Kidney Plasma Membranes Research Articles

Glycoprotein: glycosyl transferase activities of rat kidney plasma membranes.

Rat kidney plasma membrane preparations and suspensions of intact glomeruli were used to study glycoprotein: glycosyl transferase activities present on cell surfaces. Collagen: glucosyl, fetuin: galac

Properties of a synthetic plasma membrane marker: fluorescent-mercury-dextran.

Aminoethyl-Dextran T 10 (mol wt approx. 10,000) was conjugated withp-Chloromercuribenzoic acid (pCMB) and labeled with fluorescein isothiocyanate (FITC). This coupling procedure does not affect the mercurial function ofpCMB moiety of Fluorescent Mercury Dextran T 10 (FMD) since on the basis of mercury content itsK i-value for the (Na+−K+)-ATPase from rat kidney plasma membranes is identical with theK i -value of unconjugatedpCMB (3×10−6 m). FMD binds to plasma membranes if applied in vivo, which could be shown in experiments in which rat kidneys were perfused with FMD and the plasma membranes isolated after the perfusion. The membrane-FMD complex is stable during common isolation steps such as differential centrifugation, sucrose density gradient centrifugation and free-flow electrophoresis. This was shown by in vitro binding studies of FMD with isolated plasma membranes from rat kidney cortex. FMD may be removed from the plasma membranes by the addition of 1×10−4 m dithiotreitol. Since F-aminoethyl-Dextran T 10 (withoutpCMB) does not interact with the plasma membranes, it is suggested that the binding of FMD to plasma membranes may involve a Hg-SH reaction. FMD does not penetrate into rat kidney cells in contrast topCMB, but can cross capillaries. Thus, FMD seems to be suitable (a) to label luminal and contraluminal surfaces of plasma membranes of epithelial structures, and (b) to be used as a fluorescent marker for plasma membranes during succeeding isolation procedures.

Effects of some organic solvents on the reactivity of sodium plus potassium ion-transport ATPase

1. 1. Organic solvents including aliphatic alcohols, acetone, diethyl ether and chloroform inhibited (Na + + K +)-ATPase (EC 3.6.1.3) of the plasma membranes of guinea pig kidney. Inhibition was uncompetitive with respect to ATP if the ATP concentration was larger than 5 μM. Below this concentration of ATP, K + inhibits rather than stimulates the Na +-dependent splitting of ATP by the native enzyme. The tested organic solvents converted this K + inhibition into K + stimulation. In the presence of these solvents, the apparent affinity of the enzyme increased for Na + and ATP and decreased for K + as estimated by the kinetics of ATP hydrolysis. Apparent homotropic allosteric interaction between the Na + and K + sites increased. 2. 2. Organic solvents interfered with different steps of the transient phosphorylation of the enzyme. As estimated by pulse labeling, organic solvents enhanced interaction of ATP with the enzyme prior to phosphorylation both in the absence or in the presence of K +. The solvents decreased the rate of dephosphorylation of the phospho-enzyme either in the presence of K + (native enzyme) or in the presence of ADP ( N-ethylmaleimide-treated enzyme) but they did not affect the ratio of the ADP-sensitive form of the native phospho-enzyme to the K +-sensitive form. Rephosphorylation by ATP of the dephospho-enzyme, formed by adding Rb +, was accelerated by organic solvents. 3. 3. Interaction of the enzyme with these organic solvents appeared to be primarily hydrophobic, as the half-maximal inhibitory concentrations of these solvents correlated with their octanol-water partition coefficients and with the length of the hydrophobic side chain in the series of homologous aliphatic alcohols. 4. 4. It is considered that these modifications of (Na + + K +)-ATPase were due partly to conformational changes of the enzyme, and partly to changes in the water structure around the active center which is proposed to be in a hydrophobic environment.

Acidification of urine by the isolated urinary bladder of the toad.

Acidification of urine by the isolated urinary bladder of the toad.

