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R Bertrand

Publications and source records attributed to R Bertrand.

At least 73 records · Page 4Linked to original sources
PubMed1989

Coupling of nonpolymerizable monomeric actin to the F-actin binding region of the myosin head.

Polymerizations of skeletal G-actin induced by salt and myosin subfragment 1 (S-1) were suppressed by reaction of G-actin with m-maleimidobenzoyl-N-hydroxysuccinimide ester. The G-actin derivative, containing few intramolecular crosslinks and a free maleimide group, was covalently coupled in solution to the S-1 heavy chain. The resulting complex could no longer bind to F-actin. The SH-1 and SH-2 thiols of S-1 were not involved in the complexation and the covalent link was shown to be exclusively on the 50-kDa segment of the S-1 heavy chain. The specific conjugation of the two proteins followed formation of a reversibly associated pyrophosphate-sensitive binary complex which was characterized by different approaches. Potentially, these complexes may be useful in developing the crystallography of actin-bound S-1.

Actins↗
PubMed1988

Cross-linking of the skeletal myosin subfragment 1 heavy chain to the N-terminal actin segment of residues 40-113.

Glutaraldehyde (GA) and N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ), a hydrophobic, carboxyl group directed, zero-length protein cross-linker, were employed for the chemical cross-linking of the rigor complex between F-actin and the skeletal myosin S-1. The enzymatic properties and structure of the new covalent complexes obtained with both reagents were determined and compared to those known for the EDC-acto-S-1 complex. The GA- or EEDQ-catalyzed covalent attachment of F-actin to the S-1 heavy chain induced an elevated Mg2+-ATPase activity. The turnover rates of the isolated cross-linked complexes were similar to those for EDC-acto-S-1 (30 s-1). The solution stability of the new complexes is also comparable to that exhibited by EDC-acto-S-1. The proteolytic digestion of the isolated AEDANS-labeled covalent complexes and direct cross-linking experiments between actin and various preformed proteolytic S-1 derivatives indicated that, as observed with EDC, the COOH-terminal 20K and the central 50K heavy chain fragments are involved in the cross-linking reactions of GA and EEDQ. KI-depolymerized acto-S-1 complexes cross-linked by EDC, GA, or EEDQ were digested by thrombin which cuts only actin, releasing S-1 heavy chain-actin peptide cross-linked complexes migrating on acrylamide gels with Mr 100K (EDC), 110K and 105K (GA), and 102K (EEDQ); these were fluorescent only when fluorescent S-1 was used. They were identified by immunostaining with specific antibodies directed against selected parts of he NH2-terminal actin segment of residues 1-113.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1987

Comparative structure of the protease-sensitive regions of the subfragment-1 heavy chain from smooth and skeletal myosins.

The heavy chain fragments generated by restricted proteolysis of the smooth chicken gizzard myosin subfragment-1 (S-1) with trypsin, Staphylococcus aureus V8 protease, and chymotrypsin were isolated and submitted to partial amino acid sequencing. The comparison between the smooth and striated muscle myosin sequences permitted the unambiguous structural characterization of the two protease-vulnerable segments joining the three putative domain-like regions of the smooth head heavy chain. The smooth carboxyl-terminal connector is a serine-rich region located around positions 632-640 of the rabbit skeletal sequence and would represent the "A" site that is conformationally sensitive to the myosin 10 S-6 transition and to its interaction with actin (Ikebe, M., and Hartshorne, D. J. (1986) Biochemistry 25, 6177-6185). A third site which undergoes a nucleotide-dependent chymotryptic cleavage which inactivates the Mg2+-ATPase (Okamoto, Y., and Sekine, T. (1981) J. Biochem. (Tokyo) 90, 833-842, 843-849) was identified at Trp-31/Ser-32. It is vicinal to Lys-34 that is monomethylated in the skeletal heavy chain but not at all in the smooth sequence. However, the two trimethyl lysine residues present in the skeletal sequence are conserved in the same regions of the smooth S-1 and may play a general functional role in myosin. The smooth central 50-kDa segment could be selectively destroyed by a mild tryptic digestion in the absence of any unfolding agent, with a concomitant inhibition of the ATPase activities. This feature is in line with the proposed domain structure of the S-1 heavy chain and also suggests a relationship between the specific biochemical properties of the smooth S-1 and the particular conformation of its 50-kDa region.

