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ABSTRACT: Cleavage of the S-carboxymethylated arginine kinase with cyanogen bromide gives rise to eight peptides of 11, 17, 23, 39, 60, 81, 87, 103 amino-acid residues, one of which is an overlap. Ion-exchange chromatography on sulfopropyl-Sephadex and gel filtration on Sephadex G-50 fine, in urea medium, as well as electrochromatography have been used with success for their separation. Their amino-acid composition and end-group structure have been determined and compared to those of the whole protein. All peptides have COOH-terminal homoserine as expected from the presence of methionine at the carboxyl end of the native protein. One acetylated fragment was identified as the N-terminal portion of the protein. The seven major fragments account for the amino-acid composition of the entire protein; from the sum of their amino-acid composition, a molecular weight of 37687 was calculated.
European Journal of Biochemistry. 03/2005; 44(1):67 - 79.
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ABSTRACT: Of the dimeric phosphagen kinases (Mr= 80000) studied, lombricine kinase is the only one which contains only one essential thiol group and one binding site for ADP-Mg. These facts suggest a non-identity between the two subunits of this enzyme.The reversible labeling of the essential thiol group of this protein with 2,4-dinitrofluorobenzene, followed by alkylation of the remaining thiol groups, allowed the separation of the two subunits by means of a Sepharose-mercurial column. The Sepharose-mercurial was prepared by treatment of Sepharose 4B with p-aminophenyl mercuric acetate.Amino-acid analyses, N- and C-terminal determinations and fingerprintings were performed to detect the differences in the primary structure between the two subunits.Furthermore, the Sepharose-mercurial column was used to purify the tryptic peptide containing the essential thiol group of lombricine kinase. By this process, it was possible to obtain, in one step, the peptide in a pure state.It is interesting to point out the applicability of this method for the purification of peptides or proteins containing essential thiol groups which can be specifically labelled by reversible inhibitors.
European Journal of Biochemistry. 03/2005; 45(1):243 - 251.
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ABSTRACT: The smooth muscle basic calponin interacts with F-actin and inhibits the actomyosin ATPase in a calmodulin or phosphorylation modulated manner. It also binds in vitro to microtubules and its acidic isoform, present in nonmuscle cells, and co-localizes with microfilaments and microtubules in cultured neurons. To assess the physiological significance and the molecular basis of the calponin-microtubule interaction, we have first studied the solution binding of recombinant acidic calponin to microtubules using quantitative cosedimentation analyses. We have also characterized, for the first time, the ability of both calponin isoforms to induce the inhibition of the microtubule-stimulated ATPase activity of the cytoskeletal, kinesin-related nonclaret dysjunctional motor protein (ncd) and the abolition of this effect by calcium calmodulin. This property makes calponin a potent inhibitor of all filament-activated motor ATPases and, therefore, a potential regulatory factor of many motor-based biological events. By combining the enzymatic measurements of the ncd-microtubules system with various in vitro binding assays employing proteolytic, recombinant and synthetic fragments of basic calponin, we further unambiguously identified the interaction of microtubules at two distinct calponin sites. One is inhibitory and resides in the segment 145-182, which also binds F-actin and calmodulin. The other one is noninhibitory, specific for microtubules, and is located on the COOH-terminal repeat-containing region 183-292. Finally, quantitative fluorescence studies of the binding of basic calponin to the skeletal pyrenyl F-actin in the presence of microtubules did not reveal a noticeable competition between the two sets of filaments for calponin. This result implies that calponin undergoes a concomitant binding to both F-actin and microtubules by interaction at the former site with actin and at the second site with microtubules. Thus, in the living cells, calponin could potentially behave as a cross-linking protein between the two major cytoskeletal filaments.
