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D Mornet

Publications and source records attributed to D Mornet.

At least 109 records · Page 6Linked to original sources
PubMed1986

Properties of the alkali light-chain-20-kilodalton fragment complex from skeletal myosin heads.

We have developed a rapid and reproducible procedure widely applicable to the preparation of pure aqueous solutions of the complex between an alkali light chain and the COOH-terminal heavy-chain fragments of skeletal myosin chymotryptic subfragment 1 (S-1) split by various proteases. It was founded on the remarkable ethanol solubility of these complexes. A systematic study of the ethanol fractionation of the tryptic (27K-50K-20K)-S-1 (A2) showed the NH2-terminal 27K fragment to behave like a specific protein entity being quantitatively precipitated at a relatively low ethanol concentration. Only the 20K peptide-A2 complex remained in solution when the S-1 derivative was treated with exactly 4 volumes of ethanol in the presence of 6 M guanidinium chloride. At a lower ethanol concentration, a soluble mixture of 50K and 20K peptides together with the light chain was obtained. The isolated 20K fragment-A2 system containing a 1:1 molar ratio of each component was investigated by biochemical and 1H nuclear magnetic resonance (NMR) techniques to highlight its structure and the interaction of the 20K heavy-chain segment with F-actin and with the light chain. During the treatment of the complex with alpha-chymotrypsin, only the 20K peptide was fragmented in contrast to its stability within the whole S-1. The binding of F-actin to the complex led, however, to a strong inhibition of its chymotryptic degradation. 1-Ethyl-3-[3-(dimethylamino)propyl]carbodiimide cross-linking of F-actin to the complex produced covalent actin-20K peptide only, the amount of which was lower relative to that observed with the entire split S-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1986

Abolition of ATPase activities of skeletal myosin subfragment 1 by a new selective proteolytic cleavage within the 50-kilodalton heavy chain segment.

We have isolated and chemically characterized several 5-thio-2-nitrobenzoate-subfragment 1 derivatives (TNB-S-1) generated by the reaction of 5,5'-dithiobis(2-nitrobenzoic acid) (DNTB, up to 10-fold molar excess) with native S-1, N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine-S-1 (AEDANS-S-1), and N,N'-p-phenylenedimaleimide-S-1 (pPDM-S-1) at 4 degrees C, pH 8.0. The reaction of the reagent with AEDANS-S-1, which has a blocked -SH1 group, induced the formation of an intramolecular cystine disulfide between two vicinal -SH groups in S-1; in contrast, the treatment of pPDM-S-1 with DTNB resulted in the formation of TNB mixed disulfides only. The incorporation of the TNB groups (up to 3 mol/mol of S-1) into the native or premodified S-1 led to a local conformational change in the 50K heavy chain region that was fully reversed upon disulfide reduction. Exploiting this peculiarity of the DTNB-modified S-1's, we have realized a highly selective proteolysis of the S-1 heavy chain by thrombin and chymotrypsin, which do not act at all on the normal S-1. The 95K heavy chain was cut by thrombin into two fragments with apparent masses of 68K and 30K, whereas the "connector segments" and the light chains were unaffected. The two new fragments were issued from a primary peptide-bound cleavage between Lys-560 and Ser-561 within the amino acid sequence of the 50K region (M. Elzinga, personal communication).(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphatases↗
PubMed1986

Alteration of the ATP hydrolysis and actin binding properties of thrombin-cut myosin subfragment 1.

