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1.
Abstract– Ca2+-stimulated ATPase activity was studied in membrane enriched preparations from the brains of audiogenic seizure-prone (DBA) and control (C57 and C3H) mice. The animals ranged in age from 7 to 60 days. Na+, K + -ATPase, 5'-nucleotidase and p -nitrophenylphosphatase were assayed to evaluate membrane integrity.
Ca2+-ATPase was significantly lower in DBA mice; notably during the period of maximal seizure sensitivity. Mg2+ -ATPase somewhat followed the pattern shown by Ca2+ -ATPase. Na+, K+ -ATPase in DBA did not differ significantly from controls and there were no differences in either 5'-nucleotidase or p-nitrophenylphosphatase activities.
Ca2+-ATPase kinetics experiments showed even more clearly the difference between DBA and control preparations. Vmax was consistently lower in DBA than in controls. The Km values appeared to fall into groupings suggestive of sequential synthesis of isozymes. Differences in the patterns of DBA and C57 just prior to the time of maximal seizure sensitivity are interpreted as reflecting failure to synthesize an isozyme or delay of its synthesis. The genesis of seizures through such an enzymatic defect may be related to the action of translocated ATP on the plasma membrane.  相似文献   

2.
Abstract— The effects of two sulfhydryl reagents, PCMBS ( p -chloromercuribenzene sulfonic acid) and NEM ( N -ethylmaleimide) on microtubule-associated Mg2+ -and Ca2+ -ATPase activity were studied in a MTP (microtubule proteins) preparation and in a MAP (microtubule-associated proteins) fraction. In the MTP preparation at pH 6.8, PCMBS stimulated the Mg2+ -ATPase activity at low concentrations and inhibited at higher, whereas the Ca2+ -ATPdse activity was only inhibited. NEM affected the activity in a similar way. At pH 8.0 PCMBS was only inhibitory. NEM showed stimulatory effects over a broader concentration range.
Preincubation in the presence of ATP counteracted the stimulatory effects of both PCMBS and NEM on Mg2+ -ATPase at pH 6.8.
In the MAP fraction at pH 6.8 PCMBS and NEM caused similar but less pronounced effects on the Mg2+ -and Ca2+ -ATPase.
The results show that brain microtubule-associated ATPase activity is similar to dynein and myosin ATPases with respect to biphasic alteration by sulfhydryl reagents.  相似文献   

3.
Abstract— The hypothesis that the ATPase and phosphatidyhnositol (PI) kinase activities of chromaffin vesicle membranes are catalysed by same enzyme was investigated. The two activities exhibited entirely different responses to variations in Mg2+ or Mn2+ concentrations. In the presence of 1 mM ATP, maximal ATPase activity occurred with 1 mM Mg2+ while maximal PI kinase activity required 100 mM Mg2+ Similar differences were observed with Mn2+ with the exception that maximal ATPase activity occurred with 0.5 mM Mn2+ and maximal PI kinase activity occurred with 5 mM Mn2+ Mn2+ was more effective than Mg2+ in stimulating PI kinase activity at low concentrations, but at optimal concentrations of each, the maximal activity obtained with Mg2+ was 5-fold greater than the maximal activity obtained with Mn2+ The heat stabilities of the two enzymes are vastly different. At 50°C the ATPase activity of the intact membranes was stable for up to 20 min while the t l/2 of PI kinase was less than 2 min. After solubilization in Lubrol PX or at higher temperatures both enzymes were less heat stable, but PI kinase was still inactivated at a much greater rate than the ATPase. The evidence suggests that the ATPase and the PI kinase are different proteins.
The major phosphorylated product was diphosphatidylinositol and once formed, it was stable. Phosphorylation of membrane protein accounted for less than 10% of the total 32P-incorporated into chromaffin vesicles. SDS gel electrophoresis of the solubilized membranes showed the presence of at least 2 major phosphorylated high molecular weight components.  相似文献   

