鯉魚頭腎多功能性蛋白激?抑制物之純化

Abstract

本文利用Reactive Blue72 染劑親和性管柱色層分析，Superdex G-75 分子篩選管柱色層分析及HPLC C18逆相疏水性管柱色層分析等一步步管柱色層分析，從鯉魚頭腎中純化出一多功能性抑制蛋白，這抑制蛋白(PKI-16)無論是在還原性或非還原性SDS-PAGE上，表現的分子質量為16,000 Da。從in vitro蛋白質磷酯化分析，我們發現PKI-16能抑制多種不同的蛋白激?，這些蛋白激?包括蛋白激?C，蛋白激?A，肝糖磷酸化?b激?，細胞質液中的蛋白激?，腦脊髓液中的蛋白激?及大腸菌中的蛋白激?。由磷酯化胺基酸分析法中，得知PKI-16主要抑制Ser/Thr蛋白激?。PKI-16的50％有效抑制濃度約為40nM，且可能藉由競爭ATP來抑制蛋白激?的作用。這抑制蛋白以頭腎，腎臟及脾臟含量較高，且在頭腎及脾臟中以高濃度鉀離子造成細胞膜去極化下，這抑制蛋白能從細胞中被釋放出來。有趣的是，PKI-16在in vivo於Tyrosine位置被磷酯化，且在in vitro下也能被腦脊髓液中Ser/Thr蛋白激?所磷酯化。

A multifunctional protein kinase inhibitor has been purified from carp headkidney by sequential column chromatograghy on Reative Blue 72 affinity column, Superdex-G 75 gel filtration column and HPLC C18 reverse phase column. The inhibitor (PKI-16) migrates as a protein of apparent Mr 16,000 on reduced and non-reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We have found that PKI-16 inhibits several protein kinases including protein kinase C, protein kinase A, phosphorylase b kinase, cytosolic protein kinases, cerebrospinal fluid (CSF) kinase and protein kinases from E.coli. by in vitro protein phosphorylation assay. By phosphoamino acid analysis, PKI-16 mainly inhibits Ser/Thr protein kinases. Under our assay conditions, PKI-16 inhibits protein phosphorylation with an EC50 of about 40 nM and the inhibition appears to be by competition of ATP. The protein is found to be present in high amounts in headkidney, spleen and kidney. In addition, the protein is secreted by headkidney and spleen and the secretion can be stimulated by K+-induced membrane depolarization. Interestingly, PKI-16 itself can be Serine-phosphorylated in vitro by the CSF kinase however it is Tyrosine-phosphorylated in vivo.