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1.
An impure preparation of acetylcholinesterase from electroplax of the electric eel can be incorporated into a bimolecular lipid membrane. The acetylcholinesterase-modified bimolecular lipid membrane shows a concentration-dependent increase in membrane conductance elicited by several agonists (acetylcholine, carbamylcholine, phenyltrimethylammonium ion, tetraethylammonium ion, decamethonium ion, and nicotine) added to the compartment opposite that to which acetylcholinesterase was originally added. Affinity and efficacy of the various agonists in generating the conductance increase were measured from dose-response curves; these are in good quantitative agreement with corresponding values observed for depolarization of intact eel electroplax. The ion conduction pathways induced by agonists in the modified bimolecular lipid membrane show a slight cation selectivity, Na ? K > Cl (3:3:1), similar to that observed for the depolarized electroplax membrane. Evidence is presented that suggests that some components other than acetylcholinesterase induce the acetylcholine receptor response in the bimolecular lipid membrane.  相似文献   

2.
A potent inhibitor of (Na+ + K+)-ATPase activity was purified from Sigma equine muscle ATP by cation- and anion-exchange chromatography. The isolated inhibitor was identified by atomic absorption spectroscopy and proton resonance spectroscopy to be an inorganic vanadate. The isolated vanadate and a solution of V2O5 inhibit sarcolemma (Na+ + K+)-ATPase with an I50 of 1 micrometer in the presence of 1 mM ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA), 145 mM NaCl, 6mM MgCl2, 15 mM KCl and 2 mM synthetic ATP. The potency of the isolated vanadate is increased by free Mg2+. The inhibition is half maximally reversed by 250 micrometer epinephrine. Equine muscle ATP was also found to contain a second (Na+ + K+)-ATPase inhibitor which depends on the sulfhydryl-reducing agent dithioerythritol for inhibition. This unknown inhibitor does not depend on free Mg2+ and is half maximally reversed by 2 micrometer epinephrine. Prolonged storage or freeze-thawing of enzyme preparations decreases the susceptibility of the (Na+ + K+)-ATPase to this inhibitor. The adrenergic blocking agents, propranolol and phentolamine, do not block the catecholamine reactivation. The inhibitors in equine muscle ATP also inhibit highly purified (Na+ + K+)-ATPase from shark rectal gland and eel electroplax. The inhibitors in equine muscle ATP have no effect on the other sarcolemmal ATPases, Mg2+-ATPase, Ca2+-ATPase and (Ca2+ + Mg2+)-ATPase.  相似文献   

3.
Papaverine (1-[(3,4-Dimethoxyphenyl) methyl]-6,7-dimethoxyisoquinoline) and nantenine (O-methyldomesticine) are chemically related isoquinoline alkaloids displaying similar dose-dependent sedative or convulsant effects, but seem to act differentially on synaptosomal membrane enzymes. Na+, K+-, Mg2+- and Ca2+-ATPase activities were inhibited by nantenine but not by papaverine, whereas acetylcholinesterase activity remained unchanged by nantenine but slightly enhanced by papaverine. Nantenine inhibited roughly both 20-50% Ca2+- and Mg2+-ATPase activities but 40-90% Na+, K+-ATPase activity. Kinetic analysis indicated that nantenine interacts with the substrate ATP for Ca2+-ATPase activity but that it competes with K+ for Na+, K+-ATPase activity. Given the roles of Na+, K+-ATPase and Ca2+-ATPase in cation transport and [Ca2+]i regulation, respectively, the inhibitory effect of nantenine upon these enzymes may explain its convulsant effect though not its sedative activity. The sedative action of both nantenine and papaverine is hardly attributable to an effect on the synaptosomal membrane enzymes assayed.  相似文献   

