Role of particle interaction on distribution of liver lysosomes in colloidal silica

Abstract

Rat liver mitochondrial-lysosomal fractions were separated on gradients of colloidal silica. Lysosomal enzymes were distributed bimodally. The dense peak (1.117 g/ml) was nearly free from contaminants; beta-N-acetyl-D-glucosaminidase was enriched nearly 60-fold. By contrast, the buoyant peak (1.085 g/ml) co-sedimented with mitochondria, microsomes, peroxisomes, and Golgi particles. Decreasing the amount of protein layered on the gradient medium or dispersing a full sample through it shifted lysosomal marker from the buoyant to the dense peak. Thus the majority of lysosomes in the two peaks appeared to have equivalent densities. Electron microscopic examination of particles separated from gradients with layered samples showed that the dense peak contained most of the dense bodies, whereas the buoyant peak was relatively enriched in autophagic vacuoles. Dispersion, however, shifted autophagic vacuoles from the buoyant to the dense peak without affecting the distribution of dense bodies. We conclude that the bulk of buoyant particles act as a sieve to retard the density equilibration of autophagic vacuoles without specifically affecting other lysosomal enzyme-containing components.

Cathepsin B: association with plasma membrane in metastatic tumors.

“…The population of greater density was shown to consist of secondary lysosomes containing mature lysosomal enzymes (29)(30)(31). The population oflesser density was shown to contain elements of the Golgi apparatus and endoplasmic reticulum by Rome et al (31) and autophagic vacuoles by Surmacz et al (32). Several studies (11,(29)(30)(31)(32)(33) suggest that a bimodal distribution of lysosomal enzymes may reflect the intracellular synthesis and processing of lysosomal enzymes-i.e., precursor or latent forms of the enzymes are found in the lighter fraction and mature enzymes in the heavier.…”

Identification of Autolysosomes Directly Associated with Proteolysis on the Density Gradients in Isolated Rat Hepatocytes

“…A mixture of 60% Percoll and 0.75% PVP was finally chosen, in which the profile consisted of a major buoyant peak (d= 1.090) and a minor dense peak (d= 1.131), quite similar to the profile obtained previously (19). However, this /9-hexosaminidase profile obtained from isolated hepatocytes was quite different from that in liver perfusion study (10,11,20). In order to test if this was the result of a difference in gradient media or lysosomal population in these two preparations, they were directly compared under the same gradient conditions (Fig.…”

“…For the present study, which aims to separate subpopulations among autophagy-related vacuoles rather than to purify them from other organelles such as mitochondria, a colloidal silica was chosen. Previous studies (10,11) demonstrated that autophagic vacuoles (AV) induced in the perfused liver could be effectively separated from DB using Ludox, a colloidal silica, mixed with polyvinylpyrrolidone (PVP) , and this has been regarded as the best separation technique of AVd and DB, intermediate and late compartments of autophagy, respectively. We applied this method to freshly isolated rat hepatocytes using Percoll, a more readily available colloidal silica than Ludox.…”

Inhibition of intracellular protein degradation by ethanol in perfused rat liver

“…Colloidal-silica/PVP gradients For measurement of the distribution of lysosomes in colloidal-silica gradients, livers were homogenized after the 40 min single-pass perfusion as in Neely et al (1974). Mitochondrial and lysosomal fractions, separated from the total homogenate by differential centrifugation, were layered on colloidal silica/PVP; centrifugation and fractionation of the gradients were as in Surmacz et al (1983). Analytical Valine in perfusate plasma was analysed chromatographically (Mortimore & Mondon, 1970).…”

“…Plasma ammonia was measured enzymically without protein precipitation, with the use of an assay kit from Boehringer (Mannheim, Germany) and a Gilford 3500 analyser. Hexosaminidase in the colloidal-silica/PVP gradient fractions and protein were determined as in Surmacz et al (1983). Electron microscopy Livers were fixed and processed for electron microscopy as in Mortimore & Schworer (1977).…”