Myometrial (Na+ + K+)-activated ATPase and its Ca2+ sensitivity

Ouabain-sensitive (Na+ + K+)-ATPase activity in the rat myometrial microsome fraction could only be determined following detergent treatment. The (Na+ + K+)-ATPase activity manifested by detergent treatment proved very stable even to high concentrations of NaN3, in contrast Mg+-ATPase activity was reduced to about 30 percent of the control. The major part of the Mg2+-ATPase in the myometrial membrane preparation was found to be identical with the NaN3-sensitive ATP diphosphohydrolase capable of ATP and ADP hydrolysis. This monovalent-cation-insensitive ATP hydrolysis could be extensively reduced by DMSO. Furthermore DMSO prevented the inactivation of the (Na+ + K+)-ATPase activity. 10-100 microM Ca2+ inhibited the (Na+ + K+)-ATPase activity obtained in the presence of SDS by 15-50 percent. The Ca2+ sensitivity of the enzyme was considerably decreased if the proteins solubilized by the detergent had been separated from the membrane fragments by ultracentrifugation. The inhibitory effect could be regained by combining the supernatant with the pellet. Ca2+ sensitivity of the (Na+ + K+)-ATPase activity was preserved even after removal of the solubilized proteins provided that DMSO had been applied. It appears that a factor in the plasma membrane solubilized by SDS may be responsible for the loss of Ca2+ sensitivity of the (Na+ + K+)-ATPase activity, the solubilization of which can be prevented by DMSO.     

Partial purification and characterization of the (Ca2+ + Mg2+)-ATPase from squid optic nerve plasma membrane

A membrane fraction enriched in axolemma was obtained from optic nerves of the squid (Sepiotheutis sepioidea) by differential centrifugation and density gradient fractionation. The preparation showed an oligomycin- and NaN3-insensitive (Ca2+ + Mg2+)-ATPase activity. The dependence of the ATPase activity on calcium concentration revealed the presence of two saturable components. One had a high affinity for calcium (K1 1/2 = 0.12 microM) and the second had a comparatively low affinity (K2 1/2 = 49.5 microM). Only the high-affinity component was specifically inhibited by vanadate (K1 = 35 microM). Calmodulin (12.5 micrograms/ml) stimulated the (Ca2+ + Mg2+)-ATPase by approx. 50%, and this stimulation was abolished by trifluoperazine (10 microM). Further treatment of the membrane fraction with 1% Nonidet P-40 resulted in a partial purification of the ATPase about 15-fold compared to the initial homogenate. This (Ca2+ + Mg2+)-ATPase from squid optic nerve displays some properties similar to those of the uncoupled Ca2+-pump described in internally dialyzed squid axons, suggesting that it could be its enzymatic basis.     

Comparative study of calixarene effect on Na+, K+ -ATPase activity in plasma membrane of contractile and mobile cells

The paper is devoted to comparative analysis of the influence of a new class of macrocyclic compounds - calixarens on enzymatic activity of two ATP-hydrolase systems localized in the plasmatic membrane of contractile (myocytes of the uterus) and mobile (spermatozoids) cells--Na+, K+ -ATPase and basal Mg2+ -ATPase. The experiments performed on plasmatic membrane suspensions of myometrium and spermatozoids treated with detergent the authors studied the influence of calixarens C-97, C-99, C-107 (identified by the codes), functionalized with fragments of alpha-hydroxyphosphonic, alpha-aminophosphonic and methylenbisphosphonic acids accordingly, on enzymatic activity. The results have shown that C-97 and C-107 calixarenphosphonic acids in 100 microM concentration (97-99%) inhibit Na+, K+ -ATPase activity in both cases almost completely. C-99 (100 microM) calixaren appeared to be less effective with regard of its influence on the enzymatic systems under study: in the case of plasmatic membranes of myometrium suspension the activity of Na+, K+ -ATPase was decreased by 84-88%, and in the case of spermatozoids suspension--just by 15-20% of the control. All the studied calixarens (for both objects) in the maximal concentration (100 microM) practically did not influence the activity of basal Mg2+ -ATP-ase. The calixarens inhibited the enzymatic activity of Na+, K+ -ATPase more effectively than ouabain: in the first case the value of apparent inhibition constant I(0,5) was 25-100 nM, and in the second case--20-100 microM. The inhibition influence of calixarens on Na+, K+ -ATPase activity is characterized by the phenomenon of negative cooperativity (Hill's coefficient nH <1); the influence of ouabain in the case of plasmatic membranes of myometrium suspension is also characterized by negative cooperativity (nH < 1), and in case of spermatozoids suspension--by positive cooperativity (nH >1). The above results show that the studied calixarens are effective inhibitors of Na+, K+ -ATPase plasmatic membrane of contractive and mobile cells (C-97, C-99, C-107 calixarens in case of myocytes of uterus, and C-97, C-107 calixarens in case of spermatozoids).     

