Search PubMed⌕ Search

Biomedical subjects

M F Morales

Publications and source records attributed to M F Morales.

At least 37 records · Page 2Linked to original sources
PubMed1988

Orientation of actin monomer in the F-actin filament: radial coordinate of glutamine-41 and effect of myosin subfragment 1 binding on the monomer orientation.

We have employed the method of radial distance measurements in order to orient the actin monomer in the F-actin filament. This method utilizes fluorescence resonance energy transfer measurements of the distance between two equivalent chemical points located on two different monomers. The interprobe distance obtained this way is used to compute the radial coordinate of the labeled amino acid [Taylor, D. L., Reidler, J., Spudich, J. A., & Stryer, L. (1981) J. Cell Biol. 89, 362-367]. Theoretical analysis has indicated that if radial coordinates of four points are determined and six intramolecular distances are known, one can, within symmetry limits, position the monomer about the filament axis. The radial distance of Gln-41 that had been enzymatically modified with dansyl, rhodamine, and fluorescein derivatives of cadaverine was found to be approximately 40-42 A. The determination of the radial distance of Cys-374 was accomplished by using monobromobimane and N-[[(iodoacetyl)amino]ethyl]-5- naphthylamine-1-sulfonate as donors and N-[4-[[4-(dimethylamino)phenyl]azo]phenyl]maleimide as acceptor; the results were consistent with a radial coordinate for this residue of 20-25 A. The effect of myosin subfragment 1 (S1) binding on the radial coordinates of (1) Gln-41, (2) Cys-374, and (3) the nucleotide binding site was also examined. S1 had a small effect on the radial coordinate of Gln-41, increasing it to 44-47 A. In the two remaining lases the change in the radial coordinate due to the S1 binding was negligible. This finding excludes certain models of the interaction between actin and S1 in which actin monomer rotates by a large angle when subfragment 1 binds to it.

Actins↗
PubMed1988

The myosin SH2-50-kilodalton fragment cross-link: location and consequences.

Some of us recently described a new interthiol cross-link which occurs in the skeletal myosin subfragment 1-MgADP complex between the reactive sulfhydryl group "SH2" (Cys-697) and a thiol (named SH chi) of the 50-kilodalton (kDa) central domain of the heavy chain; this link leads to the entrapment of the nucleotide at the active site [Chaussepied, P., Mornet, D., & Kassab, R. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 2037-2041]. In the present study, we identify SH chi as Cys-540 of the 50-kDa fragment. The portion of the heavy chain including this residue and also extending to Cys-522 that is cross-linkable to the "SH1" thiol [Ue, K. (1987) Biochemistry 26, 1889-1894] is near the SH2-SH1 region. Furthermore, various spectral and enzymatic properties of the (Cys697-Cys540)-N,N'-p-phenylenedimaleimide (pPDM)-cross-linked myosin chymotryptic subfragment 1 (S-1) were established and compared to those for the well-known (SH1-SH2)-pPDM-cross-linked S-1. The circular dichroism spectra of the new derivative were similar to those of native S-1 complexed to MgADP. At 15 mM ionic strength, (Cys697-Cys540)-S-1 binds very strongly to unregulated actin (Ka = 7 X 10(6) M-1), and the actin binding is very weakly affected by ionic strength. Joining actin with the (Cys697-Cys540)-S-1 heavy chain, using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide, produces different species than does joining unmodified S-1 with actin.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1988

Modifying preselected sites on proteins: the stretch of residues 633-642 of the myosin heavy chain is part of the actin-binding site.

We have designed an "antipeptide" capable of firmly and specifically interacting with a preselected stretch of myosin S-1 heavy chain. Covalent attachment of this antipeptide to its target stretch, residues 633-642, does not affect the intrinsic ATPase activities of the protein but significantly reduces the actin-binding capabilities of the myosin head.

Actins↗
PubMed1988

The micromechanics of contraction.

