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#17641091   2007/07/20 Save this To Up

A novel pyruvate kinase (PK-S) from boar spermatozoa is localized at the fibrous sheath and the acrosome.

Boar spermatozoa contain a novel pyruvate kinase (PK-S) that is tightly bound at the acrosome of the sperm head and at the fibrous sheath in the principal piece of the flagellum, while the midpiece contains a soluble pyruvate kinase (PK). PK-S could not be solubilized by detergents, but by trypsin with no loss of activity. Purified PK-S as well as PK-S still bound to cell structures and soluble sperm PK have all kinetics similar to those of rabbit muscle PK-M1. The PK-S subunit had a relative molecular mass of 64 +/- 1 x 10(3) (n = 3), i.e. slightly higher than that of PK-M1, and carried an N-terminal extension (NH(2)-TSEAM-COOH) that is lacking in native PK-M1. Evidence is provided that PK-S is encoded by the PKM gene. Antibodies produced against the N-terminus of purified PK-S (NH(2)-TSEAMPKAHMDAG-COOH) were specific for PK-S as they did not react with somatic PKs or soluble sperm PK, while anti-PK-M1 recognized both sperm PKs. Immunofluorescence microscopy showed anti-PK-S to label the acrosome and the flagellar principal piece, whereas the midpiece containing the mitochondria was labelled only by anti-PK-M1. Immunogold labelling confirmed the localization of PK-S at the acrosome. In the principal piece, both polyclonal anti-PK-M1 and anti-PK-S were found at the fibrous sheath. Our results suggest that PK-S is a major component in the structural organization of glycolysis in boar spermatozoa.

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#15982968   2005/09/30 Save this To Up

Concentrations of glycolytic enzymes and other cytosolic proteins in the diffusible fraction of a vertebrate muscle proteome.

We used a novel microvolumetric technique based on protein diffusion to characterize the subproteome of muscle that consists of diffusible proteins, including those involved in cell metabolism. Muscle fiber segments were mechanically demembranated under mineral oil and transferred into drops of relaxing solution. After the fiber segment was depleted of diffusible proteins, the content of each drop and residual segment was analyzed by one-dimensional polyacrylamide gel electrophoresis. Proteins were identified through peptide mass fingerprinting and quantified using purified protein standards. Ten of the most abundant cytosolic proteins, distinguished by their ability to readily diffuse out of the skinned fiber, were glycolytic enzymes whose concentrations ranged from 2.6+/-1.0 g liter-1 (phosphoglucose isomerase) to 12.8+/-1.1 g liter-1 fiber volume (pyruvate kinase). The concentrations of the other five most abundant cytosolic proteins were as follows: glycogen phosphorylase, 6.0+/-2.3 g liter-1; phosphoglucose mutase, 2.2+/-0.2 g liter-1; adenylate kinase, 1.6+/-1.3 g liter-1; phosphocreatine kinase, 6.6+/-2.6 g liter-1; and parvalbumin, 0.7+/-0.4 g liter-1. Given the molecular weight and subunit number of each enzyme, the combined concentration of the 15 most abundant cytosolic proteins was 82.3 g liter-1; the volume fraction was 0.093. The large volume fraction of diffusible proteins favors nonspecific interactions and associations, particularly if the glycolytic enzymes and diffusible phosphocreatine kinase are restricted to the I-band as previous studies suggest. The relative molar concentration of glycolytic enzymes is roughly consistent with a stoichiometry of 1:2 for enzymes catalyzing the hexose and triose sugar reactions, respectively, a stoichiometry that may favor metabolic channeling of intermediates during glycolysis. Our results indicate that subcellular fractionation of muscle proteins, in which cytosolic constituents are distinguished by their ability to diffuse readily from demembranated cells, is a promising microvolumetric technique that allows conclusions to be drawn about native protein-protein interactions based on concentration and stoichiometry.

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#10542061   2000/01/10 Save this To Up

Subunit dissociation and inactivation of pyruvate kinase by hydrostatic pressure oxidation of sulfhydryl groups and ligand effects on enzyme stability.