S-adenosyl-L-methionine modulates Na+ + K+-ATPase activity in rat colonic basolateral membranes.

Rat colonic basolateral membranes were incubated with S-adenosyl-L-[methyl-3H]methionine (0.3 mM) at 37 degrees C for 2 h at pH 9.0. This resulted in an increase in the specific activity of Na+ + K+-ATPase by 60%. Kinetic parameter analysis revealed a 2-fold increase in the Vmax. of this enzymatic activity, whereas the Km for ATP was unchanged. The methylation inhibitor S-adenosyl-L-homocysteine (2 mM) significantly reduced these S-adenosyl-L-methionine-stimulated increases in specific activity and the Vmax. of Na+ + K+-ATPase. S-Adenosyl-L-methionine treatment of basolateral membranes was also found to significantly increase the fluidity of these preparations, as assessed by steady-state fluorescence polarization techniques using the fluorophore 1,6-diphenyl-1,3,5-hexatriene; S-adenosyl-L-homocysteine (2 mM) again markedly reduced this S-adenosyl-L-methionine-induced increase in fluidity. While transmethylation reactions involving phospholipids, non-polar lipids and proteins were all found to exist in rat colonic basolateral membranes, based on a number of observations, the results of the present studies suggest that transmethylation of membrane phospholipids, but not membrane non-polar lipids or proteins, influenced the fluidity of basolateral membranes which, in turn, modified Na+ + K+-ATPase activity in these membranes.     

Modifications induced by general anesthetics on Na+/K+ ATPase obtained from human placenta

Previously it was demonstrated that thiopental in vivo anesthesia didn't affect the Na+/K(+)-ATPase activity of syncythiotrophoblast plasma membrane, while affecting other enzymatic activity. The aim of the present work was to investigate if this lack of effect of thiopental on the Na+/K+ ATPase activity might be due to its specificity of action on definite membrane proteins or if the binding sites of the anesthetic to this enzyme might be masked within the membrane. Temperature dependence of the Na+/K(+)-ATPase activity and of a spin label paramagnetic maleimide derivative (MSL,2,2,6,6-tetramethylpiperidin-1-oxyl-4-maleimide), which shows a selective binding to the reduced sulfhydryl groups of proteins were investigated. This report shows that a Na+/K(+)-ATPase membranous preparation obtained from placental tissue is strongly inhibited by thiopental.     

Kinetic studies on the (Na+ + K+)-dependent ATPase evidence for coexisting sites for Na+, K+ and Mg2+

Na+-ATPase activity of a dog kidney (Na+ + K+)-ATPase enzyme preparation was inhibited by a high concentration of NaCl (100 mM) in the presence of 30 microM ATP and 50 microM MgCl2, but stimulated by 100 mM NaCl in the presence of 30 microM ATP and 3 mM MgCl2. The K0.5 for the effect of MgCl2 was near 0.5 mM. Treatment of the enzyme with the organic mercurial thimerosal had little effect on Na+ -ATPase activity with 10 mM NaCl but lessened inhibition by 100 mM NaCl in the presence of 50 microM MgCl2. Similar thimerosal treatment reduced (Na+ + K+)-ATPase activity by half but did not appreciably affect the K0.5 for activation by either Na+ or K+, although it reduced inhibition by high Na+ concentrations. These data are interpreted in terms of two classes of extracellularly-available low-affinity sites for Na+: Na+-discharge sites at which Na+-binding can drive E2-P back to E1-P, thereby inhibiting Na+-ATPase activity, and sites activating E2-P hydrolysis and thereby stimulating Na+-ATPase activity, corresponding to the K+-acceptance sites. Since these two classes of sites cannot be identical, the data favor co-existing Na+-discharge and K+-acceptance sites. Mg2+ may stimulate Na+-ATPase activity by favoring E2-P over E1-P, through occupying intracellular sites distinct from the phosphorylation site or Na+-acceptance sites, perhaps at a coexisting low-affinity substrate site. Among other effects, thimerosal treatment appears to stimulate the Na+-ATPase reaction and lessen Na+-inhibition of the (Na+ + K+)-ATPase reaction by increasing the efficacy of Na+ in activating E2-P hydrolysis.     