4.
The main electric organ of Electrophorus electricus is particularly rich in thiamine triphosphate, which represents 87% of the total thiamine content in this tissue. The thiamine pyrophosphate concentration, however, is very low in the eel electric organ and skeletal muscle as compared with other eel or rat tissues. Furthermore, electroplax membranes contain a whole set of enzymes responsible for the dephosphorylation of thiamine tri-, pyro- and monophosphate. Thiamine triphosphatase has a pH optimum of 6.8 and is dependent on Mg2+. The real substrate of the enzyme is probably a 1:1 complex of Mg2+ and thiamine triphosphate. Thiamine pyrophosphatase is activated by Ca2+. The apparent Km for thiamine triphosphate and Vmax are found to be, respectively, 1.76 mM and 5.95 nmol/mg of protein/min. Thiamine triphosphatase activity is inhibited at physiological K+ concentrations (up to 90 mM) and increasing Na+ concentrations (50% inhibition at 300 mM). ZnCl2 (10 mM) inhibits 90% of the enzyme activity. ATP and ITP are also strongly inhibitory. No significant effect of neurotoxins is seen. Membrane-associated thiamine triphosphatase is affected differently by proteolytic enzymes and is partially inactivated by pretreatment with phospholipase C and neuraminidase. The physiological significance of thiamine triphosphatase is discussed in relation to a specific role of thiamine in the nervous system.  相似文献   

5.
Regulatory effects of polyamines on membrane-bound acetylcholinesterase   总被引:5,自引:1,他引:4  
The effects of putrescene, spermidine and spermine on membrane-bound acetylcholinesterase from human erythrocyte ;ghosts' and the solubilized enzyme of the electric organ of the electric eel were studied by kinetic methods. Measurements were made by using a photometric method which made it possible to record the enzyme reaction in the steady-state phase. Substrate-concentration-dependent activation and inhibition of acetylcholinesterase by polyamines is similar to that by Na(+), K(+), Ca(2+), Mg(2+) and certain quaternary and bisquaternary amines. The kinetics suggest an allosteric reaction mechanism. On the basis of the kinetic results a role for the polyamines as modulators of synaptic acetylcholinesterase is proposed.  相似文献   

6.
Calcium-Activated ATPases in Presynaptic Nerve Endings   总被引:7,自引:5,他引:2  
We studied the properties of calcium-activated ATPases present in preparations of isolated presynaptic nerve ending (synaptosome) and its subfractions from mouse brain. ATPase activity in the preparation was stimulated by Ca2+ and by Mg2+, but not by Na+ and K+, when each was added alone. The substrate specificities were found to be similar. The ATPases hydrolyzed only the high-energy phosphate bond and similar activity was exhibited for all nucleoside triphosphates tested (ATP, CTP, GTP, UTP). Moreover, the enzymes were insensitive to mitochondrial markers and to ouabain, but were inhibited by La3+. La3+ produced uncompetitive inhibition of Ca2+-ATPase in intact synaptosomes. Inhibition by La3+ was greatly increased after lysis of the synaptosomes, suggesting that the active sites of the enzymes may be on the cytosolic face of the membranes. The Ca2+-ATPase activity in synaptosomes was increased by increasing concentrations of external K+, suggesting that Ca2+ influx may be involved The Ca2+-ATPase in synaptosomal plasma membranes and synaptic vesicles had higher specific activities than those of intact synaptosomes and were activated, both in the presence and the absence of Mg2+, by Ca2+ concentrations approximating the intracellular level (10(-7) M). It is concluded that the nonmitochondrial synaptosomal Ca2+-ATPase may play an important role in the regulation of intracellular Ca2+.  相似文献   

7.
Inorganic lead ion in micromolar concentrations inhibits Electrophorus electroplax microsomal (Na+ + K+)-adenosine triphosphatase ((Na+ + K+)-ATPase) and K+-p-nitrophenylphosphatase (NPPase). Under the same conditions, the same concentrations of PbCl2 that inhibit ATPase activity also stimulate the phosphorylation of electroplax microsomes in the absence of added Na+. Enzyme activity is protected from inhibition by increasing concentrations of microsomes, ATP, and other metal ion chelators. The kinetics follow the pattern of a reversible noncompetitive inhibitor. No kinetic evidence is elicited for interactions of Pb2+ with Na+, K+, Mg2+, ATP, or p-nitrophenylphosphate. Na+- ATPase, in the absence of K+, and (Na+ + K+)-NPPase activity at low [K+] are also inhibited. ATP inhibition of NPPase is not reversed by Pb2+. The calculated concentrations of free [Pb2+] that produce 50% inhibition are similar for ATPase and NPPase activities. Pb2+ may act at a single independent binding site to produce both stimulation of the kinase and inhibition of the phosphatase activities.  相似文献   