4.
Abstract— The effects of 10−5 m -noradrenaline (NA), 5-hydroxytryptamine (5-HT) and dopamine (DA) on the activities of Na+-K+ ATPase (EC 3.6.1.3) were studied in synaptic membranes from 6 regions of the rabbit brain. NA and 5-HT stimulated the synaptic membrane Na+-K+ ATPase from the cerebrum, but none of the amines influenced the activity of this enzyme in the other brain regions. The Na+-K+ ATPase activity of the cerebral synaptic membrane isolated at the 0.8/0.9 m & 0.9/1.0 m interphase of a sucrose density gradient was increased two-fold by 10−5 m -NA and 5-HT. The Na+-K + ATPase recovered at the 1.0/1.2 m interphase was not influenced by NA, DA or 5-HT. NA, DA and 5-HT did not activate the Mg ATPase of synaptic membranes from any of the 6 brain regions or whole brain synaptic vesicles. The cortex synaptic membrane (Na+-K+) ATPase is postulated to have a direct role in the uptake of the biogenic amines. An indirect role is proposed for this enzyme in amine uptake into brain stem.  相似文献   

5.
Plasma membranes of the marine cyanobacterium Spirulina subsalsa were tested for ATPase activity, and for involvement in salt stress. Transition of cells from saline to hypersaline medium enhances the respiratory activity associated with extrusion of Na+ and Cl, and persisting salt stress induces synthesis of respiratory enzymes in the plasma membranes. The membranes possess an ATPase, specific for ATP and Mg2+ and sensitive to orthovanadate and dicyclohexylcarbodiimide. Immunoblot analysis of plasma membrane polypeptides from Spirulina subsalsa with anti- Arabidopsis H+-ATPase serum identified a single polypeptide of 100 kDa, which cross-reacted with the antibodies. An unusual feature of this ATPase is a specific stimulation by Na+ ions. Prolonged adaptation of S. subsals cells to hypersaline conditions induced an increase in ATPase activity in subsequent plasma membrane preparations, as well as a higher content of the 100 kDa polypeptide. It is suggested that the ATPase investigated is an H+-pump, which is involved in extrusion of Na+ and in conferring resistance to salt stress.  相似文献   

6.
Plasma membrane vesicles were purified from 8-day-old oat ( Avena sativa L. cv. Brighton) roots in an aqueous polymer two-phase system. The plasma membranes possessed high specific ATPase activity [ca 4 μmol P1 (mg protein)−1 min−1 at 37°C]. Addition of lysophosphatidylcholine (lyso-PC) produced a 2–3 fold activation of the plasma membrane ATPase, an effect due both to exposure of latent ATP binding sites and to a true activation of the enzyme. Lipid activation increased the affinity for ATP and caused a shift of the pH optimum of the H+ -ATPase activity to 6.75 as compared to pH 6.45 for the negative H+-ATPase. Activation was dependent on the chain length of the acyl group of the lyso-PC, with maximal activition obtained by palmitoyl lyso-PC. Free fatty acids also activated the membrane-bound H+-ATPase. This activation was also dependent on chain length and to the degree of unsaturation, with linolenic and arachidonic acid as the most efficient fatty acids. Exogenously added PC was hydrolyzed to lyso-PC and free fatty acids by an enzyme in the plasma membrane preparation, presumably of the phospholipase A type. Both lyso-PC and free fatty acids are products of phospholipase A2 (EC 3.1.1.4) action, and addition of phospholipase A2 from animal sources increased the H+-ATPase activity within seconds. Interaction with lipids and fatty acids could thus be part of the regulatory system for H+-ATPase activity in vivo, and the endogenous phospholipase may be involved in the regulation of the H+-ATPase activity in the plasma membranne.  相似文献   

