Studies on the fractionation of mucosal homogenates from the small intestine

Hübscher, G.; West, Gwen R.; Brindley, David N.

doi:10.1042/bj0970629

Cited by 224 publications

(53 citation statements)

References 42 publications

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“…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 .…”

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confidence: 99%

Isolation and Biochemical Characterization of Brush Borders From Rabbit Kidney

Berger

Sacktor

1970

The Journal of Cell Biology

152

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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

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confidence: 99%

Isolation and Biochemical Characterization of Brush Borders From Rabbit Kidney

Berger

Sacktor

1970

The Journal of Cell Biology

152

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“…The substrates used were p-nitrophenyl- (Hubscher, West and Brindley, 1965). Crystalline bovine serum albumin was used as a standard.…”

Section: Methodsmentioning

confidence: 99%

Glycosidases Heterogeneity Among Dimethylhydrazine Induced Rat Colonic Tumours

Mian¹,

Cowen²,

Nutman³

1974

Br J Cancer

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Summary.-Activities of N-acetyl-fl-D-glucosaminidase, N-acetyl-fl-D-galactosaminidase, 3-D-galactosidase and a-L-fucosidase were measured in rat colonic tumours induced by 1,2-dimethylhydrazine. Tumours varied considerably in their enzyme content, not only from different animals but also from the same animals. Enzymatic heterogeneity among tumours appeared to be related to their site of origin in the colon. The descending colon, which after the DMH treatment showed a significant increase in the levels of glycosidases, also gave rise to a larger number of adenocarcinomata than other parts of the colon. The relative changes in the activities of four glycosidases seemed to show a good correlation.IN GENERAL, rat colonic tumours induced bv 1,2-dimethylhydrazine (DM1H) indicated a significant increase in the levels of some glycosidases compared with the remaining colonic mucosa of the treated animals (Mian and Cowen, 1974). Evidence of a generalized change, as indicated by an overall reduction of 35S uptake, in parts of the DMH treated rat colon where tumours were commonly developed has been given (Springer, Springer and Oehlert, 1970). The site where tumours would arise in the mouse colon after DMH treatment could be predicted with great certainty (Haase et al., 1973). These authors observed that in the experimental mice the last 4 cm of the colon was always the site of at least one polyp. A gradient in the cell turnover times from the proximal to the distal end of the colon and a considerable variation in the cell proliferation rates in the DMH induced mouse colonic tumours and in the primary adenocarcinomata of the human large bowel have already been observed (Sawicki and Rowinski, 1970;Haase et al., 1973;Bottomley and Cooper, 1973;Camplejohn, Bone and Aherne, 1973).The present work was carried out to assess the enzymatic heterogeneity among the DMH induced rat colonic tumours with reference to the site specificity and variations in tumour morphology and cell kinetics. MATERIALS AND METHODSAnimals.-Wistar male rats, about 12 weeks old, were obtained from Bantin and Kingman, Hull, England. The animals were fed Oxoid 41B (Oxo Ltd, London, England) and water ad libitum. The experimental animals were injected subcutaneously for 26-32 weeks with a weekly dose of 20 mg/kg body weight of 1,2-dimethylhydrazine dihydrochloride (DMH) in 4% solution of Na2.EDTA made freshly each time as described by Haase et al. (1973). The control animals were given Na2. EDTA in saline solution. Injection of animals was stopped one week before they were used for the experiment.Preparation of tissue homogenates.-Animals were starved overnight before the experiment but had unrestricted access to water. They were killed by cervical dislocation. Colons were removed, opened by a longitudinal slit and washed with ice cold saline. Total length of the colon, location of the tumours and their number were recorded. Tumours were excised and kept individually. The remaining colons of the tumour bearing animals and the control colons were cut into 8 equ...

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“…supernates were spun at 4VC in an 873 rotor at 15,000 rpm for 15 min (345,000 g-min); the sedimented mitochondria (20) were resuspended in buffer and were centrifuged at 345,000 g-min. 4 ml of the combined supernates, which had been raised to a density of 1.052 g/ml with NaCl, were overlayered with a NaCl density gradient (21) and were spun 2 X 10' g-min in an SW 41 Ti rotor' which concentrates Sf > 20 plasma lipoproteins in the upper 0.5 ml of the tube (21).…”

Section: Methodsmentioning

confidence: 99%