Biochemistry 03/2003; 42(5):1274-82. · 3.42 Impact Factor
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ABSTRACT: The fluorescence parameters of the environment-sensitive acrylodan, selectively attached to Cys273 in the C-terminal domain of smooth muscle calponin, were studied in the presence of F-actin and using varying salt concentrations. The formation of the F-actin acrylodan labeled calponin complex at 75 mm NaCl resulted in a 21-nm blue shift of the maximum emission wavelength from 496 nm to 474 nm and a twofold increase of the fluorescent quantum yield at 460 nm. These spectral changes were observed at the low ionic strengths (< 110 mm) where the calponin : F-actin stoichiometry is 1 : 1 as well as at the high ionic strengths (> 110 mm) where the binding stoichiometry is a 1 : 2 ratio of calponin : actin monomers. On the basis of previous three-dimensional reconstruction and chemical crosslinking of the F-actin–calponin complex, the actin effect is shown to derive from the low ionic strength interaction of calponin with the bottom of subdomain-1 of an upper actin monomer in F-actin and not from its further association with the subdomain-1 of the adjacent lower monomer which occurs at the high ionic strength. Remarkably, the F-actin-dependent fluorescence change of acrylodan is qualitatively but not quantitatively similar to that earlier reported for the complexes of calponin and Ca2+-calmodulin or Ca2+-caltropin. As the three calponin ligands bind to the same segment of the protein, encompassing residues 145–182, the acrylodan can be considered as a sensitive probe of the functioning of this critical region. A distance of 29 Å was measured by fluorescence resonance energy transfer between Cys273 of calponin and Cys374 of actin in the 1 : 1 F-actin–calponin complex suggesting that the F-actin effect was allosteric reflecting a global conformational change in the C-terminal domain of calponin.
European Journal of Biochemistry. 05/1999; 262(2):335 - 341.
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Journal of Biological Chemistry 04/1995; 270(15):8867-8876. · 4.77 Impact Factor
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ABSTRACT: The cDNA coding for human skeletal muscle β-tropomyosin was expressed in Escherichia coli to produce an unacetylated β-tropomyosin. This cDNA was deleted from the sequence corresponding to the exon 9 and expressed in E. coli to produce an unacetylated β-tropomyosin mutant lacking the C-terminal residues 254–284. The main structural and functional properties of the two isolated proteins, designated tropomyosin-1 and des-(254–284)-tropomyosin, respectively, were characterized in comparison with those of the genuine rabbit skeletal muscle αβ-tropomyosin. The folding and thermal stability of the three tropomyosins were indistinguishable. Tropomyosin-1, but not des-(254–284)-tropomyosin, was polymerized in the presence of troponin and did bind to actin in the presence of the troponin complex. Despite its weak binding to actin, des-(254–284)-tropomyosin displayed a regulatory function in the presence of troponin with a marked activation of the actomyosin subfragment-1 ATPase in the presence of Ca2+ and low concentrations of subfragment-1. The data were interpreted in the light of the allosteric models of regulation and suggest the involvement of the sequence coded by exon 9 in the stabilization by tropomyosin of the off state of the thin filament.
European Journal of Biochemistry. 11/1990; 194(3):845 - 852.
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ABSTRACT: A 140-kDa polypeptide present in the striated muscle of Pecten maximus and Sepia officinalis was purified to homogeneity and its main properties were investigated using biochemical and cytochemical approaches. The protein was found to be similar to chicken gizzard caldesmon. It is a heat-stable protein. It cross-reacts immuno-logically with anti-(gizzard caldesmon) antibody, binds to calmodulin-Sepharose in a Ca2+-dependent manner, cosediments with F-actin filaments and acts in the absence and presence of tropomyosin as a potent inhibitor of rabbit skeletal actomyosin Mg2+-ATPase.The immunocytochemistry of ultrathin sections revealed, at the light microscopy resolution level, that caldesmon-like protein is present in all types of muscles hitherto examined from invertebrates and vertebrates. However, according to the distribution and the intensity of the fluorescent reaction, we concluded that, under our experimental conditions, caldesmon is not homogeneously distributed and not located in the myofibrillar bands of striated muscles but rather in the sarcoplasmic elements, at the periphery of the fibres.