7.
Red beet ( Beta vulgaris L., cv. Detroit Dark Red) plasma membrane ATPase solubilized from a deoxycholate-extracted plasma membrane fraction with Zwittergent 3–14 was reconstituted into liposomes. Detergent removal and reconstitution was carried out by column chromatography on Sephadex G-200 followed by centrifugation at 100 000 g for I h. Prior to reconstitution, optimal activity in the solubilized preparation was observed when dormant red beet tissue was used in the extraction/solubilization procedure. Following reconstitution into liposomes, ATP-dependent proton transport could be demonstrated by measuring the quenching of acridine orange fluorescence. Proton transport and ATPase activity in the reconstituted enzyme preparation were inhibited by orthovandate but stimulated by KNO3. This stimulation most likely results from a reduction in the membrane potential generated during electrogenic proton transport by the reconstituted ATPase. The ATPase activity of the reconstituted ATPase was further characterized and found to have a pH optimum of 6.5 in the presence of both Mg2+ and K+. The activity was specific for ATP, insensitive to ouabain and azide but inhibited by N;N-dicyclohexylcarbodiimide and diethylstilbestrol. Stimulation of ATP hydrolytic activity occurred in the sequence: K+ Rb+ Na+ Cs+ Li+ and the kinetics of K+ stimulation of ATPase activity followed non-Michaelis-Menten kinetics as observed for both the membrane-bound and solubilized forms of the enzyme. Reconstitution of the plasma membrane ATPase from red beet allowed a substantial purification of the enzyme and resulted in the enrichment of a 100 kDa polypeptide representing the ATPase catalytic subunit.  相似文献   

8.
The effect of Mg2+, Na+, K+, ouabain and pH on ATPase activity of purified membrane fractions enriched in plasmalemma fragments from Hordeum vulgare L. (glycophyte) and Halocnemum strobilaceum L. (halophyte) was studied. Membrane ATPases from both plants were synergistically activated by K+ and Na+ in the presence of Mg2+. The maximum activity of the enzymes were observed at the ratio Na/K = 2–3. Ouabain (10-4 M) almost completely eliminated the (Na++ K+)-stimulated component of the ATPase activity. The Na, K, Mg-ATPase of Hordeum had a single pH optimum (pH 8), but that of the Halocnemum had two optima(pH 6 and 8). It appears that similar enzymes operate in the cells of both plants studied. The higher Na, K, Mg-ATPase activity of the halophyte compared to that of the glycophyte suggests the involvement of the enzyme in the extrusion of Na+ from the cytoplasm of cells of both plants.  相似文献   

9.
Coccolithophorids are marine unicellular algae characterized by their ability to carry out controlled, subcellular calcification. The biochemical and kinetic features of membrane-bound Ca2+-stimulated ATPases have been examined. Membranes and organelles from axenic cultures of Pleurochrysis sp. (CCMP299) were isolated by means of sucrose density centrifugation. High levels of Ca2+-stimulated ATPase were detected in chloroplasts, Golgi apparatus, plasma membrane, and coccolith vesicles. The sensitivity of the enzyme activity in the organelles and membranes was assessed with pharmacologic agents that are known to be specific for the several isoforms of Ca2+-stimulated ATPase. The Ca2+-stimulated ATPase activity in the Golgi and coccolith vesicle preparations was sensitive to nitrate, thiocyanate, and sodium azide and insensitive to vanadate, cyclopiazonic acid, and thapsigargin. ATP-dependent H+ movement, but not 45Ca2+ transport, across the coccolith vesicle was demonstrated. The Ca2+-stimulated ATPase in the plasma membrane preparation was sensitive to vanadate. ATP-dependent, vanadate-sensitive efflux of 45Ca2+ was demonstrated for microsomal material derived from gradient-isolated plasma membrane. Polypeptides from isolated Golgi and coccolith vesicle preparations cross-reacted to an antibody raised against a subunit of the oat root proton pump, whereas polypeptides from the chloroplast preparations did not cross-react. These findings show that a V-type Ca2+-stimulated ATPase is located on the coccolith vesicle membrane and a P-type Ca2+-stimulated ATPase is located on the plasma membrane.  相似文献   

10.
To better understand migratory divergences among Anguilla anguilla glass eels, the behaviour of individuals caught at the time of their estuary entrance was studied through their response to a light:dark cycle and then to both water current reversal and light:dark cycle. In a first experiment, fish moving with the flow in response to dusk (M+ fish) and fish that had not exhibited any movement (M fish) were distinguished. Anguilla anguilla from these two groups were then individually marked and their response to water current reversal compared. M+ individuals mainly exhibited negative rheotaxis with a tidal periodicity, whereas positive rheotaxis was mainly exhibited by M individuals. Thus, M+ A. anguilla glass eels showing negative rheotaxis appear to have the strongest propensity to migrate, the converse applies to M ones showing positive rheotaxis. A small percentage of individuals (5%) were hyperactive, alternately swimming with and against the current with almost no resting phase. These fish lost c . 2 mg wet mass day−1, whereas individuals which were almost inactive lost c . 1 mg day−1. Wet and dry mass changes in relation to activity levels were compared with previous experiments and it was concluded that A. anguilla glass eel energy status might be involved in differences in migratory tendencies but other factors that might be important are discussed. It is proposed that any decrease in A. anguilla glass eel energy stores associated with global warming might lead to an increase in the proportion of sedentary individuals and thus be involved in the decrease in the recruitment to freshwater habitats.  相似文献   

