Precipitation of Ca oxalate in the sarcoplasmic reticulum of chemically skinned rabbit psoas fibers caused an increase in light scattering which was proportional to the amount of Ca accumulated per unit fiber volume. The increase in scattering was used to measure net accumulation rates and steady-state Ca capacities of the sarcoplasmic reticulum in single fibers. The data obtained were qualitatively and quantitatively similar to those reported for isolated vesicle preparations. Under conditions in which Ca was not depleted from the medium, Ca accumulation was linear with time over much of its course. Steady-state capacities were independent of the Ca concentration; uptake rates were half-maximal at 0.5 microM Ca++ and saturated above about 1.0 microM. Both rate and capacity varied with the oxalate concentration, being maximal at oxalate concentrations greater than or equal to mM and decreasing in proportion to one another at lower concentrations, with a threshold near 0.25 mM. At the lower loads, electron micrographs showed many sarcoplasmic reticulum elements empty of precipitate alongside others that were full, whereas virtually all were filled in maximally loaded fibers. These data indicate that the Ca oxalate capacity of each fiber varies with the number and volume of elements in which Ca oxalate crystals can form at a given oxalate concentration, and that individual regions of the sarcoplasmic reticulum within each sarcomere differ in their ability to support Ca oxalate precipitation. Our working hypothesis is that this range in ability to form Ca oxalate crystals involves differences in ability to accumulate and retain ionized Ca inside the sarcoplasmic reticulum.
The nonionic detergent Brij 58 eliminates irreversibly the capability of the sarcoplasmic reticulum (SR) of skinned crayfish muscle fibers to sequester Ca and to release it under appropriate stimulation. In contrast to deoxycholate (DOC) which causes an irreversible diminution of tension as well, Brij 58 does not affect the contractile proteins. Comparison of the timecourse of tension development before and after Brij treatment demonstrates that Ca is accessible to the contractile proteins more rapidly after the SR is destroyed but, nevertheless, much more slowly than is predicted for free diffusion of Ca in the myoplasm. Slowing apparently results because of the presence of ca 1 mmol/kg fiber of myoplasmic Ca-binding sites that remain after Ca uptake of the SR is eliminated. A theoretical model is presented which allows for the effects of binding sites and of an unstirred layer in the vicinity of the fiber on Ca diffusion into the myoplasm.
The control of tension in skinned fibers by Mg-ATP and Ca described in previous publications has been studied at high substrate concentrations over a wide range of temperature and salt concentration. Curves of tension versus pCa shift systemically to the right as [Mg-ATP] increases. The maximum Ca-activated tension of a skinned fiber declines at sufficiently high substrate concentrations. This behavior is described by a generalization of the scheme given in the earlier reports.
In intact single crayfish muscle fibers and frog semitendinosus muscles we have studied the tension response to sinusoidal length changes in the frequency range of 0.25-133 Hz. By this method we have resolved three processes in the interaction of myosin cross-bridges with actin in fully activated preparations. They are (A) a low-frequency phase advance, (B) a middle-frequency delay, and (C) a high-frequency advance. These processes can be used as probes to study the chemomechanical coupling of contractility. Process (B) represents net power output from the muscle preparation (oscillatory work). With maximal K or caffeine activation of crayfish muscle at 20 degrees C, it decreases to zero in the initial 45 s of maintained tension. Similar results were obtained with frog semitendinosus whole muscles. We interpret this decrease of (B) with time as a gradual decrease in actomyosin ATP-hydrolysis rate.
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