Composition of Cellular Membranes in the Pancreas of the Guinea Pig

Abstract

1 3 0The lipid composition of rough and smooth microsomal membranes, zymogen granule membranes, and a plasmalemmal fraction from the guinea pig pancreatic exocrine cell has been determined . As a group, membranes of the smooth variety (i .e ., smooth microsomes, zymogen granule membranes, and the plasmalemma) were similar in their content of phospholipids, cholesterol and neutral lipids, and in the ratio of total lipids to membrane proteins . In contrast, rough microsomal membranes contained much less sphingomyelin and cholesterol and possessed a smaller lipid/protein ratio . All membrane fractions were unusually high in their content of lysolecithin (up to _~20% of the total phospholipids) and of neutral lipids, especially fatty acids . The lysolecithin content was shown to be due to the hydrolysis of membrane lecithin by pancreatic lipase ; the fatty acids, liberated by the action of lipase on endogenous triglyceride stores, are apparently scavenged by the membranes from the suspending media . Similar artifactually high levels of lysolecithin and fatty acids were noted in hepatic microsomes incubated with pancreatic postmicrosomal supernatant . E 600, an inhibitor of lipase, largely prevented the appearance of lysolecithin and fatty acids in pancreatic microsomes and in liver microsomes treated with pancreatic supernatant .In the first article of this series, we have described the separation of membrane subfractions from rough microsome-, smooth microsome-, and zymogen granule fractions isolated from homogenates of guinea pig pancreas . We have also shown that a cell membrane fraction can be obtained from the same source (1) .These fractions represent membrane-bounded compartments which are involved in the synthesis, segregation, intracellular transport, and discharge of secretory proteins . In an attempt to understand how the membranes of these compartments interact with one another during the secretory process, we have investigated their chemical composition and enzyme activities . In this paper the lipid composition of the membranes is described .At the onset of this study, we anticipated that a small fraction of the digestive enzymes stored in the gland, particularly in zymogen granules and in the duct system, could be released during tissue homogenization and fractionation and thereupon cause artifactual degradation and alteration of membrane components . In this respect, it has been reported that pancreatic lipase is active in the cold (2) and that at least some proteases may become activated during cell fractionation (3) . Indeed, our studies showed that released lipolytic enzymes extensively affect the lipid composition of the isolated membranes . These alterations have been evaluated and the data on membrane lipids have been corrected accordingly .

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

“…Neutral lipase assayed using as substrate fl-naphthyl nonanoate in the presence of sodium taurocholate (8) gave a positive reaction barely above the colorimetric level of the blank only when a large fraction (one-fourth to one-third the quantity of a single experiment) of the total granule protein was assayed as a single sample. These assays confirmed the initial assumption that the parotid might represent a promising exocrine system in contrast to the guinea pig exocrine pancreas (49,50) for the study of undegraded intracellular membranes.…”

“…4 a, this rabbit parotid granule fraction contains recognizable immature secretion granules (13), and the higher magnification (Fig. 4 b) further indicates that the limiting membranes of the granules are well preserved, showing a reduced incidence of myelin figure formation (at the expense of membrane phospholipids) when compared to pancreatic zymogen granules (49,50).…”

“…These membranes, which had accumulated free fatty acid secondary to the action of endogenous pancreatic lipase on tissue triglycerides and membrane phospholipids (50), banded at a 0.7-1.0 M sucrose interface, while residual mitochondria pelleted through the 1.0 M sucrose during centrifugation. Since rabbit parotid secretion granules contain little or no lipase activity, the membranes of lysed granules would not be expected to undergo any significant shift to lower density.…”

“…To lyse secretion granules we sought an alternative to the lysis at mildly alkaline pH used for pancreatic zymogen granules (21,25,(49)(50)(51) since rabbit parotid granules were found to be reasonably stable in the pH range 7.5-8.0. Instead, we exploited the instability of rabbit parotid granules to hypotonic media in developing a lysis procedure, which together with the subsequent separation of granule membranes and secretory content is presented in detail in the Results.…”

Immunological similarity of the NADH—cytochrome c electron transport system in microsomes, Golgi complex and mitochondrial outer membrane of rat liver cells

“…Although different membranes may be functionally related, they have been shown to be biochemically distinguishable entities in various systems [1][2][3][4]. Some enzymes, however, have been demonstrated to be localized in more than one type of membrane.…”