Characterization of a beta-actinin-like protein in purified non-muscle cell membranes. Its activity on (Na+ + K+)-ATPase

Treatment by EDTA of purified plasma membranes from MF2S cells (a variant of the murine plasmacytoma MOPC 173) solubilized proteins and increased by a 1000-fold the sensitivity of (Na+ + K+)-ATPase to ouabain. When added back with Ca2+ to treated plasma membranes, these EDTA-solubilized proteins restored the initial sensitivity of the enzyme to its inhibitor. We report the purification of a protein of Mr 32000, isolated from the EDTA-treated membrane supernatant. This protein was purified by a one-step procedure involving a preparative polyacrylamide gel electrophoresis without detergent. In the presence of Ca2+ it was able to restore the original sensitivity to ouabain of (Na+ + K+)-ATPase from EDTA-treated membrane. This protein was shown to be similar to the beta-actinin described by Maruyama by the following criteria: (1) molecular weight and amino acid composition; (2) cross-reactivity with their respective antisera; (3) in the presence of Ca2+ the same quantitative biological activity on ouabain sensitivity of the (Na+ + K+)-ATPase. A possible interaction between beta-actinin, calmodulin and membrane-bound (Na+ + K+)-ATPase is discussed.     

Studies of (Na+ + K+)-sensitive ATPase activity in pig lymphocytes. Effects of concanavalin A.

(Na+ + K+)-ATPase activity is demonstrated in plasma membranes from pig mesenteric lymph nodes. After dodecyl sulfate treatment plasma membranes have an 18-fold higher (Na+ + K+)-ATPase activity, while their ouabain-insensitive Mg2+-ATPase is markedly lowered. A solubilized (Na+ +K+)-ATPase fraction, obtained by Lubrol WX treatment of the membranes, has very high specific activity (21 mumol Pi/h per mg protein). Concanavalin A has no effect on these partially purified (Na+ + K+)-ATPase, while inhibits (40%) this activity in less purified fractions which still contain Mg2+-ATPase activity.     

Effect of calixarene-phosphonic acid on Na+, K+-ATPase activity in plasma membranes of the smooth-muscle cells

Effect of calix[4]arenes C-97, C-99, C-107, functionalized by fragments of alpha-hydroxy-phosphonic, alpha-aminophosphonic- and methylene-bisphosphonic acid on enzymatic activity of oubaine-sensitive Na+, K+-ATPase and oubaine-resistant basal Mg2+- ATPase (specific activity - 10.6 +/- 0.9 and 18.1 +/- 1.2 micromol Pi/h per 1 mg of protein, respectively; n = 7) was studied in experiments made on the suspension of myometrium cell plasma membranes treated by 0.1% solution of digitonin. It was found that calixarene-phosphonic acids in concentration of 100 microM inhibited enzymatic activity of Na+, K+-ATPase by 86-98% and did not practically affect activity of Mg2+-ATPase. These calixarenes were more efficient than oubaine in suppressing enzymatic activity of the sodium pump: in case of the effect of calixerenes the value of the appearence constant of inhibition I0.5 was < 0.1 microM. Calixarene-methylene-bisphosphonic acid (calixarene C-97; I0.5 =33 +/- 4 microM (n = 6) takes the most efficient inhibitory effect on Na+,K+-ATPase activity among the studied calixarenes. A phenomenon of negative cooperation: the Hill coefficient value etaH =0.1-0.5<1 is characteristic of both the inhibiting effect of calixarenes and oubaine. Reguliarities of calixarenes C-97 effect on enzymatic activity of Na+,K+-ATPase were studied. As it appeared its inhibiting effect cannot be caused by trivial factors - potentially possible binding of Mg ions by it and (or) this substance effect on Mg2+ interaction with ATP4- in the incubation medium. Calixerene C-97 does not also decrease the enzyme affinity for Mg ions or ATP. However this calixerenes decreases the affinity of Na+,K+-ATPase for Na ions (the value of activation constant K(Na+)) from 50 +/- 4 (control) to 76 +/- 6 microM in the control and under the effect of calixerene, respectively). A conclusion is made that calixerene C-97 is highly-efficient (with respect to oubaine) and selective (with respect to lack of its effect on basal Mg2+-ATPase) inhibitor of Na+,K+-ATPase of plasma membrane. In the practical aspect it may be used in concentration of 1-10 microM in biochemical membranology when testing and studying kinetic and catalytic properties of the sodium pump in case of such experimental model, as the plasma membrane fraction.     