Amino Acid Sequence↗
PubMed1987

Human liver methenyltetrahydrofolate synthetase: improved purification and increased affinity for folate polyglutamate substrates.

Methenyltetrahydrofolate synthetase (5-formyltetrahydrofolate cyclodehydrase (cyclo-ligase) (ADP-forming) EC 6.3.3.2) catalyzes the ATP- and Mg2+-dependent transformation of 5-formyltetrahydrofolate (leucovorin) to 5,10-methenyltetrahydrofolate. The enzyme has been purified 49,000-fold from human liver by a two-column procedure with Blue Sepharose followed by folinate-Sepharose chromatography. It appears as a single band both on SDS-polyacrylamide gel electrophoresis (Mr 27,000) and on isoelectric focusing (pI = 7.0) and is monomeric, with a molecular weight of 27,000 on gel filtration. Initial-velocity studies suggest that the enzyme catalyzes a sequential mechanism and at 30 degrees C and pH 6.0 the turnover number is 1000 min-1. The enzyme has a higher affinity for its pentaglutamate substrate (Km = 0.6 microM) than for the monoglutamate (Km = 2 microM). The antifolate methotrexate has no inhibitory effect at concentrations up to 350 microM, while methotrexate pentaglutamate is a competitive inhibitor with a Ki = 15 microM. Similarly, dihydrofolate monoglutamate is a weak inhibitor with a Ki = 50 microM, while the pentaglutamate is a potent competitive inhibitor with a Ki of 3.8 microM. Thus, dihydrofolate and methotrexate pentaglutamates could regulate enzyme activity and help explain why leucovorin fails to rescue cells from high concentrations of methotrexate.

Adenosine Triphosphate↗
PubMed1986

Specific interactions of the alkali light chain 1 in skeletal myosin heads probed by chemical cross-linking.

We have investigated the enzymatic properties of the 120K cross-linked heavy-chain-light-chain derivative formed upon reaction of chymotryptic myosin subfragment 1 (S-1) isoenzymes with the bis(imido esters) dimethyl 3,3'-dithiobis(propionimidate) and dimethyl suberimidate. The formation of the 120K product was accompanied for S-1(A1) but not for S-1(A2) by a loss of the actin-activated ATPase without alteration of the Ca2+-ATPase whereas the Mg2+-ATPase was increased 2-fold. Up to 70%, the inhibition of the acto-S-1(A1) ATPase activity was closely correlated with the extent of cross-linking of the A1 light chain; this activity could be largely restored upon cleavage of the cross-link using the reversible cross-linker dimethyl 3,3'-dithiobis(propionimidate). The covalent link affected the acto-S-1(A1) Mg2+-ATPase activity by reducing 3-fold the Vmax and increasing 2-fold the Kapp. On reacting for the first time the hydrophobic, carboxyl group directed cross-linker N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) with the acto-S-1(A1 + A2) complex, we found that the N-terminal tail of the A1 light chain was cross-linked to actin to an extent much larger than observed earlier with the water-soluble 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide; like the latter agent, EEDQ elicited the covalent union of the A1 subunit to the COOH-terminal part of actin. This cross-linker appears to be a valuable chemical probe of the F-actin-A1 light-chain interaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1985

Transient kinetics of adenosine 5'-triphosphate hydrolysis by covalently cross-linked actomyosin complex in water and 40% ethylene glycol by the rapid flow quench method.