11.
Insertional mutagenesis was used to identify genes involved in mating and/or zygote formation in the unicellular green alga Chlamydomonas reinhardtii Dangeard. Approximately 800 insertionally mutagenized transformants were examined, and a single nonagglutinating mutant was identified. Plasmid rescue was used to clone a genomic fragment containing transforming DNA. This fragment was then used to identify the wild-type copy of the gene disrupted during mutagenesis. The wild-type gene is transcribed during all stages of the life cycle and, based on sequence similarity, encodes a P2-type proton transporting ATPase. The gene is referred to as Pmh1 for plasma membrane H + -ATPase. PMH1 displays the greatest sequence similarity to ATPases from two parasitic flagellates and a raphidophytic alga but not to the ATPase from a closely related green alga. We propose that PMH1 represents a distinct H + -ATPase isoform expressed in flagellates.  相似文献   

12.
CATION MODULATION OF SYNAPTOSOMAL RESPIRATION   总被引:16,自引:14,他引:2  
Abstract— Synaptosomes were prepared from the cerebral cortex of the adult rat by a rapid technique, involving the use of centrifugation in a Ficoll-sucrose discontinuous gradient. Adequate respiratory control ratios were obtained with glutamate and succinate plus rotenone. The addition of Na+ to the incubation medium stimulated synaptosomal, State-4 respiration, with a half-maximal response at 15 mM Na+. The stimulation by Na+ was inhibited by atractylate, oligomycin, ouabain or EDTA. A cooperative interaction between Na+ and low concentrations of Mg2+ was observed. A significant proportion (39 per cent) of the total Na-K ATPase (EC 3.6.1.4) activity in the discontinuous gradient was localized in the synaptosomal fraction. In the absence of exogenous Mg2+, Na+ induced a 64 per cent stimulation of the synaptosomal ATPase activity which was sensitive to ouabain. Such stimulation of ATP hydrolysis would account for the formation of increased amounts of ADP, with consequent recycling to ATP through adequately controlled oxidative phosphorylation. These observations demonstrate a significant role for transmembrane cationic gradients in the control of synaptosomal respiration and mitochondrial oxidative phosphorylation. The preparation exhibits moderate respiratory control and should prove useful in studies of integrated mitochondrial oxidative metabolism and neuronal membrane function.  相似文献   

13.
As water and nutrient uptake should be related in the response of plants to salinity, the aim of this paper is to establish whether or not aquaporin functionality is related to H+-ATPase activity in root cells of pepper ( Capsicum annuum L.) plants. Thus, H+-ATPase activity was measured in plasma membrane vesicles isolated from roots and aquaporin functionality was measured using a cell pressure probe in intact roots. Salinity was applied as 60 m M NaCl or 60 m M KCl, to determine which ion (Na+, K+ or Cl) is producing the effects. We also investigated whether the effects of both salts were ameliorated by Ca2+. Similar results were obtained for cell hydraulic conductivity, Lpc, and H+-ATPase activity, large reductions in the presence at NaCl or KCl and an ameliorative effect of Ca2+. However, fusicoccin (an activator of H+-ATPase) did not alter osmotic water permeability of protoplasts isolated from roots. Addition of Hg2+ inhibited both ATPase and aquaporins, but ATPase also contains Hg-binding sites. Therefore, the results indicate that H+-ATPase and aquaporin activities may not be related in pepper plants.  相似文献   

