Oxidative stability and radical scavenging activity of extra virgin olive oils: an electron paramagnetic resonance spectroscopy study.

The oxidative stability of extra virgin olive oils (EVOO) from the Greek island of Crete was evaluated by electron paramagnetic resonance (EPR) spectroscopy and the spin trapping technique. The spin trap N-t-butyl-alpha-phenylnitrone (PBN) was added to the olive oil samples and the production of free radicals was monitored during heating at 70 degrees C. Induction time for the accelerated oxidation of virgin olive oils at 70 degrees C was determined. The EPR results were compared with the oxidative stability values provided by the Rancimat method at 110 degrees C and high linear correlations were found (r=0.922). EPR spin trapping provides a sensitive and rapid method for evaluating the oxidative stability of EVOO. The same samples of Greek extra virgin olive oils were also examined for their radical scavenging activity (RSA) toward the stable galvinoxyl radical by EPR spectroscopy. The decrease of the intensity of the EPR signal upon incubation time was followed. Both oxidative stability and radical scavenging activity of EVOO samples were correlated to their content in polyphenols and tocopherols.

A new homogeneous enzyme immunoassay for thyroxine using glycogen phosphorylase b-thyroxine conjugates.

BACKGROUND: [corrected] Measurement of serum thyroxine (T(4)) concentration is important for diagnosis of thyroid gland diseases. We developed a practical homogeneous enzyme immunoassay for thyroxine analysis in unextracted sera. METHODS: A thyroxine derivative conjugated to a reactive sulfhydryl group of glycogen phosphorylase b (GPb). Conjugation caused inhibition of enzyme activity and the enzyme conjugate was re-activated upon the binding of a polyclonal anti-T(4) antibody. Antibody-activation was blocked by the presence of free T(4). RESULTS: Conjugation affected the allosteric character of the enzyme and the K(m) for the allosteric activator AMP was increased 28 times, while anti-T(4) antibody partially reversed this effect. The optimum concentration ratio of enzyme conjugate to anti-T(4) antibody was determined, and T(4) was measured with desired sensitivity and accuracy in the range between 10 and 240 microg/l. Furosemide was used to inhibit the interaction of thyroxine with serum T(4)-binding sites. Human serum T(4) values obtained by this method correlated well with those obtained by a radioimmunoassay (y=1.9+1.0x, r=0.97, N=72). CONCLUSIONS: Chemical modification of glycogen phosphorylase b with a T(4) derivative led to the development of a simple homogenous enzyme immunoassay for T(4) analysis with the desired sensitivity and accuracy.

Homogeneous enzyme immunoassay for triiodothyronine in serum.

BACKGROUND: The concentration of triiodothyronine (T3) in human serum is extremely low and can be determined only by very sensitive methods. We developed a homogeneous enzyme immunoassay for T3 analysis in unextracted serum. METHODS: A T(3) derivative was conjugated to the -SH groups of glycogen phosphorylase b (GPb) from rabbit muscle. Conjugation caused inhibition of enzyme activity, and the enzyme conjugate was reactivated upon binding of anti-T3 antibody. Activation was blocked by the presence of non-antibody-bound T3; this was the basis for the development of the homogeneous enzyme immunoassay for T3 by determining GPb activity fluorometrically. RESULTS: We used furosemide to block the interaction of T3 with serum proteins with T3-binding sites, avoiding any serum treatment step. T3 was measured in the range 0.3-8 microg/L. T3 values obtained by this assay correlated well with those obtained by a RIA (y = 0.97x - 0.07 microg/L; r = 0.96; n = 92). Within- and between-run imprecision (CV) was 5-9% for normal and high concentrations and 16-20% for low concentrations. CONCLUSIONS: Chemical modification of GPb with a T3 derivative allows the development of a simple homogeneous enzyme immunoassay for T3 in unextracted serum.

Immunocytochemical localization of glycogen phosphorylase kinase in rat brain sections and in glial and neuronal primary cultures.

