scholarly journals Localization of the membrane-associated thiol oxidase of rat kidney to the basal-lateral plasma membrane

1982 ◽  
Vol 203 (2) ◽  
pp. 371-376 ◽  
Author(s):  
L H Lash ◽  
D P Jones
Keyword(s):  
Plasma Membrane ◽  
Brush Border ◽  
Specific Activity ◽  
Rat Kidney ◽  
Gradient Centrifugation ◽  
Kidney Cortex ◽  
Marker Enzyme ◽  
Marker Enzymes ◽  
Lateral Membrane ◽  
Thiol Oxidase

The localization of the membrane-associated thiol oxidase in rat kidney was investigated. Fractionation of the kidney cortex by differential centrifugation demonstrated that the enzyme is found in the plasma membrane. The crude plasma membrane was fractionated by density-gradient centrifugation on Percoll to obtain purified brush-border and basal-lateral membranes. Gamma-Glutamyltransferase, alkaline phosphatase and aminopeptidase M were assayed as brush-border marker enzymes, and (Na+ + K+)-stimulated ATPase was assayed as a basal-lateral-membrane marker enzyme. Thiol oxidase activity and distribution were determined and compared with those of the marker enzymes. Its specific activity was enriched 18-fold in the basal-lateral membrane fraction relative to its activity in the cortical homogenate, and its distribution paralleled that of (Na+ + K+)-stimulated ATPase. This association indicates that thiol oxidase is localized in the same fraction as (Na+ + K+)-stimulated ATPase, i.e. the basal-lateral region of the plasma membrane of the kidney tubular epithelium.

scholarly journals Phosphatidylinositol kinase and diphosphoinositide kinase of rat kidney cortex: properties and subcellular localization

1976 ◽  
Vol 160 (1) ◽  
pp. 97-105 ◽  
Author(s):  
P H Cooper ◽  
J N Hawthorne
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Brush Border ◽  
Rat Kidney ◽  
Subcellular Fractionation ◽  
Kidney Cortex ◽  
Marker Enzymes ◽  
Rat Kidney Cortex ◽  
Phosphatidylinositol Kinase ◽  
Golgi Membranes

The properties of phosphatidylinositol kinase and diphosphoinositide kinase from rat kidney cortex were studied. The enzymes were completely Mg2+-dependent. Cutscum detergent activated phosphatidylinositol kinase, but diphosphoinositide kinase was inhibited by all detergents tested. The pH optima were 7.7 for phosphatidylinositol kinase and 6.5 for diphosphoinositide kinase. On subcellular fractionation of kidney-cortex homogenates by differential centriflgation, the distribution of phosphatidylinositol kinase resembled that of the marker enzymes for brush-border, endoplasmic-reticulum and Golgi membranes. Diphosphoinositide kinase distribution resembled that of thiamin pyrophosphatase (assayed in the absence of ATP), diphosphoinositide phosphatase and triphosphoinositide phosphatase. Activities of both kinases were low in purified brush-border fragments. Diphosphoinositide kinase is probably localized in the Golgi complex.

Localization of Phosphate-Independent Glutaminase in the Brush Border of Rat Kidney Cortex

1974 ◽  
Vol 52 (9) ◽  
pp. 762-766 ◽  
Author(s):  
J. Kalra ◽  
John T. Brosnan
Keyword(s):  
Alkaline Phosphatase ◽  
Brush Border ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Rat Kidney ◽  
Sucrose Density Gradient ◽  
Kidney Cortex ◽  
Salt Treatment ◽  
Rat Kidney Cortex ◽  
Nadph Cytochrome C Reductase

A microsomal fraction that contains the highly enriched activities of NADPH – cytochrome c reductase, 5′-nucleotidase, phosphate-independent glutaminase, and alkaline phosphatase was isolated by differential centrifugation from rat kidney cortex. Continuous sucrose density gradient studies on this fraction have shown that the distribution pattern of phosphate-independent glutaminase is identical with that of alkaline phosphatase and the specific activity of these enzymes in peak fractions were 13- to 17-fold higher than in the whole homogenate. These results indicate that the phosphate-independent glutaminase is localized in the brush border of rat kidney cortex. The enzyme is truly membranous as it could not be removed by sonication, salt treatment, or pH alterations.

scholarly journals Renal inactivation of substance P in the rat

1978 ◽  
Vol 171 (1) ◽  
pp. 143-148 ◽  
Author(s):  
P E Ward ◽  
A R Johnson
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Angiotensin Ii ◽  
Substance P ◽  
Brush Border ◽  
Rat Kidney ◽  
Proximal Tubules ◽  
Marker Enzymes ◽  
Morphological Examination ◽  
Isolated Glomeruli