8.
We here describe a protocol for fusing vesicles into large structures suitable for patch clamp recording. The method may be used with native membrane vesicles or with liposomes containing reconstituted/purified ion channels. The resulting unilamellar membranes exhibit high channel surface abundance, yielding multiple channels in the average excised patch. The procedure has been used to record voltage-sensitive Na channels from three native membrane preparations (eel electroplax, rat skeletal muscle, squid optic nerve), and from reconstituted protein purified from eel electroplax. Channels treated with batrachotoxin (BTX) displayed characteristic activation voltage dependence, conductances, selectivity, and sensitivity to saxitoxin (STX).  相似文献   

9.
Batrachotoxinin-A [3H]benzoate ([3H]BTX-B) binds specifically and with high affinity (KD 48 nM) to sites (Bmax 2.1 pmol/mg protein) associated with voltage-dependent sodium channels in rodent brain vesicular preparations. High affinity binding requires the presence of scorpion (Leiurus) venom and a membrane potential. Local anesthetics antagonize the binding. Nonspecific binding is defined in the presence of veratridine. In particulate preparations from electroplax of the eel Electrophorus electricus, [3H]BTX-B binds with a KD of about 140 nM and a Bmax of 2.5 pmol/mg protein in the presence of scorpion venom. Higher concentrations of scorpion venom are required to enhance binding in Electrophorus preparations than in brain preparations. Local anesthetics antagonize binding in Electrophorus preparations with potencies similar to those in brain preparations. Veratridine and batrachotoxin are less potent in blocking binding in Electrophorus than in brain preparations. It appears likely that binding in Electrophorus preparations is primarily to membrane fragments rather than vesicular entities as in brain. Binding of [3H]BTX-B to particulate preparations from electroplax of the ray Torpedo californica and the catfish Malapterurus electricus is mainly nonspecific. Scorpion venom does not enhance total binding and local anesthetics are not effective in antagonizing binding.  相似文献   

10.
The inhibition of soluble guanylyl cyclase by Ca2+ has been kinetically characterized and the results support a two-metal-ion catalytic mechanism for formation of cGMP. Ca2+ reversibly inhibits both the basal and NO-stimulated forms of bovine lung soluble guanylyl cyclase. Inhibition is independent of the activator identity and concentration, revealing that Ca2+ interacts with a site independent of the heme regulatory site. Inhibition by Ca2+ is competitive with respect to Mg2+ in excess of substrate, with Kis values of 29 +/- 4 and 6.6 +/- 0.6 microM for the basal and activated states, respectively. Ca2+ inhibits noncompetitively with respect to the substrate MgGTP in both activity states. The qualitatively similar inhibition pattern and quantitatively different Ki values between the basal and NO-stimulated states suggest that the Ca2+ binding site undergoes some structural modification upon activation of the enzyme. The competitive nature of Ca2+ inhibition with respect to excess Mg2+ is consistent with a two-metal-ion mechanism for cyclization.  相似文献   

11.
Paraquat inhibited the acetylcholinesterase activity of human erythrocytes and electric organs of Electrophorus electricus. The inhibition of acetylcholinesterase activity was reversible, as shown from the following two experimental results: [I] The degree of inhibition was not affected by changing the preincubation time of the enzyme and paraquat before the addition of the substrate. [II] The enzyme, preincubated with paraquat and subsequently freed from inhibitor by gel filtration on Sephadex G-25, showed the same activity as the untreated enzyme. Paraquat gave effective protection against the inhibition by an irreversible anionic site inhibitor, dibenamine, but not by irreversible esteratic site inhibitors, dichlorvos and methanesulfonyl chloride. These results indicate that paraquat functions as a reversible inhibitor for the anionic site. The inhibitory powers and Hill coefficients of paraquat and diquat were compared with the other quaternary ammonium compounds. Although secondary to edrophonium, paraquat strongly inhibited acetylcholinesterases of human erythrocytes and electric eel, and showed higher inhibition selectivity for both acetylcholinesterases than for human plasma butyrylcholinesterase. The Hill coefficients concerning the interaction of paraquat with acetylcholinesterases of human erythrocytes and electric eel were given as 0.83 and 0.73, respectively. This indicates negative cooperativity between these enzymes and paraquat, which is similar to the case with d-tubocurarine. On the other hand, diquat showed weak inhibitory power and low inhibition selectivity, and its Hill coefficients were almost 1.0, indicating a competitive inhibition mode.  相似文献   

