Insulin stimulates the activity of a protamine kinase in isolated rat hepatocytes.
Treatment of isolated rat hepatocytes with 10-100 nM insulin for 5-10 min increased by about 2-fold the activity of a protamine kinase which exhibited properties similar to those of a protamine kinase from bovine kidney (Damuni, Z., Amick, G. D., and Sneed, T. R. (1989) J. Biol. Chem. 264, 6412-6416). Half-maximal increase in protamine kinase activity occurred at about 1 nM insulin. This effect of insulin was detected only when 25 mM NaF or 50 mM KPO4 were included in the homogenization buffers and was not prevented by preincubation of the hepatocytes with 10 microM cycloheximide. Insulin stimulation of protamine kinase was maintained following chromatography of extracts on protamine-agarose, DEAE-cellulose, and Sephacryl S-200 gel filtration. The apparent Mr of the protamine kinase from control and insulin-treated hepatocytes was 45,000 as estimated by gel permeation chromatography. Experiments utilizing partially purified protamine kinase from control and insulin-treated hepatocytes indicated that insulin did not affect the apparent Km for protamine, Mg2+, or ATP, but increased the Vmax for the protamine kinase reaction by 1.6-2-fold. Incubation with the catalytic subunit of protein phosphatase 2A completely inactivated the protamine kinase from control and insulin-treated cells. The results indicate that the insulin-stimulated increase in protamine kinase activity may be due to a covalent modification, possibly phosphorylation, of the protamine kinase.
Reddy
S A
SA
Department of Biological Sciences, University of South Carolina, Columbia 29208.
Amick
G D
GD
Cooper
R H
RH
Damuni
Z
Z
D000818:Animals; D002384:Catalysis; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004789:Enzyme Activation / Q000187:drug effects; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D008970:Molecular Weight; D010710:Phosphates / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D017680:Potassium Compounds; D006656:Protamine Kinase; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D054648:Protein Phosphatase 2; D051381:Rats; D011919:Rats, Inbred Strains; D012969:Sodium Fluoride / Q000494:pharmacology; D013379:Substrate Specificity
D007328:Insulin; D010710:Phosphates; D017680:Potassium Compounds; D047428:Protein Kinase Inhibitors; D012969:Sodium Fluoride; C013216:potassium phosphate; D011494:Protein Kinases; D006656:Protamine Kinase; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D011188:Potassium