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Biomedical subjects

R J Solaro

Publications and source records attributed to R J Solaro.

At least 145 records · Page 8Linked to original sources
PubMed1979

Isolation of cardiac myofibrils and myosin light chains with in vivo levels of light chain phosphorylation.

Conditions are described for the preparation of functional myofibrils and myosin light chains from freeze-clamped beating hearts with the state of light chain phosphorylation chemically 'frozen' during the extraction procedure. Myofibrils were shown to be functionally intact by measurement of Ca2+ binding and ATPase activity. Highly purified cardiac myosin light chains could be routinely isolated from myofibrillar preparations using ethanol fractionation together with ion-exchange chromatography. Analysis of light chains for covalent phosphate indicated that basal levels of phosphorylation of the 18--20 000 dalton light chain of myosin in rabbit hearts beating in situ or in a perfusion apparatus were 0.3--0.4 mol/mol. Covalent phosphate content of the light chain fraction did not change during perfusion of hearts with 10 microM epinephrine.

Animals↗
PubMed1979

The calcium binding properties of phosphorylated and unphosphorylated cardiac and skeletal myosins.

Porcine left ventricular cardiac myosin and rabbit white skeletal myosin were phosphorylated by rabbit skeletal myosin light chain kinase and their Ca2+ binding properties were examined by equilibrium dialysis techniques. No significant effect of phosphorylation on the Ca2+ binding properties of these myosins was observed. Both types of striated muscle myosins bound approximately 2 mol of Ca2+/mol of myosin with similar affinities of 3 x 10(7) M-1. In the presence of 3 x 10(-4) M Mg2+ the myosins bound Ca2+ with a reduced affinity of 3 to 4 x 10(5) M-1. Assuming competition between Mg2+ and Ca2+ for the binding sites on myosin, the changes in Ca2+ binding can be accounted for by a Mg2+ affinity of 2.5 to 3.0 x 10(5) M-1.

Animals↗
PubMed1979

Nature of the calcium regulatory system of bovine arterial actomyosin.

A calcium-sensitive actomyosin was prepared from bovine aortic muscularis. The results of applying filament displacement tests indicate that bovine arterial actomyosin possesses predominantly a calcium regulatory system associated with the myosin filament, but also may possess a calcium regulatory system associated with the filament. Calcium binding to highly purified myosin preparations could not account for the calcium binding to calcium-sensitive actomyosin. A calcium-insensitive actomyosin preparation, prepared by omitting dithiothreitol from the isolation procedure, showed a definite loss of tropomyosin but no change in calcium binding relative to the calcium-sensitive actomyosin.

Actins↗
PubMed1977

Relative capabilities of sarcoplasmic reticulum in fast and slow mammalian skeletal muscles.

1. The calcium uptake capabilities of the sarcoplasmic reticulum (SR) of the fat-twitch muscles extensor digitorum longus (EDL) and tibialis anterior (TA) of the rat and the extensor digitorum longus of the cat have been compared with the same capabilities of the slow-twitch soleus muscles of the rat and cat. 2. For the ra the Vmax values of sarcoplasmic reticulum from tibialis anterior, extensor digitorum longus and from soleus muscles were 50, 51, and 10 micronmole Ca2+/g per minute, respectively. 3. For the extensor digitorum longus and soleus muscles of the cat the Vmax values were 34 and 5-6 micronmole Ca2+/g per minute, respectively. 4. These data were compared with mechanical data as reported in the literature for the same muscles. The relative calcium uptake capabilities of sarcoplasmic reticulum from slow and fast muscles corresponded closely to the relative rates of relaxation of these muscles.

Animals↗
PubMed1976

Effects of ionic strength on calcium binding to rabbit skeletal myofibrils, thin filaments and myosin.

Calcium binding by rabbit skeletal myosin, thin filaments and myofibrils was measured in solutions with and without 2 mM MgATP and with ionic strengths adjusted with KCl to 0.05, 0.10 and 0.14 M. Free Mg2+ was held constant at 1 mM, pH at 7.0 and temperature at 25 degrees C. In the presence of MgATP, the relation between free Ca2+ and myofibrillar bound calcium shifted to the left as ionic strength was decreased from 0.14 to 0.05 M. In the absence of MgAPT, myofibrillar calcium binding was enhanced over a wide range of free Ca2+ concentration, but calcium binding was no longer a function of ionic strength. Similarly, calcium binding by thin filaments and myosin was unaffected by changes in ionic strength from 0.05 to 0.14 M. In view of evidence that cross-bridge connections between thick and thin filaments increase as ionic strength decreases, our results suggest that these connections enhance myofibrillar calcium binding. These results thus confirm previous data of Bremel and Weber (Bremel, R. D. and Weber, A. (1972) Nature New Biol. 238, 97-101) who first showed that nucleotide-free cross-bridge connections enhance thin filament calcium binding.

Animals↗
PubMed1976

Modulation of Ca2+ control of dog and rabbit cardiac myofibrils by Mg2+. Comparison with rabbit skeletal myofibrils.

Increases in free Mg2+ from 0.04 to 10.0 mM with constant pH 7.0 TO 0.10 M ionic strength, and 2 mM MgATP2- caused a rightward shift of the free Ca-relative ATPase relation for both cardiac skeletal myofibrils. The specific activity of cardiac myofibrillar ATPase over a wide range of free Ca2+ was, however, depressed in 0.04 vs. 1.0 mM Mg2+, whereas a similar decrease in free Mg2+ slightly enhanced skeletal myofibrillar ATPase. Lowering free Mg2+ from 1.0 to 0.04 mM caused similar increases in cardiac and skeletal myofibrillar bound calcium, which were largely attributable to increased calcium binding to myofibrillar myosin. Raising free Mg2+ from 1.0 to 10.0 mM caused only a slight decrease of skeletal myofibrillar bound calcium, and this change was attributable to myofibrillar myosin. The same increase in free Mg2+ caused cardiac myofibrils to bind increased amounts of calcium and this change was not attributable to myofibrillar myosin. By subtracting calcium bound to myofibrillar myosin, we were able to estimate calcium binding by myofibrillar troponin. The transition between basal and maximal ATPase in 1.0 and 10 mM Mg2+ was found to be assocciated with binding of an additional 2 mol/mol of either skeletal or cardiac myofibrillar troponin.

Adenosine Triphosphatases↗
PubMed1975

Calcium regulation of cardiac myofibrillar activation: effects of MgATP.

In 2 mM MgATP, 0.08 ionic strength and 1 mM free Mg++ cardiac myofibrils bound 3.5 nmoles Ca/mg protein at maximal ATPase activation. Significant amounts of Ca were also bound to cardiac myosin with these same conditions. By subtraction of this myosin-bound Ca we obtained an estimate of 4 moles Ca bound per mole of myofibrillar troponin at maximal ATPase. We found, however, that Ca activation of myofibrillar ATPase could be estimated assuming that only two of troponin's Ca-binding sites are engaged in regulation of crossbridge activity. Increases in MgMTP from 0.3 to 5.0 mM raised the free Ca, giving half-maximal isomteric tension or ATPase. Although part of this shift is most probably due to changes in the number of rigor (nucleotide-free) actin-myosin linkages, the rightward shift of the free Ca++-activation relation with increase in MgATP from 2 to 5 mM appears to be due to effects of active (nucleotide-containing) actin-myosin linkages.

Adenosine Triphosphatases↗