Characterization of the membrane proteins of rat liver lysosomes. Composition, enzyme activities and turnover

Abstract

Lysosomes prepared from the livers of untreated rats and from the livers of rats injected with either Triton WR-1339 or dextran yielded membranes that were similar in both polypeptide composition and activities of ATPase and acid 5'-nucleotidase. The administration of Triton WR-1339 (and dextran) resulted in an increase in ATPase activity of liver homogenates that was associated with a parallel increase in the ATPase activity of the lysosomal membrane. On the other hand, plasma membranes appear to be different from lysosomal membranes with respect to polypeptide composition and enzyme activities. The ATPase activity of lysosomal membranes is not affected by ouabain and suramin, inhibitors of the plasma-membrane ATPase. The plasma-membrane alkaline 5'-nucleotidase has little activity at acid pH. Pulse-labelling of lysosomal membranes with [3H]fucose and with [3H]- and [14C]-leucine occurred rapidly, faster than labelling of plasma membranes. The labelling kinetics indicate that lysosomal membranes may be assembled independently of plasma membranes. These data suggest that, in liver, little bulk transport of plasma membrane to lysosomes takes place, and lysosomal-membrane proteins may not be derived from those of plasma membranes.

Determination of Intracellular Unbound Concentrations and Subcellular Localization of Drugs in Rat Sandwich-Cultured Hepatocytes Compared with Liver Tissue

“…2A) is an example of potential cross-contamination. Lysosomes are quite fragile, a characteristic that is noted and/or exploited in traditional fractionation methods (Burnside and Schneider, 1982;Ohsumi et al, 1983); therefore, it is likely that a portion of the lysosomes were disrupted during the homogenization process in both SCH and whole-tissue matrices. 2) Potential disruption of binding equilibrium between cellular compartments occurs during the fractionation process.…”

A Proteomic Analysis of Lysosomal Integral Membrane Proteins Reveals the Diverse Composition of the Organelle

“…While many procedures have been used for lysosomal purification, we chose one of the most reliable and best studied, namely, Triton WR1339-filled rat liver lysosomes (tritosomes) as described by Leighton et al (12). Although tritosomes are considered modified lysosomes as they contain a quantity of non-physiological material (Triton WR1339), tritosomes retain lysosomal enzymatic activities (6,(12)(13)(14) and perform identically to lysosomes in endosomal and phagosomal fusion assays (15). In this study we identified 215 separate proteins in the integral membrane protein fraction of the lysosome, many not previously associated with the lysosome and several that are still only identified as uncharacterized cDNAs.…”

“…have been extensively characterized as functional secondary (late stage) lysosomes (12,14,15,19). We have previously demonstrated the enrichment of lysosomal markers (␤-hexosaminidase activity, Rab7, and LAMP-1) in our tritosome preparations (70-fold enrichment of ␤-hexosaminidase) and the absence of markers from mitochondria (citrate synthase activity) and ER (calnexin protein) (Refs.…”

The potentials of MS‐based subproteomic approaches in medical science: The case of lysosomes and breast cancer

“…Tritosomes are lysosomes that have ingested Triton. They are a wellestablished model of rat liver lysosomes distinct from early endosomes, late endosomes, and phagosomes, as previously shown by electron microscopy, enzyme assays, Western blots of marker proteins, and phagosome-fusion assays (Leighton et al, 1968;Khan et al, 1981;Burnside & Schneider, 1982;Wattiaux et al, 1996). Briefly, Tritosomes are obtained by injecting Triton WR1339 (Tyloxapol) to rats 5 days prior to sacrifice.…”