Rat intestinal microvillus membranes. Purification and biochemical characterization

Abstract

1. A technique is described for the removal of subcellular contaminants from intact rat intestinal brush borders, and for the subsequent separation of a microvillus membrane fraction from a fibrillar residue. 2. Increments in invertase activity, microscopic homogeneity and low nucleic acid content indicate that the microvillus plasma membrane has been extensively purified. Multiple membrane preparations have been shown to be highly reproducible with respect to their invertase specific activity, cholesterol content and phospholipid content. Alkaline phosphatase, leucine aminopeptidase, Mg(2+)- and Ca(2+)-dependent adenosine triphosphatase and seven separate disaccharidases were shown to be predominantly confined to the membrane fraction. 3. The fibrillar fraction has been shown to contain approximately 30% of the total protein of purified brush borders, plus most of the residual nucleic acid contaminant. No evidence was found for the localization of any specific enzyme in this fraction.

Glucose- and phlorrhizin-protected thiol groups in pig intestinal brush-border membranes.

“…Contamination from nuclei was negligible. Alkaline phosphatase activity, measured with 1 mM-p-nitrophenyl phosphate as substrate in the presence of 20mM-MgCI2 at pH9.6, was high and comparable with values reported previously (3.0± 0.6,umol ofp-nitrophenyl phosphate hydrolysed/min per mg of pig brush-border protein; 4.9 for the rat; Forstner et al, 1968).…”

Composition of Cellular Membranes in the Pancreas of the Guinea Pig

“…' Finally, the cell membrane fraction, morphologically the least satisfactory of our preparations, has the usual type of plasmalemmal enzyme activities and little else . In contrast to the situation in other tissues (7,25,33,34,36,37) it has no alkaline p-nitrophenylphosphatase activity, and its NADH-cytochrome c reductase, as well as part of the Mgt+-ATPase activity could be accounted for by mitochondrial contamination as indicated by experiments and calculations similar to those described in the case of zymogen granule membranes . ' Smooth microsomes, zymogen granule membranes, and plasmalemmal fractions have a rather similar lipid composition characterized by high sphingomyelin and cholesterol content (2) .…”

“…5'-nucleotidase (5, 9, 20-23, 25, 33-35), ß-leucyl naphthylamidase (22,25,33,36,37), and Mgt+-ATPase (5,20,22,(34)(35)(36)(37)(38) (Table II), are also highly concentrated in the pancreatic plasmalemmal fraction . In addition, they are present in different concentrations in smooth microsomes and zymogen granule membranes .…”

Isolation and Biochemical Characterization of Brush Borders From Rabbit Kidney

“…A characterization of the biochemical properties of the isolated brush border, therefore, should provide significant information on the molecular mechanisms underlying tubular reabsorption . Brush borders from intestinal epithelial cells have been isolated as a discrete subcellular fraction, and several enzymes are now known to be associated with this membranous structure (1)(2)(3)(4) . In contrast, little has been done on the isolation or biochemical characterization of the renal brush border .…”