An azide-insensitive low-affinity ATPase stimulated by Ca2+ or Mg2+ in basal-lateral and brush border membranes of kidney cortex

Basal-lateral and brush border membranes from pig kidney cortex were prepared by differential centrifugation followed by free-flow electrophoresis. In each type of membrane, azide-insensitive, low-affinity Ca2+-ATPase and Mg2+-ATPase activities are demonstrated. A comparative study for both membranes further reveals the following analogies between these ATPases: (a) they show maximal activity between pH 8 and 8.5; (b) they exhibit Km values for Ca-ATP or Mg-ATP in the millimolar range and have a comparable low substrate specificity; (c) they are insensitive to 10 microM of vanadate, N,N'-dicyclohexylcarbodiimide, e diethylstilbestrol, quercetin, harmaline and amiloride. The partial inhibition by 1 mM of the various compounds is rather aspecific. In view of these similarities it is concluded that only one enzyme entity is responsible for the activity which is measured in both membrane types. The HCO3-stimulated Mg2+-ATPase activity in pig kidney cortex was also studied. This enzyme, however, is clearly of mitochondrial origin since the HCO3-stimulation coincides with the distribution profile of succinate dehydrogenase, a mitochondrial marker; and since it is inhibited by azide.     

Localization and properties of a high-affinity (Ca2+ + Mg2+)-ATPase in isolated kidney cortex plasma membranes

Surface membrane fractions from Paramecium tetraurelia cells contain a calmodulin-stimulated Ca²⁺-ATPase responding to low levels of free Ca²⁺ and with features characteristic of a membrane-bound ATPase responding to low levels of free Ca²⁺ and with features characteristics of a membrane-bound ATPase. Among the different strains analyzed this enzyme was practically absent selectively from the ‘non-discharge” mutant nd9—28°C (from J. Beisson); if cultured at a permissive temperature (18°C), this strain showed identical values of calmodulin-stimulated Ca²⁺-ATPase activity as wild-type cells (7S) or strains with mutations which do not affect exocytosis performance. We conclude that this calmodulin-stimulated Ca²⁺-activated ATPase might be a prerequisite for membrane fusion in the course of exocytosis performance.     

Effect of anions on folate binding by isolated brush border membranes from rat kidney

The characteristics of folate binding by brush border membranes from rat kidney homogenates were investigated. At pH 7.4, binding of [3′, 5′, 9-³H]-pteroylglutamic acid to membranes containing endogenous folate is inhibited by anions, with chloride being most effective followed by bromide, thiocyanate, iodide, phosphate and sulfate. A maximum inhibition of 70–75% is attained at a concentration of 0.1 M chloride and an incubation time of 30 min. The inhibition diminishes with increased incubation time and at 24 h is negligible. The binding of [3′,5′,9-³H]pteroylglutamic acid to brush border membranes stripped of endogenous folate by acid treatment is not inhibited by anions. Anion sensitivity can be restored to these treated membranes by reconstitution with membrane-derived folate, particularly 5-methyltetrahydropteroylglutamic acid, or by preincubation with synthetic 5-methyltetrahydropteroylglutamic acid. Inhibition of [3′,5′,9-³H]pteroylglutamic acid binding by anions in membranes with endogenous folate is best explained by an anion-induced stabilization of endogenous folate-binding protein complex resulting in a decreased rate of exchange with exogenous [3′,5′,9-³H]pteroylglutamic acid.     

Cell volume-sensitive Na+-ATPase activity in rat kidney cortex cell membranes

A ouabain-insensitive, K+-independent, sodium pump, has been demonstrated in guinea-pig and rat kidney proximal tubular cells. This pump is thought to be distinct from the ouabain-sensitive Na+/K+ pump. We present evidence here indicating the modulation of the biochemical expression of the Na+ pump, i.e. the ouabain-insensitive Na+-ATPase, by the cell volume in rat kidney proximal tubular cells. Thus, basolateral plasma membranes from swollen cells show a ouabain-insensitive Na+-ATPase activity 10-times higher than that in membranes from control cells. If the swollen cells recover their volume, the activity decreases ten times to control values. The ouabain-sensitive Na+/K+-ATPase is not affected by changes in the cell volume.     

Binding of lysozyme to brush border membranes of rat kidney

The binding of ¹²⁵I-labelled egg-white lysozyme to isolated brush border membranes of rat kidney cortex was investigated. The lysozyme binding was reversible and saturable. The Scatchard plot revealed a one-component binding type with a dissociation constant of 7.8 μM and 15.6 nmol/mg membrane protein for the number of binding sites. The binding of the basic lysozyme could be reduced by basic amino acids such as l-lysine, l-ornithine or l-arginine, while neutral amino acids such as l-citrulline or l-alanine had no effect. The inhibitory effect of lysine was competitive.     

Synthesis and brush border expression of intrinsic factor-cobalamin receptor from rat renal cortex.

