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1.
The nucleoside 5'-triphosphate (NTP) substrate specificities for Ca-stimulated ATPase and ATP-dependent Ca2+ uptake activities have been examined in cardiac sarcolemma (SL) and sarcoplasmic (SR) membrane vesicles. The results indicate that SL membrane vesicles exhibit a much narrower range of NTP substrate specificities than SR membranes. In SR membrane vesicles, the Ca-stimulated Mg-dependent hydrolysis of ATP and dATP occurred at nearly equivalent rates, whereas the rates of hydrolysis of GTP, ITP, CTP, and UTP ranged from 16-33% of that for ATP. All of the above nucleotides also supported Ca2+ transport into SR vesicles; dATP was somewhat more effective than ATP while GTP, ITP, CTP, and UTP ranged from 28-30% of the activity for ATP. In the presence of oxalate, the initial rate of Ca accumulation with dATP was 4-fold higher than for ATP, whereas the activity for GTP, ITP, CTP, and UTP ranged from 35-45% of that for ATP. For the SL membranes, Ca-activated dATP hydrolysis occurred at 60% of the rate for ATP; GTP, ITP, CTP, and UTP were hydrolyzed by the SL preparations at only 7-9% of the rate for ATP. NTP-dependent Ca2+ uptake in SL membranes was supported only by ATP and dATP, with dATP 60% as effective as ATP. GTP, ITP, CTP, and UTP did not support the transport of Ca2+ by SL vesicles. The results indicate that the SL and SR membranes contain distinctly different ATP-dependent Ca2+ transport systems.  相似文献   

2.
Hydrolysis of umbelliferone phosphate is inhibited by sodium ions and ATP added to the incubation medium together with Na+ activates it, CTP, ITP, UTP and GTP produce the same effect. Acetyl phosphate, p-nitrophenyl phosphate, ADP and adenylyl imidodiphosphate beta, gamma-NH-ATP, ATP nonhydrolyzable analog, have no activating effect. ATP produces a mixed inhibition of umbelliferone phosphate hydrolysis both in the presence and absence of Na+. A scheme is suggested which explains the mechanism of the nucleotide activating effect.  相似文献   

3.
A partially purified soluble ATPase (ATP phosphohydrolase, EC 3.6.1.3) from pea cotyledon mitochondria was characterized. Inhibition patterns with azide, NaF, and cold, and a stimulation by 2,4-dinitrophenol were typical of F1-ATPases from mammalian mitochondria. The enzyme hydrolysed GTP, ITP, and ATP, but not CTP, UTP, ADP, or IDP. ATPase and ITPase activities were strongly inhibited by ADP and to a lesser extent by IDP. Distinctive properties of the pea mitochondrial enzyme were activation by high concentrations of CaCl2 and stimulation by NaCl.  相似文献   

4.
Choleragen-dependent ADP ribosylation of soluble proteins from bovine thymus, using [32P]NAD as substrate, was increased 3- to 4-fold by GTP. The effect was specific for nucleoside triphosphate, with GTP approximately equal to ITP greater than CTP greater than ATP greater than UTP. Half-maximal enhancement was observed with 0.5 mM GTP. The magnitude of the GTP effect decreased with increasing NAD concentration; GTP had no effect on hydrolysis of NAD at low NAD concentrations. Digestion of ADP-ribosylated proteins with snake venom phosphodiesterase yielded primarily 5'-AMP. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of soluble proteins from thymus after incubation with choleragen and [32P]NAD separated numerous ADP-ribosylated proteins; radioactivity in all bands was increased by nucleoside triphosphate. Choleragen catalyzed the ADP ribosylation of several purified proteins; depending on the protein, GTP either increased, decreased, or had no effect on the extent of ADP ribosylation. Choleragen-dependent ADP ribosylation of a wide variety of proteins is consistent with the possibility that intoxication results in covalent modification of more than one cellular protein and perhaps alters the activity of other enzymes in addition to adenylate cyclase.  相似文献   

5.
The Accumulation of Calcium Ions by Sarcotubular Vesicles   总被引:5,自引:1,他引:4       下载免费PDF全文
The accumulation of Ca++ by microsomal (sarcotubular) preparations of rabbit skeletal muscle in the presence of oxalate, and the concurrent splitting of nucleoside triphosphate, displayed moderate nucleotide specificity in the sequence ATP > GTP, CTP, ITP > UTP > (ADP) > ATetraP for the former, ATP > (ADP) > ITP > GTP > CTP > UTP > ATetraP for the latter process. The "calcium pump" was weakly inhibited by caffeine, and was inhibited together with the ATPase by pyridoxalphosphate. Carnosine had no effect as such nor in the presence of pyridoxalphosphate except at high concentration; thiourea and p-chloromercuribenzoate were inhibiting while iodoacetate was inactive. Ca++ accumulation and ATPase were inhibited by atabrine (not tested on ATPase), dinitrophenol, and amytal. High concentrations of oligomycin and rutamycin inhibited Ca++ uptake while slightly stimulating ATPase. Antimycin A stimulated the Ca++ uptake. These results are discussed in the light of their possible relation to partial reactions in oxidative phosphorylation. The Ca++ uptake and relaxing factor activities did not behave identically throughout. This is in part ascribed to changes in reactivity of actomyosin in the relaxation test, in part to the participation of relaxing substances other than the calcium pump.  相似文献   

