A Reconsideration of the Isolation in Presence of EDTA and the Swelling-Contraction Characteristics of Rat Heart Sarcosomes. Metabolic Topography of the Sarcosomal Contractile Protein^*

Abstract

The yield of rat heart sarcosomes, but not of liver mitochondria, is considerably decreased when ethylenediaminetetraacetate is included in the 0.44 m sucrose isolation medium. The sensitivity of sarcosomes to this agent is also shown by the appreciable inhibition of hypotonicity-initiated swelling and by the inhibition of oxygen uptake. Sarcosomes isolated in a 0.44 M sucrose medium containing no ethylenediaminetetraacetate were found to be intact by phase-contrast microscopic standards. The hypotonicity-initiated swelling of heart sarcosomes, unlike that of liver mitochondria, shows a series of minima and maxima when the total concentration of the medium is varied between 0.01 and 2.00 m. The pH dependence of swelling of heart sarcosomes in 0.04 m sucrose shows a sharp maximum at pH 7.4, which is absent in 0.44 M sucrose.The kinetics of swelling induced by NAD, Ca2+, dodecylsulfate, and desoxycholate follow the kinetics of drug-receptor complex formation, while those induced by thyroxine, pentachlorophenol, and p-mercuribenzoate show linear relationships. The swelling induced in heart sarcosomes by various agents is inhibited and reversed by ATP + Mg2 + and by reduced nicotinamideadenine dinucleotide (NADH2). Both types of inhibition and reversal are strongly pH de-

Effect of Thiamine Deficiency on the Swelling, Electron Transport, and Oxidative Phosphorylation of Rat Heart Sarcosomes*

“…caused by thiamine deficiency and may not be attributed to leakage of NAD from the particles. Decreased inhibition is, in fact, not influenced by the addition of 0.07 Mmole of this nucleotide to the test systems, which is a level below the minimum concentration required for swelling-inducer action of NAD (Arcos and Argus, 1964). Change in the Rate of Electron Transport.-Figure 2 shows the NADH2-cytochrome c reductase and diaphorase activities in a 0.44 M sucrose medium (buffered with phosphate) during the time course of thiamine depletion.…”

“…The use of the pyruvate-plus-fumarate substrate combination corresponds to maximal loading of the oxidative system (Aisenberg and Potter, 1955). Inhibition of sarcosomal swelling by the two substrates indicates the resynthesis of ATP from the inorganic phosphate and ADP present in small equimolar amounts (resulting from the hydrolysis of ATP carried in the particles through isolation), since ATP inhibits and reverses the swelling of liver mitochondria (Raaflaub, 1953;Lehninger, 1959), and of heart sarcosomes (Nakamura et al, 1961;Arcos and Argus, 1964).…”

“…Swelling and Oxidative Phosphorylation.-Sarcosomes were isolated, the swelling tests were carried out, and oxygen uptake was measured as described previously (Arcos and Argus, 1964). However, in the present studies on the inhibition of swelling by pyruvate plus fumarate (both at 1 X 10 ~2 m) the cells were tilted at frequent regular intervals for oxygenation of the medium.…”