The initial steps of the ATPase of covalently cross-linked actomyosin subfragment 1 (acto-SF-1) were studied by the rapid flow quench method, and the results obtained were compared with those with reversible (i.e., non-cross-linked) acto-SF-1 and SF-1 under identical conditions. Cross-linked acto-SF-1 plus [gamma-32P]ATP reaction mixture milliseconds old was quenched either in a large excess of unlabeled ATP (ATP chase) or in acid (Pi burst). The conditions were pH 8 and 15 degrees C at 5 mM or 0.15 M KCl and with or without 40% ethylene glycol. In 40% ethylene glycol (5 mM KCl), as with SF-1 and reversible acto-SF-1, the ATP chase was used to titrate active sites and to study the kinetics of ATP binding. Unlike those with SF-1 or reversible acto-SF-1, saturation kinetics were not obtained. The second-order rate constant for ATP binding was 3.1 X 10(6) M-1 s-1 for cross-linked acto-SF-1, 1.8 X 10(6) M-1 s-1 for reversible acto-SF-1, and 2 X 10(6) M-1 s-1 for SF-1. In Pi burst experiments, a transient phase could not be discerned. Because of a high kcat, cross-linked acto-SF-1 was difficult to study in aqueous solution, but at 5 mM KCl, the ATP chase and Pi burst curves were similar to those obtained in 40% ethylene glycol. At 0.15 M KCl the ATP chase curve was difficult to interpret (small amplitude), and there was a small Pi burst.(ABSTRACT TRUNCATED AT 250 WORDS)

Actomyosin↗
PubMed1985

Interaction of the heavy chain of gizzard myosin heads with skeletal F-actin.

To probe the molecular properties of the actin recognition site on the smooth muscle myosin heavy chain, the rigor complexes between skeletal F-actin and chicken gizzard myosin subfragments 1 (S1) were investigated by limited proteolysis and by chemical cross-linking with 1-ethyl-3-[3-(dimethyl-amino)propyl]carbodiimide. Earlier, these approaches were used to analyze the actin site on the skeletal muscle myosin heads [Mornet, D., Bertrand, R., Pantel, P., Audemard, E., & Kassab, R. (1981) Biochemistry 20, 2110-2120; Labbé, J.P., Mornet, D., Roseau, G., & Kassab, R. (1982) Biochemistry 21, 6897-6902]. In contrast to the case of the skeletal S1, the cleavage with trypsin or papain of the sensitive COOH-terminal 50K-26K junction of the head heavy chain had no effect on the actin-stimulated Mg2+-ATPase activity of the smooth S1. Moreover, actin binding had no significant influence on the proteolysis at this site whereas it abolished the scission of the skeletal S1 heavy chain. The COOH-terminal 26K segment of the smooth papain S1 heavy chain was converted by trypsin into a 25K peptide derivative, but it remained intact in the actin-S1 complex. A single actin monomer was cross-linked with the carbodiimide reagent to the intact 97K heavy chain of the smooth papain S1. Experiments performed on the complexes between F-actin and the fragmented S1 indicated that the site of cross-linking resides within the COOH-terminal 25K fragment of the S1 heavy chain. Thus, for both the striated and smooth muscle myosins, this region appears to be in contact with F-actin.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1985

Conformational changes induced by Mg2+ on actin monomers. An immunologic attempt to localize the affected region.

The effect of Mg2+ ions on the conformation of G-actin and in particular on the accessibility of its antigenic regions has been tested. Experiments were performed with G-actin coupled to Sepharose 4B which was, therefore, maintained in the monomeric state. The results presented her show that the 2mM MgCl2-perturbed antigenic site is located in a central region of the actin sequence.

Actins↗
PubMed1985

Further characterization of the structural and functional properties of the cross-linked complex between F-actin and myosin S-1.