European Journal of Biochemistry. 10/1989; 185(3):589 - 595.
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ABSTRACT: The active site of ATP: guanidine phosphotransferases. II. Evidence for a critical histidine residue through use of a specific reagent, diethylpyrocarbonate1.1. ATP-creatine phosphotransferase (EC 2.7.3.2) and ATP:l-arginine phosphotransferase (EC 2.7.3.3) are inhibited at pH 6.1 by diethylpyrocarbonate, their reactive sulfhydryl groups being reversibly masked or not. The extent of inactivation is first-order with respect to time and inhibitor concentration. The ability of the substrates, used separately or mixed, to protect arginine kinase against inhibition, suggests the involvement of the modified group in the enzymic reaction. Nevertheless, in the case of creatine kinase no protection has been observed.2.2. The carbethoxylated enzymes develop a difference ultraviolet spectrum with a maximum peak at 240 mμ, a specific feature of N-carbethoxyimidazole formation. From the kinetic data of spectrophotometric titrations and loss of activity, it is concluded that one histidine residue and two histidine residues are modified per mole of arginine kinase and creatine kinase respectively, causing total inactivation.3.3. Advantages of diethylpyrocarbonate as a protein-histidyl reagent are demonstrated by the specific titration of the histidine content in both denaturated enzymes. Furthermore it is shown that the number and reactivity of -SH groups are not modified in the carbethoxylated inhibited phosphokinases.Résumé1.1. L'ATP:créatine phosphotransférase (EC 2.7.3.2) et l'ATP:l-arginine phosphotransférase (EC 2.7.3.3), employées sous la forme native ou après masquage de leurs groupes -SH essentiels, sont inhibées, à pH 6.1, par le pyrocarbonate d'éthyle. Le degré d'inhibition varie avec le temps et la concentration en inhibiteur. Les substrats de la réaction de transphosphorylation, employés séparément ou mélangés, protègent efficacement l'arginine kinase, mais ne sont d'aucun effet vis-à-vis de la créatine kinase.2.2. Les enzymes carbéthoxylés présentent un spectre d'absorption différentiel avec un maximum d'absorption à 240 mμ, caractéristique de la formation de . Les déterminations spectrophotométriques effectuées parallèlement aux mesures de la perte d'activité démontrent l'implication de deux résidus histidine actifs dans la créatine kinase et d'un résidu histidine essentiel dans l'arginine kinase.3.3. La spécificité du pyrocarbonate d'éthyle à l'égard des résidus histidine a été mise en relief par l'étude de la carbéthoxylation de ces aminoacides dans les phosphagène kinases dénaturées. Cette spécificité a été encore soulignée par la non-modification du nombre et de la réactivité des groupes -SH dans les enzymes inhibés.
Biochimica et Biophysica Acta (BBA) - Enzymology. 167(2):317-325.
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ABSTRACT: Initial velocity and partial exchange studies are performed on arginine kinase (ATP:l-arginine phosphotransferase, EC 2.7.3.3) from Homarus vulgaris muscle. The steady-state kinetic patterns suggest a reaction mechanism proceeding via interconversion of ternary complexes, but we can also observe partial exchange reactions between ATP and ADP or arginine phosphate and arginine. Properties of these exchange reactions are investigated. In addition, arginine kinase is the only phosphagen kinase studied which catalyses this partial exchange.Comparison between partial exchange rates related to native and specifically inhibited arginine kinase leads to confirmation of our results previously obtained concerning the role of several essential amino acid residues in the active site: dansylation of one lysyl residue or carboxymethylation of one cysteinyl residue prevents the formation of guanidine—enzyme complexes, thus only the arginine phosphate—arginine exchange is abolished. Carbethoxylation of the histidyl residue implicated in the catalytic process suppresses the two partial exchange reactions.
Biochimica et Biophysica Acta (BBA) - Enzymology.
