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PMID: 4634836 Published · ppublish English Journal Article

Preparation and properties of nexuses and lipid-enriched vesicles from mouse liver plasma membranes.

The Biochemical journal ·Vol. 128 ·No. 3 ·1972-07-00 ·Pages 691-700

Evans WH, Gurd JW

Abstract

Extraction of mouse liver plasma membranes with 4% (w/v) N-laurylsarcosinate-tris buffer, pH7.8, solubilized 80-90% of the protein and 60% of the 5'-nucleotidase activity. The membrane residue remaining after extraction was resolved on sucrose gradients into two fractions: a vesicular membrane fraction and a fraction characterized by the presence of large numbers of nexuses in an amorphous background. The vesicular fraction had a phospholipid/protein weight ratio of 7:1, it contained most of the plasma-membrane glycolipids, and polyacrylamide-gel electrophoresis indicated the presence of only five to eight proteins, including two or three glycoproteins. The 5'-nucleotidase and leucine naphthylamidase specific activities were 23- and 6-fold higher respectively than in the plasma membranes. Electron microscopy of thin sections and negatively stained preparations indicated that the nexuses present in the second fraction closely resembled gap junctions present in tissue sections and isolated plasma membranes. The nexus fraction contained a distinctive protein pattern, and of the 20 proteins present about four were identified as glycoproteins by Schiff-periodate staining. Examination of the lipid composition of the fractions by t.l.c. showed that in the nexus fraction, phospholipids and glycolipids were present in small amounts compared with triglycerides and cholesterol. Amino sugar analyses confirmed the t.l.c. results and amino acid analysis showed the fractions to have characteristic protein compositions. A ;reconstituted' membranous fraction prepared by dialysis against MgCl(2) of membrane components soluble in N-laurylsarcosinate-tris buffers, pH7.8, lacked the trilaminar image characteristic of the two other membrane fractions isolated and was devoid of enzyme activities. The results indicate that proteins and glycoproteins play an important role in the structural maintenance of the nexuses isolated from the liver by the present procedure.

MeSH Terms
Amino Acids/analysis Aminopeptidases/analysis Animals Cell Membrane/analysis,enzymology Centrifugation, Density Gradient Cholesterol/analysis Chromatography, Thin Layer Electrophoresis, Polyacrylamide Gel Glycolipids/analysis Glycoproteins/analysis Histocytochemistry Leucine Lipids/analysis Liver/cytology Mice Microscopy, Electron Naphthalenes/analysis Nucleotidases/analysis Phospholipids/analysis Phosphorus Isotopes Proteins/analysis Subcellular Fractions Triglycerides/analysis
Chemicals
Amino Acids Glycolipids Glycoproteins Lipids Naphthalenes Phospholipids Phosphorus Isotopes Proteins Triglycerides Cholesterol Nucleotidases Aminopeptidases Leucine
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Evans W H
Gurd J W
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48 references, click to expand
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Article Info
Journal
The Biochemical journal
Abbr.
Biochem J
ISSN
0264-6021
DOI
10.1042/bj1280691
Published
1972-07-00
Pages
691-700
Language
English
Region
England
NLM ID
2984726R
PMCID
PMC1173820
Subset
IM
External Links
PubMed source DOI: 10.1042/bj1280691 PMC Full Text View journal details
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