An enriched preparation of basal-lateral plasma membranes from gastric glandular cells

Summary A procedure is described for the preparation of a membrane fraction enriched in basal-lateral plasma membranes from gastric mucosa. Gastric glands isolated from rabbit were employed as starting material, greatly reducing contamination from nonglandular cell types. The distribution of cellular components during the fractionation procedure was monitored with specific marker enzymes. (Na⁺+K⁺)-ATPase, ouabain-sensitive K⁺-stimulatedp-nitrophenyl-phosphatase and histamine-stimulated adenylate cyclase were used as markers for basal-lateral membranes. These three markers were similarly distributed during both differential and equilibrium density gradient centrifugation. The enriched membrane fraction contained more than 30% of the total initial activities of the three basal-lateral membrane markers which were purified better than 11-fold with respect to protein. (Na⁺+K⁺)-ATPase activity was resolved from the activities of acid phosphatase, pepsin, Mg²⁺-ATPase, cytochromec oxidase, NADPH-cytochromec reductase, glucose-6-phosphatase, (K⁺+H⁺)-ATPase, DNA and RNA.     

Effect of concanavalin A on membrane-bound enzymes from mouse lymphocytes

The ionic influence and ouabain sensitivity of lymphocyte Mg²⁺-ATPase and Mg²⁺-(Na⁺ + K⁺)-activated ATPase were studied in intact cells, microsomal fraction and isolated plasma membranes. The active site of 5′-nucleotidase and Mg²⁺-ATPase seemed to be localized on the external side of the plasma membrane whereas the ATP binding site of (Na⁺ + K⁺)-ATPase was located inside the membrane.Concanavalin A induced an early stimulation of Mg²⁺-ATPase and (Na⁺ + K⁺)-ATPase both on intact cells and purified plasma membranes. In contrast, 5′-nucleotidase activity was not affected by the mitogen. Although the thymocyte Mg²⁺-ATPase activity was 3–5 times lower than in spleen lymphocytes, it was much more stimulated in the former cells (about 40 versus 20 %). (Na⁺ + K⁺)-ATPase activity was undetectable in thymocytes. However, in spleen lymphocytes (Na⁺ + K⁺)-ATPase activity can be detected and was 30 % increased by concanavalin A. Several aspects of this enzymic stimulation had also characteristic features of blast transformation induced by concanavalin A, suggesting a possible role of these enzymes, especially Mg²⁺-ATPase, in lymphocyte stimulation.     

Preparation of renal cortex basal-lateral and brush 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 γ-glutamyltranspeptidase, whereas the basal-lateral membrane preparation was enriched in (Na⁺ + K⁺)-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.     

Cytochemical demonstration of NPPase activity for detecting proton-translocating ATPase of Golgi complex in rat pancreatic acinar cells

Summary An attempt at cytochemical demonstration of acidification proton-translocating ATPase (H⁺-ATPase) of Golgi complex in rat pancreatic acinar cells has been made by using p-nitrophenylphosphatase (NPPase) cytochemistry which is used for detecting of Na⁺-K⁺-ATPase (Mayahara et al. 1980) and gastric H⁺-K⁺-ATPase (Fujimoto et al. 1986). K⁺-independent NPPase activity was observed on the membrane of the trans cisternae of Golgi complex, but not inside of cisternae. The localization of NPPase activity is different from that of acid phosphatase activity where reaction products were seen on the inside of the trans Golgi cisternae. Since this activity was insensitive to vanadate, ouabain and independent of potassium ions, it was distinct from plasma membranous ATPases such as Na⁺-K⁺-ATPase and Ca²⁺-ATPase. The K⁺-independent NPPase activity was diminished by the inhibitors of H⁺-ATPase such as N-ethylmaleimide (NEM) and 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). The NPPase reaction products were also seen on the membranes of other acidic organelles, i.e., lysosomes, endosomes, autophagosomes and coated vesicles. These results suggest that NPPase activity on the membrane of the Golgi complex and other acidic organelles corresponds with H⁺-ATPase which plays a role in acidification.     

