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P Pantel

Publications and source records attributed to P Pantel.

12 recordsLinked to original sources
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↗
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↗
PubMed1979

Involvement of an arginyl residue in the catalytic activity of myosin heads.

1. Phenylglyoxal reacts rapidly with isolated myosin heads (subfragment 1) and induces two successive and distinguishable effects on their enzymic properties: first, a twofold activation of the Ca2+ and Mg2+-dependent ATPases with no effect onthe K+-ATPase followed by inhibition of the K+, Ca2+ and actin-activated Mg2+-ATPases. A specific protein-reagent reagent complex is formed during the second phase of the modification reaction (Ki approximately 5 x 10(-3) M). 2. ADP and ATP with or without cations provide efficient protection only against the loss of ATPase activities, suggesting that the second inhibitory process is occurring at or close to the active site. 3. On the basis of [14C]phenylglyoxal-labelling experiments and the composition of modified subfragment-1 derivatives, it is demonstrated that the sequential modification of two reactive arginyl residues is responsible for the observed activation-inhibition phenomena. Blocking of the first reactive residue produces a shift in the pH/activity curves related to the Ca2+ and Mg2+-dependent ATPases with an apparent activation effect. Modification of the second guanidino group does not destroy the affinity of the protein for the nucleotide substrates but does alter the nucleotide binding site as reflected in the inability of Mg2+. ATP to dissociate the modified subfragment-1--actin complex. It is concluded that electrostatic interactions between this positively charged group and the negatively charged ATP and ADP molecules may be critical for the hydrolytic efficiency of myosin heads. 4. After dissociation and separation of the polypeptide constituents of the protein in acetic acid medium, both labelled sites are found to reside in the heavy chain.

Adenosine Triphosphatases↗