The activity and distribution of substance P-catabolizing enzyme(s) were studied in the rat kidney. Kidney homogenates inactive substance P 5-20 times as fast as do homogenates of intestine, liver, lung, heart or brain. The catabolizing activity was highest in the cortex and decreased progressively down the papilla. Cortex of rat kidney was homogenized and fractions enriched in microsomal membrane, final supernatant, plasma membrane, endoplasmic reticulum, brush border and intact glomeruli were prepared. The identity and homogeneity of the preparations were determined by assaying marker enzymes and by morphological examination. Substance P was catabolized most rapidly by the microsomal and plasma-membrane-enriched fractions, and least rapidly by endoplasmic reticulum or final supernatant fractions. Purified brush border of proximal tubules inactivated substance P more than 10 times as fast as isolated glomeruli. Our experiments show that substance P is catabolized at a rate that is similar to the rates of inactivation of bradykinin and angiotensin II. Further, the distribution of substance P-catabolizing activity in various kidney fractions is similar to the distribution of kininase and angiotensinase activities previously reported.

Isolation of Renal Membranes that Contain Kallikrein, Angiotensin I-Converting Enzyme (Kininase II) and Angiotensinase in the Rat

1976 ◽  
Vol 51 (s3) ◽  
pp. 267s-270s
Author(s):  
P. E. Ward ◽  
E. G. Erdös ◽  
C. D. Gedney ◽  
R. M. Dowben ◽  
R. C. Reynolds
Keyword(s):  
Plasma Membrane ◽  
Brush Border ◽  
Rat Kidney ◽  
Kidney Cortex ◽  
Converting Enzyme ◽  
Angiotensin I ◽  
Rat Kidney Cortex ◽  
Kininase Ii ◽  
Angiotensin I Converting Enzyme ◽  
Proximal Tubular

1. Fractions highly enriched in plasma membrane, endoplasmic reticulum or brush border were prepared from rat kidney cortex. Kallikrein was concentrated in the plasma membrane fraction, but not in the brush border fraction. Angiotensin I-converting enzyme (kininase II) and angiotensinase were localized in the brush border membrane. 2. It is suggested that kallikrein in the urine may originate from plasma membrane distal to the brush border of proximal tubules and the conversion of angiotensin I and the inactivation of bradykinin and angiotensin II may occur on the lumen membrane of the proximal tubular cells.

scholarly journals Isolation of rat liver lysosomes by isopycnic centrifugation in a metrizamide gradient.

1978 ◽  
Vol 78 (2) ◽  
pp. 349-368 ◽  
Author(s):  
R Wattiaux ◽  
S Wattiaux-De Coninck ◽  
M F Ronveaux-dupal ◽  
F Dubois
Keyword(s):  
Enzyme Activity ◽  
Plasma Membrane ◽  
Rat Liver ◽  
Specific Activity ◽  
Mitochondrial Fraction ◽  
Marker Enzyme ◽  
Marker Enzymes ◽  
Liver Lysosomes ◽  
Rat Liver Lysosomes ◽  
Isopycnic Centrifugation

A preparation, similar to the light mitochondrial fraction of rat liver (L fraction of de Duve et al, (1955, Biochem. J. 60: 604-617), was subfractionated by isopycnic centrifugation in a metrizamide gradient and the distribution of several marker enzymes was established. The granules were layered at the top or bottom of the gradient. In both cases, as ascertained by the enzyme distributions, the lysosomes are well separated from the peroxisomes. A good separation from mitochondria is obtained only when the L fraction if set down underneath the gradient. Taking into account the analytical centrifugation results, a procedure was devised to purify lysosomes from several grams of liver by centrifugation of an L fraction in a discontinuous metrizamide gradient. By this method, a fraction containing 10--12% of the whole liver lysosomes can be prepared. As inferred from the relative specific activity of marker enzymes, it can be estimated that lysosomes are purified between 66 and 80 times in this fraction. As ascertained by plasma membrane marker enzyme activity, the main contaminant could be the plasma membrane components. However, cytochemical tests for 5'AMPase and for acid phosphatase suggest that a large part of the plasma membrane marker enzyme activity present in the purified lysosome preparation could be associated with the lysosomal membrane. The procedure for the isolation of rat liver lysosomes described in this paper is compared with the already existing methods.