The physiological function of brain glycogen and the role of phosphorylase kinase as a regulatory enzyme in the cascade of reactions associated with glycogenolysis in the brain have not been fully elucidated. As a first step toward elucidating such a function, we studied the localization of phosphorylase kinase in glial and neuronal primary cell cultures, and in adult rat brain slices, using a rabbit polyclonal antibody against skeletal muscle glycogen phosphorylase kinase. Immunocytochemical examination of rat astroglia-rich primary cultures revealed that a large number of cells were positive for glycogen phosphorylase kinase immunoreactivity. These cells were also positive for vimentin, a marker for immature glia, while they were negative for glial fibrillary acidic protein, a marker for mature astroglia, and for galactocerebroside, an oligodendroglial marker. Neurons in rat neuron-rich primary cultures did not show any kinase-positive staining. In paraformaldehyde-fixed adult rat brain sections, phosphorylase kinase immunoreactivity was detected in glial-like cells throughout the brain, with relatively high staining found in the cerebral cortex, the cerebellum, and the medulla oblongata. Phosphorylase kinase immunoreactivity could not be detected in neurons, with the exception of a group of large neurons in the brain stem, most likely belonging to the mesencephalic trigeminal nucleus. Phosphorylase kinase was also localized in the choroid plexus and to a lesser degree in the ependymal cells lining the ventricles. Phosphorylase kinase thus appears to have the same cellular distribution in nervous tissue as its substrates, i.e. glycogen phosphorylase and glycogen, which suggests that the physiological role of brain phosphorylase kinase is the mobilization of glycogen stores to fuel the increased metabolic demands of neurons and astrocytes.

The association of phosphorylase kinase with membranes of rat liver smooth endoplasmic reticulum.

Upon fractionation of a post mitochondrial supernatant from rat liver, phosphorylase kinase activity was largely recovered in the cytosol and the smooth endoplasmic reticulum (SER) fraction. The presence of phosphorylase kinase in SER vesicles was not due to an interaction of the enzyme with glycogen particles, since previous elimination of SER glycogen either by 48 h animal starvation or by treatment of the membrane fraction with alpha-amylase did not significantly alter phosphorylase kinase activity content. Washing of the initial pellet of SER fraction (crude SER) by dilution and recentrifugation, released in the supernatant an amount of phosphorylase kinase activity, which is dependent on: i) the degree of dilution, ii) the number of washes, iii) the ionic strength of the washing solution and iii) the presence or absence of Ca2+. Crude SER-associated phosphorylase kinase was marginally affected by increased concentrations of antibody against rabbit skeletal muscle holoenzyme which nevertheless drastically inhibited cytosolic enzyme activity, while it showed a higher resistance to partial proteolysis and a different Western blotting profile with anti-phosphorylase kinase when compared with the soluble kinase. A small but significant fraction of SER phosphorylase kinase was strongly associated with the microsomal fraction being partly extractable only in presence of detergents. This membrane-bound enzyme form exhibited an alkaline pH optimum, in contrast to the neutral pH optima of both soluble and weakly associated phosphorylase kinase.

Subcellular distribution of phosphorylase kinase in rat brain. Association of the enzyme with mitochondria and membranes.

The evaluation of glycogen phosphorylase kinase in rat brain subcellular fractions was undertaken in order to get further insight into the association of this kinase with specific neuronal cell compartments. The enzyme was found to be primarily soluble, but considerable latent specific activities were observed in particulate fractions, especially in microsomes, mitochondria and synaptosomes, which could be unmasked by treatment with Triton-X-100. The submitochondrial and subsynaptic distribution patterns of phosphorylase kinase revealed high overt activity in the mitochondrial intermembrane space and high latent activities in mitochondrial membranes, and synaptic vesicles, membranes and mitochondria. The Ca(2+)-dependency of soluble phosphorylase kinase was similar to that of microsomal enzyme but higher than that of other particulate enzyme forms. Mitochondrial phosphorylase kinase showed a higher pH 6.8:8.2 activity ratio than the soluble and the microsomal enzyme. The rate of inactivation of cytosolic phosphorylase kinase by proteinase K was higher than that of microsomal and mitochondrial enzymes. Antibodies against rabbit skeletal muscle phosphorylase kinase effectively inhibited both cytosolic and microsomal enzyme but failed to significantly affect the kinase activity present in intact mitochondria and intermembrane space. Western blotting with anti-phosphorylase kinase showed that rat brain mitochondria exhibited a significantly lower immunoreactivity compared to soluble cytosol. In conclusion, the presence of phosphorylase kinase activity in a variety of particulate fractions of rat brain suggests a multiplicity of actions of this kinase in neuronal tissues.