The occurrence of vitamin A in biological membranes

1. 1. Rats maintained on a low vitamin A intake were intraperitoneally injected with 50.7 μg [11,12- 3H 2]retinyl acetate. 15 h later, the animals were killed and liver and kidney plasma membranes, kidney endoplasmic reticulum and erythrocyte ghosts were prepared. 2. 2. The labeled vitamin A content of the membrane preparations varied consistently between 0.003–0.005 μg retinol/mg N, while the level in the homogenate varied from 0.001–0.009 μg retinol/mg N. The labeled vitamin A content of the erythrocyte ghosts was low (max. 0.0001 μg retinol/mg N) probably due to heavy hemoglobin contamination. 3. 3. The radioactive vitamin A compounds were strongly bound to the membranes: n- hexane removed only 7% of the radioactive compounds, whereas chloroform-methanol (2:1, v/v) extraction of the membrane preparations removed 90% of the radioactive compounds. 4. 4. Thin-layer chromatography of the chloroform-methanol (2:1, v/v) extract of the kidney endoplasmic reticulum, in three different solvent systems, revealed that retinol and retinoic acid were the dominant forms of vitamin A. This was confirmed by combined column and thin-layer chromatography (using a fourth solvent system) of the chloroform-methanol (2:1, v/v) extract of endoplasmic reticulum.

The characterization of rat kidney plasma membranes prepared by zonal centrifugation.

1. Plasma membranes were isolated from a 10000g-min pellet prepared from a renal cortical homogenate in 20mm-NaHCO(3) by isopycnic centrifugation in a linear sucrose gradient in an ;A'-type zonal rotor. 2. The preparation was characterized by electron microscopy, and alkaline phosphatase, 5'-nucleotidase, l-leucine beta-naphthylamidase and l-leucine p-nitroanilidase activities were found to be selectively associated with the renal plasma membrane. 3. The preparation had a high degree of purity, as indicated by the presence of low activities of marker enzymes associated with subcellular organelles. A preliminary chemical analysis indicated that the chemical composition resembled that of plasma membranes of other tissues. 4. Plasma membranes were also prepared from tubular fragments and their enzyme contents were found to be similar to those of plasma membranes prepared from cortical homogenates. 5. l-Leucine beta-naphthylamidase, l-leucine p-nitroanilidase and 5'-nucleotidase were not enriched to the same extent as alkaline phosphatase in the preparation of plasma membranes from tubular fragments. A possible explanation for this finding is discussed.

Spin exchange of spin labeled probes in a natural membrane

Spin exchange can be seen in kidney plasma membrane fragments with spin labeled probes at concentrations of the labels in the membrane which do not inhibit the (Na + + K +)-ATPase. The second order rate constants for exchange are on the order of 10 7 M −1 sec −1. The labels must be fairly uniformly distributed throughout the membrane and able to diffuse freely. The viscosity for lateral diffusion in the membrane is estimated to be 100 to 1000 times that for water.

Ethacrynic acid and kidney cell metabolism

Cellular effects of ethacrynic acid were examined in the rabbit and rat kidney. Emphasis was placed on levels of the compound in vitro lower than those employed in earlier studies. In the present study in vitro, ethacrynic acid is without effect on sodium-potassium-dependent adenosine triphosphatase (ATPase) activity. When 0.1 mM ethacrynic acid is incubated with rabbit kidney cortex slices, it inhibits the maintenance of intracellular potassium but has no effect on cellular respiration or cell volume. Ethacrynic acid (0.05 mM) inhibits the respiration of isolated mitochondria and suppresses membrane ATPase stimulation of mitochondrial respiration. Ethacrynic acid administered in vivo at a dose sufficient to elicit a diuretic response does not affect kidney mitochondrial respiration or inhibit the sodium-potassium-dependent ATPase activity. Ethacrynic acid labeled with carbon-14 localizes in part in the kidney plasma membrane. About 90 per cent of the plasma membrane-bound ethacrynic acid is found in association with proteins comprising the low molecular weight fraction. The compound is not bound significantly to cell membrane macromolecules associated with the ATPase system.

Effect of mineralocorticoid agonists and antagonists on binding of 3H-aldosterone to adrenalectomized rat kidney plasma membranes

Binding of 3H-aldosterone to plasma membranes of adrenalectomized rat kidney has been demonstrated in vivo and in vitro . Steroids with mineralocorticoid activity, deoxycorticosterone or 9-α-fluorohydrocortisone (agonists), or aldosterone antagonists (spironolactone, progesterone) markedly reduced the binding of 3H-aldosterone to renal plasma membranes. Steroids without agonist or antagonist properties (testosterone, 17-β-estradiol) were not effective. The 3H-aldosterone-membrane complex was resistent to solubilization and subsequent chromatography employing Sephadex G-25. These experiments indicate that binding of 3H-aldosterone to adrenalectomized rat kidney plasma membranes may be involved in the cellular mode of action of aldosterone.

