Purification of diacylglycerol kinase from Microsporum gypseum and its phosphorylation by the catalytic subunit of protein kinase A.
Diacylglycerol (DG) kinase (EC 2.7.1.107) was purified to homogeneity from the soluble extract of Microsporum gypseum, a dermatophyte. Purified enzyme showed a final specific activity of 2172 pmol/min/mg protein and its apparent molecular weight on SDS-PAGE was found to be 93 kDa. The activity of purified enzyme was inhibited in a dose-dependent manner in the presence of DG-kinase inhibitor (D5919, Sigma). DG-kinase activity was found to be stimulated in the presence of phosphatidylcholine, phosphatidylethanolamine, and cardiolipin while the activity was alleviated in the presence of phosphatidic acid and arachidonic acid. Kinase activity was partially inhibited when assayed after prior treatment with alkaline phosphatase. Treatment of DG-kinase with the catalytic subunit of protein kinase A (PKA)-stimulated DG-kinase activity in a dose-dependent manner. Incubation of DG-kinase with the catalytic subunit of PKA led to the phosphorylation of DG-kinase as revealed by autoradiography. The phosphorylated band disappeared completely in the presence of specific PKA inhibitor. Increased activity of DG-kinase on incubation with the catalytic subunit of PKA was possibly due to the phosphorylation of the former by the latter. Whether this in vitro phosphorylation and activation of DG-kinase occurs under physiological conditions remains to be elucidated.
Haq
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Department of Biochemistry, Postgraduate Institute of Medical Education & Research, Chandigarh-160 012, India.
Sharma
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S
Khuller
G K
GK
D000255:Adenosine Triphosphate / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000494:pharmacology; D002308:Cardiolipins / Q000494:pharmacology; D020134:Catalytic Domain; D002845:Chromatography; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D019852:Diacylglycerol Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008864:Microsporum / Q000201:enzymology*; D010713:Phosphatidylcholines / Q000494:pharmacology; D010714:Phosphatidylethanolamines / Q000494:pharmacology; D010766:Phosphorylation; D012965:Sodium Chloride / Q000494:pharmacology
D002308:Cardiolipins; D004791:Enzyme Inhibitors; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D012965:Sodium Chloride; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D019852:Diacylglycerol Kinase; D017868:Cyclic AMP-Dependent Protein Kinases; D000469:Alkaline Phosphatase