Evidence for mitochondrial origin of the HCO3(-)-ATPase in brush border membranes of rat proximal tubules

1985 ◽  
Vol 248 (3) ◽  
pp. F389-F395
Author(s):  
H. Knauf ◽  
M. Sellinger ◽  
K. Haag ◽  
U. Wais
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Brush Border ◽  
Rat Kidney ◽  
Proximal Tubules ◽  
Precipitation Method ◽  
Kidney Cortex ◽  
Marker Enzyme ◽  
Rat Kidney Cortex ◽  
Renal Tubular Cells

High HCO3(-)-ATPase activity is known to exist in mitochondria of renal tubular cells. In brush border membrane (BBM) preparations of proximal tubules such an anion-stimulated enzyme was also found. However, these preparations always contained mitochondrial markers. The putative localization and the role of this ATPase in BBM is still controversial. Some authors consider the HCO3(-)-ATPase in the BBM to be a mitochondrial contamination; others attribute to this ATPase a key role in H+ transport in the proximal tubule. To reinvestigate this problem, BBMs from rat kidney cortex were isolated by a simple, rapid (1.5-h) Ca2+-precipitation method, yielding a BBM fraction enriched 12.4-fold with respect to the marker enzyme leucine aminopeptidase (LAP). There was no basolateral Na+-K+-ATPase and no mitochondrial succinate dehydrogenase detectable. Cytochrome c oxidase was drastically reduced to 7 +/- 1% of that observed in the homogenate (TH). The activity of HCO3(-)-ATPase in the BBM fraction was 19 +/- 4 IU/g protein, i.e., 27% that of the homogenate. As sonication of the TH exclusively increases the activity of HCO3(-)-ATPase, its relative activity was 7.5% and thus equal to that of the mitochondrial marker. In many BBM preparations no HCO3(-)-ATPase was detectable. In those BBM preparations in which traces of HCO3(-)-ATPase were found, this activity coincided with that of cytochrome c oxidase in the respective preparation. There was a constant activity ratio of cytochrome c oxidase/HCO3(-)-ATPase in the TH, BBM, and pellet 1. The activity of HCO3(-)-ATPase in BBM did not depend on the activity of LAP.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Isolation of membrane-bound renal kallikrein and kininase

1975 ◽  
Vol 151 (3) ◽  
pp. 755-758 ◽  
Author(s):  
P E Ward ◽  
C D Gedney ◽  
R M Dowben ◽  
E G Erdös
Keyword(s):  
Plasma Membrane ◽  
Brush Border ◽  
Rat Kidney ◽  
Kidney Cortex ◽  
Angiotensin I ◽  
Rat Kidney Cortex ◽  
Angiotensin I Converting Enzyme ◽  
Renal Kallikrein ◽  
Proximal Tubular ◽  
Membrane Bound

Fractions highly enriched in plasma membrane, endoplasmic reticulum or brush border were prepared from homogenized rat kidney cortex. Kallikrein was concentrated in the plasma-membrane fraction, but not in the brush border of the proximal tubules. Kininase II or angiotensin I-converting enzyme was localized in the brush-border membrane. It is suggested that kallikrein in the urine may originate from the plasma membrane of the distal tubules and the conversion of angiotensin I and the inactivation of bradykinin may occur on the lumen membrane of the proximal tubular cells.

scholarly journals Studies on the orientation of brush-border membrane vesicles

1978 ◽  
Vol 172 (1) ◽  
pp. 57-62 ◽  
Author(s):  
W Haase ◽  
A Schäfer ◽  
H Murer ◽  
R Kinne
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Rat Kidney ◽  
Freeze Fracture ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Immunological Methods ◽  
Kidney Cortex ◽  
Asymmetric Distribution ◽  
Rat Kidney Cortex

Orientation of rat renal and intestinal brush-border membrane vesicles was studied with two independent methods: electron-microscopic freeze-fracture technique and immunological methods. With the freeze-fracture technique a distinct asymmetric distribution of particles on the two membrane fracture faces was demonstrated; this was used as a criterion for orientation of the isolated membrane vesicles. For the immunological approach the accessibility or inaccessibility of aminopeptidase M localized on the outer surface of the cell membrane to antibodies was used. With both methods we showed that the brush-border membrane vesicles isolated from rat kidney cortex and from rat small intestine for transport studies are predominantly orientated right-side out.

scholarly journals THE POLARITY OF THE PROXIMAL TUBULE CELL IN RAT KIDNEY

1972 ◽  
Vol 54 (2) ◽  
pp. 232-245 ◽  
Author(s):  
Hans-G Heidrich ◽  
Rolf Kinne ◽  
Eva Kinne-Saffran ◽  
Kurt Hannig
Keyword(s):  
Proximal Tubule ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Rat Kidney ◽  
High Specific Activity ◽  
Kidney Cortex ◽  
Proximal Tubule Cell ◽  
Surface Charges ◽  
Tubule Cell ◽  
Rat Kidney Cortex

Two different membrane fractions were obtained from a brush-border fraction of rat kidney cortex by using their different electrical surface charges in preparative free-flow electrophoresis. One membrane fraction contained only morphologically intact microvilli and was characterized by a high specific activity of alkaline phosphatase. The other fraction morphologically resembled classical plasma membranes by possessing junctional complexes and a high Na-K-ATPase activity The contamination of the isolated membrane fractions by other cell organelles was extremely low These two fractions represent the apical (luminal) and the basal (interstitial) area of the renal proximal tubule cell membrane and clearly demonstrate the polarity of this cell.

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