6.
Membranes from Halobacterium saccharovorum contained a cryptic ATPase which required Mg2+ or Mn2+ and was activated by Triton X-100. The optimal pH for ATP hydrolysis was 9-10. ATP or GTP were hydrolyzed at the same rate while ITP, CTP, and UTP were hydrolyzed at about half that rate. The products of ATP hydrolysis were ADP and phosphate. The ATPase required high concentrations (3.5 M) of NaCl for maximum activity. ADP was a competitive inhibitor of the activity, with an apparent Ki of 50 microM. Dicyclohexylcarbodiimide (DCCD) inhibited ATP hydrolysis. The inhibition was marginal at the optimum pH of the enzyme. When the ATPase was preincubated with DCCD at varying pH values, but assayed at the optimal pH for activity, DCCD inhibition was observed to increase with increasing acidity of the preincubation medium. DCCD inhibition was also dependent on time of preincubation, and protein and DCCD concentrations. When preincubated at pH 6.0 for 4 h at a protein:DCCD ratio of 40 (w/w), ATPase activity was inhibited 90%.  相似文献   

7.
H+ transport into synaptosomal membrane vesicles of the rat brain was stimulated by ATP and to a lesser extent by GTP, but not by ITP, CTP, UTP, ADP, AMP or beta, gamma-methylene ATP. ATP at concentrations up to 200 mM concentration-dependently stimulated the rate of H+ transport with a Km value of 0.6 mM, but at higher concentrations of this nucleotide the rate decreased. Other nucleotides such as CTP, UTP, GTP and AMP, or products of ATP hydrolysis i.e. ADP and Pi also reduced the ATP-stimulated H+ transport. The inhibition by GTP and ADP was not affected by the ATP concentration. These findings suggest that plasma membranes of nerve endings transport H+ from inside to outside of the cells utilizing energy from ATP hydrolysis, and that this transport is regulated by the intracellular concentration of nucleotides and Pi on sites other than those involved in substrate binding.  相似文献   

8.
The ATPase of avian myeloblastosis virus (AMV) is not a recognizable cellular enzyme. It hydrolyzes ATP, GTP, ITP, UTP, and dCTP at equal rates, is inhibited by high concentrations of dithiothreitol, and is partially inhibited by 1 × 10?5mp-chloromercuribenzoic acid (PCMB) and p-chloromercuribenzene sulfonate acid (PCMBS). The inhibition by the mercurials is reversed by increasing the concentration of PCMB or PCMBS to 1 × 10?3m. The enzyme requires phospholipid for activity. Incubation with phospholipase C inhibits activity and subsequent addition of lecithin-containing saturated fatty acids partially restores activity, whereas lecithin-containing unsaturated fatty acids further inhibit activity.  相似文献   

9.
Several nucleotide triphosphates (NTPs) were tested as energy source for the Ca2+ uptake by human platelet membrane vesicles. The Ca2+ uptake by these membranes was driven by ATP, GTP, ITP, UTP and CTP. The steady-state level of accumulated Ca2+ was equal with the different NTPs. The highest uptake velocity was found with ATP, but about 40–80% of the velocity with ATP could be accomplished with the other nucleotides. The highest affinity was also found with ATP (Km apparent  15 μM). The liberation of Pi from the various NTPs was measured simultaneously with the Ca2+ uptake. The coupling ratio (moles of Ca2+ taken up/moles of Pi liberated) varied from 0.4 for ATP to 2.3 for UTP and was almost independent of the NTP concentration. The enzyme activity with ATP as substrate is strongly dependent on the Ca2+ concentration in contrast to the activity with GTP, ITP, UTP or CTP.  相似文献   

10.
Effect of anions on the ATPase activity of submitochondrial particles   总被引:1,自引:0,他引:1  
The effects of anions on the ATPase activity of submitochondrial particles from mouse liver cells were investigated. Thiocyanite decreased the ATP hydrolysis, acting as a competitive inhibitor with respect to sulfite. All the anions tested changed the ATPase activity noncompetitively towards Mg-ATP. The hydrolysis of CTP, GTP, ITP and UTP was insensitive to sulfite and thiocyanate. In the presence of Mn2+, Ca2+, Co2+, Zn2+ and Ba2+ an anion-dependent hydrolysis of ATP took place. It was assumed that the anions control the rate of the limiting step of the ATPase reaction, since sulfite and thiocyanate change the activation energy of ATP hydrolysis. The data obtained are discussed in terms of a previously proposed mechanism of the anions effect on the activity of mitochondrial ATPase.  相似文献   

