Abstract
Abstract: :
Purpose: Protein phosphorylation / dephosphorylation are a common and important regulatory modification of proteins. The phosphorylation of bovine interphotoreceptor retinoid–binding protein (IRBP) has been examined in crude bovine interphotoreceptor matrix (IPM) washes using [γ–32P]ATP (Wiggert et al., 1988). In that study, it was found that the phosphorylated IRBP was bound tightly to Con–A sepharose and not eluted by 50 mM α–methyl–D–mannoside. During the purification of IRBP from crude bovine IPM preparations, approximately 18% of the IRBP in the initial wash is tightly bound to Con–A. The purpose of these experiments was to characterize the phosphorylation sites of the two fractions of IRBP, fraction I that is eluted from Con–A with 50 mM α–methyl–D–mannoside and fraction II that is bound tightly to Con–A. Methods:Interphotoreceptor retinoid–binding protein, fractions I and II, was subjected to SDS polyacrylamide gel electrophoresis. In–gel detection of phosphate groups was achieved using the Pro–Q® Diamond phosphoprotein gel stain. Total protein was stained with Sypro® Ruby. Immobilized metal ion affinity chromatography was used for the isolation of phosphorylated peptides from proteolytic digests of bovine IRBP, fractions I and II. An aliquot of the sample, containing either phosphorylated or non–phosphorylated peptides, was spotted onto a target plate. An equal volume of α–cyano–4–hydroxycinnamic acid (10 mg/ml in 50% acetonitrile/0.1% TFA) was applied on top of the sample. Linear and reflectron MALDI mass spectra were recorded on an Applied BioSystems Voyager DE sSTR mass spectrometer. Samples were also subjected to ß–elimination followed by alkylation with methylamine prior to mass spectrometry to distinguish between phosphorylated serine, threonine, and tyrosine residues. Results:Both IRBP fractions, I and II, were stained with the phosphoprotein specific fluorescent stain, an indication that IRBP is phosphorylated. Mass spectrometric analysis of the phosphopeptides isolated from proteolytic digests revealed that the sites of phosphorylation were different for IRBP fractions I and II. In the case IRBP fraction I, the phosphorylated peptides are localized toward the C–terminus, whereas, the phosphorylated peptides isolated from IRBP fraction II are localized closer to the N–terminus. Conclusions:Both IRBP fractions I and II are phosphorylated. IRBP fraction I contains phosphopeptides localized toward the C–terminus, whereas, IRBP fraction II contains phosphopeptides localized toward the N–terminus. Phosphorylation of IRBP may be important in the function of IRBP in the IPM.
Keywords: phosphorylation • protein purification and characterization • protein modifications–post translational