Physicochemical approaches to the alcohol-membrane interaction in brain

The effects of ethanol on physicochemical and enzymatic perturbations of neuronal membranes were examined. Using synaptic plasma membrane (SPM) isolated from cerebral cortex of Sprague-Dawley rats, a biphasic mode of action for ethanol was observed with (Na⁺+K⁺)-ATPase, but not with Ca²⁺-ATPase or acetylcholinesterase. (Na⁺+K⁺)-ATPase was found to be more sensitive to low concentration of sodium deoxycholate treatment than Ca²⁺-ATPase. A sharp transition break of (Na⁺+K⁺)-ATPase activity in response to temperature changes was found with SPM preparation. Arrhenius plots of the response also indicated that (Na⁺+K⁺)-ATPase is more sensitive to temperature changes than Ca²⁺-ATPase. The fluorescence polarization of TNS-membrane complex decreases as ethanol concentration increases, indicating an increase in membrane fluidity. However, ethanol, at low concentration (<0.3%) appears to elevate TNS fluorescence, but a hhigher concentration (3%) ethanol tends to lower the intensity of maximal emission. The results of this study indicate that ethanol may interact with the synaptic plasma membranes and elicit specific biochemical responses depending on the concentration of the alcohol used.     

A mutant of Arabidopsis thaliana partially resistant to fusicoccin has reduced plasma membrane H+-ATPase

5-2 is a mutant of Arabidopsis thaliana which is partially resistant to fusicoccin in vivo. We have analysed fusicoccin binding and the activity and amount of H⁺-ATPase in plasma membrane isolated from mature leaves of the wild type and of mutant 5-2. Fusicoccin binding was similar in plasma membrane from the two genotypes, while H⁺-ATPase activity was markedly (c. 50%) lower in plasma membrane from mutant 5-2 than in that from the wild type. The H⁺-ATPase of mutant 5-2 was activated by fusicoccin as much as that of the wild type. In plasma membrane from mutant 5-2, the amount of immunodetectable H⁺-ATPase, quantified by densitometry of Western blots, was about half that in the wild type. These results indicate that the major defect of mutant 5-2 detectable at the plasma membrane level is a reduction in the amount of H⁺-ATPase.     

Solubilization and reconstitution of ca pump from corn leaf plasma membrane

The Ca²⁺ transport system of corn (Zea mays) leaf plasma membrane is composed of Ca²⁺ pump and Ca²⁺/H⁺ antiporter driven by H⁺ gradient imposed by a H⁺ pump (M Kasai, S Muto [1990] J Membr Biol 114: 133-142). It is necessary for characterization of these Ca²⁺ transporters to establish the procedure for their solubilization, isolation, and reconstitution into liposomes. We attempted to solubilize and reconstitute the Ca²⁺ pump in the present study. A nonionic detergent octaethyleneglycol monododecyl ether (C₁₂E₈) was the most effective detergent for a series of extraction and functional reconstitution of the Ca²⁺ pump among seven detergents examined. This was judged from activities of ATP-dependent ⁴⁵Ca²⁺ uptake into liposomes reconstituted with the respective detergent-extract of the plasma membrane by the detergent dilution method. C₁₂E₈-extract of the plasma membrane was subjected to high performance liquid chromatography using a DEAE anion exchange column. Ca²⁺-ATPase was separated from VO₄³⁻-sensitive Mg²⁺-ATPase. These ATPases were separately reconstituted into liposomes, and their ATP-dependent Ca²⁺ uptake was measured. The liposomes reconstituted with the Ca²⁺-ATPase, but not with the VO₄³⁻-sensitive Mg²⁺-ATPase, showed ATP-dependent Ca²⁺ uptake. Nigericin-induced pH gradient (acid inside) caused only a little Ca²⁺ uptake into liposomes reconstituted with the Ca²⁺-ATPase, suggesting that the Ca²⁺/H⁺ antiporter was not present in the preparation. These results indicate that the Ca²⁺-ATPase actually functions as Ca²⁺ pump in the corn leaf plasma membrane.     

