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.

Phosphorylase kinase from bovine stomach smooth muscle: a Ca2(+)-dependent protein kinase associated with an actin-like molecule.

Phosphorylase kinase was purified (110-fold) from bovine stomach smooth muscle by a procedure involving DEAE-cellulose chromatography, ammonium sulfate fractionation and glycerol density ultracentrifugation. On sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) the final enzyme preparation shows a single protein band of 43 kDa. The purified protein exhibits a close similarity with bovine aortic actin, as revealed by amino acid analysis and sequencing of a tryptic decapeptide fragment, although it differs widely from actin in several respects. In our effort to separate phosphorylase kinase activity from the 43 kDa protein we used a variety of chromatographic procedures, but in all cases the catalytic activity (when eluted) was accompanied by the 43 kDa protein band. Bovine stomach phosphorylase kinase exhibits an apparent molecular mass of 950 kDa, it shows a low Vmax value for phosphorylase b (85 nmol.min-1.mg-1), a pH 6.8/8.2 activity ratio of 0.23, it has an absolute requirement for Ca2+ and it is activated 1.8-fold by Ca2+/calmodulin. Furthermore, the protein kinase activity is neither inhibited by antibodies against rabbit skeletal muscle phosphorylase kinase nor activated by protein phosphorylation. These results suggest that bovine stomach phosphorylase kinase is tightly bound to an aggregate of actin-like molecules.

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.

Effects of polymyxin B on the sarcoplasmic reticulum membrane.

Polymyxin B, a cyclic peptide antibiotic, inhibits Ca2+-ATPase, p-nitrophenyl phosphatase and phosphorylase kinase activities associated with rabbit skeletal muscle sarcoplasmic reticulum membranes; 50% inhibition is induced by 100 microM, 130 microM and 550 microM of polymyxin respectively. The fluorescence intensity of fluorescein isothiocyanate-labeled Ca2+-ATPase, decreases in the presence of polymyxin (50% of the total decrease at 70 microM polymyxin). On the other hand, the polypeptide inhibits calmodulin-dependent endogenous phosphorylation of 60 kDa, 20 kDa and 14 kDa membrane proteins, while an increase of calmodulin-dependent phosphorylation is observed in 132 kDa and 86 kDa proteins.

Modulation of phosphorylase kinase activity by sphingolipids.

Psychosine (galactosyl sphingosine) potently inhibits the activity of both nonactivated and activated by covalent modification (autophosphorylation and limited proteolysis) rabbit skeletal muscle phosphorylase kinase. Half-maximal inhibition was observed at 44 microM or 66 microM when the kinase activity was assayed at pH 6.8 or 8.2 respectively. Sphingosine was also inhibitory, but only at pH 6.8 (half-maximal inhibition was observed at 130 microM). In this respect, sphingomyelin, cerebroside and cerebroside sulfate were ineffective. On the other hand, a number of gangliosides stimulated the activity of nonactivated phosphorylase kinase at neutral pH. Among the individual gangliosides tested the activation potency was GD1a greater than GT1b greater than GM1, while GM3 was without effect. Most important, GD1a dramatically increases the activity of the kinase at low Ca2+ concentrations. Both psychosine and GD1a increased the rate of kinase autophosphorylation on alpha- subunit only, but although ganglioside-induced stimulation of autophosphorylation was accompanied with an enhancement of the rate of autoactivation at pH 6.8, psychosine completely blocked autoactivation.

Ca2+- and Mg2+-dependent association of phosphorylase kinase with human erythrocyte membranes.

The interaction of rabbit muscle phosphorylase kinase (EC 2.7.1.38) with human erythrocyte membranes was investigated. It was found that at pH 7.0 the kinase binds to the inner face of the erythrocyte membrane (inside-out vesicles) and that this binding is Ca2+- and Mg2+-dependent. The sharpest increase in the binding reaction occurs at concentrations between 70 and 550 nM free Ca2+. Erythrocyte ghost or right-side out erythrocyte vesicles showed a significantly lower capacity to interact with phosphorylase kinase. Autophosphorylated phosphorylase kinase shows a similar Ca2+-dependent binding profile, while trypsin activation of the kinase and calmodulin decrease the original binding capacity by about 50%. Heparin (200 micrograms/ml) and high ionic strength (50 mM NaCl) almost completely blocks enzyme-membrane interaction; glycogen does not affect the interaction.

Presence of sialic acids in Lactobacillus plantarum.

It was found that Lactobacillus plantarum (strain BA 11) is able to synthesize sialic acids during its growth in MRS medium and that these molecules are located mainly on the surface of the bacterium. It was demonstrated also that the addition externally of N-acetylneuraminic acid in concentrations ranged from 10 to 500 microM into the culture medium, resulted to a substantial increase of the growth rate of the bacterium. Bacterial cultures in presence of added sialic acid (100 microM) for 24 hours, resulted to a two fold increase of the final bacterial mass compared to the cultures in absence of sialic acid. Maximum levels of sialic acids were observed after 48 h of bacterial growth. It was also found that neuraminic acids production was increased when Mn++ and Mg++ ions were added in the culture medium, while the addition of Co++, Ca++, Ba++, Cu++ and Ni++ had a negative effect.

Spin-label studies of glycogen phosphorylase hosted in microemulsion droplets.