12.
The effects of two lectins, wheat germ agglutinin and concanavalin A, were studied on a variety of parameters of two highly purified (Na+ + K+)-ATPases (ATP phosphohydrolase, EC 3.6.1.3), from the rectal salt gland of Squalus acanthias and from the electroplax of Electrophorus electricus. Both lectins agglutinated the rectal gland enzyme equally, but wheat germ agglutinin inhibited (Na+ + K+)-ATPase activity much more. The electroplax enzyme was only marginally agglutinated and inhibited by the lectins. Neuraminidase treatment of the rectal gland (Na+ + K+)-ATPase had no effect on germ agglutinin inhibition. The inhibition of the rectal gland (Na+ + K+)-ATPase by wheat germ agglutinin could be reversed by N,N'-diacetylchitobiose, which has a high affinity for wheat germ agglutinin. Neither ouabain inhibition nor ouabain binding to the rectal gland enzyme was affected by wheat germ agglutinin. The p-nitrophenylphosphatase activity of the rectal gland enzyme was not inhibited by wheat germ agglutinin. Na+-ATPase activity, which reflects ATP binding and phosphorylation at the substrate site was inhibited by wheat germ agglutinin and this inhibition was reversed by potassium. Evidence is cited (Pennington, J. and Hokin, L.E., in preparation) that the inhibition of the (Na+ + K+)-ATPase by wheat germ agglutinin is due to binding to the glycoprotein subunit.  相似文献   

13.
Bass gill microsomal preparations contain a Mg2+-dependent Na+-stimulated ATPase activity in the absence of K+, whose characteristics are compared with those of the (Na+ + K+)-ATPase of the same preparations. The activity at 30 degrees C is 11.3 mumol Pi X mg-1 protein X hr-1 under optimal conditions (5 mM MgATP, 75 mM Na+, 75 mM HEPES, pH 6.0) and exhibits a lower pH optimum than the (Na+ + K+)-ATPase. The Na+ stimulation of ATPase is only 17% inhibited by 10-3M ouabain and completely abolished by 2.5 mM ethacrinic acid which on the contrary cause, respectively, 100% and 34% inhibition of the (Na+ + K+)-ATPase. Both Na+-and (Na+ + K+)-stimulated activities can hydrolyze nucleotides other than ATP in the efficiency order ATP greater than CTP greater than UTP greater than GTP and ATP greater than CTP greater than GPT greater than UTP, respectively. In the presence of 10(-3)M ouabain millimolar concentrations of K+ ion lower the Na+ activation (90% inhibition at 40 mM K+). The Na+-ATPase is less sensitive than (Na+ + K+)-ATPase to the Ca2+ induced inhibition as the former is only 57.5% inhibited by a concentration of 1 X 10(-2)M which completely suppresses the latter. The thermosensitivity follows the order Mg2+--greater than (Na+ + K+)--greater than Na+-ATPase. A similar break of the Arrhenius plot of the three enzymes is found. Only some of these characteristics do coincide with those of a Na+-ATPase described elsewhere. A presumptive physiological role of Na+-ATPase activity in seawater adapted teleost gills is suggested.  相似文献   