Separation of basolateral plasma membranes from smooth endoplasmic reticulum of the rat enterocyte by zonal electrophoresis on density gradients

Basolateral plasma membranes of rat small intestinal epithelium were purified by density gradient centrifugation followed by zonal electrophoresis on density gradients. Crude basolateral membranes were obtained by centrifugation in which the marker enzyme, (Na+ + K+)-ATPase, was enriched 10-fold with respect to the initial homogenate. The major contaminant was a membrane fraction derived from smooth endoplasmic reticulum, rich in NADPH-cytochrome c reductase activity. The crude basolateral membrane preparation could be resolved into the two major components by subjecting it to zonal electrophoresis on density gradients. The result was that (Na+ + K+)-ATPase was purified 22-fold with respect to the initial homogenate. Purification with respect to mitochondria and brush border membranes was 35- and 42-fold, respectively. Resolution of (Na+ + K+)-ATPase from NADPH-cytochrome c reductase by electrophoresis was best with membrane material from adult rats between 180 and 250 g. No resolution between the two marker enzymes occurred with material from young rats of 125 to 140 g. These results demonstrate that zonal electrophoresis on density gradients, a simple and inexpensive technique, has a similar potential to free-flow electrophoresis.     

Regulation of Na+ transport in brown adipose tissue.

In order to test the hypothesis that Na+, K+-ATPase (Na+,K+-dependent ATPase) is involved in the noradrenaline-mediated stimulation of respiration in brown adipose tissue, the effects of noradrenaline on Na+,K+-ATPase in isolated brown-fat-cell membrane vesicles, and on 22Na+ and K+ (86Rb+) fluxes across the membranes of intact isolated cells, were measured. The ouabain-sensitive fraction of the K+-dependent ATPase activity in the isolated membrane-vesicle preparation was small and was not affected by the presence of noradrenaline in the incubation media. The uptake of 86Rb+ into intact hormone-sensitive cells was inhibited by 80% by ouabain, but it was insensitive to the presence of noradrenaline. 22Na+ uptake and efflux measured in the intact cells were 8 times more rapid than the 86Rb+ fluxes and were unaffected by ouabain. This indicated the presence of a separate, more active, transport system for Na+ than the Na+,K+-ATPase. This is likely to be a Na+/Na+ exchange activity under normal aerobic conditions. However, under anaerobic conditions, or conditions simulating anaerobiosis (2 mM-NaCN), the unidirectional uptake of Na+ increased dramatically, while efflux was unaltered.     

Preparation of active enzyme samples for IR studies of Na+/K+-ATPase

In the case of the integral membrane protein Na+/K+-ATPase, preparation of highly concentrated samples for IR difference spectroscopy often leads to inactivation of the enzyme. Therefore, we compared the activity of Na+/K+-ATPase using different techniques of sample preparation. The loss of activity can be minimized by cooling the sample to 10 degrees C and by the addition of glycerol and dithiothreitol. The activity of Na+/K+-ATPase isolated from pig kidney is independent of the protein concentration whereas the enzyme from shark rectal gland is inactivated at concentrations above 1 microg/microL and is thus unsuitable for IR experiments.     

Preparation of renal cortex basal-lateral and bursh border membranes. Localization of adenylate cyclase and guanylate cyclase activities.

Luminal brush border and contraluminal basal-lateral segments of the plasma membrane from the same kidney cortex were prepared. The brush border membrane preparation was enriched in trehalase and gamma-glutamyltranspeptidase, whereas the basal-lateral membrane preparation was enriched in (Na+ + K+1)-ATPase. However, the specific activity of (Na+ + K+)-ATPase in brush border membranes also increased relative to that in the crude plasma membrane fraction, suggesting that (Na+ + K+)-ATPase may be an intrinsic constituent of the renal brush border membrane in addition to being prevalent in the basal-lateral membrane. Adenylate cyclase had the same distribution pattern as (Na+ + K+)-ATPase, i.e. higher specific activity in basal-lateral membranes and present in brush border membranes. Adenylate cyclase in both membrane preparations was stimulated by parathyroid hormone, calcitonin, epinephrine, prostaglandins and 5'-guanylylimidodiphosphate. When the agonists were used in combination enhancements were additive. In contrast to the distribution of adenylate cyclase, guanylate cyclase was found in the cytosol and in basal-lateral membranes with a maximal specific activity (NaN3 plus Triton X-100) 10-fold that in brush border membranes. ATP enhanced guanylate cyclase activity only in basal-lateral membranes. It is proposed that guanylate cyclase, in addition to (Na+ + K+)-ATPase, be used as an enzyme "marker" for the renal basal-lateral membrane.     

