Purification and characterization of a sea urchin egg Ca2+-calmodulin-dependent kinase with myosin light chain phosphorylating activity.
The crude actomyosin precipitate from sea urchin (Arbacia punctulata) egg extracts contains Ca2+-sensitive myosin light chain kinase activity. Activity can be further increased by exogenous calmodulin (CaM). Egg myosin light chain kinase activity is purified from total egg extract by fractionating on three different chromatographic columns: DEAE ion exchange, gel filtration on Sephacryl-300, and Affi-Gel-CaM affinity. The purified egg kinase depends totally on Ca2+ and CaM for activity. Unphosphorylated egg myosin has very little actin-activated ATPase. After phosphorylation of the phosphorylable light chain by either egg kinase or gizzard myosin light chain kinase, the actin-activated ATPase of egg myosin is enhanced several fold. However, the egg kinase bears some unique characteristics which are very different from conventional myosin light chain kinases of differentiated tissues. The purified egg kinase has a native molecular mass of 405 kDa, while on sodium dodecyl sulfate-polyacrylamide electrophoresis it shows a single subunit of 56 kDa. The affinity of egg kinase for CaM (Ka = 0.4 microM) is relatively weaker than that of the gizzard myosin light chain kinase. The egg kinase autophosphorylates in the presence of Ca2+ and CaM and has a rather broad substrate specificity. The possible relationship between this egg Ca2+-CaM-dependent kinase and the Ca2+-CaM-dependent kinases from brain and liver is discussed.
D000205:Actomyosin / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D000070559:Gizzard, Non-avian / Q000201:enzymology; D007700:Kinetics; D009219:Myosin-Light-Chain Kinase; D009218:Myosins / Q000378:metabolism; D010063:Ovum / Q000201:enzymology*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D012617:Sea Urchins; D013379:Substrate Specificity
D002147:Calmodulin; D000205:Actomyosin; D011494:Protein Kinases; D009219:Myosin-Light-Chain Kinase; D000251:Adenosine Triphosphatases; D009218:Myosins; D002118:Calcium