Reversible changes of extracellular pH during action potential generation in a higher plant Cucurbita pepo

A pH-sensitive electrode was applied to measure activity of H⁺ ions in the medium surrounding excitable cells of pumpkin (Cucurbita pepo L.) seedlings during cooling-induced generation of action potential (AP). Reversible alkalization shifts were found to occur synchronously with AP, which could be due to the influx of H⁺ ions from external medium into excitable cells. Ethacrynic acid (an anion channel blocker) reduced the AP amplitude but had no effect on the transient alkalization of the medium. An inhibitor of plasma membrane H⁺-ATPase, N,N’-dicyclohexylcarbodiimide suppressed both the AP amplitude and the extent of alkalization. In experiments with plasma membrane vesicles, the hydrolytic H⁺-ATPase activity was subjected to inhibition by Ca²⁺ concentrations in the range characteristic of cytosolic changes during AP generation. The addition of a calcium channel blocker verapamil and a chelating agent EGTA to inhibit Ca²⁺ influx from the medium eliminated the AP spike and diminished reversible alkalization of the external solution. An inhibitor of protein kinase, H-7 alleviated the inhibitory effect of Ca²⁺ on hydrolytic H⁺-ATPase activity in plasma membrane vesicles and suppressed the reversible alkalization of the medium during AP generation. The results provide evidence that the depolarization phase of AP is associated not only with activation of chloride channels and Cl^? efflux but also with temporary suppression of plasma membrane H⁺-ATPase manifested as H⁺ influx. The Ca²⁺-induced inhibition of the plasma membrane H⁺-ATPase is supposedly mediated by protein kinases.     

Characterization of native and reconstituted plasma membrane H+-ATPase from the plasma membrane ofBeta vulgaris

Summary Characteristics of the native and reconstituted H⁺-ATPase from the plasma membrane of red beet (Beta vulgaris L.) were examined. The partially purified, reconstituted H⁺-ATPase retained characteristics similar to those of the native plasma membrane H⁺-ATPase following reconstitution into proteoliposomes. ATPase activity and H⁺ transport of both enzymes were inhibited by vanadate, DCCD, DES and mersalyl. Slight inhibition of ATPase activity associated with native plasma membranes by oligomycin, azide, molybdate or NO ₃ ^– was eliminated during solubilization and reconstitution, indicating the loss of contaminating ATPase activities. Both native and reconstituted ATPase activities and H⁺ transport showed a pH optimum of 6.5, required a divalent cation (Co²⁺>Mg²⁺>Mn²⁺>Zn²⁺>Ca²⁺), and preferred ATP as substrate. The Mg:ATP kinetics of the two ATPase activities were similar, showing simple Michaelis-Menten kinetics. Saturation occurred between 3 and 5mM Mg: ATP, with aK _m of 0.33 and 0.46mM Mg: ATP for the native and reconstituted enzymes, respectively. The temperature optimum for the ATPase was shifted from 45 to 35°C following reconstitution. Both native and reconstituted H⁺-ATPases were stimulated by monovalent ions. Native plasma membrane H⁺-ATPase showed an order of cation preference of K⁺>NH ₄ ⁺ >Rb⁺>Na⁺>Cs⁺>Li⁺>choline⁺. This basic order was unchanged following reconstitution, with K⁺, NH ₄ ⁺ , Rb⁺ and Cs⁺ being the preferred cations. Both enzymes were also stimulated by anions although to a lesser degree. The order of anion preference differed between the two enzymes. Salt stimulation of ATPase activity was enhanced greatly following reconstitution. Stimulation by KCl was 26% for native ATPase activity, increasing to 228% for reconstituted ATPase activity. In terms of H⁺ transport, both enzymes required a cation such as K⁺ for maximal transport activity, but were stimulated preferentially by Cl^– even in the presence of valinomycin. This suggests that the stimulatory effect of anions on enzyme activity is not simply as a permeant anion, dissipating a positive interior membrane potential, but may involve a direct anion activation of the plasma membrane H⁺-ATPase.     

