Inorganic Pyrophosphatase of Rat Liver Mitochondria

Abstract

Stimulation of Mg2 +-dependent inorganic pyrophosphatase activity several fold by disruption of mitochondrial membranes does not appreciably alter the catalytic properties of the enzyme . Stimulation is due to increased accessibility of substrate to the enzyme, which is not solublized on activation . The enzyme is attached to the inside of the inner membrane, and under physiological conditions probably hydrolyzes only intramitochondrially-produced PP i .Disruption of the membranes of isolated rat liver mitochondria stimulates Mgt+-dependent inorganic pyrophosphatase (PPase) activity (Enzyme Commission 3 .6.1 .1 .) several fold (I) . This latency is shared in varying degrees by other mitochondrial enzymes . We have compared some of the catalytic properties of the overt (intact mitochondria) and latent (disrupted mitochondria) PPase, and determined the intramitochondrial location of the activity . The results bear upon the question of the basis of the latency phenomenon, and reemphasize the importance of mitochondrial structural integrity in the maintenance of physiological enzyme activity. METHODSLiver mitochondria were prepared by the method of Schneider (2) from 200g Wistar rats which had been fasted overnight. Mitochondria were washed three times by resuspension in one-fourth of the original volume of medium (9 ml/g liver) followed by recentrifugation . For studies of stimulation and inhibition of PPase activity the suspending medium contained 0.88 M sucrose, 0 .02 M Tris acetate (pH 7 .2), and 0 .1 mm ethylenediamine-tetraacetic acid (EDTA) . For mitochondria which were to be subfractionated the suspending medium was 0 .25 M sucrose .Washed mitochondria were subfractionated by a separation technique which involves successively a mild osmotic shock, ATP-driven "contraction" of the inner membranes, and centrifugation of the insoluble membranous material through a discontinuous sucrose gradient (3) . Mitochondria from 15 g of liver were centrifuged at 7000g for 10 min and the pellet was suspended in 18 ml of 10 mm Tris acetate buffer, pH 7.5, with a Teflon pestle fitted into the centrifuge tube . After 10 min at 0°the suspension was diluted with one-third vol of 1 .8 M sucrose containing 2 mm adenosine triphosphate (ATP) and 2 mm MgC12 . After 5 min further at 0°, the suspension was centrifuged at 44,000g for 15 min . The sedimented material, which contained approximately 90% of the Mgt+-dependent PPase activity, was resuspended in 0.45 M sucrose using the Teflon pestle, and 10 ml of the suspension containing approximately 90 mg protein was layered on top of a sucrose bilayer composed of 5 ml of 0 .76 M sucrose over 10 ml of 1 .32 M sucrose. After centrifugation (in the same angle head rotor used in the mitochondrial preparation) at 44,000g for 3 hr, fractions were collected by applying vacuum to an horizontally bent-tip pipette successive1" lowered into the tube, which emptied into a test tube 2 3 5 on