Purification and characterization of a protein kinase from goat sperm plasma membrane.
A protein kinase that causes phosphorylation of serine and threonine residues of casein has been partially purified from goat cauda-epididymal sperm plasma membrane and characterized. The kinase, solubilized from the membrane with 1.0% Triton X-100, was purified to 480-fold by using DEAE-cellulose and casein-Sepharose affinity chromatographic techniques. The kinase is a strongly basic protein with pI of 9.5. The enzyme has a molecular mass of 310 kilodaltons as estimated by Sephacryl S-300 gel exclusion. The kinase showed affinity for protein substrates in the order membrane proteins > casein > phosvitin > histone > protamine. The apparent Km values of the kinase for casein and membrane proteins were 1 and 0.15 mg/mL, respectively. The synthetic peptides Kemptide and poly(Glu80Tyr20) did not serve as substrates of the enzyme. ATP, rather than GTP or PP(i), is the donor of phosphate for the phosphorylation reaction. Cyclic AMP and GMP, NaCl (0.25 M), KCl (0.25 M), Ca2+, calmodulin, phosphatidylserine, and muscle protein kinase inhibitor had no appreciable effect on the kinase activity. Heparin (0.5 microgram/mL) showed high affinity for inhibiting only 40% of the kinase activity, whereas polyamines at a relatively high concentration (5 mM) inhibited 40-50% of the enzymic activity. The kinase appears to be distinct from other protein kinases including casein kinases. The activity of the kinase derived from the purified sperm plasma membrane was markedly (approximately 90%) lost when the intact spermatozoa were pretreated with diazonium salt of sulfanilic acid, a membrane nonpenetrating surface probe.(ABSTRACT TRUNCATED AT 250 WORDS)
Indian Institute of Chemical Biology, Calcutta.
S
S
Mitra
D
D
Nath
G C
GC
Majumder
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000818:Animals; D002364:Caseins / Q000378:metabolism; D002413:Cations, Divalent; D002462:Cell Membrane / Q000201:enzymology*; D006041:Goats; D006493:Heparin / Q000494:pharmacology; D007526:Isoelectric Point; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008970:Molecular Weight; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D011073:Polyamines / Q000494:pharmacology; D011494:Protein Kinases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013094:Spermatozoa / Q000201:enzymology*; D013379:Substrate Specificity
D002364:Caseins; D002413:Cations, Divalent; D011073:Polyamines; D010769:Phosphothreonine; D010768:Phosphoserine; D002731:4-Chloromercuribenzenesulfonate; D006493:Heparin; D011494:Protein Kinases; D008274:Magnesium