Comparative study of properties of Na+, K+-ATPase and Mg2+-ATPase of the myometrium plasma membrane

The comparative research of catalytic properties of two ATP-hydrolases of the sarcolemma of the smooth muscle of the uterus--ouabaine-sensitive Na+,K+-ATPase and ouabaine-resistent Mg2+-ATPase is carried out. The specific enzymatic activity of Na+,K+-ATPase and Mg2+-ATPase makes 10.2 +/- 0.7 and 18.1 +/- 1.2 mmol P/mg of protein for 1 hour, accordingly. The action of ouabaine on Na+,K+-ATPase is characterized by magnitude of quotient of inhibition I0.5=21.3 +/- 1.5 mkM. Processing of the sarcolemma fraction by digitonin in concentrations 0.001 +/- 0.1% promotes an activation of Na+,K+ATPase and Mg2+- ATPase, and in the first case much more efficiently than in the second. The kinetics of accumulation of the product of ATP-hydrolase reactions of phosphate satisfies the laws of the zero order reaction (incubation time--about 10 min). Na+,K+-ATPase is highly specific concerning the univalent cations--Na+, K+, however Li+ can partially substitute K+. Activity of Mg2+-ATPase is not specific concerning univalent cations. The dependence of Na+,K+-ATPase activity on pH in the range of 6.0-8.0 is characterized by the bell-shaped curve, at the same time the linear dependence on pH is peculiar to Mg2+-ATPase. The functioning of Na+,K+-ATPase is provided only by ATP, in the case of Mg2+-ATPase ATP can be successfully replaced with other nucleotidetriphosphates. It is supposed that the obtained experimental data can be beneficial in further research of membranous mechanisms underlying the cation exchange in the smooth muscles, in particular when studying the role of the plasma membrane in the maintenance of electromechanical coupling in them, and also in the regulation of ionic homeostasis in myocytes.     

Effect of a NaCl gradient on the transport of para-aminohippuric acid into the vesicles of the basolateral membrane of the kidney cortex

Basolateral membrane vesicles were isolated from the rat kidney cortex by a modified method of cation precipitation. Different steps of preparation were analysed using the marker enzymes: Na+,K+-ATPase (for basolateral membrane), alkaline phosphatase (for apical membrane), glucose-6-phosphatase (for membranes of endoplasmic reticulum) and succinate dehydrogenase (for mitochondria). The basolateral membrane was purified by a 8-9-fold treatment with Na+,K+-ATPase, while other membrane contaminations were as low as 2% (as compared to homogenate). The transport of 3H-p-aminohippurate (3H-PAH) by basolateral membrane vesicles was measured under different experimental conditions. The 3H-PAH uptake was found to be Na-gradient dependent. The initial rate of 3H-PAH uptake in the presence of NaCl gradient (500 pM/mg X min) was higher than without the gradient (88 pM/mg X min). It is concluded that the PAH transfer across the basolateral membrane may be energized by the Na+ chemical gradient.     

Identification of N-[p-(2-benzimidazolyl)phenyl]maleimide-modified residue participating in dynamic fluorescence changes accompanying Na+,K+-dependent ATP hydrolysis

Na+,K+-ATPase from pig kidney was specifically modified with a sulfhydryl fluorescent reagent, N-[p-(2-benzimidazolyl)phenyl]maleimide (BIPM), by pretreatment of N-ethylmaleimide. The preparation thus obtained retained 100% of initial Na+,K+-ATPase activity and contained 1 BIPM residue/alpha-chain, and it showed almost 2-fold larger fluorescence changes accompanying ATP hydrolysis than the previous preparations which retained 60% of initial activity and contained 3-4 BIPM residues/alpha-chain (Taniguchi, K., Suzuki, K., and Iida, S. (1982) J. Biol. Chem. 257, 10659-10667). Extensive trypsin (Sigma type I) treatment of the new preparation produced mainly two different fluorescent peptide peaks in both ion-exchange and reverse-phase chromatography. Amino acid sequence analysis of both peptides showed that they had the same common sequence, Ser-Tyr-X-Pro-Gly-Met-Gly-Val, except that the larger one contained Ala-Leu next to the Val residue. From the comparison of the amino acid sequence deduced from cDNA from sheep kidney (Shull, G. E., Schwartz, A., and Lingrel, J. B. (1985) Nature 316, 691-695), X was shown to correspond to Cys-964 of the alpha-chain in Na+,K+-ATPase. The data suggest that the microenvironment of the BIPM residue covalently bound to the sulfhydryl group of Cys-964 changes accompanying sequential appearance of reaction intermediates of Na+,K+-ATPase.     