Characterization of the adenyl cyclase of rat kidney plasma membranes

1. 1. This paper examines the characteristics of a renal plasma membrane adenyl cyclase with respect to NaF and hormonal activation, enzyme kinetic parameters, effects of solubilization procedures on adenyl cyclase activity, and gel-filtration chromatography of a “soluble” membrane adenyl cyclase. 2. 2. Renal membrane adenyl cyclase was stimulated by NaF, vasopressin and parathyroid hormone in a concentration-dependent manner. 3. 3. A K m for ATP of 0.1 mM was obtained for both the renal cortex and medulla membrane adenyl cyclase whereas the divalent cation dependency was found to be: cortex, Mg 2+ > Mn 2+ > Co 2+; medulla, Mg 2+ = Mn 2+ > Co 2+, for sustaining adenyl cyclase activity in the presence of NaF. 4. 4. The particulate plasma membrane adenyl cyclase was solubilized by use of the detergent, Lubrol WX or by intense shear forces with a French pressure cell. Addition of NaF to the membrane suspension prior to solubilization was necessary for protection of adenyl cyclase activity. Gel-filtration chromatography with 6 % agarose gave two adenyl cyclase peaks of different specific activity and molecular size.

Micropuncture study of renal tubular hydrogen ion transport in the rat.

Micropuncture study of renal tubular hydrogen ion transport in the rat.

Binding of Adenosine Triphosphate to Sodium and Potassium Ion-stimulated Adenosine Triphosphatase

Abstract In order to characterize a step in the reaction sequence of sodium and potassium ion-dependent adenosine triphosphatase (EC 3.6.1.3), the binding of radioactive adenosine triphosphate to kidney cell membranes was estimated by flow dialysis in the presence of a chelator of divalent cations, which prevented hydrolysis of the adenosine triphosphate. Adenosine triphosphate was bound to a single site on sodium and potassium ion-stimulated adenosine triphosphatase (a) because binding capacity of the membranes was equal, one for one, to the phosphorylation capacity of this enzyme, (b) because binding was inhibited by pretreatment of the membranes with the specific inhibitor, ouabain, and (c) because binding was inhibited by potassium ion with antagonism of this inhibition by sodium ion, which had no direct effect. The dissociation constant was 0.22 µm. Other nucleotides displaced ATP including, in order of decreasing effectiveness: deoxyadenosine triphosphate, β,γ-methylene adenosine triphosphate, cytidine triphosphate, and inosine triphosphate. Monovalent cations reduced the binding affinity at progressively higher cation concentrations, as follows: potassium, rubidium, thallous, ammonium, and cesium ions. Lithium ion had no effect. Binding was stable between pH 5.6 and 7.6, but declined sharply above pH 8.0. In the presence of potassium ion alone, there appeared to be one or more binding sites on this enzyme with a much lower affinity for adenosine triphosphate. These may be regulatory sites.

Characterization of Plasma Membrane Proteins in Mammalian Kidney: II. The Binding of Organic Mercurials to Rat Kidney Plasma Membrane Proteins in vivo

Abstract The binding of organic mercurial compounds to the proteins of kidney plasma membrane preparations has been examined following the administration in vivo of organic mercurial compounds labeled with 203Hg. When the kidney cells are fractionated, plasma membrane radioactivity is lower than radioactivity in the cytoplasm. The radioactivity appears to be bound to membrane protein and experiments with 14C-labeled p-chloromercuribenzoate and 3H-labeled chlormerodrin indicate that the binding 2 hours after administration is mostly that of free mercury. When plasma membranes are solubilized and chromatographed on 6% agarose gel, two major protein peaks are eluted. p-Chloromercuribenzoate label is localized primarily in the high molecular weight proteins of the initial void volume peak. Chlormerodrin, a compound with diuretic activity in the kidney, is distributed fairly evenly in the protein fractions of both column peaks. Electrophoretic data, however, suggest heterogeneity of the protein labeling by chlormerodrin. Sodium-potassium-dependent ATPase activity is found in several fractions of the first peak eluted from the agarose gel column. When ouabain, a known inhibitor of the ATPase system, is administered in vivo, it depresses the chlormerodrin radioactivity of the first peak.