Isolation and characterization of cardiac sarcolemma

A procedure was developed for the isolation of cardiac sarcolemmal vesicles. These vesicles are enriched about ten-fold (with respect to the tissue homogenate) in K⁺-stimulated p-nitrophenylphosphatase, (Na⁺ + K⁺)-ATPase, 5'-nucleotidase activities and sialic acid content, all of which are believed to be components of the sarcolemma. The sarcolemma of tissue culture cardiac cells were radioiodinated and the distribution of this radioiodine paralleled the distribution of the other membrane markers above. There was very little contamination of the sarcolemmal fraction by sarcoplasmic reticulum (as judged by Ca²⁺-ATPase and glucose-6-phosphatase activities) or inner mitochondrial membranes (as judged by succinate dehydrogenase activity). There may, however, be some contamination by outer mitochondrial membranes (as judged by monoamine oxidase and rotenone-insensitive NADH cytochrome c reductase activities) which have rarely been monitored in cardiac sarcolemmal preparations. The purity of this preparation is good when compared with other cardiac sarcolemmal preparations. This preparation should be very useful in studying the roles of the cardiac sarcolemma (e.g. in excitation contraction coupling and Ca²⁺ binding).     

Subfractionation of rat liver plasma membrane: Uneven distribution of plasma membrane-bound enzymes on the liver cell surface

Plasma membranes were islotaed from rat liver mainly under isotonic conditions. As marker enzymes for the plasma membrane, 5′-nucleotidase and (Na⁺+K⁺)-ATPase were used. The yield of plasma membrane was 0.6–0.9 mg protein per g wet weight of liver. The recovery of 5′-nucleotidase and (Na⁺+K⁺)-ATPase activity was 18 and 48% of the total activity of the whole-liver homogenate, respectively. Judged from the acitvity of glucose-6 phosphatase and succinate dehydrogenase in the plasma membrane, and from the electron microscopic observation of it, the contamination by microsomes and mitochondria was very low. A further homogenization of the plasma membrane yielded two fractions, the light and heavy fractions, in a discontinuous sucrose gradient centrifugation. The light fraction showed higher specific activities of 5′-nucleotidase, alkaline phosphatase, (Na⁺+K⁺)-ATPase and Mg²⁺-ATPase, whereas the heavy one showed a higher specific activity of adenylate cyclase. Ligation of the bile duct for 48 h decreased the specific activities of (Na⁺+K⁺)-ATPase and Mg²⁺-ATPase in the light fraction, whereas it had no significant influence on the activities of these enzymes in the heavy fraction. The specific activity of alkaline phosphatase was elevated in both fractions by the obstruction of the bile flow. Electron microscopy on sections of the plasma membrane subfractions showed that the light fraction consisted of vesicles of various sizes and that the heavy fractions contained membrane sheets and paired membrane strips connected by junctional complexes, as well as vesicles. The origin of these two fractions is discussed and it is suggested that the light fraction was derived from the bile front of the liver cell surface and the heavy one contained the blood front and the lateral surface of it.     

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.     

Body Plasma Membrane Vesicles from Paramecium Contain a Vanadate-Sensitive Ca--ATPase

Paramecia are an excellent model system for studying the mechanisms involved in sensory transductions and intracellular Ca²⁺ regulation. These cells have two functionally distinct plasma membrane domains, body and cilia. The body plasma membrane is responsible for transduction of sensory stimuli into receptor potentials and the ciliary membrane is required for Ca²⁺ action potentials. Although ciliary membrane vesicles (cmv) have been purified and well characterized, body plasma membranes have not. We have generated body plasma membrane vesicles (bmv) by homogenization of deciliated cells and purified them from the microsome fraction by a two-phase aqueous polymer separation. The major criteria for purity of the bmv fraction are: (i) It is enriched 15-fold for a known plasma membrane marker (immobilization antigen) while the marker activities for other membranes were all decreased. The protein banding pattern of bmv is generally similar to cmv on SDS-PAGE. (ii) It contains a vanadate-sensitive Ca²⁺-ATPase activity that has been suggested to be a plasma membrane Ca²⁺ pump. The specific activity of this bmv Ca²⁺-ATPase is increased 4-fold over that of the homogenate. (iii) The phospholipid, fatty acid, and sterol composition of the bmv fraction are indicative of plasma membranes because they are qualitatively similar to cmv. The bmv also contains a membrane-bound NADPH-dependent cytochrome c reductase activity, suggesting that it may play a role in body plasma membrane function. This purified bmv preparation is useful for studying the role of the body plasma membrane in Ca²⁺ regulation, sensory transduction, protein and lipid trafficking, and plasma membrane fusion events.     