Several structural and functional properties of the covalent complex, formed upon cross-linking of the myosin heads (S-1) to F-actin with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, were characterized. The elevated Mg2+-ATPase activity was measured during a 1-month storage of the complex under various conditions. In aqueous medium it showed a rapid time-dependent decrease but it was significantly more stable in the presence of 50% ethylene glycol at -20 degrees C. The ATPase loss most likely reflects a progressive conformational change within the S-1 ATPase site resulting from its greater exposure to the medium, induced by the permanently bound F-actin. The covalent acto-S1 complex was submitted to depolymerization-repolymerization experiments using different depolymerizing agents (0.6 M KI; 4.7 M NH4Cl; low-ionic-strength solution). The depolymerization led to an immediate loss of the enhanced Mg2+-ATPase activity; this activity was almost entirely recovered upon repolymerization of the complex. The protein material formed upon depolymerization of the covalent acto-S1 was analyzed by gel chromatography, gel electrophoresis, analytical ultracentrifugation and electron microscopy. It comprised mainly small-sized actin oligomers associated with the covalently bound S-1 and only a limited amount of free G-actin. The results illustrate the relationships between the filamentous state of actin and its ability to stimulate the Mg2+-ATPase activity of S-1. They also indicate that the binding of S-1 to F-actin is transmitted to several neighbouring actin subunits and strengthens the interactions between actin monomers. Acto-S1 cross-linked complexes were prepared in the presence of tropomyosin and the tropomyosin-troponin system. Under the conditions employed, the regulatory proteins were not cross-linked to actin or S-1 and did not affect the extent or the pattern of S-1 cross-linking to F-actin. Measurements of the elevated Mg2+-ATPase activity of the cross-linked preparations revealed that tropomyosin and the tropomyosin-troponin complex, in the absence of Ca2+, inhibit ATP hydrolysis; the extent of ATPase inhibition (up to 50%) was dependent on the amount of covalently bound S-1, being larger at low level of S-1 cross-linking; the addition of Ca2+ restored the ATPase activity to the control value. The data provide direct evidence that the regulatory proteins can modulate directly the kinetics of ATP hydrolysis by the covalent acto-S1 complex as has earlier been suggested for the reversible complex [Chalovich, J. M. and Eisenberg, E. (1982) J. Biol. Chem. 257, 2432-2437].(ABSTRACT TRUNCATED AT 400 WORDS)

Actins↗
PubMed1984

The interaction of skeletal myosin subfragment 1 with the polyanion, heparin.

The association between chymotryptic skeletal muscle myosin subfragment 1 (S1) and the polyanion, heparin, was investigated as an experimental approach in probing the functional importance of the cationic sites on S1 and their involvement in ionic interactions within the myosin head during energy transduction. The direct binding of heparin, used at micromolar concentrations, and its influence on the structural and functional properties of S1 were followed by gel chromatography, electron microscopy, chemical cross-linking techniques and limited digestion studies. 1. The limited tryptic digestion of S1 showed that the presence of heparin, as well as of the homopolymer, poly-(L-glutamic acid) causes a specific structural change in the 50-kDa heavy chain region of S1 and accelerates the breakdown of this segment into a 45-kDa species by a proteolytic cleavage restricted to its COOH-terminal portion. Under similar experimental conditions, the binding of MgATP and MgADP to S1 led also to the 50-kDa----45-kDa conversion, suggesting that the S1-nucleotide interactions exhibit some resemblances to the polyanion-S1 binding of polyanionic ligands to S1. This particular area is adjacent to the actin site containing the 45-kDa and 20-kDa segments of the S1 heavy chain. On the other hand, the polyanions as well as nucleotides induced changes in the interface between the heavy chain and the alkali light chains. 2. Moreover, the binding of heparin to S1 resulted in the self-association of the enzyme and the production of stable small S1 oligomers, most likely dimers, which were demonstrated by the alteration of the size of the S1 particles examined by electron microscopy and their freezing by chemical cross-linking agents. These findings are relevant to the recently reported property of skeletal chymotryptic S1 to form dimers under convenient ionic conditions, in particular in the presence of Mg-nucleotides. The interaction of cationic sites on S1 and possibly on the 50-kDa region of the heavy chain with polyanions promotes the dimerization of the S1 molecules. The binding of S1 to F-actin abolished S1 aggregation.

Actins↗
PubMed1983

Selective cleavage of the connector segments within the myosin-S1 heavy chain by staphylococcal protease.

