A preparation, similar to the light mitochondrial fraction of rat liver (L fraction of de Duve et al. [1955, Biochem. J. 60: 604-617]), was subfractionated by isopycnic centrifugation in a metrizamide gradient and the distribution of several marker enzymes was established. The granules were layered at the top or bottom of the gradient. In both cases, as ascertained by the enzyme distributions, the lysosomes are well separated from the peroxisomes. A good separation from mitochondria is obtained only when the L fraction is set down underneath the gradient. Taking into account the analytical centrifugation results, a procedure was devised to purify lysosomes from several grams of liver by centrifugation of an L fraction in a discontinuous metrizamide gradient. By this method, a fraction containing 10-12% of the whole liver lysosomes can be prepared. As inferred from the relative specific activity of marker enzymes, it can be estimated that lysosomes are purified between 66 and 80 times in this fraction. As ascertained by plasma membrane marker enzyme activity, the main contaminant could be the plasma membrane components. However, cytochemical tests for 5'AMPase and for acid phosphatase suggest that a large part of the plasma membrane marker enzyme activity present in the purified lysosome preparation could be associated with the lysosomal membrane.The procedure for the isolation of rat liver lysosomes described in this paper is compared with the already existing methods.KEY WORDS metrizamide centrifugation rat liver lysosomes 9 plasma membrane gradient Isopycnic centrifugation in a metrizamide gradient of a total mitochondrial fraction of rat liver (M + L according to de Duve et al. [15]), allows a good separation of peroxisomes from the other major constituents of the fraction, mitochondria, and lysosomes (13). On the other hand, lysosomes are poorly separated from mitochondria as ascertained by the distribution curves of their reference enzymes (13). In the present paper, we show that by using a light mitochondrial fraction (L fraction of de Duve et al. [15]), in certain conditions it is possible to purify rat liver lysosomes extensively by centrifugation in a metrizamide gradient, with satisfactory yield. MATERIALS AND METHODS Tissue FractionationExperiments were performed on male Wistar rats weighing 200-250 g. The animals were decapitated after J. CELL BmLO~V 9 The Rockefeller University Press 9 0021-9525/78/0801-034951.00 349 being starved for 20 h. The livers were removed, chilled in ice-cold 0.25 M sucrose, and homogenized in the same medium by means of a smooth glass tube fitted with a Teflon pestle (Arthur H. Thomas Co., Philadelphia, Pa.) rotating at 3,000 rpm. The homogenate was made up to a volume of 7 ml/g of liver. After filtration on two layers of cheesecloth, a small sample was kept for enzyme and protein determination; the remainder was centrifuged at an integrated force of 30,000 g. min in the N ~ 40 rotor (ravg 5.9 cm) of the Spinco preparative ultracentrifuge (Beckman Instruments, Inc....

Oxygen free radicals (OFR) are thought to mediate ischemia-reperfusion injury to endothelium of heart, lung, brain, liver, and kidney and contribute to development of atherosclerosis, pulmonary O2 toxicity, and adult respiratory distress syndrome. Increased cytosolic free Ca2+ (Cai2+) has been proposed as a mechanism of injury from oxidative stress, yet the pathways by which an increase in Cai2+ may cause OFR-mediated endothelial cell injury remain unknown. Using multiparameter digitized video microscopy and the fluorescent probes, fura-2 acetoxymethyl ester and propidium iodide, we measured Cai2+ and cell viability in human umbilical endothelial cells during oxidative stress with xanthine (50 microM) plus xanthine oxidase (40 mU/ml). Oxidative stress caused a sustained increase in Cai2+ from a resting level of 90-100 nM to near 500 nM, which was preceded by formation of plasma membrane blebs. The increase in Cai2+ was prevented by removal of extracellular Ca2+ (Cao2+). Prevention of the increase in Cai2+ was associated with prolonged cell viability. Readdition of Cao2+ resulted in an immediate large increase in Cai2+ and rapid onset of cell death. The protease inhibitors, leupeptin and pepstatin, delayed the increase in Cai2+ and prolonged cell viability. The results are consistent with the hypothesis that endothelial cell injury due to oxidative stress may be the result of Cai2+ influx and resultant activation of Ca(2+)-dependent proteases.

The distribution of mitochondrial enzymes after isopycnic centrifugation of a rat-liver mitochondrial fraction in a sucrose gradient depends on the speed of centrifugation. From the distribution changes, it may be deduced that when centrifugation reaches a certain speed there is an increase in the equilibrium density of some mitochondria; after this the mitochondria undergo disruption. The hydrostatic pressure to which the granules are subjected in the gradient seems to be the main cause of these phenomena. The results of morphological examinations are in good agreement with those suggested by biochemical determinations.As we have briefly reported [l] when a rat-liver mitochondrial fraction undergoes isopycnic centrifugation in a sucrose gradient, the enzyme distributions differ depending on whether centrifugation was performed at 39000 rev./& or a t 50000 rev./min in the Spinco SW 50.1 rotor. This finding is best explained by postulating that mitochondria undergo damage when they are centrifuged a t too high a speed. I n this study we shall show how this phenomenom varies with the speed of centrifugation and with the hydrostatic pressure to which the mitochondria are exposed during centrifugation. Some morphological results will also be presented. METHODS Centrifugation Experiments Morphological ExaminationsSmall aliquots of selected fractions of the gradient were poured into an ice cold solution containing 1.5O/, (wlv) of glutaraldehyde in 0.05 M phosphate buffer pH 7.4. After 10 min they were diluted in order to obtain about 120pg of protein per amount of filtered material. After this, according to the procedure described by Baudhuin et al. [9], 0.75 ml of each fixed suspension was filtered through Millipore membranes of 0.025 pm pore size. Filtration time was approximately half an hour. The Millipore filters were then washed in buffer and postfixed with 1 O/, (wlv) osmium tetroxide in 0.05 M phosphate buffer pH 7.4 for 1 h. After dehydration through graded alcohol, the filters were transferred to propylene oxide for about 18 h and the films of filtered materials were embedded in Epon 812. Sections were obtained with a LKB Ultratome I11 microtome and mounted on unsupported grids. They were stained with 2O/, (wlv) uranyl acetate in alcohol solution for 1-2 min followed by lead citrate [lo] for 5 min and examined with a Philips EM 300 electron microscope under a 60 kV accelerating voltage. Calculation of Hydrostatic PressureThe variation of the hydrostatic pressure as a function of the radial distance is given by the following relationship : dP dx where P is the hydrostatic pressure, x the radial distance, Q the solution density and w the angular velocity. If Q is constant, Equation (1) can be integrated to give the pressure P at the radial distance x: where x,, is the radial distance at the top of the liquid column. If Q linearly varies as a function of the radial distance (linear gradient) :where a is the slope of the gradient, Rmin the radial distance a t the meniscus and b the ordinate a t origin.only sm...

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