Purification and properties of a protamine kinase from bovine kidney microsomes.
About an eightfold increase in protamine kinase activity was detected following extraction of highly purified microsomes from bovine kidney with 1% Triton X-100. Relative to the soluble fraction, the microsomes contained about 30% protamine kinase activity. The microsomal protamine kinase was purified to apparent homogeneity. The purified enzyme exhibited an apparent M(r) approximately 45,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel permeation chromatography on Sephacryl S-200. Relative to protamine, the purified kinase exhibited about 100% activity with the synthetic peptide RRLSSLRA and about 5, 8, and less than 0.1% activity with casein, histone H2B, and histone H1, respectively. The purified kinase phosphorylated several 40 S ribosome polypeptides. One of these polypeptides was identified as ribosomal protein S6 by N-terminal sequencing. About 2.5 mol of phosphoryl groups was incorporated per mole of ribosomal protein S6 following incubation of the 40 S ribosomes with the purified kinase. Following incubation with protein phosphatase 2A2, purified preparations of the protamine kinase were inactivated. These properties were identical to those of purified preparations of a protamine kinase from extracts of bovine kidney cytosol (Z. Damuni, G.D. Amick, and T.R. Sneed, 1989, J. Biol. Chem. 264, 6412-6418). Near identical peptide patterns were obtained following incubation of purified preparations of the microsomal and cytosolic protamine kinases with Staphylococcus aureus V8 proteinase. The results indicate that a form of the cytosolic protamine kinase is present in microsomes.
Amick
G D
GD
Department of Biological Sciences, University of South Carolina, Columbia 29208.
Reddy
S A
SA
Damuni
Z
Z
D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology*; D008970:Molecular Weight; D010766:Phosphorylation; D006656:Protamine Kinase; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism; D013379:Substrate Specificity
D011494:Protein Kinases; D006656:Protamine Kinase