Lipids of plasma membranes of monkey and hamster kidney cells and of parainfluenza virions grown in these cells

Plasma membranes have been isolated from primary rhesus monkey kidney (MK), baby hamster kidney (BHK21-F), and adult hamster kidney (HaK) cells, and detailed lipid analyses of the membranes, the whole cells, and parainfluenza virus SV5 grown in MK and BHK21-F cells have been carried out. The lipid compositions of the plasma membranes differ from those of the respective whole cells, and show characteristic features, including a high cholesterol and sphingomyelin content, and a cholesterol to phospholipid ratio which is higher than that of the whole cell. The lipid patterns of the membranes of the various cell types are significantly different from each other, especially with regard to phospholipids. In MK membranes, e.g., phos-phatidylethanolamine is the major phospholipid, and there is a relatively high content of phosphatidylserine, whereas in BHK21-F membranes, phosphatidylcholine is present in highest amount, and there is relatively little phosphatidylethanolamine and phosphatidylserine. Previous studies have shown that the plasma membranes of these cells exhibit significant biological differences, particularly in their interactions with SV5. SV5 virions grown in MK or BHK21-F cells have a lipid composition that is closely similar to that of the plasma membrane of the particular cell type in which the virus was grown. The data support the hypothesis that during the maturation of the virion by budding from the cell surface, the lipids of the plasma membrane are incorporated quantitatively into the viral envelope.

(Na +-K +)-activated ATPase in kidney cell membranes of normal and methylprednisolone-treated rats

A method is described for the isolation of a purified preparation of plasma membranes from rat kidney tissue, with a high specific activity of (Na +-K +)-activated ATPase. Treatment of rats for 4–5 days with methylprednisolone produced an increase in the specific activity of (Na +-K +)-ATPase in kidney homogenates, but not of Mg 2+-dependent ATPase or succinate dehydrogenase (EC 1.3.99.1). Methylprednisolone did not, however, increase the (Na +-K +)-ATPase per mg of protein in kidney plasma membranes. The data are interpreted to support the hypothesis that glucocorticoids increase (Na +-K +)-ATPase activity in the kidney by increasing the quantity of plasma membrane per cell, rather than by increasing the activity of enzyme per unit of plasma membrane.

Evidence for an interaction between kidney cell membrane and certain phosphate-transferring enzymes

As a facet of our investigation of the role of adenosine-3′,5′-monophosphate in the mechanism of action of antidiuretic hormone we have explored the possibility of an interaction between kidney cell membrane and certain phosphate-transferring enzymes. This communication is concerned with evidence for a membrane-enzyme interaction as derived from optical density studies, enzyme activation and inhibition effects, and optical rotatory dispersion data. We have found that phosphoglucomutase, phosphorylase, and pyruvic kinase appear to interact with kidney cell membrane. These enzymes are unique with respect to those studied in requiring a substrate which bears a phosphorylated carbonyl moiety.

Interaction of aldosterone with liver and kidney cell membranes and subcellular fractions of kidney.

1. A simplified method for isolation of kidney and liver cell membranes is reported. 2. Aldosterone is weakly bound to kidney subcellular fractions as demonstrated by the techniques of equilibrium fractionation and equilibrium dialysis. 3. The liver and kidney cell membranes studied were found to either take up the steroids according to their lipid solubility or to bind them in accordance with the polarity rule for steroid-protein interaction. 4. Spirolactone failed to displace aldosterone from the kidney cell membranes.

Cellular lipoproteins: IV. The insoluble lipoproteins of various tissues

1. 1. The insoluble lipoprotein was prepared from the liver, kidney, lung, brain, muscle, and red cell membranes of the rabbit and rat and in the livers of bull frogs and trout. 2. 2. The insoluble lipoproteins prepared from the various tissues differ from each other. 3. 3. There is a species difference in the insoluble lipoprotein prepared from the same tissues from different animals. 4. 4. The lipoprotein prepared from red cell membranes seems to be an excellent one to study the protein, lipid, and carbohydrate components comprising the lipoprotein.

Oxygen consumption and active transport in separated renal tubules

A method for the separation of intact viable cells from rabbit renal cortex has been developed. Separation is achieved by treatment of the excised kidney with the proteolytic enzyme collagenase. The resultant suspension consists principally of short lengths of proximal tubules. Survival of the tissues is indicated by maintenance of oxygen consumption, PAH uptake, and electrolyte composition. Since, in contrast to renal cortical slices, the cells in the tubule suspension are bathed directly by a solution of uniform composition, there is more rapid accumulation of PAH and a higher rate of oxygen consumption in this preparation than in slices. The technique affords a method for the direct measurement of fluxes across membranes of kidney cells.