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A N Martonosi

Publications and source records attributed to A N Martonosi.

At least 19 recordsLinked to original sources
PubMed2000

Animal electricity, Ca2+ and muscle contraction. A brief history of muscle research.

This brief review attempts to summarize some of the major phases of muscle research from Leeuwenhoek's description of sarcomeres in 1674, through Galvani's observation of "animal electricity" in 1791, to the discovery of Ca2+ as the key messenger in the coupling of nerve excitation to muscle contraction. The emerging molecular mechanism of the contraction process is one of the great achievements of biology, reflecting the intimate links between physics, chemistry and the life Sciences in the solution of biological problems.

Animals↗
PubMed1995

The structure and interactions of Ca(2+)-ATPase.

Electron crystallographic studies on membrane crystals of Ca(2+)-ATPase reveal different patterns of ATPase-ATPase interactions depending on enzyme conformation. Physiologically relevant changes in Ca2+ concentration and membrane potential affect these interactions. Ca2+ induced difference FTIR spectra of Ca(2+)-ATPase triggered by photolysis of caged Ca2+ are consistent with changes in secondary structure and carboxylate groups upon Ca2+ binding; the changes are reversed during ATP hydrolysis suggesting that a phosphorylated enzyme form of low Ca2+ affinity is the dominant intermediate during Ca2+ transport. A two-channel model of Ca2+ translocation is proposed involving the membrane-spanning helices M2-M5 and M4, M5, M6 and M8 respectively, with separate but interacting Ca2+ binding sites.

Animals↗
PubMed1990

Emerging views on the structure and dynamics of the Ca2(+)-ATPase in sarcoplasmic reticulum.

The ATP-dependent Ca2+ transport in sarcoplasmic reticulum involves transitions between several structural states of the Ca2(+)-ATPase, that occur without major changes in the secondary structure. The rates of these transitions are modulated by the lipid environment and by interactions between ATPase molecules. Although the Ca2(+)-ATPase restricts the rotational mobility of a population of lipids, there is no evidence for specific interaction of the Ca2(+)-ATPase with phospholipids. Fluorescence polarization and energy transfer (FET) studies, using site specific fluorescent indicators, combined with crystallographic, immunological and chemical modification data, yielded a structural model of Ca2(+)-ATPase in which the binding sites of Ca2+ and ATP are tentatively identified. The temperature dependence of FET between fluorophores attached to different regions of the ATPase indicates the existence of 'rigid' and 'flexible' regions within the molecule characterized, by different degrees of thermally induced structural fluctuations.

Animals↗
PubMed1990

The binding of monoclonal and polyclonal antibodies to the Ca2(+)-ATPase of sarcoplasmic reticulum: effects on interactions between ATPase molecules.

We analyzed the interaction of 14 monoclonal and 5 polyclonal anti-ATPase antibodies with the Ca2(+)-ATPase of rabbit sarcoplasmic reticulum and correlated the location of their epitopes with their effects on ATPase-ATPase interactions and Ca2+ transport activity. All antibodies were found to bind with high affinity to the denatured Ca2(+)-ATPase, but the binding to the native enzyme showed significant differences, depending on the location of antigenic sites within the ATPase molecule. Of the seven monoclonal antibodies directed against epitopes on the B tryptic fragment of the Ca2(+)-ATPase, all except one (VIE8) reacted with the enzyme in native sarcoplasmic reticulum vesicles in both the E1 and E2V conformations. Therefore these regions of the Ca2(+)-ATPase molecule are freely accessible in the native enzyme. The monoclonal antibody VIE8 bound with high affinity to the Ca2(+)-ATPase only in the E1 conformation stabilized by 0.5 mM Ca2+ but not in the E2V conformation stabilized by 0.5 mM EGTA and 5 mM vanadate. Several antibodies that reacted with the B fragment interfered with the crystallization of Ca2(+)-ATPase in the presence of EGTA and vanadate and at least two of them destabilized preformed Ca2(+)-ATPase crystals, suggesting inhibition of interactions between ATPase molecules. Of five monoclonal antibodies with epitopes on the A1 tryptic fragment of the Ca2(+)-ATPase only one gave strong reaction with the native enzyme, and none interfered with ATPase-ATPase interactions as measured by the polarization of fluorescence of FITC-labeled Ca2(+)-ATPase. Therefore the regions of the molecule containing these epitopes are relatively inaccessible in the native structure. Partial tryptic cleavage of the Ca2(+)-ATPase into the A1, A2 and B fragments did not promote the reaction of anti-A1 antibodies with sarcoplasmic reticulum vesicles, but solubilization of the membrane with C12E8 rendered the antigenic site fully accessible to several of them, suggesting that their epitopes are located in areas of contacts between ATPase molecules. Two monoclonal anti-B antibodies that interfered with ATPase-ATPase interactions, produced close to 50% inhibition of the rate of ATP-dependent Ca2+ transport, with significant inhibition of ATPase; this may suggest a role for ATPase oligomers in the regulation of Ca2+ transport. The other antibodies that interact with the native Ca2(+)-ATPase produced no significant inhibition of ATPase activity even at saturating concentrations; therefore their antigenic sites do not undergo major movements during Ca2+ transport.