ESR spectra of Phosphorylase b labelled with two different paramagnetic probes were recorded in microemulsions of various compositions, formulated with AOT and isooctane. Measurements of the hyperfine coupling constant aN and of the degree of anisotropy H1/H0, showed that: i) the polarity of the microenvironment of both spin-labels bound to the enzyme, expressed in terms of aN, is increased with increasing the hydration ratio of the reversed micelles, R; ii) a transition of the polarity between bound and free water is observed at R = 10; iii) the mobilities of the spin-labelled moieties of the enzyme are different, depending on the hydrophobicity of the site where the probe is bound and on the size of the microdroplets.

Stimulation of glycogen phosphorylase kinase by phospholipids.

The acidic phospholipids phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-biphosphate (PIP2) and the neutral phospholipid lysophosphatidylcholine (LPC) were found to stimulate (3 to 8-fold) the activity of nonactivated rabbit skeletal muscle phosphorylase kinase at pH 6.8, without significantly affecting the activity at pH 8.2. In this respect, phosphatidylcholine and phosphatidylethanolamine were ineffective, while the anionic detergent sodium dodecyl sulfate (SDS) and the anionic steroid dehydroisoandrosterone sulfate (DIAS) were able to mimic the action of phospholipids. SDS was also found to be a very efficient activator of the autophosphorylation of phosphorylase kinase (20-fold activation at 200 microM). The activating effect of phospholipids largely depends on the size of lipid vesicles, which is connected with the procedure of their preparation. These results suggest that phosphorylase kinase belongs to the class of Ca2+-dependent enzymes, which are sensitive to stimulation by calmodulin, limited proteolysis and anionic amphiphiles.

Interaction of flavonoids with rabbit muscle phosphorylase kinase.

We have examined the effect of several flavonoids on the activity of phosphorylase kinase from rabbit skeletal muscle. From 14 flavonoids tested, the flavones quercetin and fisetin were found to be efficient inhibitors of nonactivated phosphorylase kinase when assayed at pH 8.2, causing 50% inhibition at a concentration of about 50 microM, while the flavanone hesperetin stimulated phosphorylase kinase activity about 2-fold when tested at 250 microM. The efficiency of quercetin in inhibiting the kinase is higher when the enzyme is stimulated either by ethanol or by alkaline pH. Both casein and troponin phosphorylation by phosphorylase kinase and the autophosphorylation of the kinase were inhibited by quercetin. In addition, quercetin was found to be a competitive inhibitor of ATP for the phosphorylation of phosphorylase b at pH 8.2. These observations suggest that the inhibitory effect of the flavone is directly on the phosphorylase kinase molecule. Trypsin-activated phosphorylase kinase was inhibited by quercetin and stimulated by hesperetin, as for the native enzyme.

Interaction of phosphorylase kinase with polymyxins.

Nonactivated rabbit skeletal muscle phosphorylase kinase is inhibited by the polymyxins A, B, D and E when assayed at pH 8.6. Polymyxin B is the most effective inhibitor, causing 50% inhibition at 0.3 mM. Following the effect of polymyxin B on the kinase activity toward troponin, no inhibition was observed. In contrast, polymyxin B was found to greatly stimulate the autophosphorylation of phosphorylase kinase. About 10 mol of phosphate per tetramer (alpha beta nu delta) were incorporated in presence of polymyxin B (full autophosphorylation). This incorporation was about 6-fold higher than that observed without polymyxin. The stimulation of autophosphorylation by polymyxin B was accompanied with enhancement of the rate of autoactivation at pH 6.8.

Effect of sodium cholate on the catalytic and structural properties of phosphorylase b.

Sodium cholate at millimolar concentration is able to induce activity in rabbit muscle phosphorylase b in the absence of AMP. The maximum activation of the enzyme in presence of 7 mM sodium cholate was 24% of that achieved by 1 mM AMP. Other bile salts tested showed a negligible activating effect. The Ka for AMP was lowered fivefold by 5 mM of the steroid detergent, while the cooperative binding of the nucleotide was abolished. Phosphorylase b', a modified form of phosphorylase in which the phosphorylation site has been removed by limited tryptic attack, presented an activation profile similar to that of phosphorylase b. In contrast, phosphorylase a was inhibited by the bile salt, while the activity of liver phosphorylase b was not significantly affected. Modification of the AMP site of the enzyme with 2,3-butanedione could not inhibit sodium-cholate-induced activity. tert-Butanol, an organic solvent activator of phosphorylase b, was found to enhance the activity induced by sodium cholate. The interaction of sodium cholate and phosphorylase b was also followed by difference spectroscopy using a fluorescein isothiocyanate--phosphorylase b conjugate. Furthermore, measurements of electron spin resonance demonstrated that the mobility of a spin-label bound at buried--NH2 groups of phosphorylase b decreases cooperatively with increasing bile salt concentration.

Interaction of aliphatic amines with glycogen phosphorylase.

A number of aliphatic amines was shown to stimulate AMP-dependent activity of phosphorylase b. The extent of stimulation depends on the molecular structure of amines. For linear amines, the longer the linear chain, the greater the stimulation observed. High concentrations of amines were able to induce a small activation of phosphorylase b in the absence of AMP. Kinetic studies of phosphorylase b indicated that the presence of n-hexylamine (a) results in lowering Km values for AMP and glucose 1-phosphate, (b) increases maximal velocity of the enzyme, and (c) modifies the glucose 6-phosphate, ATP, caffeine, and glucose binding sites of the enzyme by increasing the inhibition constants for these inhibitors. In contrast, the activity of phosphorylase b' is not altered by n-hexylamine. This fact suggests the possibility that amines interact with the N-terminal tail of phosphorylase b chain.