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G Inesi

Publications and source records attributed to G Inesi.

At least 73 records · Page 4Linked to original sources
PubMed1979

Rapid kinetics of calcium ion transport and ATPase activity in the sarcoplasmic reticulum of dystrophic muscle.

Vesicular fragments of sarcoplasmic reticulum were isolated from pectoralis muscle of normal and dystrophic chicken. Purification of both preparations was equally satisfactory, as shown by a prominent ATPase band in electrophoresis gels. Measurements of ATPase phosphorylation, Ca2+ transport and Pi cleavage by rapid quench methods revealed a lower specific activity of the dystrophic vesicles with respect to all of these functions. On the other hand, Ca2+-independent ATPase activity was found to be increased in dystrophic vesicles. It is suggested that a fraction of ATPase units of dystrophic sarcoplasmic reticulum is not activated by Ca2+, owing to an altered protein assembly within the membrane bilayer. In fact, when the membrane structure is perturbed by detergents normal and dystropic preparations acquire an equally high Ca2+-dependent ATPase.

Adenosine Triphosphatases↗
PubMed1979

Sarcomere motion in isolated cardiac cells.

Computerized image-analysis techniques have been employed to examine the sarcomere dynamics of isolated mammalian cardiac myocytes. The cells were prepared by perfusion of adult rabbit hearts with hyaluronidase-collagenase solutions; they exhibited phasic contractions in the presence of 10(-6) M Ca2+. The dissociated cells were visualized by phase microscopy and a video camera interfaced in a minicomputer. Digitized cell images were processed by an algorithm utilizing signal averaging and contrast enhancement to yield data showing individual sarcomere position and shortening vs. time, so that patterns of sarcomere activation could be observed in spontaneously contracting cells. Compared to records of whole-cell shortening and of striation displacement, computerized image analysis provided a much more faithful indication of time course and sequence of sarcomere shortening. Spontaneously contracting cells showed sequential sarcomere shortening beginning at one end and propagating longitudinally with a constant velocity, typically at 100--150 micron/s for beat rates of 40 min-1. Velocities of initial sarcomere shortening appeared to increase with elevated Ca2+. These observations are consistent with a regenerative mechanism of calcium-induced calcium release.

Animals↗
PubMed1979

Calcium transport and contractile activity in dissociated mammalian heart cells.

A homogeneous population of dissociated myocytes can be obtained by enzymic perfusion of adult rabbit hearts. The external membrane of the dissociated cells is freely permeable to electrolytes and is electrochemically shunted. Nevertheless, in the presence of 0.2--0.6 muM Ca2+, the myocytes undergo phasic contractions. Within the range of Ca2+ concentrations eliciting phasic contractions, the dissociated myocytes exhibit Ca2+ uptake activity that is sustained by endogenous ATP in the presence of oxygen and metabolites, or by hexogenous ATP in the presence of oligomycin and antimycin. The uptake is greatly enhanced by oxalate, an effect that is attributed to the activity of sarcoplasmic reticulum. Addition of the calcium ionophore A23187 prevents the occurrence of both Ca2+ accumulation and phasic contractions. In the presence of the ionophore the myocytes remain relaxed at Ca2+ concentrations (less than 1.0 mM) normally sustaining phasic contractile activity, and undergo irreversible contracture at Ca2+ concentrations (greater than 1.0 muM) sufficient to produce direct activation of the myofibrils. It is concluded that Ca2+ transport by sarcoplasmic reticulum is directly related to the occurrence of phasic contractions in dissociated myocytes prepared from rabbit heart. On the other hand, metabolic activity is required for ATP regeneration.

Adenosine Triphosphate↗
PubMed1978

The effect of propranolol and its analogs on Ca++ transport by sarcoplasmic reticulum vesicles.

Vesicular fragments of sarcoplasmic reticulum were used as a model system to investigate the mechanism of propranolol inhibition on various steps of the Ca++ transport cycle, It was found that Ca++-dependent transfer of ATP terminal phosphate to the sarcoplasmic reticulum protein and formation of the phosphorylated enzyme intermediate are not inhibited by propranolol concentrations effective on Ca++ transport. Rather, a specific step following enzyme phosphorylation and corresponding to Ca++ translocation across the membrane is primarily inhibited. The consequent hydrolysis of phosphorylated enzyme is then secondarily inhibited, while "Ca++ independent" ATP hydrolysis remains unchanged. Comparison of the relative potencies of several propranolol analogs yields similar patterns for Ca++ transport inhibition in sarcoplasmic reticulum vesicles and negative inotropic effects on cardiac muscle. These patterns are at variant with those displayed by these agents with respect to beta adrenergic blockade.