The effect of hydrostatic pressure on the stability of tetrameric rabbit muscle pyruvate kinase was investigated by enzyme activity measurements, size-exclusion chromatography, circular dichroism and fluorescence spectroscopies. Under nonreducing conditions, enzyme activity was irreversibly inhibited by increasing pressure and was completely abolished at 350 MPa. Inhibition was dependent on the concentration of pyruvate kinase, indicating that it was related to pressure-induced subunit dissociation. Size-exclusion chromatography of pressurized samples confirmed a decrease in the proportion of tetramers and an increase in monomers relative to native samples. Addition of dithiothreitol immediately following pressure release led to full recovery of both enzyme activity and of native tetramers. Furthermore, no irreversible inhibition of pyruvate kinase was observed if pressure treatment was carried out in the presence of dithiothreitol. These data suggest that pressure-dissociated monomers undergo conformational changes leading to oxidation of sulfhydryl groups, which prevents correct refolding of native tetramers on decompression. These conformational changes are relatively subtle, as indicated by the lack of significant changes in far-UV circular dichroism and intrinsic fluorescence emission spectra of previously pressurized samples. The effects of various physiological ligands on the pressure stability of pyruvate kinase were also investigated. A slight protection against inhibition was observed in the simultaneous presence of K+, Mg2+ and ADP. Both phosphoenolpyruvate and the allosteric inhibitor, phenylalanine, caused marked stabilization against pressure, suggesting significant energy coupling between binding of these ligands and stabilization of the tetramer.

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#9169614   1997/06/27 Save this To Up

Biochemical characterization of the mouse muscle-specific enolase: developmental changes in electrophoretic variants and selective binding to other proteins.

The glycolytic enzyme enolase (EC 4.2.1.11) is active as dimers formed from three subunits encoded by different genes. The embryonic alphaalpha isoform remains distributed in many adult cell types, whereas a transition towards betabeta and gammagamma isoforms occurs in striated muscle cells and neurons respectively. It is not understood why enolase exhibits tissue-specific isoforms with very close functional properties. We approached this problem by the purification of native betabeta-enolase from mouse hindlimb muscles and by raising specific antibodies of high titre against this protein. These reagents have been useful in revealing a heterogeneity of the beta-enolase subunit that changes with in vivo and in vitro maturation. A basic carboxypeptidase appears to be involved in generating an acidic beta-enolase variant, and may regulate plasminogen binding by this subunit. We show for the first time that pure betabeta-enolase binds with high affinity the adjacent enzymes in the glycolytic pathway (pyruvate kinase and phosphoglycerate mutase), favouring the hypothesis that these three enzymes form a functional glycolytic segment. betabeta-Enolase binds with high affinity sarcomeric troponin but not actin and tropomyosin. Some of these binding properties are shared by the alphaalpha-isoenolase, which is also expressed in striated muscle, but not by the neuron-specific gammagamma-enolase. These results support the idea that specific interactions with macromolecules will address muscle enolase isoforms at the subcellular site where ATP, produced through glycolysis, is most needed for contraction. Such a specific targeting could be modulated by post-translational modifications.

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#9028878   1997/03/13 Save this To Up

A glycolytic enzyme binding domain on tubulin.

Cleavage of tubulin at tryptophan residues yielded several peptides, one of which strongly interacted with aldolase as determined by inhibition of aldolase activity. This peptide was identified as the C-terminal, residues 408-451, of the alpha-subunit of tubulin. Peptides with identical sequences to the C-terminal regions of the alpha- and beta-subunits of tubulin were synthesized to further characterize interactions with glycolytic enzymes. A 43-amino-acid C-terminal peptide from alpha-tubulin (residues 409-451) was found to have binding properties similar to those of native tubulin and was designated the tubulin glycolytic enzyme binding domain (T-GEBD-43mer).

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#8292653   1994/03/02 Save this To Up

[Structure-functional properties of conjugates of proteins with polyalkylene oxides: study by a fluorescence method].

A fluorescent study of some structural and functional properties of conjugates of a number of proteins (bovine serum albumin, pyruvate kinase, alpha-chymotrypsin, and the two toxic proteins of plant origin--ricin and viscumin) with polyalkylene oxides (polyethylene glycol and pluronic) has been carried out. Analysis of the intrinsic protein fluorescence showed that the structure and stability of various protein conjugates to denaturing agents change only slightly: the conformational mobility of tryptophan residues accessible to the solvent decreases, whereas that of tryptophan residues localized in the protein regions of low polarity remains unchanged. Besides, the conjugates display a higher thermal stability in comparison with their native proteins. The fluorescence of 1-anilinonaphthalene-8-sulfonic acid and water insoluble 2',3',4',5'-tetrabenzoylriboflavin bound to the native and modified proteins indicated that modification of the proteins with polyalkylene oxides decreased the polarity and increased the viscosity of the microenvironment. Hence, this modification makes it possible to change some functional characteristics of the protein without causing any significant changes in its structure.

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#8218391   1993/12/14 Save this To Up

A kinetic study of an unstable enzyme measured through coupling reactions. Application to the self-inactivation of detergent-solubilized Ca(2+)-ATPase from sarcoplasmic reticulum.

