Analysis of Calcium Binding and Release by Canine Cardiac Relaxing System (Sarcoplasmic Reticulum): THE USE OF SPECIFIC INHIBITORS TO CONSTRUCT A TWO-COMPONENT MODEL FOR CALCIUM BINDING AND TRANSPORT

Abstract

Abstract ATP induces calcium binding to a cardiac relaxing system preparation (sarcoplasmic reticulum; CRS) under specific conditions. This is followed by spontaneous calcium release, a thermodynamically and kinetically distinct process that represents a separate phase of an ATP-dependent binding-release cycle. Inhibitors of calcium binding studied include X537A, a broadly specific antibiotic ionophore, and arsenate. These inhibitors were significantly more potent on calcium uptake (accumulation in the presence of oxalate) than on binding (accumulation in the absence of oxalate). Inhibition decreased with decreasing magnesium concentration suggesting that the agents were acting primarily on a magnesium-dependent process. Computer analysis of the kinetics of ATP-dependent calcium binding to CRS suggested that there are two distinct binding sites or components that differ in response to magnesium, pH, and in their sensitivity to the actions of inhibitors. Under conditions of optimal ATP and calcium concentrations, a continuous curve for calcium binding adhered to a pseudo-first order model for occupation of free calcium binding sites: A + B = A0e-kat + B0e-kbt. Site A required very low magnesium (40% of maximal binding at 9 µm magnesium), was ATP-specific and was less sensitive to the inhibitors of calcium binding, X537A (Ki, 10 to 15 µg per mg) and arsenate (unaffected by concentrations up to 10 mm). Calcium was released spontaneously from Site A in the presence or absence of oxalate, therefore, this pool of calcium does not appear to be connected to an oxalate space (vesicular lumen). In contrast, Site B was related to calcium transport (i.e. uptake, see above) in that it required a relatively high concentration of magnesium (Km, 2.7 to 4.7 mm) and was much more sensitive to the noncompetitive inhibitors, X537A (Ki, 3.7 µg per mg) and arsenate (Ki, 1 to 2 mm). The predicted initial rates of calcium binding to Site B (rate = kB0) were similar to the average range of rates of calcium uptake in the presence of 5 mm oxalate. Nucleoside triphosphates other than ATP activated only Site B suboptimally (50 to 60%). When Site B was activated by nucleotides other than ATP, its sensitivity to pH, arsenate, and X537A was unchanged; however, the magnesium requirement of Site B disappeared. Nucleotides other than ATP did not activate the calcium-dependent ATPase and did not support continuous calcium uptake. Sodium azide specifically inhibited the rate of calcium release without affecting binding. All of the inhibitors were specific for the described individual phases and did not affect calcium-dependent ATPase.