Purification and properties of the phosphorylated form of guanylate cyclase.
Guanylate cyclase is dephosphorylated in response to the interaction of egg peptides with a spermatozoan surface receptor (Suzuki, N., Shimomura, H., Radany, E. W., Ramarao, C. S., Ward, G. E., Bentley, J. K., and Garbers, D. L. (1984) J. Biol. Chem. 259, 14874-14879). Here, the phosphorylated form of guanylate cyclase was purified to apparent homogeneity from detergent-solubilized spermatozoan membranes by the use of GTP-agarose, DEAE-Sephacel, and concanavalin A-Sepharose chromatography. To prevent dephosphorylation of the enzyme during purification, glycerol (35%) was required in all buffers. Following purification, a single protein-staining band of Mr 160,000 was obtained on sodium dodecyl sulfate-polyacrylamide gels. The final specific activity of the purified enzyme was 83 mumol of cyclic GMP formed/min/mg of protein at 30 degrees C, an activity 5-fold higher than that observed with the purified, dephosphorylated form of guanylate cyclase. A preparation containing protein phosphatase from spermatozoa, or highly purified alkaline phosphatase (from Escherichia coli), catalyzed the dephosphorylation of the enzyme; this resulted in a subsequent decrease in guanylate cyclase activity and a shift in the Mr from 160,000 to 150,000. The phosphate content of the high Mr form of the enzyme was 14.6 mol/mol protein whereas the phosphate content of the low Mr form was 1.6 mol/mol protein. All phosphate was localized on serine residues. The Mr 160,000 form of guanylate cyclase demonstrated positive cooperative kinetics with respect to MnGTP while the Mr 150,000 form displayed linear, Michaelis-Menten type kinetics. The phosphorylation state of the membrane form of guanylate cyclase, therefore, appears to dictate not only the absolute activity of the enzyme but also the degree of cooperative interaction between catalytic or GTP-binding sites.
D000818:Animals; D005990:Glycerol; D006160:Guanosine Triphosphate / Q000378:metabolism; D006162:Guanylate Cyclase / Q000302:isolation & purification* / Q000378:metabolism; D008297:Male; D008345:Manganese / Q000378:metabolism; D008970:Molecular Weight; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D012617:Sea Urchins; D013094:Spermatozoa / Q000201:enzymology
D008345:Manganese; C037047:manganese GTP; D006160:Guanosine Triphosphate; D010749:Phosphoprotein Phosphatases; D006162:Guanylate Cyclase; D005990:Glycerol