14.
The Na(+)-Ca2+ exchanger from Drosophila was expressed in Xenopus and characterized electrophysiologically using the giant excised patch technique. This protein, termed Calx, shares 49% amino acid identity to the canine cardiac Na(+)-Ca2+ exchanger, NCX1. Calx exhibits properties similar to previously characterized Na(+)-Ca2+ exchangers including intracellular Na+ affinities, current-voltage relationships, and sensitivity to the peptide inhibitor, XIP. However, the Drosophila Na(+)-Ca2+ exchanger shows a completely opposite response to cytoplasmic Ca2+. Previously cloned Na(+)-Ca2+ exchangers (NCX1 and NCX2) are stimulated by cytoplasmic Ca2+ in the micromolar range (0.1- 10 microM). This stimulation of exchange current is mediated by occupancy of a regulatory Ca2+ binding site separate from the Ca2+ transport site. In contrast, Calx is inhibited by cytoplasmic Ca2+ over this same concentration range. The inhibition of exchange current is evident for both forward and reverse modes of transport. The characteristics of the inhibition are consistent with the binding of Ca2+ at a regulatory site distinct from the transport site. These data provide a rational basis for subsequent structure-function studies targeting the intracellular Ca2+ regulatory mechanism.  相似文献   

15.
The causes of inhibition of Escherichia coli inorganic pyrophosphatase (PPase) by Ca2+ were investigated. The interactions of several mutant pyrophosphatases with Ca2+ in the absence of substrate were analyzed by equilibrium dialysis. The kinetics of Ca2+ inhibition of hydrolysis of the substrates MgPPi and LaPPi by the native PPase and three mutant enzymes (Asp-42-Asn, Ala, and Glu) were studied. X-Ray data on E. coli PPase complexed with Ca2+ or CaPPi solved at atomic resolution were analyzed. It was shown that, in the course of the catalytic reaction, Ca2+ replaces Mg2+ at the M2 site, which shows higher affinity for Ca2+ than for Mg2+. Different properties of these cations account for active site deformation. Our findings indicate that the filling of the M2 site with Ca2+ is sufficient for PPase inhibition. This fact proves that Ca2+ is incapable of properly activating the H2O molecule for nucleophilic attack on PPi. It was also demonstrated that Ca2+, as a constituent of the non-hydrolyzable substrate analog CaPPi, competes with MgPPi at the M3 binding site. As a result, Ca2+ is a powerful inhibitor of all known PPases. Other possible reasons for the inhibitory effect of Ca2+ on the enzyme activity are also considered.  相似文献   

16.
Scorpion toxins, the basic miniproteins of scorpion venom, stimulated the passive uptake of Na+ and Ca2+ in chick embryo heart cells. Half-maximum stimulation was obtained for 20-30 nM Na+ and 40-50 nM Ca2+. Scorpion toxin-activated Na+ and Ca2+ uptakes were fully inhibited by tetrodotoxin, a specific inhibitor of the action potential Na+ ionophore in excitable membranes. Half-maximum inhibition was obtained with the same concentration of tetrodotoxin (10 nM) for both Na+ and Ca2+. Scorpion toxin-stimulated Ca2+ uptake was dependent on extracellular Na+ concentration and was not inhibited by Ca2+ channel blocking drugs which are inactive on heart cell action potential. Thus, in heart cells scorpion toxin affects the passive Ca2+ transport, which is coupled to passive Na+ ionphore. Other results suggest that (1) tetrodotoxin and scorpion toxin bind to different sites of the sarcolemma and (2) binding of scorpion toxin to its specific sites may unmask latent tetrodotoxin - sensitive fast channels.  相似文献   

17.
The bee and cobra venom phospholipases A2 as well as partially acetylated cobra venom phospholipase A2 are studied for their effect on phospholipid composition of synaptosomes and their Mg2+- and Na+,K+-ATPase activity. It is established that these phospholipases induce the splitting of phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine, inhibition of the Na+,K+-ATPase activity and activation of Mg2+-ATPase. Bee venom phospholipase A2 is more effective than cobra venom phospholipase A2, the both phospholipases splitting phosphatidylethanolamine most intensively. The ATPase activity may be partially or completely restored by exogenic phosphatidylcholine and phosphatidylserine; exogenic phosphatidylethanolamine is not efficient in this respect.  相似文献   

