Isolation and Biochemical Characterization of Brush Borders From Rabbit Kidney

Abstract

A technique for the isolation of intact brush borders from rabbit renal cortex was evaluated . The procedure was monitored by phase and electron microscopy and marker enzymes, i .e . ATP :NMN adenylyl transferase, nuclear ; cytochrome oxidase, mitochondrial ; (3-glucuronidase, lysosomal ; and glucose-6-Pase, microsomal ; and indicated an essentially pure preparation of brush borders . The disaccharidase, trehalase, previously reported in renal tubules, was localized uniquely in brush borders . Maltase was also found ; the specific activities of the two enzymes in the brush borders were increased 10-to 20-fold . Other disaccharidases, such as sucrase, isomaltase, lactase, and cellobiase, were absent . It is suggested that trehalase and maltase are appropriate candidates for marker enzymes of the renal brush border. Isolated brush borders possessed a ouabain-sensitive (Na+ + K+) ATPase, an oligomycin-insensitive Mg++ ATPase, and a Ca++-activated ATPase . Alkaline phosphatases, dephosphorylating (3-glycero-P, and trehalose-6-P were also present . The specific activities of these enzymes were increased three-to-five fold in the brush-border preparations ; however, activities were found in other subcellular fractions of the renal cortex . Hexokinase, although evident in the isolated brush border, was found prominently associated with other membranous fractions . Phosphoglucomutase and UDPG pyrophosphorylase were localized in the soluble fraction of the renal cortex .The renal brush border has a prominent role in determining the specificity and rate of reabsorption from the glomerular filtrate . 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 (1-4) . In contrast, little has been done on the isolation or biochemical characterization of the renal brush border . Recently, however, Thuneberg and Rostgaard (5) described a procedure for isolating brush borders from rabbit kidney cortex, but no biochemical correlates were made, and Kinne and Kinne-Saffran (6) and

Calcium transport in canine renal basolateral membrane vesicles. Effects of parathyroid hormone.

“…Characterization of the BLMV by means of specific enzyme markers was performed as described previously (33, 34) using activities of Na+/K+-ATPase, a basolateral membrane marker (35); acid phosphatase, a lysosomal marker (36); cytochrome oxidase, a mitochondrial marker (37); maltase, a brush border marker (38); and NADPH-cytochrome-C reductase, an endoplasmic reticulum marker (39). Protein content was measured (40) using a bovine serum albumin standard.…”

Glucocorticoids increase the Na+-H+ exchange and decrease the Na+ gradient-dependent phosphate-uptake systems in renal brush border membrane vesicles.

“…Maltase was assayed as described (23). y-Glutamyltransferase activity was determined with y-glutamyl-pnitroanilide as substrate (24) and competitive inhibition ofboth these processes by amiloride (15,16).…”

Monoclonal antibodies to renal brush border membrane maltase: age-associated antigenic alterations.

“…(The Animal Resources Facility, Gerontology Research Center, is fully accredited by the American Association for Accreditation of Laboratory Animal Care.) Maltase activity was determined by a coupled enzymatic assay (2). lodination of Maltase.…”

“…Maltase (a-D-glucoside glucohydrolase, EC 3.2.1.20) is the major disaccharidase in rat kidney brush border membranes (1,2). Maltase-specific activities in renal cortex homogenates and brush border membranes prepared from aged rats decrease 30-40% relative to the activities of the enzyme from young adult animals (3,4).…”