14.
Abstract. An investigation has been made of methods for isolating membrane vesicles from corn ( Zea mays L.) roots active in calcium transport and K+-stimulated ATPase. Pretreating and grinding the roots at room temperature with EGTA and fusicoccin increases basal ATPase activity. Improvement in Ca2+ uptake requires isolation of a scaled vesicle fraction by the method of Sze(1980). Sorbitol is superior to sucrose as an osmoticant. The pH optimum for Ca2+ uptake is 7.5. whereas that for associated ATPase activity is 6.5. Calmodulin strongly stimulates Ca2+ uptake in a process little affected by uncouplers and ATPase inhibitors, but blocked by chlorpromazine. Fusicoccin gives less stimulation of Ca2+ uptake which is sensitive to uncouplers, and is dependent upon isolation with fusicoccin present. It appears that the sealed vesicle fraction may possess two Ca2+ transport systems: a calmodulin-activated Ca2+-transporting ATPase, and a Ca2+/H+ antiport coupled through the protonmotive force to a fusicoccin-stimulated H+-ATPase.  相似文献   

15.
Abstract Monoclonal antibodies (mAbs) have been produced by immunizing BALB/C mice with whole M+ bacteria in incomplete Freund adjuvant and the resulting mAbs for M3 protein have been selected by an indirect immuno-fluorescent technique using formaldehyde-fixed M+ and M bacteria. Four mAbs reacted with a 65 kDa protein in an extract obtained from the cell wall of M+ bacteria after treatment with N -acetyl muramidase and lysozyme. The purified 65 kDa protein neutralized the phagocytic activity of rabbit anti-M3 antibody. The N-terminal amino acid sequence of the 65 kDa protein was identical with that of protein generated by the M3 gene which has been previously cloned and sequenced. The evidence indicates that the 65 kDa protein is M3 protein. The M3 protein bound not only human fibrinogen but also human serum albumin (HSA). When the M3 protein was purified by gel-filtration and ion-exchange chromatography in the absence of phenylmethyl sulfonyl fluoride (PMSF), four fragments (35 kDa, 32 kDa, 30 kDa, and 25 kDa) in addition to the intact molecule appeared. N-terminal amino acid sequence analysis showed that 35 kDa and 25 kDa fragments were ANAAD and DARSV, respectively, being identical at positions 1–5 and 198–202 to the M3 gene derived protein. Therefore, the 35 kDa and 25 kDa fragments, which were presumed to be cleavage products, may be derived from the C-terminal part and N-terminal part of the intact molecule, respectively. When the effect of purified M3 protein in the bactericidal activity of normal human blood in the presence of M bacteria was investigated, the M3 protein was responsible for the organism's resistance to attack by phagocytic cells.  相似文献   

16.
Cichlids of the genus Oreochromis are fish of economic importance in African countries. They tolerate brackish water, however, with great variations between species. In this work, two species, both from the Ivory Coast but of different origins, O. niloticus (field and laboratory strains) and O. aureus (field strain) were compared during osmotic challenges (10, 20 and 30%o salinity) in order to provide physiological support for their specific behaviour when confronted with natural hypertonic environments. Tolerance to salinity was assessed by correlated observations on gill structure, plasma sodium levels and gill Na+/K+ ATPase activity. In fresh water (FW), all fish presented a gill epithelium structure characteristic of FW stenohaline fish: no chloride cells (CC) on the lamellae and few CC on the filaments. An increase in external salinity induced the proliferation of CC on filaments, a feature typical of seawater teleosts. This change in gill structure was accompanied by an increase of gill Na+/K+ ATPase activity. In the most tolerant strains, plasma Na+ did not change, indicating successful ion regulation in the hypertonic media. With regard to potential interest of field strains in fish culture, O. aureus acclimated more easily to brackish water than O. niloticus . Interestingly, O. niloticus , kept for several generations in the laboratory, performed best in our challenge studies. Plasma Na+ levels and gill CC proliferation upon transfer to an isotonic medium may be the parameters of choice when testing these fish for their response to a salinity change.  相似文献   