The existence of the two connector segments linking the tryptic 50 kDa fragment of skeletal S1 heavy chain to the adjacent 27 kDa and 20 kDa peptides was ascertained by digestion of S1 with staphylococcal protease which was found to act specifically at these particular regions. Three new peptides of Mr 28000, 48000 and 22000 were produced and the novel S1 derivative formed had an intact actin-activated ATPase activity. Amino acid sequence analyses indicated that the 48 kDa and 22 kDa peptides overlap the two connector elements.

Amino Acid Sequence↗
PubMed1981

Structure of the actin-myosin interface.

The topography of the rigor complex between F-actin and myosin heads (S1) has been investigated by carbodiimide zero-length cross-linking. The results demonstrate for the first time that the 95,000-molecular weight (95K) heavy chain of the myosin head enters into van der Waals contact with two neighbouring actin monomers; one is bound to the 50K domain and the other to the 20K domain of the myosin chain. The covalent F-actin-S1 complex can be isolated; it shows a vastly elevated Mg2+-ATPase. Each pair of actin subunits in the thin filament seems to act as a functional unit for specific binding of a myosin head and stimulation of its Mg2+-ATPase activity.

Actins↗
PubMed1981

Structural aspects of actomyosin interaction.

Actin binding to myosin-S1 modulates the limited tryptic cleavage of the COOH-terminal region of the 95K heavy chain at the joint connecting the 75K and 20K peptide units; concomitantly actin affords total protection against the resulting loss of acto-S1 Mg2+-ATPase activity. The specificity of the actin effect is illustrated by the fact that it exerts itself not only on free S1 but also on the intact myosin molecule. Mg2+-ATP and Mg2+-ADP impair the protective action of actin to an extent closely related to their respective affinity for the acto-S1 complex. Tryptic fragmentation of S1 heavy chain under highly controlled conditions, using trypsin to S1 weight ratios in the range 1:1000 - 1:1500 led us to establish that peptide bond cleavage at the 75K-20K junction is a sequential process giving rise first to a 22K peptide intermediate which is subsequently converted to the stable 20K fragment. Most importantly, it is also demonstrated that the loss of S1 activation by actin is not due to the initial scission of the 75K-22K linkage but is intimately associated with the breakdown of the 22K precursor into its 20K moiety. Three trypsin-modified S1 derivatives, the heavy chain of which is a complex of two or three fragments, were purified. A detailed analysis of the C-termini of these fragments, as compared to the C-terminal structure of the intact heavy chain, indicated that the 20K fragment is formed mainly through the degradation of a NH2-terminal 2K segment in the 22K precursor and that this proteolytic event is the only one accounting for the acto-S1 ATPase loss. Cross-linking experiments exploiting the reaction of a carbodiimide reagent with rigor complexes containing either fluorescent actin or fluorescent fragmented S1 revealed unequivocally the attachment of the actin monomer to recognition sites on the 20K and 50K units of S1 heavy chain. Specific interactions between the C-terminal 20K domain and light chain LC2 are proposed as being part of the molecular mechanism of the myosin-linked regulation of actomyosin interaction.

Actins↗
PubMed1980

Fibrous glass: carcinogenicity and dimensional characteristics.

Pleural responses in rats after the implantation of 17 samples of fibrous glass allowed Stanton et al. (1977) to formulate a statistical interpretation of the relationship between the carcinogenicity of this material and its dimensional characteristics. Using the data from that study, a new statistical approach has been developed, using correspondence analysis, multiple regression on the length and diameter of the fibres and simple regression on the average aspect ratio of the fibres, i.e., their average length:diameter ratio. Relationships obtained indicate that carcinogenicity is a continuous, increasing function of the aspect ratio of the fibres, and thus show that it is not possible to separate the effects of the two variables, length and diameter. A hypothesis is presented according to which the fibres could act as an active support for the transformation of adsorbed precarcinogenic molecules. This role would be dependent on the dimensional characteristics of the fibres.

Animals↗