Subcellular distribution of renal tripeptide-releasing exopeptidases active on collagen-like sequences

1987 ◽  
Vol 252 (5) ◽  
pp. F890-F898
Author(s):  
K. J. Andersen ◽  
J. K. McDonald
Keyword(s):  
Subcellular Distribution ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Rat Kidney ◽  
Broad Band ◽  
Distribution Patterns ◽  
Kidney Cortex ◽  
Rat Kidney Cortex ◽  
Tripeptidyl Peptidase ◽  
N Terminus

The rat kidney cortex was found to contain two N-terminal exopeptidases of the tripeptidyl peptidase (TPP) class. Each required a free N-terminus to catalyze the release of collagen-related (Gly-Pro-X) "triplets." In accordance with their apparent pH optima, activities were routinely determined fluorimetrically at pH 4.0 (TPP 4) and at pH 7.0 (TPP 7) on Gly-Pro-Met-2-naphthylamide. The specific activity in both the homogenate and the classical subfractions was much greater at pH 7 than at pH 4. Subfractionation of the microsomal fraction by equilibrium banding in sucrose did not separate the TPP 4 and TPP 7 activities. The banding density (1.18 g/ml) and the distribution patterns for TPP 7 in the microsomal subfractions, and also in the subfractions of the small lysosomes in the mitochondrial-lysosomal (ML) fraction, demonstrate that TPP 7 is associated with smooth membranes. The TPP 4 and TPP 7 activities were clearly separated during subfractionation of the ML fraction. Rate sedimentation demonstrated that TPP 4 was present in the large, fast-sedimenting lysosomes (protein droplets) and in a heterogeneous broad band of smaller lysosomes. Equilibrium banding of the small lysosomes gave two distinct TPP 4-containing populations at densities 1.20 and 1.235 g/ml. Notably, dipeptidyl peptidase II (DPP II) gave identical banding densities and showed distributions very similar to TPP 4.(ABSTRACT TRUNCATED AT 250 WORDS)

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 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.

scholarly journals Renin substrate in granules from rat kidney cortex

1976 ◽  
Vol 154 (3) ◽  
pp. 625-637 ◽  
Author(s):  
B J. Morris ◽  
C I. Johnston
Keyword(s):  
Microsomal Fraction ◽  
Rat Kidney ◽  
Gradient Centrifugation ◽  
Rat Plasma ◽  
Reaction Scheme ◽  
Kidney Cortex ◽  
Angiotensin I ◽  
Renin Substrate ◽  
Rat Kidney Cortex ◽  
Molecular Weights

1. Subcellular fractions of rat kidney cortex generated angiotensin I continuously over 2h when incubated at 37degreesC with rat renin, indicating the presence of renin substrate within cells in the renal cortex. 2. Renin substrate was located in highest specific concentration in particulate fractions. The particles containing renin substrate had a sedimentation velocity slightly lower than mitochondria and renin granules but greater than the microsomal fraction. 3. Isopycnic gradient centrifugation indicated a density of 1.190g/ml for the particles containing renin substrate, compared with 1.201 for renin granules, 1.177 for mitochondria, and 1.170 and 1.230 for lysosomes in the heavy-granule fraction. 4. In the liver, renin substrate was also found in particles, but these had a lower sedimentation rate than those from the kidney. 5. The molecular weights of renin substrate in kidney and liver granules and rat plasma were similar, namely 61000-62000. 6. On the basis of these biochemical findings, a mechanism for the intrarenal production of angiotensin, incorporating a subcellular reaction scheme, is proposed.

In vivo generation of hydrogen peroxide by rat kidney cortex and glomeruli

1989 ◽  
Vol 256 (1) ◽  
pp. F158-F164 ◽  
Author(s):  
B. R. Guidet ◽  
S. V. Shah
Keyword(s):  
Hydrogen Peroxide ◽  
Catalase Activity ◽  
Renal Cortex ◽  
Specific Activity ◽  
Rat Kidney ◽  
Kidney Cortex ◽  
Compound I ◽  
Rat Kidney Cortex ◽  
Hydrogen Peroxide Generation

The purpose of this study was to demonstrate in vivo generation of hydrogen peroxide by rat renal cortex and glomeruli. Aminotriazole irreversibly inactivates catalase only in the presence of hydrogen peroxide, and previous studies have shown that aminotriazole-mediated inhibition of catalase is a measure of in vivo changes in the hydrogen peroxide generation. Aminotriazole injected intraperitoneally caused a dose-dependent (0.1-1 g/kg) and a time-dependent (15, 30, 60, 90, 120 min) inhibition of the catalase activity in renal cortex. We confirmed that catalase inactivation by aminotriazole was due to formation of a catalase-hydrogen peroxide intermediate (compound I) because catalase inactivation was prevented by ethanol (2 g/kg), a competitive substrate for compound I. The specific activity of catalase in the glomeruli [0.27 +/- 0.026 k/mg protein (where k is the first-order reaction rate constant), n = 5] was significantly lower than the specific activity in the tubules (1.04 +/- 0.15 k/mg protein, n = 5) obtained from the same rats. The residual catalase activity (RCA) in the glomeruli (0.05 +/- 0.01 k/mg protein) was 19% of control values at 90 min after aminotriazole injection (1 g/kg). Taken together these data provide evidence for in vivo generation of hydrogen peroxide by rat renal cortex and glomeruli under normal conditions. Aminotriazole-mediated inhibition of catalase has been used in previous studies as a measure of in vivo changes in the hydrogen peroxide generation.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Studies on the isolation and properties of renin granules from the rat kidney cortex

