scholarly journals Asymmetrical distribution of l-isoaspartyl protein carboxyl methyltransferases in the plasma membranes of rat kidney cortex

1994 ◽  
Vol 297 (1) ◽  
pp. 145-150 ◽  
Author(s):  
D Gingras ◽  
D Boivin ◽  
R Beliveau
Keyword(s):  
Plasma Membranes ◽  
Rat Kidney ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Kidney Cortex ◽  
Rat Kidney Cortex ◽  
Phase Partitioning ◽  
Membrane Attachment ◽  
Endogenous Substrates ◽  
Triton X

We have studied the distribution of membrane-associated L-isoaspartyl protein carboxyl methyltransferases (PCMTs) in plasma membranes purified from rat kidney cortex. Addition of CHAPS to brush-border membranes (BBM) and basolateral membranes (BLM) was required to measure optimal membrane-dependent methylation of ovalbumin and TS-isoD-YSKY, substrates of L-isoaspartyl PCMTs. Extraction of both membrane-associated enzymes was achieved with detergents, but not with high-salt solutions, suggesting a strong membrane attachment. However, upon phase partitioning using Triton X-114, both enzymes were predominantly associated with the detergent-poor phase, suggesting a relatively hydrophilic nature. The enzymes showed similar catalytic properties such as substrate recognition and affinity towards the methyl donor, S-adenosyl-L-methionine. The activity of the BBM enzyme, however, was about 2-fold higher than that of the BLM enzyme. Identification of the endogenous substrates located in the two plasma membranes by acidic gel electrophoresis in the presence of a cationic detergent revealed significant differences in the methyl-accepting proteins of both membranes. The BBM-methylated proteins had sizes of 35, 50 and 54 kDa, whereas the major BLM-methylated substrates were of 97 and 100 kDa. The enzymes showed distinct behaviour on Mono Q anion-exchange chromatography. The BBM-associated PCMT did not bind to the column, being eluted in the flow-through, whereas the BLM enzyme bound to the column and was eluted at 0.15 M NaCl. Moreover, the two enzymes had different molecular masses under both denaturing and nondenaturing conditions, the BLM PCMT migrating at an apparent molecular mass of 29 kDa, compared with 27 kDa for the BBM enzyme. Taken together, these results show the presence of two distinct L-isoaspartyl PCMTs in the plasma membranes of the kidney cortex.

Tyrosine protein phosphorylation in plasma membranes of rat kidney cortex

1994 ◽  
Vol 267 (3) ◽  
pp. F415-F422
Author(s):  
L. Tremblay ◽  
R. Beliveau
Keyword(s):  
Protein Phosphorylation ◽  
Tyrosine Phosphorylation ◽  
Plasma Membranes ◽  
Rat Kidney ◽  
Specific Protein ◽  
Ammonium Molybdate ◽  
Kidney Cortex ◽  
Protein Kinase Activity ◽  
Rat Kidney Cortex ◽  
Endogenous Substrates

The endogenous tyrosine protein kinase activity (TPKA) associated with brush-border (BBM) and basolateral (BLM) membranes of rat kidney cortex was studied with an anti-phosphotyrosine monoclonal antibody (PY20). Distinct major phosphotyrosine-containing proteins were associated with BBM (50, 54, and 120 kDa) and BLM (37, 90, 130, and 170 kDa). For both plasma membranes, tyrosine phosphorylation leveled off after 10 min of incubation. Endogenous phosphotyrosine-specific protein phosphatases (PT-Pases) were active in both membranes, since the presence of sodium vanadate or ammonium molybdate, which are inhibitors of PTPases, was essential to detect endogenous phosphorylation. Substrates and/or tyrosine protein kinases (TPKs) seem to be differently distributed in these plasma membranes, since phosphorylation of endogenous substrates in BLM and BBM was differently sensitive to competitive inhibitors of TPKs. Moreover, insulin- and insulin-like growth factor I-stimulated tyrosine phosphorylation of a 90-kDa substrate was only observed in solubilized BLM proteins. However, similar p60v-src-related TPKs appear to be present in the BBM and BLM, since an antibody raised against p60v-src recognized proteins of 52, 58, and 75 kDa by immunoblotting and could immunoprecipitate the TPKs associated with both plasma membranes. These data provide evidence that the endogenous tyrosine protein phosphorylation observed in the BLM is catalyzed by nonreceptor TPKs as well as receptor TPKs, whereas that observed in the BBM is exclusively due to nonreceptor TPKs.

