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M F Morales

Publications and source records attributed to M F Morales.

At least 19 recordsLinked to original sources
PubMed1992

The sequence location of the actin metal.

We have incorporated Fe2+ into the high-affinity metal-ion-binding site of actin. By supplying the system with oxygen from air and a reductant (dithiothreitol or ascorbate), we have induced free-radical generation, with the intent of causing peptide cleavage at the metal-ion-binding site. By analysis of the resulting fragments from actin in the F-form, we have deduced that cuts occurred at positions 159-160 and 301-302 (at the latter location we could not be sure if more than one cut occurred). We considered that these two cuts occurred in the chain strand coursing from the outer to the inner domain and vice-versa. Our results harmonize very well with the recently reported atomic structure of actin [Kabsch, W., Mannherz, H.G., Suck, D., Pai, E.F. & Holmes, K.C. (1990) Nature 347, 37-44] and remove ambiguities that had remained in the structure. The results partly bear out the homology-based prediction of Strzelecka-Golaszewska et al. [Strzelecka-Golaszewska, H., Boguta, G., Zmorzynshi, S. & Moraczcwska, J. (1989) Eur. J. Biochem. 182, 299-305].

Actins↗
PubMed1991

A search for protein structural changes accompanying the contractile interaction.

It appears that small movements (detected hitherto only by fluorescence resonance energy transfer measurements and crosslinking studies) in a region of the myosin S-1 particle may mediate chemomechanical energy transduction in the contractile system. Here we find under conditions of high precision at 10 degrees C and 20 degrees C that ATP binding to S-1 causes small (0.4%) changes in CD signal, delta epsilon 222, as do temperature changes in the regime below 16 degrees C. ATP binding perturbs tryptophan residues that we now think are in the mobile region, and we find here that temperature affects tryptophan fluorescence in much the same way that it affects the CD signal, so we believe that the CD signal reports transduction-related movements in S-1. If S-1 is exposed to the range 16-30 degrees C, CD signal falls with temperature; ATP counteracts this fall. Analysis of vacuum-UV CD spectra yields 42% alpha-helix, 9% antiparallel beta-sheet, 7% parallel beta-sheet, 14% beta-turns, and 29% other structures.

Adenosine Triphosphate↗
PubMed1990

Nucleotide regulation of vasoactive intestinal peptide binding to bovine thyroid plasma membranes.

The specific binding of vasoactive intestinal peptide (VIP) to bovine thyroid plasma membranes is inhibited by guanine nucleotides. Guanosine 5'-triphosphate (GTP) and the non-hydrolyzable GTP analogs guanosine 5'-beta,gamma-imidotriphosphate (Gpp(NH)p) and guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) inhibited markedly the binding of VIP to its receptors. This inhibition was higher with GTP than with Gpp(NH)p and GTP-gamma-S and was due to an increase of the rate of dissociation of peptide bound to membranes. Other nucleotides did not show any effect.

Animals↗
PubMed1989

Location of a contact site between actin and myosin in the three-dimensional structure of the acto-S1 complex.

Using fluorescence resonance energy transfer (FRET), we measured distances from chromophores located at or near the actin-binding stretch of amino acids 633-642 of myosin subfragment 1 (S1), to five points in the acto-S1 complex. Specific labeling of this site was achieved by first attaching the desired chromophore to an "antipeptide" that by means of its charge complementarity specifically binds to this segment of S1 [Chaussepied & Morales (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7471] and then cross-linking the fluorescent peptide to the protein. According to this technique, antipeptides containing three different labels, viz., N-dansylaziridine, (iodoacetamido)fluorescein, and monobromobimane, were purified and covalently bound to S1. A second chromophoric group, required for FRET measurements, was selected in such a way as to provide a good spectral overlap with the corresponding peptide chromophore. Cys-707 (SH1) and Cys-697 (SH2) on S1 were modified by using iodoacetamido and maleimido derivatives of rhodamine, 1,N6-ethenoadenosine 5'-diphosphate was trapped at the S1 active site with orthovanadate, Cys-374 on actin was modified with either N-[4-[4-(dimethylamino)phenyl]azo]phenyl]maleimide or N-[(iodoacetyl)-amino]ethyl]-5-naphthylamine-1-sulfonate, and ADP bound to F-actin was exchanged with the fluorescent etheno analogue. By use of excited-state lifetime fluorometry, the following distances from the stretch 633-642 of S1 to other points on S1 or actin have been measured: Cys-707 (S1), 50.3 A; Cys-697 (S1), 49.4 A; active site of S1, greater than or equal to 44 A; nucleotide binding site (actin), 41.1 A; and Cys-374 (actin), approximately 53 A.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1989

Effect of chronic intake of ethanol on the binding of vasoactive intestinal peptide to rat spleen lymphoid cells.