This paper suggests the molecular mechanism in muscle whereby some of the delta G of ATP hydrolysis is converted to mechanical work. There is good evidence for thinking that all mechanical, thermal, and chemical observations of muscle result from the summated, repetitive, operation of identical unitary "engines". Such an engine is the S-1 moiety of a myosin crossbridge and the adjacent actin. The operational principle is evident on realizing that S-1 is a particle with two interactive ligand (ATP and actin) binding sites. The change resulting from binding nucleotide is propagated to the actin-binding site, there specifying affinity and angles of attachment. Repetition follows because stepwise enzymatic degradation through intermediates is equivalent to cyclical changing of the site occupant. Trans- (S-1) propagation ensures a work cycle because actin held at a succession of relative positions can generate external work. Proximity mapping and protein chemical studies of S-1 suggest that variation in the position at which actin is held results from the simultaneous operation of a continuing (S-1)-actin contact and a polyphosphate charge-modulated coulombic contact.

Actins↗
PubMed1987

Two kinds of transducer.

After recognizing that there are two kinds of biotransducers, attention is focused on the kind that transduces energy, as in muscle or in ion pumps. Using muscle as the example, it is suggested that the essential elements in the transducer (S-1 segment of myosin) are (1) An ATPase site that, in time, is occupied by a succesion of ligands ("intermediates"); (2) A spatially separate site for binding a second ligand (actin, in the case of muscle) that can be held in a variety of spatial relations; and (3) A transmitting mechanism (electric field, propagated structural distortion) that allows the conformation impressed by the ligand of (1) to be conducted over space and impose a specific relation to the ligand of (2).

Biophysical Phenomena↗
PubMed1986

Modification of the actin interface of skeletal myosin subfragment-1 by treatment with dibromobimane.

Recently, by treating the head portion of skeletal myosin subfragment-1 (S1) with the bifunctional agent dibromobimane, we introduced an intramolecular covalent cross-link which resulted in the stabilisation of an internal loop in the heavy chain structure of the head [Mornet et al. (1984) Proc. Natl Acad. Sci. USA 82, 1658-1662]. In order to define the functional properties of this new S1 conformational state, we have first determined the experimental conditions for the optimum modification of S1 by dibromobimane. We finally settled on a 60% yield of cross-linked S1. Because the modification occurs between the 50-kDa and the 20-kDa tryptic heavy chain fragments which have been postulated to be involved in the interaction of native S1 with actin, we have investigated the association of dibromobimane-treated S1 with actin, using chemical cross-linking of their rigor complex with 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide. The cross-linked species obtained were analyzed by polyacrylamide gel electrophoresis and compared with those known for unmodified S1. The carbodiimide-catalyzed linkage between actin and dibromobimane-modified S1 led to a singlet protein band migrating with an apparent molecular mass of 155 kDa, in contrast to the usual doublet bands of 175 kDa and 185 kDa produced with native S1. This result suggests that a change has occurred at the actin interface on the dibromobimane-treated S1 heavy chain. The covalent complex generated by carbodiimide cross-linking between actin and dibromobimane-modified S1 (27-kDa + 50-kDa + 20-kDa fragments) was submitted to chemical hydrolysis with hydroxylamine. The nature of the products identified is consistent with the conclusion that the internal freezing of the heavy chain structure by dibromobimane induces the loss of the ability to cross-linkage of the actin site on the 20-kDa domain but does not affect the conformation of the second site on the 50-kDa segment, which becomes the unique actin region cross-linkable by actin.

Actins↗
PubMed1985

Exposure of actin thiols by the removal of tightly held calcium ions.

The removal of bound metal ions from G-actin uncovered two thiols, Cys-10 and Cys-257. The uncovering of these thiols requires a free calcium concentration lower than 10 nM. Therefore, participation of one or both thiols in Ca2+ binding is suggested. Actin labeled with N-(5-fluoresceinyl)maleimide in the absence of calcium moves as a doublet in NaDodSO4/PAGE. It is suggested that two conformers are induced by metal removal and labeling.

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

Application of the Dale-Eisinger analysis to proximity mapping in the contractile system.

Fluorescence anisotropy decays were used to quantify the degree of rapid librational motion associated with several fluorescent probes attached to contractile proteins. This information allows an analysis of how far kappa 2, the resonance energy transfer orientation factor, may deviate from the dynamically averaged value of 2/3. Extrema can then be set on R0, the critical transfer distance, and hence on the interprobe distance. These results set maximum ranges on possible distances between several probe pairs used in the mapping of contractile protein structure by resonance energy transfer.

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

Isolation and partial renaturation of proteolytic fragments of the myosin head.

