No abstract

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....

Glycyl-L-phenylalanine 2-naphthylamide (Gly-L-Phe-2-NNap), a cathepsin C substrate, induces an increase of the free and unsedimentable activities of this enzyme when incubated with a total mitochondrial fraction of rat liver. 1 mM-ZnSO4 considerably inhibits the cathepsin C total activity, measured with Gly-L-Phe-2-NNap as the substrate, in the presence of Triton X-100. The inhibition is markedly less pronounced when the free activity is determined; a high activity remains that depends on the integrity of the lysosomes; it decreases as the free activity of N-acetylglucosaminidase increases when lysosomes are subjected to treatments able to disrupt their membrane. Cathepsin C activity is reduced when thioethylamine hydrochloride is omitted from the incubation medium. Under these conditions at 37 degrees C, the free activity equals the total activity, although the lysosomes are intact, as indicated by the low free activity of N-acetylglucosaminidase. 1 mM-ZnSO4 strikingly inhibits the total activity, whereas more than 80% of the free activity remains. These observations are presented as evidence that Gly-L-Phe-2-NNap can possibly cause a disruption of the lysosomes as a result of its hydrolysis inside these organelles. In the presence of ZnSO4, intralysosomal hydrolysis becomes apparent, owing to a preferential inhibition by Zn2+ of extralysosomal hydrolysis; in the absence of thioethylamine hydrochloride, it is measurable because the disruption of lysosomes by Gly-L-Phe-2-NNap is delayed as a result of a slow-down of the reaction. The usefulness of Gly-L-Phe-2-NNap and related dipeptidyl naphthylamides in lysosomal-membrane-permeability studies is emphasized.

The fact that after an hydrodynamic injection (35)S-DNA remains bound to the outside face of the plasma membrane for at least 1 h indicates that it is not, or very slowly, internalised during that period. The relatively small difference in the amount of DNA picked up by hepatocytes depending on the type of injection could not explain the absence of expression after a conventional injection and the strong expression after a hydrodynamic injection. If DNA enters the cells by endocytosis, even after an hydrodynamic injection, its persistence at the outside face of the plasma membrane could favour transfection by allowing hepatocytes to dispose for a relatively long time of a reservoir of intact DNA.

Our results show that the efficiency of hydrodynamics-based transfection depends on a process that takes place very quickly after injection and is not linked to a delay of DNA degradation and the persistence of a large proportion of DNA bound to hepatocytes of the plasma membrane, strongly suggesting that expression after a hydrodynamic injection is caused by a small proportion of DNA molecules that rapidly enter the cytosol probably by plasma membrane pores generated by the hydrodynamic pressure.