The main objective of the current study was to investigate the factors that affect brush border membrane expression of intrinsic factor-cobalamin receptor (IFCR). Because of high levels of IFCR expression (Seetharam, B., Levine, J. S., Ramasamy, M., and Alpers, D. H. (1988) J. Biol. Chem. 263, 4443-4449) in the rat kidney, we have studied the synthesis and expression of IFCR using rat cortical slices in culture. The IFCR activity in the renal apical brush border was maximum from rats between the age of 20-24 days and about 75% of the activity was lost from the isolated apical surface membranes following culture of cortical slices with nonradioactive intrinsic factor-cobalamin. However, the membrane IFCR activity recovered to 100 or 75%, respectively, when the slices were cultured with intrinsic factor-cobalamin mixed with either leupeptin or chloroquine. When these lysosomotropic agents were added during the metabolic labeling of the cortical slices with trans-35S-label neither the synthesis nor the amount of [35S]IFCR transported to the apical membrane was inhibited. However, with the addition of colchicine, the apical membrane expression of [35S]IFCR was inhibited by 75-80%. Metabolic labeling of cortical slices with trans-35S-label and immunoprecipitation of the Triton X-100 extract from the total, internal, and apical membranes revealed the presence of a 230-kDa band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With either continuous or pulse-chase labeling of the cortical slices, the amount of 230-kDa [35S]IFCR recovered in the apical membrane did not exceed 10-15% of the total labeled receptor synthesized. Based on these and our recent studies (Seetharam, S., Dahms, N., Li, N., Ramanujam, K.S., and Seetharam, B. (1991) Biochem. Biophys. Res. Commun. 177, 751-756), we propose that rat renal IFCR is synthesized as a single polypeptide chain of 220 kDa and is transported slowly to the apical membrane during which four or five N-linked oligosaccharides are processed to the complex type. Moreover, the brush border expression of IFCR is regulated by the biosynthetic and not by the endocytic pathway.     

Characterization of Mg2+-ATPase from slow-twitch muscle membranes

SR vesicles from rabbit slow-twitch muscle reveal high activity (0.7-0.9 mumol/mg X min) of "basic" or Mg2+-ATPase. This enzyme differs in its biochemical properties from the well characterized Ca2+ pump ATPase. It is active in millimolar concentration of magnesium or calcium. The activity is inhibited by various detergents except for digitonin. This enzyme seems to be an integral membrane protein since it remains in the membrane after removal of peripheral proteins with EDTA. It can be partially solubilized from the membrane using digitonin without a decrease in specific activity. Ion exchange chromatography on DEAE-Sephacel of the post digitonin supernatant allows us to obtain a 5-fold increase in Mg2+-ATPase specific activity concomitantly with the enrichment in two proteins of Mr = 30,000 and 150,000.     

A water-soluble Mg2+-ATPase from erythrocyte membranes.

A ouabain-insensitive ATPase activity associated with the water-soluble proteins of the human and bovine erythrocyte membrane is demonstrated by means of activity-staining in polyacrylamide gels. The ATPase activity from both sources had an absolute requirement for Mg2+, activity becoming easily detectable at 0.2 mM Mg2+. At low Mg2+ concentrations added Ca2+ appeared to decrease the intensity of the ATPase stain. The activity is unaffected by monovalent cations, does not hydrolyse p-nitrophenyl phosphate and is not inhibited by 2 : 4 dinitrophenol. The ATPase has an apparent molecular weight of approximately 100 000 as determined by electrophoresis in acrylamide gels containing dodecyl sulphate.     

Binding of nicotinamide adenine dinucleotide by the renal brush border membrane from rat kidney cortex

The characteristics of nicotinamide adenine dinucleotide (NAD) binding on brush border membranes prepared from rat renal cortex were investigated with the use of radioactively labelled NAD, [adenine-2,8-3H]NAD+, as a ligand. (1) We found that NAD binds on brush border membrane and that the extent of NAD binding is linearly proportional to the brush border membrane protein, and progressively increases with concentration of NAD in the medium. (2) The rate of NAD binding was dependent on temperature. At 20 degrees C, the equilibrium binding was obtained at 15 min, while NAD binding at 0 degree C was slower, but the final level of binding reached at 120 min was similar to that plateau of binding observed at 20 degrees C. Brush border membrane inactivated by heating at 95 degrees C for 3 min did not bind NAD. Binding of NAD on brush border membranes was reversed by simple dilution or by the addition of unlabelled NAD. Both alpha-NAD and beta-NAD stereoisomers displaced bound [3H]NAD. Reduced NAD (NADH) caused less displacement of bound NAD than oxidized NAD+. Adenine, nicotinamide, pyrophosphate, of 5'-AMP did not displace bound NAD. (3) The NAD binding to brush border membranes was nearly saturable, approximating saturation at 10(-4) M NAD. Kinetic analysis by Scatchard plot indicates two sets of NAD binding sites in brush border membranes: a high-affinity binding site (Kd = 1.9 . 10(-5) M) and a low-affinity binding site (Kd = 2.2 . 10(-3) M). (4) Unlike concentrative uptake of D-[14C]glucose by brush border membrane vesicles, binding of NAD was not dependent on the presence of an outside-in sodium gradient [Na+0 greater than Na+i], nor was it abolished by repeated freezing and thawing of brush border membranes. Unlike D-[14C]glucose uptake, NAD binding by brush border membranes did not change upon decrease of intravesicular volume in hypertonic media. These observations indicate that NAD association with brush border membranes is true binding rather than intravesicular uptake of this compound. (5) The presence of specific binding sites in renal brush border membrane capable of binding of NAD with a high degree of affinity suggests that such sites may be involved in previously observed (Kempson, S.A., Colon-Otero, G., Ou, S.L., Turner, S.T. and Dousa, T.P. (1981) J. Clin. Invest. 67, 1347) modulatory effect of NAD on sodium-gradient-dependent uptake of phosphate across luminal brush border membrane of proximal tubules.     