We have characterized various structural and enzymatic properties of the (68K-30K)-S-1 derivative obtained by thrombic cleavage [Chaussepied, P., Mornet, D., Audemard, E., Derancourt, J., & Kassab, R. (1986) Biochemistry (preceding paper in this issue)]. The far-ultraviolet CD spectra and thiol reactivity measurements indicated an unchanged overall polypeptide conformation of the enzyme whereas the CD spectra in the near-ultraviolet region suggested a local change in the environments of phenylalanine side chains; the latter finding was rationalized by considering the existence of about five of these amino acids in the vicinity of the cleavage sites. When the binding of Mg2+-ATP and Mg2+-ADP to the derivative was assessed by CD spectroscopy, distinct spectra were obtained with the two nucleotides as with native subfragment 1 (S-1), but some spectral features were unique to the nicked S-1. Stern-Volmer fluorescence quenching studies using acrylamide and the analogues 1,N6-ethenoadenosine 5'-triphosphate and 1,N6-ethenoadenosine 5'-diphosphate indicated that the complexes formed with the modified S-1 have a solute quencher accessibility close to that observed for the complexes with the normal S-1. However, in contrast to the parent enzyme, the thrombin-cut S-1 was unable to bind irreversibly Mg2+-ATP, nor did it form a stable Mg2+-ADP-sodium vanadate complex or achieve the entrapping of Mg2+-ADP after cross-linking of SH1 and SH2 with N,N'-p-phenylenedimaleimide. Additionally, the amplitude of the Pi burst was very low, indicating that the inactivation of the proteolyzed S-1 was linked to the suppression of the hydrolysis step in the ATPase cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1986

Nucleotide trapping at the ATPase site of myosin subfragment 1 by a new interthiol crosslinking.

When myosin subfragment 1 derivatives in which the reactive sulfhydryl SH1 has been blocked react with N,N'-p-phenylenedimaleimide or 5,5'-dithiobis(2-nitrobenzoic acid), the reactive sulfhydryl group SH2 of the 20-kDa domain is crosslinked with a thiol of the 50-kDa domain of the heavy chain. The crosslink induces the stable trapping of a significant amount of Mg2+-nucleotide in the ATPase site.

Adenine Nucleotides↗
PubMed1985

Molecular movements promoted by metal nucleotides in the heavy-chain regions of myosin heads from skeletal muscle.

Molecular movements generated in the heavy-chain regions (27-50-20(X 10(3)) Mr) of myosin S1 on interaction with nucleotides ATP, AMPPNP, ADP and PPi were investigated by limited proteolysis of several enzyme-metal nucleotide complexes in the absence and presence of reversibly bound and crosslinked F-actin. The rate and extent of the nucleotide-promoted conversion of the NH2-terminal 27 X 10(3) Mr and 50 X 10(3) Mr segments into products of 22 X 10(3) Mr and 45 X 10(3) Mr, respectively, were estimated to determine the amplitude of the molecular movements. The 22 X 10(3) Mr peptide was identified by amino acid sequence studies as being derived from cleavage of the peptide bond between Arg and Ile (at position 23 to 24). The 45 X 10(3) Mr peptide, previously shown to represent the NH2-terminal part of the 50 X 10(3) Mr region, would be connected to the adjacent C-terminal 20 X 10(3) Mr region by a pre-existing loop segment of about 5 X 10(3) Mr; the proteolytic sensitivity of the latter region is increased particularly by nucleotide binding. The tryptic reaction proved to be a sensitive indicator of the conformational state of the liganded heavy chain as the rate of peptide bond cleavage in the two regions is dependent on the nature of the bound ligand; it decreases in the order: ATP greater than AMPPNP greater than ADP greater than PPi. It depends also on the nature of the metal present, Mg2+ and Ca2+ being much more effective than K+. Binding of F-actin to the S1-MgAMPPNP complex affords significant protection against breakdown of 27 X 10(3) Mr and 50 X 10(3) Mr peptides, but with concomitant hydrolysis of the 50 X 10(3) Mr-20 X 10(3) Mr junction. Additionally, interaction of MgATP with HMM modulates the tryptic fission of the S1-S2 region. The overall data provide a molecular support for the two-state model of the myosin head and emphasize the involvement of the 50 X 10(3) Mr unit in the mechanism of coupling between the actin and nucleotide binding sites.

Actins↗
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

Incorporation of 6-carboxyfluorescein into myosin subfragment 1.