Amino Acid Sequence↗
PubMed1984

Crystallization of intramembrane particles in rabbit sarcoplasmic reticulum vesicles by vanadate.

Sarcoplasmic reticulum (SR) membranes isolated from rabbit skeletal muscle appear in freeze-fracture as 0.15-0.2 micron vesicles. The concave fracture surface (P-face) contains a dense population of 8.5 nm particles that were previously identified as the Ca2+-transport ATPase. The convex surface (E-face) is mostly smooth, displaying an occasional particle but no complementary arrays of pits. Incubation of the vesicles at 4 degrees C in calcium-free solutions containing 5 mM Na3VO4 induces the formation of two-dimensional crystalline arrays of the Ca2+, Mg2+-ATPase, accompanied by structural changes visible by freeze-etch electron microscopy. Most vesicles elongate into tubules 60-80 nm in diameter and the 8.5 nm intramembrane particles of the P-face become regularly organized into parallel ridges. The ridges are coiled around the tubules in right-handed helices, oriented at 50-60 degrees angle to the long axis of the tubules. The particles repeat along the rows at about 5.5 nm and the rows repeat at 10.5-11.0. Occasionally the ridges seem to break up into 8.5 nm particles. Parallel furrows are visible on the (convex) E-face of the tubules. In high resolution replicas, the furrows are resolved into rows of pits that are complementary images of the ridges. Deep etching and rotary shadowing reveal oblique crests on the protoplasmic surface, consisting of dimeric particles close to 8.5 X 5.5 nm in size, in which each monomer can frequently be resolved into two structural domains. These data suggest that vanadate induces a conformational change in the Ca2+-transport ATPase, with crystallization of the intramembrane particles.

Animals↗
PubMed1984

Effect of Na3VO4 and membrane potential on the structure of sarcoplasmic reticulum membrane.

Two-dimensional crystalline arrays of Ca2+-ATPase molecules develop after treatment of sarcoplasmic reticulum vesicles with Na3VO4 in a Ca2+-free medium. The influence of membrane potential upon the rate of crystallization was studied by ion substitution using oxonol VI and 3,3'-diethyl-2,2'-thiadicarbocyanine (Di-S-C2(5] to monitor inside positive or inside negative membrane potentials, respectively. Positive transmembrane potential accelerates the rate of crystallization of Ca2+-ATPase, while negative potential disrupts preformed Ca2+-ATPase crystals, suggesting an influence of transmembrane potential upon the conformation of Ca2+-ATPase.

Animals↗
PubMed1984

Mechanisms of Ca2+ release from sarcoplasmic reticulum of skeletal muscle.

Since the discovery of the ATP-dependent Ca2+ transport by SR a little over two decades ago, remarkable progress has been made in understanding the kinetic mechanism of Ca2+ transport and ATP hydrolysis and the role of phosphorylated enzyme intermediates in the energetics of active ion transport. Significant information has accumulated on the structure and composition of the SR membrane, on the primary amino acid sequence of the Ca2+-pump protein, and on the adaptive changes in the Ca2+-transport function during embryonic development and muscle activity. The discovery of the charge movement as a step in EC coupling and the use of novel optical probes for analyzing potential and calcium transients in living muscle changed the enigma of EC coupling into a well-defined problem that is clearly open to rational solutions. Studies on the structure, composition, and function of the isolated components of the T-SR system have just begun. The effectiveness of this approach will depend on successful maintenance of the functionally intact structure of the T-SR junction during the disruption of the muscle, which is required for the isolation of pure membrane elements. Reconstitution of a functionally competent junctional complex from isolated components is the ultimate aim of these studies, but the path toward that goal is so difficult that much of the mechanism of EC coupling may be solved by electrophysiologists, before reconstitution is achieved. The avalanche of information on Ca2+ releases induced by various agents under diverse and sometimes ill-defined conditions led to formulation of a series of hypothetical mechanisms. Of these, Ca2+-induced Ca2+ release promises to be an important element of the physiological Ca2+-release process, but few of the other proposed mechanisms can be eliminated from consideration at this stage. The impressive progress of the last few years has left several fundamental problems largely unsolved. Among these are the physical mode of translocation of Ca2+ across the membrane and the molecular mechanism of the coupling of Ca2+ transport to ATP hydrolysis; the regulation of the concentration of the Ca2+-pump protein and calcium in the SR of fast and slow skeletal, cardiac, and smooth muscles; the gating mechanisms that regulate the graded release of calcium from the SR and the composition and biochemical characterization of the triad; the role of SR membrane potential in the regulation of Ca2+ fluxes in vivo; the permeability of SR membranes in living muscle; the functional significance of protein-protein interactions in the SR with respect to Ca2+ transport and permeability control.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenine Nucleotides↗
PubMed1983

Membrane crystals of Ca2+-ATPase in sarcoplasmic reticulum of normal and dystrophic muscle.