Calix[4]arene C-90 selectively inhibits Ca2+,Mg2+-ATPase of myometrium cell plasma membrane

The supramolecular compound calix[4]arene C-90 (5,11,17,23-tetra(trifluoro)methyl(phenylsulfonylimino)-methylamino-25,26,27,28-tetrapropoxycalix[4]arene) is shown to efficiently inhibit the ATP hydrolase activity of Ca²⁺,Mg²⁺-ATPase in the myometrium cell plasma membrane fraction and also in a preparation of the purified enzyme solubilized from this subcellular fraction. The inhibition coefficient I _0.5 values were 20.2 ± 0.5 and 58.5 ± 6.4 μM for the membrane fraction and the solubilized enzyme, respectively. The inhibitory effect of calix[4]arene C-90 was selective comparatively to other ATPases localized in the plasma membrane: calix[4]arene C-90 did not influence the activities of Na⁺,K⁺-ATPase and “basal” Mg²⁺-ATPase. The inhibitory effect of calix[4]arene C-90 on the Ca²⁺,Mg²⁺-ATPase activity was associated with the cooperative action of four trifluoromethylphenyl sulfonylimine (sulfonylamidine) groups oriented similarly on the upper rim of the calix[4]arene macrocycle (the calix[4]arene “bowl”). The experimental findings seem to be of importance for studies, using calix[4]arene C-90, of membrane mechanisms of regulation of calcium homeostasis in smooth muscle cells and also for investigation of the participation of the plasma membrane Ca²⁺-pump in control of electro- and pharmacomechanical coupling in myocytes.     

Ca2+-dependent ATPase associated with plasma membrane from a calcareous alga, Serraticardia maxima (Corallinaceae, Rhodophyta)

The plasma membrane was isolated from a calcareous red alga, Serraticardia maxima (Yendo) Silva (Corallinaceae), by aqueous two-phase partitioning. Its purity was examined with marker enzymes, Mg²⁺-dependent ATPase, inosine diphosphatase, cytochrome c oxidase and NADH-cytochrome c reductase, as well as the sensitivity of Mg²⁺-dependent ATPase to vanadate, azide and nitrate. The results showed that the isolated plasma membrane was purified enough to study its functions. Electron microscopic observations on thin tissue sections revealed that most vesicles of the isolated plasma membrane were stained by the plasma membrane specific stain, phosphotungstic acid-chromic acid. Mg²⁺- or Ca²⁺-dependent ATPases were associated with the plasma membrane. Ca²⁺-dependent ATPase was activated at physiological cytoplasmic concentrations of Ca²⁺ (0.1–10 μmol/L). However, calmodulin (0.5 μmol/L) did not affect its activity. The pH optimum was 8.0, in contrast to 7.0 for Mg²⁺-dependent ATPase. The isolated plasma membrane vesicles were mostly right side-out. To test for H⁺-translocation, right side-out vesicles were inverted; 27% of vesicles were inside-out after treatment with Triton X-100. The inside-out plasma membrane vesicles showed reduction of quinacrine fluorescence in the presence of 1 mmol/L ATP and 100 μmol/L Ca²⁺. The reduced fluorescence was recovered with the addition of 10 mmol/L NH₄Cl, or 5 μmol/L nigericin plus 50 mmol/L KCl. UTP and CTP substituted for ATP, but ADP did not. Ca²⁺-dependent ATPase might pump H⁺ out in the physiological state. The acidification by this pump might be coupled with alkalinization at the calcifying sites, which induces calcification.     