Polyunsaturated fatty acids inhibit Mg2+ -ATPase in basolateral membranes from rat enterocytes.

It is known that certain polyunsaturated fatty acids of the n-6 family, for example linoleic and arachidonic acids, can activate both Na+, K+-ATPase and Ca2+-ATPase. These enzymes drive active absorption processes in the duodenal enterocyte. This study presents data which show a 30-50% inhibition of Mg2+-ATPase activity in enterocyte basolateral membrane preparations by linoleic and gamma-linolenic acids (also a member of the n-6 family.) Mg2+-ATPase activity has several possible roles in the enterocyte: involvement in Mg2+ and Ca2+ absorption (as part of Ca2+-ATPase and also myosin I activity) as well as control of phospholipid distribution in the membrane by a class of Mg2+-ATPases called 'flippases'. The action of linoleic and gamma-linolenic acids on basolateral membrane Mg2+-ATPase may thus modulate several cellular transport processes.     

Proton pump-linked Mg2+-ATPase activity in isolated rat liver lysosomes

ATPase activity in highly purified rat liver lysosome preparations was evaluated in the presence of other membrane cellular ATPase inhibitors, and compared with lysosome ATP-driven proton translocating activity. Replacement of 5 mM Mg2+ with equimolar Ca2+ brought about a 50% inhibition in divalent cation-dependent ATPase activity, and an 80% inactivation of ATP-linked lysosomal H+ pump activity. In the presence of optimal concentrations of Ca2+ and Mg2+, ATPase activity was similar to that seen in an Mg2+ medium. Mg2+-dependent ATPase activity was greatly inhibited (from 70 to 80%) by the platinum complexes; cis-didimethylsulfoxide dichloroplatinum(II) (CDDP) at approximately 90 microM and cis-diaminedichloroplatinum(II) at twofold higher concentrations. Less inhibition, about 30 and 45%, was obtained with N,N'-dicyclohexylcarbodiimide and N-ethylmaleimide, and the maximal effect occurred in the 50-100 microM and 0.1-1.5 mM ranges, respectively. The concentration dependence of inhibition by the above drugs was determined for both proton pumping and ATPase activities, and half-maximal inhibition concentration of each activity was found at nearly similar values. A micromolar concentration of carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) prevented ATP from setting up a pH gradient across the lysosomal membranes, but stimulated Mg2+-ATPase activity significantly. ATPase activity in Ca2+ medium was also inhibited by CDDP and stimulated by FCCP, but both effects were two- to threefold less than those observed in Mg2+ medium. FCCP failed to stimulate ATPase activity in a CDDP-supplemented medium, thus suggesting that the same ATPase activity fraction was sensitive to both CDDP and FCCP. Mg2+-ATPase activity, like the proton pump, was anion dependent. The lowest activity was recorded in a F-medium, and increased in the order of F- less than SO2-4 less than Cl- approximately equal to Br-. The CDDP-sensitive ATPase activity observed, supported by Mg2+ and less so by Ca2+, may be related to lysosome proton pump activity.     

myo-Inositol binding and transport in brush border membranes of rat kidney

Using hypotonically treated brush border membranes, binding and transport of myo-inositol were examined.By hypotonic treatment, both total and non-specific uptake decreased significantly, but specific uptake was not affected.myo-Inositol release from membranes preloaded by incubation for 2 min was very rapid and about 98% of preloaded myo-inositol was released in 5 min of incubation. However, myo-inositol release from membranes preloaded by incubation for 20 min was fairly slow and 50% of myo-inositol remained in the membranes even after 10 min of incubation.Uptake of myo-inositol decreased by the increase of osmolarity in the medium. However, effect of osmolarity on the uptake was less significant when myo-inositol concentration was lower.Under conditions in which mainly binding occurred, myo-inositol binding to the membranes was measured. Two binding systems were demonstrated and high affinity site could bind 22 pmol/mg protein at most and the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value was 8.3 μM.Both binding and transport processes were dependent on Na⁺ and enhanced by Na⁺-gradient.     

A rapid method for the isolation of kidney brush border membranes

A simple rapid method for the preparation of purified brush border membranes from rabbit kidney proximal tubules is described. The method is based on hypotonic lysis, Ca²⁺ aggregation of contaminants and differential centrifugation. In contrast to most other published methods, the brush border membranes are free of contamination by basolateral membranes.