1979 ◽  
Vol 182 (2) ◽  
pp. 301-309 ◽  
Author(s):  
G A Sagnella ◽  
W S Peart
Keyword(s):  
Incubation Temperature ◽  
Specific Activity ◽  
Rat Kidney ◽  
Kidney Cortex ◽  
Low Molecular Weight ◽  
Enzyme Release ◽  
Rat Kidney Cortex ◽  
Dense Granules ◽  
Triton X ◽  
Primary Granule

The present study was undertaken to isolate and investigate some physicochemical properties of renin granules from the rat kidney cortex. Two preparations of subcellular organelles were used: a primary-granule fraction, which allowed the properties of lysosomes to be compared simultaneously with those of renin granules, and a semi-purified preparation of the latter. The specific activity of renin in the primary-granule preparations was about 4-fold higher than in the original homogenate; that of the semi-purified renin-granule preparation was about 18-fold higher than in the homogenate, and consisted mainly of electron-dense granules but some mitochondria were also observed. Renin and acid phosphatase release from the primary-granule preparation was increased by lowering osmolality, by a low-molecular-weight solute (glucose) and by Triton X-100 or digitonin. Enzyme release was decreased by lowering the incubation temperature (4 degrees C) or the presence of CaCl2. Renin release from the partially purified granule preparation was not affected by cyclic AMP, cyclic GMP and ATP.

Presence and possible role of a renal brush-border Gly-Pro-X-releasing exopeptidase

1987 ◽  
Vol 253 (4) ◽  
pp. F649-F655
Author(s):  
Knut-Jan Andersen ◽  
J. Ken McDonald
Keyword(s):  
Brush Border ◽  
Rat Kidney ◽  
Kidney Cortex ◽  
Membrane Domains ◽  
Rat Kidney Cortex ◽  
Proximal Tubule Cells ◽  
Dpp Iv ◽  
Tripeptidyl Peptidase ◽  
Membrane Compartment

Differential pelleting of a rat renal cortical homogenate clearly demonstrated the microsomal localization of an N-terminal exopeptidase of the tripeptidyl peptidase (TPP) class that typically requires a free N-terminus to catalyze the release of collagen-related (Gly-Pro-X) “triplets” at pH 7.0 (TPP 7). Once fractionated by differential pelleting, microsomal populations of different size were subfractionated by equilibrium banding in sucrose gradients for the purpose of comparing the distribution profiles and the isopycnic banding densities of TPP 7 to those for known marker enzymes. This analytical approach permitted the localization of these enzymes to specific membrane domains in the renal cortex and provided evidence for the brush-border location of TPP 7. Notably, dipeptidyl peptidase IV (DPP IV), an established plasma membrane exopeptidase with a prolyl-bond specificity, gave banding densities and distributions that were consistent with the presence of both TPP 7 and DPP IV in the same membrane compartment. Because triplets of the Gly-Pro-X type released by TPP 7 would be ideal substrates for DPP IV, a coupled TPP 7-DPP IV exopeptidase mechanism at the luminal surface (brush border) of proximal tubule cells could therefore make a major contribution to the renal degradation and reabsorption of filtered collagen fragments. tripeptidyl peptidase; aminopeptidase; collagen; rat kidney cortex Submitted on February 11, 1987 Accepted on May 13, 1987

Subcellular distribution and guanine nucleotide dependency of COOH-terminal methylation in kidney cortex

1993 ◽  
Vol 265 (2) ◽  
pp. F316-F322
Author(s):  
D. Gingras ◽  
D. Boivin ◽  
R. Beliveau
Keyword(s):  
Subcellular Distribution ◽  
Plasma Membranes ◽  
Rat Kidney ◽  
Wide Distribution ◽  
Cysteine Residue ◽  
Kidney Cortex ◽  
Rat Kidney Cortex ◽  
Carboxyl Methyltransferase ◽  
Exogenous Substrate ◽  
Gamma S

The subcellular distribution of COOH-terminal carboxyl methyltransferase and methylated substrates was studied in purified brush-border and basolateral plasma membranes, as well as in crude intracellular membranes and the cytosolic fraction isolated from rat kidney cortex. The three membrane fractions showed intrinsic carboxyl methylation of 21- to 23-kDa proteins, whereas 18- and 41-kDa methylated proteins were observed in the cytosol. In contrast, methylation activities toward N-acetyl-S-trans,trans-farnesyl-L-cysteine (AFC), a synthetic farnesylated substrate, were found to be strictly associated with membranes, with no detectable level of activity in the cytosol. Methylation of all membrane-associated substrates was inhibited by AFC but remained unaffected by TS-isoD-YSKY, a synthetic isopeptide recognized by L-isoaspartyl methyltransferase, suggesting that the membrane-associated substrates were methylated on a COOH-terminal isoprenylated cysteine residue. The membrane-associated methylated proteins were tightly bound to the membranes as reflected by their extraction with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate but not with 1 M NaCl or 2 M urea. The nonhydrolyzable analogues of GTP and GDP, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), markedly increased the methylation of the 21- to 23-kDa substrates, whereas ATP gamma S and ADP beta S were without effect. This effect of guanine nucleotides was restricted to endogenous 21- to 23-kDa substrates with no stimulation of methylation of the exogenous substrate, AFC. Our results show a wide distribution of both COOH-terminal protein carboxyl methyltransferase activities and associated methylated substrates in the kidney cortex.(ABSTRACT TRUNCATED AT 250 WORDS)

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