11.
DCCD, an inhibitor of membrane-bound ATPases, markedly inhibitedboth the endogenous and IAA-induced growth of cucumber hypocotylsections. The inhibition was negated by the simultaneous applicationof ADP, ATP and 3',5'-cyclic AMP. The effect of these nucleotideswas similar to that of GA3. Adenine, adenosine, AMP, 2',3'-cyclicAMP, CTP, GTP, ITP and UTP were all ineffective. (Received April 26, 1976; )  相似文献   

12.
1. The purification of ATPase (EC 3.6.1.3) from human placental mitochondria is described. The yield based on mitochondrial enzyme activity was about 70% and the purification was 380-fold. 2. The rate of Mg-ATP hydrolysis was 85 mumole per min per mg of protein under optimum conditions. 3. Nucleoside triphosphates were hydrolyzed by the purified enzyme at decreasing rates in the following order: GTP greater than ITP greater than ATP greater than epsilon-ATP greater than UTP greater than CTP in Tris-HCl buffer (pH 8.0), and in the order: ATP greater than GTP greater than or equal to ITP greater than epsilon-ATP greater than UTP greater than CTP in Tris-bicarbonate buffer at pH 8.0. 4. The values of kinetic parameters are reported. The ATPase reaction deviated from typical Michaelis-Menten kinetics in Tris-HCl buffer but not in Tris-bicarbonate. Eadie-Hofstee plots for Mg-ATP hydrolysis were biphasic in Tris-HCl (Km = 0.2 mM, 0.09 mM) and monophastic in Tris-becarbonate medium (Km = 0.16 mM). 5. In the presence of Mg-ITP or Mg-GTP as substrates no curvature of the reciprocal plots was observed. 6. The results presented reflect the fact that multiple conformations of the enzyme molecule do exist and are probably involved in its regulatory functions. 7. The existence of two kinetically distinct classes of catalytic sites and of an anion-binding site on the placental ATPase is proposed.  相似文献   

13.
A procedure for the purification of Mg2+ adenosine triphosphatase (EC 3.6.1.3) from free-living and bacteroid forms of Rhizobium lupini NZP2257 is described. The enzyme was released from cell envelopes using Triton X-100 and purified by gel filtration on Ultrogel AcA 22, followed by preparative gel electrophoresis on agarose. The purified ATPase had a molecular weight of about 355,000, as determined from sedimentation coefficients on sucrose gradients. Kinetic analysis of activity of the enzyme from free-living R. lupini showed it to be typical of F1-type Mg2+ ATPases from bacteria. Mg stimulated activity at pH 7.0, although, when present as the free ion, Mg caused non-competitive inhibition (K1 = 1.5 mM). Maximum activity with ATP occurred over a broad pH range from 6.0 to 10.5. ATP, GTP, and UTP, and, to a much lesser degree, CTP and ADP, were hydrolyzed by the enzyme. Hydrolysis of glucose 6-phosphate was not observed. The Km for ATP at pH 7.0 was 0.67 and for GTP 1.4 mM. ATPase activity was inhibited by ADP, and competitive with ATP (KI = 0.18 mM). Azide also caused inhibition but fluoride and DCCD had no effect. Native and sodium dodecyl sulfate-gel electrophoretic analysis revealed no obvious differences between ATPases from free-living and bacteroid forms of R. lupini.  相似文献   

14.
An activity of Ca2+-dependent nucleotidase was detected in axenically-cultivated trophozoites of Entamoeba histolytica. The enzyme was concentrated by differential and sucrose density gradient centrifugation and catalyzed hydrolysis of nucleoside tri- and diphosphates and also thiamine pyrophosphate. Hydrolysis of nucleoside mono-phosphates was not affected by Ca2+. Among substrates tested, ATP was most active. Addition of Zn2+ or heat treatment almost abolished the enzyme activity. The enzyme exhibited almost the identical activity at acid and neutral pH. Among 6 bands isolated by polyacrylamide gel electrophoresis, 4 were stained with ATP, UTP, CTP and ADP, whereas the other 2 were stained only with ATP, UTP and CTP. The concentrated enzyme preparation, primarily composed of membrane fragments, also had activities of acid phosphatase, acid inorganic pyrophosphatase, 5'-nucleotidase and Mg2+-dependent ATPase. These observations suggest that E. histolytica has 2 Ca2+-dependent nucleotidases, i.e. one Ca2+-dependent ATPase and the other Ca2+-dependent nucleoside diphosphatase or an apyrase-like enzyme, and that these nucleotidases are at least partially associated with the plasma membrane or an organelle of lysosomal nature in this parasite.  相似文献   