Animals↗
PubMed1977

Ca2+-dependent effect of ATP on spin-labeled sarcoplasmic reticulum.

Vesicular fragments of sarcoplasmic reticulum (SR) were labeled with the --SH-directed spin label 2,2,6,6-tetra-methyl,4-amino(N-iodoacetamide). Colorimetric titrations of the remaining --SH residues and determinations of unbound spin label indicated that primarily 3 residues/enzyme molecule were labeled under saturating conditions. This labeling was accompanied by minimal losses in activity, providing precautions were taken to prevent sulfhydryl oxidation during the labeling process. Additions of ATP produced a new "highly constrained" component in the ESR spectrum of the labeled SR, an effect not noted in previous studies. It is demonstrated that the changes produced by ATP are reversible, and require both substrate binding and Ca2+ binding. However, hydrolysis of the substrate is not required. It is further demonstrated that the labeled residue(s) responsible for the spectral change is not in the immediate vicinity of the ATP binding site. It is apparent that the observed spectral change is related to a conformational effect of ATP and Ca2+ on the ATPase protein, which is associated with a large free energy change occurring on binding. It is also suggested that the conformational effect extends to a significant distance from the nucleotide binding site and may be a precursory step to Ca2+ translocation.

Adenosine Triphosphatases↗
PubMed1977

The involvement of sarcotubular membranes in genetic muscular dystrophy.

Microsomal preparations from breast muscle of normal and dystrophic chickens are characterized with regard to ultrastructural features, protein composition, Ca2+ transport and ATPase activity. Dystrophic muscle yields a greater microsomal dry weight, with a reduced protein to lipid ratio. This is related to the presence of a considerable number of low density microsomes, in addition to seemingly normal microsomes. The low density microsomes display a reduced number of protein particles on freeze fracture faces. Electrophoretic analysis reveals nearly identical patterns in normal and dystrophic microsomes. Furthermore, normal and dystrophic microsomes sustain equal rates of Ca2+ transport and ATPase, demonstrating an identical protein specific activity. However, the dystrophic microsomes have a lower capacity to retain transported Ca2+. The high yield of low density microsomes with reduced capacity for Ca2+ uptake is attributed to the presence of membranes proliferated in the junctional and tubular sarcomere regions of the dystrophic muscle. It is suggested that proliferation of such membranes accounts for the altered excitation-contraction coupling and cable properties of genetically dystrophic muscle.

Adenosine Triphosphatases↗
PubMed1976

Two functional states of sarcoplasmic reticulum ATPase.

The "total" ATPase activity of rabbit sarcoplasmic reticulum (SR) vesicles includes a Ca2+-independent component ("basic") and Ca2+-dependent component ("extra"). Only the "extra" ATPase is coupled to Ca2+ transport. These activities can be measured under conditions in which the observed rates approximate maximal velocities. The "basic" ATPase is predominant in one of the various SR fractions obtained by prolonged density-gradient centrifugation of SR preparations already purified by repeated differential centrifugations and extractions at high ionic strength. This fraction (low dnesity, high cholesterol) has a protein composition nearly identical with that of other SR fractions in which the "extra" ATPase is predominant. In these other fractions the ratio of "extra" to "basic" ATPase activities is temperature dependent, being approximately 9.0 at 40 degrees C and 0.5 at 4 degrees C. In all the fractions and at all temperatures studied, similar steady-state levels of phosphorylated SR protein are obtained in the presence of ATP and Ca2+. Furthermore, in all cases the "basic" (Ca2+-independent) ATPase acquires total Ca2+ dependence upon addition of the nonionic detergent Triton X-100. This detergent also transforms the complex substrate dependence of the SRATPase into a simple dependence, displaying a single value for the apparent Km. The experimental findings indicate that the ATPase of rabbit SR exists in two distinct functional states (E1 and E2), only one of which (E2) is coupled to Ca2+ transport. The E1 in equilibrium E2 equilibrium is temperature-dependent and entropy-driven, indicative of its relation to the physical state of the ATPase protein in its membrane environment. Thenonlinearity of Arrhenius plots of Ca2+-dependent ("extra") ATPase activity and Ca2+ transport is explained in terms of simultaneous contribtuions from both the free energy of activation of enzyme catalysis and the free energy of conversion of E1 to E2. Thermal equilibrium between the two functional states is drastically altered by factors which affect membrane structure and local viscosity.

Adenosine Triphosphatases↗