We describe for the first time the introduction of a label into the "50K" domain of myosin subfragment 1 (S-1), and we investigate the properties of this fluorescent modification in relation to the ATPase and actin-binding activities, both residing in the myosin head. The labeling consists of a major incorporation of 6-carboxyfluorescein into the "50K" domain of S-1. Using different conditions for tryptic digestion that allowed a fragmentation of the "50K" domain with a loss of 5 kilodaltons (kDa) leading to a final product of 45 kDa, we have shown that the fluorescent dye remains in the 45-kDa final product. By studying cross-linking as a function of time, we have demonstrated that the "50K" domain and the 45-kDa fluorescent peptide are equally cross-linkable to actin. We have also investigated the K+EDTA-, Ca2+-, Mg2+-, and actin-activated ATPase activities of this modified S-1 and after purification observed no enzymatic changes.

Actins↗
PubMed1985

Stabilization of a primary loop in myosin subfragment 1 with a fluorescent crosslinker.

A bifunctional fluorescent alkylating agent, dibromobimane, has been used to stabilize a preexisting primary loop in myosin subfragment 1 (S-1). The crosslink achieved joins Cys-707 (called sulfhydryl group "SH1") of the 20-kDa domain (formerly called "20K" domain) with a thiol of the 50-kDa domain and seems to place the dibromobimane near the ATP-perturbable tryptophan.

Animals↗
PubMed1984

Proteolysis and structure of skeletal muscle actin.

Under standard conditions, G-actin has been submitted to nine proteases of varying specificity, and in each case the pattern of fragments produced has been studied by NaDodSO4 gel electrophoresis. The results suggest that the actin monomer consists of a large region (ca. 33 kilodaltons) and a small, easily degraded region (ca. 9 kilodaltons). The COOH terminus is in the large region. Consideration of primary sequence homologies, medium resolution maps of actin crystals, and certain reactions of actin suggests that the NH2 terminus is in the small region, as is the negative sequence to which a divalent metal cation is normally chelated, but that the nucleotide-binding site is on the large region near the junction between the regions. From analysis of these results, numerous properties of actin are understandable.

Actins↗
PubMed1984

Proteolysis and the domain organization of myosin subfragment 1.

Because the proteolytic cleavage of a folded polypeptide depends not only on the specificity of the protease but on the nature of the folding, we investigated the cleavage of (chymotryptically produced) subfragment 1 (designated "S-1") or "head" segment of myosin by seven proteases with different specificities. All seven produced approximately the same three fragments of S-1--namely, fragments (from the NH2 terminus) of 27, 50, and 20 kilodaltons, suggesting that in intact S-1 these fragments are distinct domains. The same proteases were used to hydrolyze the MgADP complex of S-1. All failed to do so except trypsin, which, as found earlier [Hozumi, T. (1983) Biochemistry 22, 799-804], makes two additional cleavages. This result suggests that the conformational change induced by MgADP opens up only a small stretch of polypeptide chain, which stretch happens to be vulnerable to trypsin.

Animals↗
PubMed1984

On the mechanism of energy transduction in myosin subfragment 1.

It is proposed that the myosin subfragment 1 moiety of the muscle contractile apparatus is a self-contained "engine" whose operational plan is based on the interactive nature of ATP (or degradation intermediate) binding and actin binding, made possible by an intersite communication system. It is suggested that the spatial information required for examining this engine can, at least provisionally, be derived from fluorescence resonance energy transfer measurements interpreted by the Förster equation and that the existence of an intersite communication system can be deduced from piece-wise detection of interacting pairs of points.

Actins↗
PubMed1983

Structural and actin-binding properties of the trypsin-produced HMM and S1 from gizzard smooth muscle myosin.

The reaction of trypsin on the heavy chain of gizzard myosin and chymotryptic HMM was investigated under restricted fragmentation conditions. The three fragments of the head part with 29 kDa, 50 kDa and 26 kDa were isolated and identified. The 66 K heavy chain segment containing the S1-S2 junction was slowly but extensively degraded liberating a S1-like entity which lacked an intact COOH-terminal 26 kDa region; this isolated species displayed full intrinsic ATPase activities but little actin-binding ability. Tryptic HMM was also formed bearing a fragmented heavy chain and lacking the 20 kDa light chain. Its actin-activated ATPase was derepressed upon cleavage of the 66 kDa segment by papain. We propose that the integral 66 kDa heavy chain component is directly involved in the regulation of the gizzard actomyosin ATPase.

Actins↗
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↗