The effect of calcium on the respiratory and phosphorylative activities of heart-muscle sarcosomes

Abstract

Incubation of heart-muscle sarcosomes for 15 min. at 25[degree] largely inactivated the alpha-ketoglutaric oxidase and malic oxidase systems, activated the succinic oxidase system, and had little effect on oxidation of oxaloacetate. The P:O ratio was greatly decreased with malate or oxaloacetate as substrate, was less affected with succinate, and only slightly decreased with alpha-ketoglutarate. The inactivation of the alpha-ketoglutaric oxidase system was studied in greater detail. The inactivation had a high temp. coefficient, proceeded at a steady rate without any lag period, and was not markedly affected by pH or tonicity of the suspending medium. The inactivation of the alpha-ketoglutaric oxidase system was much less in the presence of the reaction mixture used in oxidative phosphorylation expts. The component mainly responsible was adenosine diphosphate; adenosine triphosphate was also effective but not adenosine monophosphate. The combined addition of Mg fluoride and phosphate also gave some protection. Ethylenediaminetetra-acetic acid (0.01 [image]) usually completely protected the alpha-ketoglutaric oxidase system against incubation of the sarcosomes for 15 min. at 25[degree], even in the absence of the reaction mixture. In the presence of ethylenediaminetetraacetic acid (EDTTA) and the reaction mixture, sarcosomes oxidized alpha-ketoglutar ate at practically a uniform rate for an hour or more at 25[degree]. Both adenosine diphosphate and EDTTA prevented activation of the succinic oxidase system (which is probably due to a decrease of the steady-state concn. of oxaloacetate) and the inactivation of the accompanying phosphorylation caused by incubation. Sarcosomes isolated in EDTTA and then freed from this substance were much more stable than those isolated in saline alone, but they were very susceptible to added Ca. Added Ca also increased rate of inactivation with normal sarcosomes. The most satisfactory prepns. of sarcosomes are obtained by including EDTTA in the isolation medium. By direct analysis it was found that isolated sarcosomes contained all the Ca in the heart muscle, even when only about 30% of the total sarcosomes were isolated. Prepns. isolated with EDTTA contained much less Ca. Both types of sarcosomes took up large amts. of Ca from solns. The localization of the Ca in the isolated sarcosomes does not, necessarily reflect the position in the intact heart. It is very probable that the stabilizing effect of EDTTA is due to removal of Ca from the sarcosomes. The mechanism of action of the Ca was not established, but different possibilities are discussed.