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.

Kinetic properties of tetrameric glycogen phosphorylase b in solution and in the crystalline state.

R-state monoclinic P2(1) crystals of phosphorylase have been shown to be catalytically active in the presence of an oligosaccharide primer and glucose-1-phosphate in 0.9 M ammonium sulfate, 10 mM beta-glycerophosphate, 0.5 mM EDTA, and 1 mM dithiothreitol, the medium in which the crystals are grown or equilibrated for crystallographic studies (Barford, D. & Johnson, L.N., 1989, Nature 360, 609-616; Barford, D., Hu, S.-H., & Johnson, L.N., 1991, J. Mol. Biol. 218, 233-260). Kinetic data suggest that the activity of crystalline tetrameric phosphorylase is similar to that determined in solution for the enzyme tetramer. However, large differences were found in the maximal velocities for both oligosaccharide or glucose-1-phosphate substrates between the soluble dimeric and crystalline tetrameric enzyme.

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.

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 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+.

Pest sequences present in the subunits of phosphorylase kinase.

We report that the amino acid sequences of all four subunits of rabbit skeletal muscle phosphorylase kinase possess one or more regions rich in proline (P), glutamic acid (E), serine (S) and threonine (T). alpha and beta subunits contain strong PEST sequences, showing PEST scores greater than 0 (Rogers et al. (1986) Science 234, 364-368), while gamma and delta subunits contain weak PEST regions (negative PEST scores greater than -5.3). In addition to PEST sequences, alpha, beta and gamma subunits contain clusters of arginine pairs. The above sequence characteristics may serve to signal rapid turnover of phosphorylase kinase.

Electrophoretic purification of the alpha and beta subunits of phosphorylase kinase and evidence in support of the deduced amino acid sequences.

A simple, rapid sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method is presented for isolating the alpha, alpha' and beta subunits of rabbit muscle phosphorylase kinase. The SDS-PAGE procedure can yield milligram amounts of alpha and beta from a single preparative gel and also allows isolation of the alpha' isozyme free of alpha. Notably the method provides the purified subunits in a form amenable to structural analysis. Edman degradation of alpha and alpha' reveal identical NH2-terminal structures. Amino acid analysis of the electrophoretically purified alpha and beta subunits are in good agreement with their deduced primary structures. The amino acid sequence of 488 residues in alpha and 713 residues in beta were determined by gas phase Edman degradation. The data support the recently deduced primary structures of alpha (Zander et al., Proc. Natl. Acad. Sci. USA, 1988, 85, 9381-9385).

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.

Reaction of fluorescein isothiocyanate with an ATP-binding site on the phosphorylase kinase alpha subunit.

Phosphorylase kinase can be labeled specifically on the alpha subunit with fluorescein 5'-isothiocyanate (FITC) which concomitantly inactivates the enzyme (T. G. Sotiroudis and S. Nikolaropoulus (1984) FEBS Lett. 176, 421-425). Labeled peptides have been purified and their primary structure has been determined. The amino acid sequence of the fluorescein-labeled tryptic peptide is Lys-Met-Gln-Asp-Gly-Tyr-Phe-Gly-Gly-Ala-Arg. The environment of this fluorescein-labeled lysine has been determined by sequencing peptides isolated from a Staphylococcus aureus V8 digest and two further cyanogen bromide fragments of the purified [14C]carboxymethylated alpha subunit. The partial sequences obtained have then been localized in the primary structure of the alpha subunit [Zander et al. (1988) Proc. Natl Acad. Sci. USA 85, 2929-2933]. Both the incorporation of the fluorescent label and enzymatic inactivation are inhibited by ATP only at pH 7.0; ADP and AMP do not protect. Kinetic analysis reveals a competition between ATP and FITC; a Ki for ATP of 728 +/- 100 microM has been determined.