Partial purification and characterization of two phosvitin phosphatases from rat brain.

The mechanism of dephosphorylation of multiphosphorylated proteins in the brain is not well understood. We have used the multiphosphorylated protein, phosvitin as a model substrate and undertaken the purification and characterization of brain phosphatases that preferentially dephosphorylate multiphosphorylated proteins. Two phosvitin phosphatase activities, termed Phosvitin Phosphatase 1 and 2 (PvP1, PvP2), which show acidic pH optima were resolved from the 33,000g supernatant fraction from rat brain by a procedure employing successive DEAE-cellulose, Sepharose 6B, second DEAE-cellulose and FPLC/Superose 6 chromatography steps. Following FPLC/Superose 6 size exclusion chromatography of PvP1 and PvP2, single peaks of phosvitin phosphatase activities were eluted in the range of 160-220 kDa with acidic pH optima. When FPLC/Sepharose 6 chromatography was performed in the presence of 0.5 M NaCl and 0.1% Triton X-100, low molecular mass protein phosphatase forms were produced in addition to the high-M, activity peak, ranging from 25 to 35 kDa (PvP1) and from 15 to 25 kDa (PvP2). Under these conditions, both high- and low-M, forms of PvP1 and PvP2 exhibited neutral pH optima. Both phosphatases dephosphorylate also (i) phosphorylase a, (ii) the alpha and beta subunits of phosphorylase kinase, and (iii) the microtubule-associated protein tau, phosphorylated by cAMP-dependent protein kinase. The present results suggest that two forms of protein phosphatases, displayed molecular and biochemical characteristics both similar and distinct from type 1 and type 2A protein phosphatases, are present in rat brain.

Interaction of aluminum ions with phosvitin.

We studied the effects of aluminum ions on the dephosphorylation of phosvitin catalyzed by acid phosphatase, and the metachromasia resulting from the interaction of phosvitin with toluidine blue. In both cases the action of Al3+ was inhibitory and the extent of inhibition was dependent on Al3+ concentration and the length of incubation of Al3+/phosvitin mixtures. The inhibition profiles of dephosphorylation of phosvitin (50 micrograms/ml) showed IC50 values of 15 and 2 microM Al3+ at 1 and 48 hr incubation time, respectively. The effect was proved to be substrate directed, while the inhibition was not reversed by EDTA. In contrast, the action of other divalent or trivalent cations on the dephosphorylation process, when inhibitory, was completely reversible by EDTA. Exposure of fluorescein 5-isothiocyanate-labeled phosvitin to Al3+ resulted in: a) the failure of the protein to migrate into sodium dodecyl sulfate containing polyacrylamide gels and b) the decrease of the fluorescence emission of the bound fluorescein. These findings suggest that phosvitin can be used as a model for studying interactions of aluminum with multiphosphorylated proteins and other polyanionic biopolymers.

A domain of the alpha-subunit of rabbit phosphorylase kinase shows homologies with regions of rabbit alpha-tropomyosin, human EGF receptor, and the alpha chain of bovine S-100 protein.

Sequence comparison of the alpha-subunit of phosphorylase kinase with alpha-tropomyosin revealed 32% identity, and 49% similarity, between the region of alpha-tropomyosin coded by exon 5 and a 39 amino acid segment of the kinase subunit. A subsequence of the alpha-subunit segment and a sequence overlapping the same alpha-subunit region are homologous with: (a) a region of the cytoplasmic domain of EGF receptor (50% identity) and (b) a Ca(2+)-binding domain of the alpha chain of S-100 protein (50% identity) respectively. Statistical analysis shows that these homologies are significant. The biological implication of the above similarities is discussed.

A malachite green colorimetric assay for protein phosphatase activity.

A simple and sensitive colorimetric assay for protein phosphatase activity based on the determination of released Pi by an improved malachite green procedure (A. A. Baykov, O. A. Evtushenko, and S.M. Avaeva, 1988, Anal. Biochem. 171, 266-270) is described. Proteins must be removed or stabilized prior to Pi determination with 0.25 N sulfuric acid or 3% (w/w) perchloric acid. Alternatively, to avoid possible acid hydrolysis of phosphate groups from organic compounds during deproteinization, the protein present in the phosphatase assay mixture can be stabilized with sodium dodecyl sulfate. In this case, the excess detergent is subsequently removed by precipitation with KCl because it colors with the malachite green reagent. The above procedure was applied to the determination of phosphorylase phosphatase activity in bovine brain extracts and the results are comparable to those obtained with the radioisotopic phosphatase assay.

A low-molecular weight acid phosphatase present in crystalline preparations of rabbit skeletal muscle glycogen phosphorylase b.

Crystalline preparations of glycogen phosphorylase b contain traces of acid phosphatase activity. Non-denaturing gel electrophoresis of phosphorylase b reveals a single band of 1-naphthyl phosphate phosphohydrolase activity which co-migrates with phosphorylase. The two enzymes can be separated by Sephadex G-200 column chromatography, where the phosphatase exhibits an apparent Mr of 17,000. The contaminant enzyme hydrolyzes effectively the phenolic ester of monoorthophosphate with optimal activity for p-nitrophenyl phosphate and L-phosphotyrosine between pH 5.5 and 6.0. The phosphatase is insensitive to inhibition by L(+)-tartrate but strongly inhibited by microM vanadate and Zn2+.