Plasma membranes from fibroblastic cells in culture isolation,morphological and enzymatic identification

Plasma membranes were isolated from murine plasmocytoma cells in culture, by a procedure involving lysis in hypoosmotic medium leaving the nuclei intact, and separation of surface membranes from the lysate constituents on a discontinuous sucrose gradient.The purity of the fractions was assessed by electron microscopy and by assaying enzymes for cross-contaminants. Phosphohydrolases, including the (Na⁺ + K⁺)-stimulated Mg²⁺-ATPase (EC 3.6.1.3) and 5′-nucleotidase (EC 3.1.3.5), were concentrated in the plasma membrane-rich fractions. These fractions were essentially free from NADH: cytochrome c reductase, lysosomes and mitochondrial membrane enzymes.     

Boric acid stimulates the plasma membrane H+-ATPase of ungerminated lily pollen grains

The stimulation of the plasma membrane (PM) H⁺-ATPase by boric acid was studied on a microsomal fraction (MF) obtained from ungerminated, boron-dependent pollen grains of Lilium longiflorum Thunb. which usually need boron for germination and tube growth. ATP hydrolysis and H+ transport activity increased by 14 and 18%, respectively, after addition of 2-4 mM boric acid. The optimum of boron stimulation was at pH 6.5-8.5 for ATP hydrolysis and at pH 6.5-7.5 for H⁺ transport. No boron stimulation was detected when vanadate was added to the MF, whereas an increase of 10-20% in ATP hydrolysis and H⁺ transport was still measured in the presence of inhibitors specific for V -type ATPase (nitrate and bafilomycin) and F-type ATPase (azide), respectively. A vanadate-sensitive increase in ATP hydrolysis activity was also observed in partially permeabilized vesicles (0.001%[w/v] Triton X-100) suggesting a direct interaction between borate and the PM H⁺-ATPase rather than a weak acid-induced stimulation. Additionally, we measured the effect of boron on membrane voltage (V_m) of ungerminated pollen grains and observed small hyperpolarizations in 48% of all experiments. Exposing pollen grains to a more acidic pH of 4 caused a depolarization, followed in some experiments by a repolarization (21%). In the presence of 2 mM boron such hyperpolarizations, perhaps caused by an enhanced activity of the H⁺-ATPase, were measured in 58% of all tested pollen grains. The effects of boron on V_m may be reduced by additional stimulation of a K⁺ inward current of opposite direction to the H⁺-ATPase. All experiments indicate that boron stimulates an electrogenic transport system in the plasma membrane which is sensitive to vanadate and has a pH optimum around 7, i.e. the plasma membrane H⁺-ATPase. A boron-increased PM H⁺-ATPase activity in turn may stimulate germination and growth of pollen tubes.     

两相法分离纯化橡胶树树皮质膜

橡胶树树皮质膜H~+-ATPase在橡胶树产排胶过程中扮演着重要角色,制备高纯度及高活性的质膜是研究质膜H~+-ATPase特性和功能的必要条件。该研究以一年生巴西橡胶树(Hevea brasiliensis)树皮为材料,利用差速离心法获得粗膜微粒体,通过两相分配法分离纯化质膜,并研究两相体系中不同浓度聚合物(5.9%、6.1%、6.3%、6.5%、6.7%,W/W)和KCl(2、5、8、11、14 mmol·L~(-1))对质膜蛋白得率和纯化效率的影响。通过Bradford法对质膜蛋白得率进行检测,同时采用酶活性检测法对质膜纯度进行检测,分析结果表明选用6.4%(W/W)聚合物浓度和5mmol·L~(-1)KCl组成的两相体系可获得较高纯度和得率的橡胶树树皮质膜。通过电镜观察法在形态学上对质膜纯度进一步评价,利用铅铀能侵染全部膜组分使其染色,而磷钨酸只能专一性地侵染质膜并使其染色这一特性,分别使用铅铀和磷钨酸对切片进行染色,并通过透射电镜对切片染色程度进行直接观察,结果表明提取的粗膜微粒体中质膜组分较少,存在大量的细胞器膜污染,而纯化后的质膜膜组分较单一,其他膜组分污染较少,而且质膜大小较均一,可以用于进行后续橡胶树树皮质膜H~+-ATPase特性和功能的研究。     