Two-dimensional crystalline arrays of the Ca2+ transport ATPase develop after treatment of sarcoplasmic reticulum vesicles with Na3VO4. The dimensions of the crystal lattice are similar in sarcoplasmic reticulum membranes isolated from normal and genetically dystrophic human, mouse, and chicken muscles. These observations indicate similar requirements for ATPase-ATPase interactions in sarcoplasmic reticulum of normal and dystrophic muscles and lessen the likelihood of a molecular defect of the Ca2+ pump in the various forms of genetic muscular dystrophies.

Animals↗
PubMed1981

The effect of Ca2+ ionophores upon the synthesis of proteins in cultured skeletal muscle.

The influence of the Ca2+ ionophores, ionomycin and A23187 upon the incorporation of [35S]methionine into proteins of cultured chicken pectoralis muscle was studied during differentiation of myoblasts into multinucleated myotubes. Fusion was reversibly arrested by growing cells in low-calcium media from the time of plating. Exposure of normal and fusion blocked cultures to 10-6-10-5 M ionomycin or A23187 for 2-6 h on the second to fourth day of growth, resulted in a selective increase in the incorporation of [35S]methionine into two proteins of about 100 000 and 80 000 dalton. When 10-5 M ionomycin or A23187 were added to older cultures, all large myotubes contracted and detached from the plate. Only the adhering myoblasts and small myotubes incorporated [35s[methionine into the muscle proteins and showed increased incorporation of label into 100 000 and 80 000 proteins. After ionophore pulse, the adhering cells retained the ability to differentiate and accumulate myosin. The effect of Ca2+ ionophores upon the rate of protein synthesis is presumably related to increased influx of extracellular Ca2+ with a rise in the Ca2+ concentration of the cytoplasm. We conclude that Ca2+ sensitive mechanisms may regulate the synthesis of a select group of muscle proteins.

Animals↗
PubMed1981

The mechanism of voltage-sensitive dye responses on sarcoplasmic reticulum.

The mechanism of voltage-sensitive dye responses was analyzed on sarcoplasmic reticulum vesicles to assess the changes in membrane potential related to Ca2+ transport. The absorbance and fluorescence responses of 3,3'-diethyl-2,2'-indodicarbocyanine and oxonol VI during ATP-dependent Ca2+ transport are influenced by the effect of accumulated Ca2+ upon the surface potential of the vesicle membrane. These observations place definite limitations on the use of these probes as indicators of ion-diffusion potential in processes which involve large fluctuations in free Ca2+ concentrations. Nile Blue A appeared to produce the cleanest optical signal to negative transmembrane potential, with least direct interference from Ca2+, encouraging the use of Nile Blue A for measurement of the membrane potential of sarcoplasmic reticulum in vivo and in vitro. 1,3-dibutylbarbituric acid (5)-1-(p-sulfophenyl)-3 methyl, 5-pyrazolone pentamethinoxonol (WW 781) gave no optical response during ATP-induced Ca2+ transport and responded primarily to changes in surface potential on the same side of the membrane where the dye was applied. Binding of these probes to the membrane plays a major role in the optical response to potential, and changes in surface potential influence the optical response by regulating the amount of membrane-bound dye. The observations are consistent with the electrogenic nature of ATP-dependent Ca2+ transport and indicate the generation of about 10 mV inside-positive membrane potential during the initial phase of Ca2+ translocation. The potential generated during Ca2+ transport is rapidly dissipated by passive ion fluxes across the membrane.

Animals↗
PubMed1981

Effect of phospholipid substitution on the mobility of spin labels bound to the ATPase of sarcoplasmic reticulum.

The mobility of spin labels covalently bound to the Ca2+-transport ATPase (ATP phosphohydrolase [EC 3.y.1.3]) was studied by electron spin-resonance spectroscopy in purified ATPase and reconstituted vesicles. The purified ATPase of sarcoplasmic reticulum of rabbit skeletal muscle was covalently labeled with maleimide spin-labels of different chain length and the phospholipids were exchanged for dipalmitoylphosphatidylcholine. The spectrum of the short-chain maleimide spin-label, bound to purified ATPase indicates reduced mobility after substitution of endogenous phospholipids with dipalmitoylphosphatidylcholine. With the long-chain maleimide derivative no difference was detected in the spectra, measured at 20-35 degrees C temperature before and after substitution with dipalmitoylphosphatidylcholine. Below 10 degrees C temperature the substitution with dipalmitoylphosphatidylcholine decreased the mobility of the prove, indicating that the microviscosity of environment in the vicinity of nitroxide groups was influenced by changes in the fatty acid composition. With both short and long chain spin-labels bound to purified ATPase adn sarcoplasmic reticulum vesicles the amplitude of weakly immobilized component sharply decreased in media containing 20-50% glycerol. Therefore, the mobility of covalently bound nitroxide group in short or long chain maleimide derivatives is also sensitive to the viscosity of the water phase.

Animals↗