Role of negatively charged phospholipids in highly purified (Na+ + K+)-ATPase from rabbit kidney outer medulla studies on (Na+ + K+)-activated ATPase, XXXIX.

1. The requirement for specific polar head groups of phospholipids for activity of purified (Na+ + K+)ATPase from rabbit kidney outer medulla has been investigated. 2. Comparison of content and composition of phospholipids in microsomes and the purified enzyme indicates that purification leads to an increase in the phospholipid/protein ratio and in phosphatidylserine content. 3. The purified preparation contains 267 molecules phospholipid per molecule (Na+ + K+)-ATPase, viz. 95 phosphatidylcholine, 74 phosphatidylethanolamine, 48 spingomyelin, 35 phosphatidylserine and 15 phosphatidylinositol. 4. Complete conversion of phosphatidylserine into phosphatidylethanolamine by the enzyme phosphatidylserine decarboxylase has no effect on the (Na+ + K+)-ATPase activity of the purified preparation. 5. Complete hydrolysis of phosphatidylinositol by a phospholipase C from Staphylococcus aureus, which is specific for this phospholipid, has no effect on the (Na+ + K+)-ATPase activity. 6. Hydrolysis of 95% of the phosphatidylcholine and 60--70% of the spingomyelin and phosphatidylethanolamine by another phospholipase C (Clostridium welchii) lowers the (Na+ + K+)-ATPase activity by about 20%. 7. Combination of the phospholipid-converting enzymes has the same effect as can be calculated from the effects of the enzymes separately. Only complete conversion of both phosphatidylserine and phosphatidylinositol results in a loss of 44% of the (NA+ + K+)-ATPase activity and 36% of the potassium 4-nitrophenylphosphatase activity. 8. These experiments indicate that there is no absolute requirement for one of the polar head groups, although in the absence of negative charges the activity is lower than in their presence.     

Cyclic AMP-dependent protein kinase phosphorylation of cardiac (Na+ + K+)-ATPases. Effect on calcium binding.

1. Calcium binding to (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) preparations from beef and pig heart preparations of varying degrees of purity was measured. 2. Binding was inhibited by Mg2+, Na+ and K+. Inhibition by Na+ and K+ appeared to be due to an ionic strength effect. 3. Four classes of binding sites were identified with Kd values for calcium of about 0.03, 1, 15 and 200 micrometer. 4. Cyclic AMP-dependent phosphorylation of the enzyme by protein kinase (ATP: protamine O-phosphotransferase, EC 2.7.1.70) had no effect on (Na+ + K+)-ATPase activity. 5. Phosphorylation also had no effect on either Kd or Bmax for calcium binding at any of the four sites whether measured in the presence of absence of NaCl or KCl. 6. It is concluded that previous reports of an effect of phosphorylation on calcium binding to a (Na+ + K+)-ATPase preparation may have been due to the presence of membrane material not directly associated with (Na+ + K+)-ATPase.     

Isolation of transporting plasma membrane vesicles from bovine tracheal epithelium

A method is described for isolating plasma membrane vesicles from bovine tracheal epithelium. The procedure yields highly purified apical membranes which are enriched 19-fold in the marker enzyme, alkaline phosphatase. Contamination of this fraction by other organelles is minimal. Basolateral membranes isolated from the same preparation have a 4-fold enrichment of (Na+ + K+)-ATPase and a 2-fold reduction in alkaline phosphatase specific activity compared to the starting material. Assays of Na+ uptake by the apical membrane vesicles demonstrate their suitability for transport studies. Transport of Na+ into an intravesicular space was demonstrated by (1) a linear inverse correlation between Na+ uptake and medium osmolarity; (2) complete release of accumulated Na+ by treatment with detergent; and (3) a marked temperature-dependence of Na+ uptake rate. Other features of Na+ transport were (1) inhibition by amiloride; (2) insensitivity to furosemide; and (3) anion-dependence of uptake rate with the following selectivity:SCN- greater than Cl- greater than gluconate-.