Some in vivo and in vitro effects of ethacrynic acid on renal Na+,K+ -ATPase

Binding of [¹⁴C]ethaerynic acid [EA]at concentrations of EA from 10^?4m to 10^?2m to a membrane preparation containing Na⁺,K⁺-ATPase activity in vitro occurred in a nonsaturable manner; binding was stimulated by Na⁺ or K⁺, but was not affected by Mg²⁺ and/or ATP. [¹⁴C]EA significantly bound to a microsomal preparation with low Na⁺,K⁺-ATPase activity as well as to a heat-denatured enzyme; this binding reaction was not stimulated by Na⁺. These observations suggest that EA binds non-specifically or to nonspecific sites on membrane preparations. Nonselective binding of [¹⁴C]EA to subcellular particles after fractionation of slices also suggested the presence of nonspecific EA binding sites in vivo. In vitro [³H]ouabain binding to medullary and cortical Na⁺,K⁺-ATPase preparations was partially reduced by pretreatment with EA. On the other hand, [¹⁴C]EA binding to Na⁺,K⁺-ATPase was not affected by pretreatment of the preparation with ouabain (10^?6m to 5 × 10^?4m). EA reduced the sensitivity of [³H]ouabain binding to the enzyme preparation to Na⁴ and K⁺.EA was infused (0.1, 1.0, and 10 mg/min) into one renal artery of hydropenic dogs. A prompt natriuresis in the infused kidney occurred. Similar changes were observed in the contralateral kidney 20 min after starting the infusion. Both kidneys were removed 30 min after the beginning of the infusion, and Na⁺,K⁺-ATPase was isolated from the cortex and the medulla. Enzyme activity from cortex and medulla of either kidney was not significantly different from enzyme activity from cortex and medulla of control, uninfused dogs, regardless of dose of EA or method of enzyme isolation. Furthermore, in vitro binding of [³H]ouabain to Na⁺,K⁺-ATPase membrane preparations from cortex and medulla was the same for experimental and control kidneys. In vitro incubation of 2 × 10^?3m EA with a membrane preparation caused the same inhibition of ATPase activity when the enzyme was isolated either from control or EA-infused dogs. The inhibition could not be reversed by recentrifugation or rehomogenization of the enzyme. Our results do not support the concept that Na⁺,K⁺-ATPase is a pharmacological receptor for ethacrynic acid.     

The plasma membrane (Mg2+)-dependent adenosine triphosphatase from the human erythrocyte is not an ion pump

Summary The plasma membrane (Mg²⁺)-dependent adenosine triphosphatase ((Mg²⁺)-ATPase) from human erythrocytes has been tested for its ability to transport ions. Using a preparation of inside-out vesicles loaded with the pH-sensitive fluorescence probe 1-hydroxypyrene-3,6,8-trisulfonic acid (HPTS), we have demonstrated the absence of proton movement during (Mg²⁺)-ATPase activity. From the rate of ATP hydrolysis and the passive proton permeability of these vesicles, an upper limit of 0.03 H⁺ transported per ATP hydrolyzed was calculated. To verify that proton pumping could be detected in this system, the intravesicular pH was monitored during (Ca²⁺)-dependent adenosine triphosphatase ((Ca²⁺)-ATPase) activity. Proton efflux associated with (Ca²⁺)-ATPase activity was observed (in agreement with a recent report of proton pumping by a reconstituted erythrocyte (Ca²⁺)-ATPase (Niggli, V., Sigel, E., Carafoli, E. (1982)J. Biol. Chem. 257:2350–2356)) and was shown to be stimulated by calmodulin. The ability of the (Mg²⁺)-ATPase to pump²⁸Mg²⁺,³⁵SO ₄ ^2– and⁸⁶Rb⁺ was also tested, with the results leading to the conclusion that the human erythrocyte enzyme does not function as an ion transport system.