17.
Abstract— Three different types of p -nitrophenyl phosphatases (NPPases) were solubilized by deoxycholate treatment from a membrane fraction of bovine cerebral cortex, and their characteristics were determined. Of these three NPPases (acid, Mg2+-activated, and K+, Mg2+-activated), only K-Mg NPPase was stimulated about two-fold by phospholipid and was inhibited by unsaturated neutral lipids and fatty acids. Unlike Na+-K+-Mga+-activated ATPase, the enzyme did not absolutely require phospholipid for its activity, but was similarly thermolabile and was protected by phospholipid from thermal inactivation. Acid NPPase was separable from the other two NPPases by ammonium sulphate fractionation, and partly solubilized by dialysis against ATP-mercaptoethanol solution. Hg2+ inhibited equally all three NPPases, but Ca2+ inhibited only Mg and K-Mg NPPases. Ouabain was effective on K-Mg NPPase alone.  相似文献   

18.
Plasmalemma was isolated from the roots of 2-week-old cucumber plants ( Cucumis sativus L. cv. Rhensk druv) by utilizing an aqueous polymer two-phase system with 6.5%:6.5% (w/w) Dextran T500 and polyethylene glycol (PEG) 3350 at pH 7.8. The plasmalemma fraction comprised ca 6% of the membrane proteins contained in the microsomal fraction. The specific activity of the plasma membrane marker enzyme (K+, Mg2+-ATPase) was 14- to 17-times higher in the upper (PEG-rich) than in the lower (Dextran-rich) phase, and the reverse was true for marker enzymes (cytochrome c oxidase, EC 1.9.3.1, and antimycin A-resistant NADPH cytochrome c reductase) of intracellular membranes. The ATPase was highly stimulated by the addition of detergent (Triton X-100), so that the isolated plasmalemma vesicles appear tightly sealed and in a right-side-out orientation. Further characterization of the ATPase activities showed a pH optimum at 6.0 in the presence of Mg2+. This optimum was shifted to pH 5.8 after addition of K+. K+ stimulated the ATPase activity below pH 6 and inhibited above pH 6. The ATPase activity was specific for ATP and sensitive to N,N-dicyclohexylcarbodiimide and sodium vanadate, with K+ enhancing the vanadate inhibition. The enzyme was insensitive to sodium molybdate, NO3, azide and oligomycin. No Ca2+-ATPase was detected, and even as little as 0.05 m M Ca2+ inhibited the Mg2+-ATPase activity.  相似文献   

19.
The effect of aluminum on dimorphic fungi Yarrowia lipolytica was investigated. High aluminum (0.5–1.0 mM AlK(SO4)2) inhibits yeast–hypha transition. Both vanadate-sensitive H+ transport and ATPase activities were increased in total membranes isolated from aluminum-treated cells, indicating that a plasma membrane H+ pump was stimulated by aluminum. Furthermore, Al-treated cells showed a stronger H+ efflux in solid medium. The present results suggest that alterations in the plasma membrane H+ transport might underline a pH signaling required for yeast/hyphal development. The data point to the cell surface pH as a determinant of morphogenesis of Y. lipolytica and the plasma membrane H+-ATPase as a key factor of this process.  相似文献   

20.
Mg2+- and Ca2+-uptake was measured in dark-grown oat seedlings ( Avena sativa L. cv. Brighton) cultivated at two levels of mineral nutrition. In addition the stimulation of the ATPase activity of the microsomal fraction of the roots by Mg2+ was measured. Ca2+-uptake by the roots was mainly passive. Mg2+-uptake mainly active; the passive component of Mg2+-uptake was accompanied by Ca2+-efflux up to 60% of the Ca2+ present in the roots.
In general Mg2+ -uptake of oat roots was biphasic. The affinity of the second phase correspond well with that of the Mg2+-stimulation of the ATPase activity, in low-salt roots as well as in high-salt roots and in roots of plants switched to the other nutritional condition. Linear relationships were observed when [phase 2] Mg2+-uptake was plotted against Mg2+-stimulation of the ATPase activity of the microsomal fraction of the roots. In 5 days old high-salt plants 1 ATP (hydrolysed in the presence of Mg2+ J corresponded with active uptake of a single Mg2+ ion, but in older high-salt roots and in low-salt roots more ATP was hydrolysed per net uptake of a Mg2+ ion. The results are discussed against the background of regulation of the Mg2+-level of the cytoplasm of root cells by transport of Mg2+ by a Mg2+-ATPase to the vacuole, to the xylem vessels, and possibly outwards.  相似文献   

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