusions:
- The 64-kDa protein in human PMN is identified as phosphoglucomutase.
- Phosphorylation of phosphoglucomutase is mediated by glucose-6-phosphate generated by hexokinase.
- Glucose-induced p64 phosphorylation is a distinct event from inflammatory signaling-induced phosphorylation in PMN.