Abstract
Abstract: :
Purpose: The reversible phosphorylation of proteins plays essential roles in regulating various cellular events, and its process is regulated by the opposite action of protein kinases and protein phosphatases. In the lens system, a number of proteins are phosphorylated by a variety of kinases, resulting in well-known regulatory effects, however, the identity and role of corresponding phosphatase in less clear. Protein phosphatases are generally divided into two main groups based on substrate specificity, namely serine/threonine phosphatases (PSP) and tyrosine phosphatases (PTP), and each is subdivided into some groups. Here we report that various kinds of protein phosphatases are present in the lens, based on assays of their phosphatase activities. Methods: Lens homogenate of chick embryos was prepared and fractionated by size exclusion chromatography. Various phosphatase activities in all fractions were assayed using corresponding specific substrates and inhibitors. Further purification and characterization of fractions containing each phosphatase activity were also conducted. Results: Using [P-32]phosphorylase a and inhibitor-1, which are a specific substrate and an inhibitor of PSP type1 (PP1), we confirmed PP1 activities in different three fractions of over 200-kDa, around 110-kDa and 45 kDa in the lens protein. When used a phosphoThr-containing peptide and okadaic acid, which is specific for PP2A, different two peaks estimated as PP2A were detected around 110-kDa and 35-kDa. Further, PSP activity that was highly dependent on magnesium or manganese ions was found around 50-kDa. It presumably belongs to PP2C. On the other hand, when phosphoTyr-containing peptides were examined at neutral pH, which are specific substrates for PTP, only one activity peak at over 200-kDa was detected, presumed to be membrane-bound PTP(s). At pH5.5, another peak of 18 kDa was found, which have been confirmed as a low-molecular-weight(LMW)-PTP. Conclusions: PSP type 1, type 2A, 2C, membrane-bound PTP and LMW-PTP do exist in the ocular lens. Recently these protein phosphatases are suggested to be involved in stress response and apoptosis. Their physiological roles in the lens are interesting.
Keywords: enzymes/enzyme inhibitors • protein purification and characterization • signal transduction