Unsaturated fatty acid-activated protein kinase (PKx) from goat testis cytosol

The cytosolic fraction of goat cauda epididymis possesses a protein kinase (PKx) activity which is stimulated by a number of unsaturated fatty acids of which arachidonic acid is the best activator in absence of cAMP or Ca 2+. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and diac...

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Bibliographic Details
Published in:Biochimica et biophysica acta 1999-09, Vol.1434 (1), p.161-169
Main Authors: Roy, Koushik, Mandal, Atin K., Sikdar, Rita, Majumdar, Subrata, Ono, Yoshitaka, Sen, Parimal C.
Format: Article
Language:English
Subjects:
Animals
Arachidonic Acid - pharmacology
Chromatography, Affinity
Chromatography, Ion Exchange
Cytosol - enzymology
DEAE-Cellulose
Electrophoresis, Polyacrylamide Gel
enzyme activation
Enzyme Activation - drug effects
Fatty Acids, Unsaturated - pharmacology
Goat testis
Goats
Male
Phosphorylation
Protein kinase
Protein Kinases - isolation & purification
Substrate Specificity
testes
Testis - enzymology
Unsaturated fatty acid
unsaturated fatty acids
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Summary:The cytosolic fraction of goat cauda epididymis possesses a protein kinase (PKx) activity which is stimulated by a number of unsaturated fatty acids of which arachidonic acid is the best activator in absence of cAMP or Ca 2+. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and diacylglycerol have no effect either alone or in combination. The membrane fraction does not show any appreciable kinase activity even after detergent treatment. PKx migrates as a single band of apparent molecular mass of 116 kDa on 10% SDS–PAGE after sequential chromatographic separation on DEAE–cellulose, phenyl–Sepharose, high-Q anion exchange and protamine–agarose affinity column. PKx phosphorylates histone H1, histone IIIs and protamine sulfate, but not casein. However, the best phosphorylation was obtained with a substrate based on PKC pseudosubstrate sequence (RFARKGSLRQKNV). The kinase phosphorylates two endogenous cytosolic proteins of 60 and 68 kDa. Ser residues are primarily phosphorylated although a low level of phosphorylation is observed on Thr residues also. Ca 2+ and Mn 2+ inhibit PKx activity in the micromolar range. Staurosporine is found to inhibit the PKx activity to a significant level at sub-nanomolar concentration. Lyso-phosphatidylcholine and certain detergents at very low concentrations (
ISSN:0167-4838
0006-3002
1879-2588
1878-2434
DOI:10.1016/S0167-4838(99)00173-9