Methods have been devised for isolating two of the tryptic fragments (those termed "20K" and "50K") of myosin "subfragment 1" in pure form. Fragment 20K was examined for renaturation after removal of denaturants used in its preparation. It generated a CD spectrum corresponding to ca. 64% formed structure (roughly what would be expected from its amino acid sequence) and a red-shifted UV spectrum such as arises when phenylalanine and tyrosine are perturbed by structural interactions. Actin affinity of fragment 20K was tested by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide cross-linking, inhibition of the actin-activated ATPase of subfragment 1 containing light chain 3, cosedimentation with actin, and light scattering; the affinity exceeded 5 X 10(6) M-1. The foregoing suggests that moiety 20K has a sovereign existence in (i.e., is a domain of) myosin subfragment 1. Preliminary work indicates that fragment 50K also binds actin, but with a lesser affinity.

Actins↗
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↗
PubMed1982

Effects of tryptic digestion on myosin subfragment 1 and its actin-activated adenosinetriphosphatase.

Myosin subfragment 1 (S-1) was fluorescently labeled at its rapidly reacting thiol ("SH1"). Short exposure to trypsin cuts the S-1 heavy chain into three still-associated fragments (20K, 50K, and 27K) [Balint, M., Wolf, L., Tarcsafalvi, A., Gergely, J., & Sreter, F.A. (1978) Arch. Biochem. Biophys. 190, 793-799] which bind F-actin to the same extent as does the uncut labeled S-1, as indicated by time-resolved fluorescence anisotropy decay (at 4 degrees C, pH 7, in 0.15 M KC1 and 5 mM MgC12, +/- 1 mM ADP). These results are thus in agreement with turbidity measurements on similar systems as reported by Mornet et al. [Mornet, D., Pantel, P., Audemard, E., & Kassab, R. (1979) Biochem. Biophys. Res. Commun. 89, 925-932]. The excited-state lifetime of the fluorescent label on cut S-1 is indistinguishable from that on normal S-1 (+/- ADP, +/- F-actin). F-Actin activation of MgATPase of cut S-1 is lower than that for normal S-1 at moderate concentrations of F-actin, as reported by Mornet et al. (1979). But as the F-actin concentration is increased, the MgATPase activities for cut S-1 approach those for uncut S-1. In terms of an eight-species steady-state kinetics scheme involving actin binding to free S-1, S-1 . ATP, S-1. ADP X P, and S-1 . ADP, actin affinity for the species S-1 . ADP X P was found to be 13.4 times greater for uncut S-1 than for cut S-1 [at 24 degrees C, pH 7.0, in 3 mM KC1, 1 mM ATP, 1 mM MgCl2, and 20 mM N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid].

Actins↗
PubMed1982

Inferences from the equations used for describing the chemistry of contraction.

Schemes describing the chemical kinetic behavior of actin/myosin/ATP (i.e., contractile) systems are often describable as circles of isomerizations. In this paper explicit formulas for the steady-state concentrations of intermediate complexes and of the net reaction rate are given, and then the power spectrum of the concentration fluctuations is derived explicitly for certain cases, in a manner applicable to experiments.

Actins↗
PubMed1980

A reexamination of the effects of creatine on muscle protein synthesis in tissue culture.

Experiments designed to test the hypothesis that intracellular creatine level regulates the synthesis of muscle specific proteins have failed to demonstrate any creatine regulatory effect. Manipulation of the extracellular creatine in culture medium over a 5,700-fold range (1.3-7.4 mM) was successful in altering intracellular total creatine by only a factor of 20 (1.4-42 mg creatine/mg protein), an indication that muscle cells are able to regulate intracellular creatine levels over a wide range of external creatine concentrations. Alterations of cell creatine had no effect on either total protein synthesis or synthesis of myosin heavy chain. Methods were perfected to measure total creatine, and incorporation of [3H]leucine into total protein and purified myosin heavy chain from the same culture dish to avoid the possibility of variation between dishes. The creatine analog 1-carboxymethyl-2-iminohexahydropyrimidine (CMIP) previously reported to stimulate myosin synthesis in culture was found to depress creatine accumulation by cells and depressed total protein synthesis and synthesis of myosin heavy chain. This inhibitory action of CMIP is consistent with the reported competitive inhibition of creatine kinase and presumed interference with energy metabolism.

Animals↗