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 Inositol lipid signalling occurs in brush-border membranes during initiation of compensatory renal growth in the rat

1993 ◽  
Vol 295 (2) ◽  
pp. 599-605 ◽  
Author(s):  
H Banfić ◽  
M Vuica ◽  
M Knotek ◽  
S Moslavac ◽  
N Divecha
Keyword(s):  
Brush Border ◽  
Plasma Membranes ◽  
Rat Kidney ◽  
Unilateral Nephrectomy ◽  
Kidney Cortex ◽  
Compensatory Renal Growth ◽  
Rat Kidney Cortex ◽  
Contralateral Kidney ◽  
Inositol Lipids ◽  
Stimulation Of

Using highly specific mass assays, concentrations of inositol lipids and 1,2-diacylglycerol (DAG) were determined in plasma membranes isolated from rat kidney cortex. Significantly higher concentrations of inositol lipids were determined in brush-border (BBM) than in basal-lateral (BLM) plasma membranes, although DAG concentrations were similar in both. After unilateral nephrectomy, a decrease in PtdIns(4,5)P2 and PtdIns4P, with a concomitant increase in DAG and translocation of protein kinase C (PKC), were observed in BBM but not in BLM isolated from the remaining kidney. On the other hand, stimulation of renal cortical slices with insulin-like growth factor II (IGF-II) or phenylephrine caused similar effects in BLM but not in BBM. Stimulation of phospholipase C activity with translocation of PKC only to BBM in one kidney was also induced by occlusion of blood flow through the contralateral kidney for 15 min. At 30 min after the occlusion was removed and reflow established, DAG concentration and the amount of PKC in BBM returned to control values. These results suggest that an early signal after unilateral nephrectomy is transmitted to cells through BBM and can be switched on and off by blood occlusion and reflow through the contralateral kidney, while hormonal signals caused by IGF-II and phenylephrine are transmitted to cells through BLM.

CHICK KIDNEY ADENYLATE CYCLASE: SENSITIVITY TO PARATHYROID HORMONE AND SYNTHETIC HUMAN AND BOVINE PEPTIDES

1974 ◽  
Vol 63 (2) ◽  
pp. 369-375 ◽  
Author(s):  
T. J. MARTIN ◽  
N. VAKAKIS ◽  
J. A. EISMAN ◽  
S. J. LIVESEY ◽  
G. W. TREGEAR
Keyword(s):  
Parathyroid Hormone ◽  
Adenylate Cyclase ◽  
Plasma Membranes ◽  
Rat Kidney ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Kidney Cortex ◽  
Rat Kidney Cortex ◽  
Similar Preparation ◽  
Chick Kidney

SUMMARY Adenylate cyclase activity of crude plasma membranes from chick kidney was stimulated by low doses of parathyroid hormone (PTH). Sensitivity to PTH was ten to twenty times greater than that of a similar preparation from rat kidney cortex. Synthetic peptides consisting of the NH2-terminal 34 amino acids of bovine PTH (BPTH) and of human PTH (HPTH) were assayed, as were several analogues of these peptides. Bovine PTH (1–34) and HPTH (1–34) were equivalent in their action on chick kidney but the human peptide had only 20% of the activity of the bovine peptide on rat kidney cortex adenylate cyclase. Bovine proPTH ( −6→ + 34) and (Tyr1)-BPTH (1–34) had less activity than BPTH (1–34). Bovine PTH (2–34) inhibited the response to BPTH (1–34). Neither salmon calcitonin nor vasopressin stimulated adenylate cyclase activity.

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.

Subcellular distribution and membrane association of Rho-related small GTP-binding proteins in kidney cortex

1995 ◽  
Vol 269 (2) ◽  
pp. F180-F189 ◽  
Author(s):  
D. Boivin ◽  
R. Beliveau
Keyword(s):  
Subcellular Distribution ◽  
Plasma Membranes ◽  
Binding Proteins ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Covalent Modification ◽  
Kidney Cortex ◽  
Gtp Binding Proteins ◽  
Gtp Binding

We have examined the subcellular distribution of Rho-related small GTP-binding proteins in the kidney. RhoA, CDC42, and Rac1 small GTP-binding proteins were found to be expressed at high levels in rat outer kidney cortex. Western blot analysis showed that these proteins were predominantly associated with brush-border and basolateral plasma membranes, with the exception of Rac1 which was localized predominantly in the mitochondria. RhoA and CDC42 were also found in the cytosol, and a small fraction was associated with cytoskeletal elements. A GDP-dissociation inhibitor specific for the Rho family (RhoGDI) was also identified and found to be located exclusively in the cytosol. Upon fractionation of kidney cytosol with anion-exchange chromatography, RhoA and CDC42 proteins eluted in two major well-resolved peaks that coeluted with the RhoGDI protein, suggesting that they form heterodimers. Association of RhoA and CDC42 with RhoGDI was further suggested by coelution of these proteins with RhoGDI at an estimated size of approximately 45 kDa after gel-filtration chromatography. However, a second peak of RhoA eluted as a 20-kDa protein, indicating that not all RhoA is complexed to RhoGDI. Addition of RhoA- and CDC42-enriched fractions to purified membranes from kidney cortex resulted in their translocation to the membranes and their carboxyl methylation. Both processes were stimulated by guanosine 5'-O-(3-thiotriphosphate). Methylation inhibitors had no effect on the translocation of RhoA to membranes, suggesting that this covalent modification is not required for association to the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

A Simple Isolation Method for Basal-Lateral Plasma Membranes from Rat Kidney Cortex

1981 ◽  
Vol 4 (1) ◽  
pp. 49-61 ◽  
Author(s):  
Vito Scalera ◽  
Yu-Kuo Huang ◽  
Bruno Hildmann ◽  
Heini Murer
Keyword(s):  
Plasma Membranes ◽  
Rat Kidney ◽  
Kidney Cortex ◽  
Isolation Method ◽  
Rat Kidney Cortex
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