Selective Release of Content From Microsomal Vesicles Without Membrane Disassembly

Abstract

Rat liver rough microsomes treated with a series of desoxycholate (DOC) concentrations from 0.003 to 0.4% were analyzed by isopycnic sucrose density gradient centrifugation in media containing high or low salt concentrations. Tritium-labeled precursors administered in vivo were used as markers for ribosomes (orotic acid, 40 h), phospholipids (choline, 4 h), membrane proteins (leucine, 3 days), and completed secretory proteins of the vesicular cavity (leucine, 30 min). Within a narrow range of DOC concentrations (0.025–0.05%), the vesicular polypeptides were selectively released from the microsomes, while ribosomes, nascent polypeptides, and microsomal enzymes of the electron transport systems were unaffected. The detergent concentration which led to leakage of content was a function of the ionic strength and of the microsome concentration. At the lowest effective DOC concentration the microsomal membranes became reversibly permeable to macromoles as shown by changes in the density of the vesicles in Dextran gradients and by the extent of proteolysis by added proteases. Incubation of rough microsomes with proteases in the presence of 0.025% DOC also led to digestion of proteins from both faces of the microsomal membranes and to a lighter isopycnic density of the membrane vesicles.

Spatial orientation of glycoproteins in membranes of rat liver rough microsomes. II. Transmembrane disposition and characterization of glycoproteins.

“…Before use, RM were washed by centrifugation in HSB for 20 min at 15,000 rpm in a Ti60 rotor (abbreviated as 20 min, 15K, Ti60). RM vesicles were made permeable to macromolecules by the addition of 0.05% DOC, a condition which leaves the vesicular membrane largely intact (22,41). Protein was determined according to Lowry et al (29).…”

In vitro synthesis of the Ca 2+ transport ATPase by ribosomes bound to sarcoplasmic reticulum membranes

“…Although peripheral membrane proteins are removed from membranes by chelation of divalent cations or incubation in media of high ionic strength (28,29), the newly synthesized ATPase remained associated with the vesicles after ribosomes were detached by treatment with EDTA in a medium containing 0.5 M KC1. Furthermore, a major fraction of the in vitro synthesized ATPase was not released from microsomes by treatments that open microsomal vesicles and release their lumen contents (24,25). Only at deoxycholate concentrations that dissolved the membranes was a substantial fraction of the labeled ATPase released.…”

“…Subfractionation by differential centrifugation (1 hr at 45,000 X g) showed that the lower deoxycholate concentration used (0.1 mg/mg of microsomal protein in 0.3 ml containing 2.11 X 105 cpm) released 40% of the total microsomal-radioactivity, but labeled ATPase represented only 0.29% of the total radioactive protein in the released fraction. The bulk of the newly synthesized ATPase (0.98% of the radioactivity) was only released at a higher deoxycholate concentration (0.5 mg/mg of protein in 0.3 ml), which is known to cause extensive membrane dissolution (24,25). The major labeled product present in immunoprecipitates obtained from in vitro translation mixtures containing bound polysomes or rough microsomes had an electrophoretic mobility similar to that of the mature ATPase (Fig.…”

“…However, immunoprecipitation showed that the ribosome-stripped vesicles, which were recovered by centrifugation (2 hr at 100,000 X g) and dissolved with detergent, contained approximately 80% of the in vitro synthesized ATPase (1.12% of the total radioactivity). The stripped rough microsomal membranes were also treated with deoxycholate (30 min at 4°C) at concentrations that either release the microsomal content or dissolve the microsomal membranes (24,25). Subfractionation by differential centrifugation (1 hr at 45,000 X g) showed that the lower deoxycholate concentration used (0.1 mg/mg of microsomal protein in 0.3 ml containing 2.11 X 105 cpm) released 40% of the total microsomal-radioactivity, but labeled ATPase represented only 0.29% of the total radioactive protein in the released fraction.…”

Secretion granules of the rabbit parotid gland. Isolation, subfractionation, and characterization of the membrane and content subfractions.

“…In the case of rough microsomes isolated from rat liver, treatment with low concentrations of sodium deoxycholate, shown to create discontinuities in the microsomal membranes (35), promotes only partial extraction of short term-labeled (30-rain postpulse) polypeptides identified as protein destined for export from the liver (37). Thus both these studies suggest that adsorption to either the cytoplasmic or cisternal surfaces of membranebounded compartments as a consequence of cell or cellular compartment disruption in nonphysiologic media is both substantial and difficult to overcome by a variety of extractants.…”