18.
Renal basal-lateral and brush border membrane preparations were phosphorylated in the presence of [gamma-32P]ATP. The 32P-labeled membrane proteins were analysed on SDS-polyacrylamide gels. The phosphorylated intermediates formed in different conditions are compared with the intermediates formed in well defined membrane preparations such as erythrocyte plasma membranes and sarcoplasmic reticulum from skeletal muscle, and with the intermediates of purified renal enzymes such as (Na+ + K+)-ATPase and alkaline phosphatase. Two Ca2+-induced, hydroxylamine-sensitive phosphoproteins are formed in the basal-lateral membrane preparations. They migrate with a molecular radius Mr of about 130 000 and 100 000. The phosphorylation of the 130 kDa protein was stimulated by La3+-ions (20 microM) in a similar way as the (Ca2+ + Mg2+)-ATPase from erythrocytes. The 130 kDa phosphoprotein also comigrated with the erythrocyte (Ca2+ + Mg2+)-ATPase. In addition in the same preparation, another hydroxylamine-sensitive 100 kDa phosphoprotein was formed in the presence of Na+. This phosphoprotein comigrates with a preparation of renal (Na+ + K+)-ATPase. In brush border membrane preparations the Ca2+-induced and the Na+-induced phosphorylation bands are absent. This is consistent with the basal-lateral localization of the renal Ca2+-pump and Na+-pump. The predominant phosphoprotein in brush border membrane preparations is a 85 kDa protein that could be identified as the phosphorylated intermediate of renal alkaline phosphatase. This phosphoprotein is also present in basal-lateral membrane preparations, but it can be accounted for by contamination of those membranes with brush border membranes.  相似文献   

19.
Summary Using the dialysable detergent CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate), the tetrodotoxin-binding protein from the electroplax of the electric eel has been purified to a high degree of both chemical homogeneity and toxin-binding activity. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the best preparations showed only a single microheterogeneous band atM r approximately 260,000, despite attempts to visualize smaller bands by sample overloading. Upon dialysis, this material became incorporated into the membranes of small unilamellar vesicles, and in this form the purified protein exhibited tetrodotoxin-binding properties similar to the component in the original electroplax membrane. Furthermore, in the presence of activator neurotoxins the vesicles were able to accumulate isotopic sodium in a manner similar to that previously described for less active or less pure preparations of vesicles containing either mammalian or eel electroplax toxinbinding proteins. Quantitative consideration of the isotopic transport activity of this pure material, along with the high degree of purity of the protein, strongly suggests that the 260-kDa glycopeptide from electroplax is necessary and sufficient to account for the sodium channel function seen in these studies, and eliminates the possible involvement of smaller peptides in the channel phenomena observed.  相似文献   

20.
Photoaffinity labeling of (Na+ + K+)-ATPase in erythrocyte membranes with cardiotonic steroid derivatives, followed by gel electrophoresis, requires a radiolabel of very high specific activity, since the enzyme represents less than 0.05% of the total membrane protein. We report the synthesis of a radioiodinated, photosensitive derivative of the cardiac glycoside, 3-beta-O-(4-amino-4,6-dideoxy-beta-D-galactosyl)digitoxigenin, with very high specific activity. The product, [125I]iodoazidogalactosyl digitoxigenin ([125I]IAGD), is carrier-free with a specific activity of 2200 Ci/mmol. Incubation of [125I]IAGD (1.8 nM) with human erythrocyte membranes (300 micrograms protein), followed by photolysis and analysis by SDS-PAGE, showed specific radiolabeling of a polypeptide that had the same molecular weight as catalytic alpha subunit (100,000 Mr) of (Na+ + K+)-ATPase in eel electroplax microsomes. Photoaffinity labeling of erythrocyte and electroplax membranes by [125I]IAGD was specific for the cardiac glycoside binding site of (Na+ + K+)-ATPase since radiolabeling of the alpha subunit was inhibited when ouabain was included in the pre-photolysis incubation. [125I]IAGD can, therefore, be used as a probe in structural studies of human erythrocyte membrane (Na+ + K+)-ATPase.  相似文献   

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