A methodology for the kinetic study of the self-inactivation of an unstable enzyme has been developed by using the transient-phase approach when the enzymatic activity is measured through a coupled enzyme system. An experimental design has been developed and applied to the inactivation of the Ca(2+)-ATPase from sarcoplasmic reticulum solubilized in the monomeric state. The catalytic activity of the ATP hydrolysis is determined in the presence of pyruvate kinase and lactate dehydrogenase as auxiliary enzymes, and the oxidation of the last substrate, NADH, is continuously monitored. The experimental results show that both substrates, ATP and calcium, protect against enzyme inactivation. This enzyme, the monomeric ATPase, fulfills the catalytic cycle of the native ATPase, and free enzyme and first-calcium bound enzyme are proposed as the intermediates which are being inactivated.

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#1815584   1992/06/26 Save this To Up

Purification and properties of pig heart pyruvate kinase.

Using essentially a two-step procedure involving phosphocellulose column chromatography followed by gel filtration on Sephadex G200, pig heart pyruvate kinase (PH PyK) was purified 267-fold to at least 97% purity. PH PyK co-sedimented with rabbit muscle PyK during sucrose density ultracentrifugation yielding an S20,w of 10 and a corresponding molecular weight of about 237,000. Sodium dodecyl sulfate polyacrylamide gel electrophoresis yielded a subunit molecular weight of approximately 59,000, suggesting that native PH PyK exists as a tetramer. The isoelectric point (pI) was determined to be 8.2, and the pH optimum (pHo) for the forward reaction is 7.2. Steady-state kinetics with phospho(enol)pyruvate (PEP) as the variable substrate show that there is a threefold decrease in the Km for PEP in the presence of 1.0 mM fructose-1,6-diphosphate (FDP), and that the activity of PH PyK is increased over fourfold by FDP at low (0.1 mM) PEP concentrations. Lineweaver-Burk plots are linear in the presence and absence of FDP, indicating that the Michaelis-Menten curves are hyperbolic. The amino acid composition for pig heart PyK shows close similarities between pig muscle and kidney PyKs, but not liver PyK. Among the data on pI, pHo, and FDP activation, only the activation by FDP is useful in tentatively designating pig heart PyK as an M2 isozyme.

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#16667885   2010/06/29 Save this To Up

Purification of leucoplast pyruvate kinase from developing castor bean endosperm.

Leucoplast pyruvate kinase from endosperm of developing castor oil seeds (Ricinus communis L.; cv Baker) has been purified 1370-fold to a specific activity of 41.1 micromoles pyruvate produced per minute per milligram protein. Nondenaturing polyacrylamide gel electrophoresis of the purified enzyme resulted in a single protein staining band that co-migrated with pyruvate kinase activity. However, following sodium dodecyl sulfate polyacrylamide electrophoresis, two major protein staining bands of 57.5 and 44 kilodaltons, which occurred in an approximate 2:1 ratio, respectively, were observed. The native molecular mass was approximately 305 kilodaltons. Rabbit antiserum raised against the final enzyme preparation effectively immunoprecipitated leucoplast pyruvate kinase. The 57.5- and 44-kilodalton polypeptides are immunologically related as both proteins cross-reacted strongly on Western blots probed with the rabbit anti-(developing castor seed endosperm leucoplast pyruvate kinase) immunoglobulin that had been affinity-purified against the 57.5-kilodalton polypeptide. In contrast, pyruvate kinases from the following sources showed no immunological cross-reactivity with the same immunoglobulin: the cytosolic enzyme from developing or germinating castor bean endosperm; chloroplastic pyruvate kinase from expanding leaves of the castor oil plant; chloroplastic or cytosolic pyruvate kinase from the green alga, Selenastrum minutum; and mammalian or bacterial pyruvate kinases.

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#2337353   1990/06/08 Save this To Up

Subunit level crosslinking of rabbit muscle pyruvate kinase by o-phthaldialdehyde.

o-Phthaldialdehyde caused irreversible inhibition of rabbit muscle pyruvate kinase following preliminary formation of an enzyme-reagent complex. At pH 7.5, 35 degrees C, the dissociation constant for the complex and the maximal pseudo-first-order rate constant for covalent modification were 0.32 +/- 0.08 mM and 2.54 +/- 0.23 min-1, respectively. The inactivation was accompanied by uv-spectral changes pointing to isoindole formation, with a limiting stoichiometry of 1 isoindole linkage per enzyme subunit. Phosphoenolpyruvate, ADP, and ATP effectively protected the enzyme against inactivation, suggesting that the active site is the target of o-phthaldialdehyde action. As native and modified enzymes were indistinguishable with respect to mobility of the major band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it was concluded that the crosslinkage was intrasubunit in character, and that the amino acid residues involved must be closely positioned in the polypeptide backbone. Lysine 366, previously shown to be selectively reactive toward 2',3'-dialdehyde ADP (Bezares et al., 1987, Arch, Biochem. Biophys. 253, 133-137), and cysteine 325 or 357 are implicated.

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