1. The effect of chronic intake of ethanol on the binding of vasoactive intestinal peptide (VIP) to rat spleen lymphoid cells was investigated. 2. The intake chronic of ethanol elicited an increase in specific VIP binding. 3. This increase was due to an increase in binding capacity of both the high and the low affinity binding sites. 4. There was a decrease in the affinities of both classes of VIP binding sites.

Alcoholism↗
PubMed1989

On the origin and transmission of force in actomyosin subfragment 1.

A proximity map showing the three-dimensional arrangement of 12 chemically defined points in actomyosin subfragment 1 is developed and roughly correlated with published electron microscope reconstruction of others. Several additional points and topological relationships in the primary polypeptide chain folding are assimilated into this model. Certain crosslinkings and distance change observations are interpreted as indicators of transmission of force/displacement between the nucleotide-binding and an actin-binding site--i.e., as indications of how energy is transduced in this system.

Microscopy, Electron↗
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↗
PubMed1979

Fluctuations in polarized fluorescence: evidence that muscle cross bridges rotate repetitively during contraction.

Particular thiols of the myosin subfragment 1 moieties of single glycerinated muscle fibers are covalently labeled with rhodamine. By using appropriate solutions such fibers can be relaxed, be in rigor, or develop active isometric tension. The rhodamine is excited by polarized 514.5-nm laser light; the greater than 580-nm fluorescence is resolved into orthogonal components and the intensity of each is measured by a computer-interfaced photon counting system. Fluctuations over-and-above noise appear in steady-state activity but not in relaxation or rigor and not when the fluorophore is actin-attached instead of myosin-attached. Fluctuations also appear in ratios of polarized intensities--quantities sensitive to fluorophore attitude but not to fluorophore number. The fluctuations are dominated by low (approximately 2 Hz) frequencies similar to separately measured ATPase frequencies. The fluctuations are ascribed to repetitive motion of the cross bridges to which the rhodamine is attached.

Actins↗
PubMed1979

On the molecular basis for chemomechanical energy transduction in muscle.

Herein it is developed that energy transduction in muscle is an activity of myosin S-1 and its ligands, actin (A) and nucleotide (N). S-1 shares with other molecular particles (e.g., hemoglobin) the property that binding events at one of its sites, the N-site, influences binding events at a remote site, the A site (specifically, influences both the actin affinity and actin attachment angle at the A site). However, there is a crucial difference between S-1 and the better-known systems. Because the N site is enzymatic, it has a temporal sequence of occupants; this imposes a temporal sequence of actin attitudes--i.e., a sequence of mechanical events.

Actins↗
PubMed1978

Investigation of the shape and size of myosin subfragment 1 using small-angle X-ray scattering.

Measurement of x-ray scattering at very small angles by solutions of myosin subfragment 1 (S1) yields a radius of gyration of 3.24 nm (mean) +/- 0.03 nm (standard error for N = 9). If S1 is assumed to be ellipsoid of revolution, of uniform electron density, with a molecular weight of 1.15 X 10(5) and a partial specific volume of 0.73 cm3 g-1, then the axial ratio of the ellipsoid is 2.89 +/- 0.06 (prolate) or 0.26 +/- 0.01 (oblate), and the major axis is 13.0 +/- 0.2 nm (prolate) or 10.1 +/- 0.1 nm (oblate). Measurements at larger angles allow models of S1 morphology to be tested; theoretical scattering curves for various ellipsoids of revolution were calculated. The observed scatterina can be approximated by the scattering from ellipsoids with axial ratio 2.0 to 3.0 (prolate), or 0.25 to 0.4 (oblate). Models that fit the data over the range of scattering angles from 0 to 30 mrad are: prolate ellipsoid with axial ratio 2.3, major axis 12 nm; and oblate ellipsoid with axial ratio 0.4 and major axis 10 nm. Thus analyses of two parts of the scattering curve, by different methods, indicate that S1 is fae from spherical, and that its morphology may be approximated by ellipsoids of the aforesaid dimensions.

Models, Chemical↗
PubMed1977

Fluctuations in tension during contraction of single muscle fibers.

We have searched for fluctuations in the steady-state tension developed by stimulated single muscle fibers. Such tension "noise" is expected to be present as a result of the statistical fluctuations in the number and/or state of myosin cross-bridges interacting with thin filament sites at any time. A sensitive electro-optical tension transducer capable of resolving the expected fluctuations in magnitude and frequency was constructed to search for the fluctuations. The noise was analyzed by computing the power spectra and amplitude of stochastic fluctuations in the photomultiplier counting rate, which was made proportional to muscle force. The optical system and electronic instrumentation together with the minicomputer software are described. Tensions were measured in single skinned glycerinated rabbit psoas muscle fibers in rigor and during contraction and relaxation. The results indicate the presence of fluctuations in contracting muscles and a complete absence of tension noise in eith rigor or relaxation. Also, a numerical method was developed to simulate the power spectra and amplitude of fluctuations, given the rate constants for association and dissociation of the cross-bridges and actin. The simulated power spectra and the frequency distributions observed experimentally are similar.

Animals↗