Garcia-Soto J, Araiza L M, Barrios M, Darszon A, Luna-Arias J P
Instituto de Investigacion en Biologia Experimental, Facultad de Quimica, Universidad de Guanajuato, Guanajuato, GTO, Mexico.
Biochem Biophys Res Commun. 1991 Nov 14;180(3):1436-45. doi: 10.1016/s0006-291x(05)81357-9.
Activity of cyclic nucleotide-dependent protein kinase was investigated in flagellar plasma membranes of sea urchin sperm (S. purpuratus). Membranes incubated with [gamma-32P]ATP showed in the presence of 1 microM cAMP an increased phosphorylation in multiple polypeptides. Half maximal response was seen at 0.6 microM of cAMP. In contrast, higher concentrations (100 microM) of cGMP were required to cause the same amount of protein phosphorylation. 80% of the protein kinase activity stimulatable by cAMP was resistant to extraction by 10 mM EGTA and sonication but it was entirely recovered in a detergent-solubilized fraction. Membranes pretreated with 200 microM cAMP, ultracentrifuged and resuspended in buffer solution did not undergo cAMP-stimulated phosphorylation in their polypeptides. This study demonstrates that flagellar plasma membranes isolated from S. purpuratus sea urchin sperm have an endogenous cAMP-dependent protein kinase, which may be bound to the membrane via its regulatory subunit.
对海胆精子（紫球海胆）鞭毛质膜中依赖环核苷酸的蛋白激酶活性进行了研究。用[γ-32P]ATP孵育的质膜在存在1微摩尔环磷酸腺苷（cAMP）的情况下，多种多肽的磷酸化作用增强。在0.6微摩尔cAMP时可见半数最大反应。相比之下，需要更高浓度（100微摩尔）的环磷酸鸟苷（cGMP）才能引起相同程度的蛋白质磷酸化。可被cAMP刺激的蛋白激酶活性的80%对10毫摩尔乙二醇双四乙酸（EGTA）提取和超声处理具有抗性，但它完全在去污剂增溶级分中回收。用200微摩尔cAMP预处理、超速离心并重新悬浮于缓冲溶液中的质膜，其多肽未发生cAMP刺激的磷酸化作用。这项研究表明，从紫球海胆精子中分离出的鞭毛质膜具有一种内源性cAMP依赖蛋白激酶，它可能通过其调节亚基与膜结合。