15.
The well-known soluble kinases are not specific for ATP (1). All these enzymes convert ATP as well as GTP, ITP, CTP, and UTP, although at different rates. The only exception is adenylate kinase (1). However, with this enzyme, a direct determination of ATP in tissue extracts which contain both the di- and mononucleotides is not possible.Phosphoglycerate kinase from various sources is specific for ATP, GTP, and ITP and does not react with the pyrimidine nucleotides (2), Now, however, it was found that phosphoglycerate kinase from the blue alga Spirulina platensis does not convert GTP and ITP. With this enzyme, therefore, it is possible to specifically determine ATP in tissue extracts or in mixtures of nucleotides. In the same test, GTP and ITP can be determined by adding phosphoglycerate kinase from yeast or from other sources (2).  相似文献   

16.
A gene coding for the novel ras protein, p21X, in which the domains of guanine binding and phosphate binding were exchanged, was constructed and expressed in Escherichia coli. The gene product, p21X, showed GTP binding activity, but no GPTase activity. In addition, p21X revealed binding activity toward ATP and CTP. In a competitive binding assay, [3H]GTP binding to p21X was inhibited in the presence of ATP, CTP and UTP, ITP as well as GDP, GTP and dGTP.  相似文献   

17.
Yoshida T  Kawaguchi R  Maruyama T 《FEBS letters》2002,514(2-3):269-274
The archaeal chaperonin-mediated folding of green fluorescent protein (GFP) was examined in the presence of various nucleotides. The recombinant alpha- and beta-subunit homo-oligomers and natural chaperonin oligomer from Thermococcus strain KS-1 exhibited folding activity with not only ATP but also with CTP, GTP, or UTP. The ADP-bound form of both recombinant and natural chaperonin had the ability to capture non-native GFP, but could not refold it in the presence of CTP, GTP or UTP until ATP was supplied. The archaeal chaperonin thus utilized ATP, but could not use other nucleoside triphosphates in the cytoplasm where ADP was present.  相似文献   

18.
J B Fagan  E Racker 《Biochemistry》1977,16(1):152-158
Adenosine triphosphate (ATP) hydrolysis catalyzed by the plasma membrane (Na+,K+)ATPase isolated from several sources was inhibited by Mg+, provided that K+ and ATP were also present. Phosphorylation of the adenosine triphosphatase (ATPase) by ATP and by inorganic phosphate was also inhibited, as was p-nitrophenyl phosphatase activity. (Ethylenedinitrilo)tetraacetic acid (EDTA) and catecholamines protected from and reversed the inhibition of ATP hydrolysis by Mg2+, K+ and ATP. EDTA was protected by chelation of Mg2+ but catecholamines acted by some other mechanism. The specificities of various nucleotides as inhibitors (in conjunction with Mg2+ and K+) and as substrates for the (Na+, K+) ATPase were strikingly different. ATP, ADP, beta,gamma-CH2-ATP and alpha,beta-CH2-ADP were active as inhibitors, whereas inosine, cytidine, uridine, and guanosine triphosphates (ITP, CTP, UTP, and GTP) and adenosine monophosphate (AMP) were not. On the other hand, ATP and CTP were substrates and beta,gamma-NH-ATP was a competitive inhibitor of ATP hydrolysis, but not an inhibitor in conjunction with Mg2+ and K+. The Ca2+-ATPase from sarcoplasmic reticulum and F1, the Mg2+-ATPase from the inner mitochondrial membrane, were also inhibited by Mg2+. Catecholamines reversed inhibition of the Ca2+-ATPase, but not that of F1.  相似文献   

19.
We have recently characterised the presence of a Ca2(+)-mobilising receptor for ATP which stimulates exocytosis in differentiated HL60 cells. Here we demonstrate that the undifferentiated HL60 cells also respond to extracellular ATP by stimulating an increase in inositol phosphates and exocytosis. Of the nucleotides (ATP, UTP, ITP, ATP gamma S, AppNHp, XTP, CTP, GTP, 8-Br-ATP and GTP gamma S) that were active in stimulating inositol phosphate formation, only UTP, ATP, ITP, ATP gamma S and AppNHp were active in stimulating secretion. On differentiation, the extent of secretion due to the purinergic agonists ATP, ITP, ATP gamma S and AppNHp remained unchanged whilst secretion due to UTP, a pyrimidine, was substantially increased. These results indicate that the effect of ATP and UTP may be mediated via separate purinergic and pyrimidinergic receptors, respectively.  相似文献   

20.
1. The addition of ATP to cultured bovine aortic endothelial cells induced the increase in intracellular free calcium concentration ([Ca2+]i) and thereby activated the sodium/proton exchanger and the prostacyclin production in a similar dose-dependent manner, as observed by the addition of ATP. 2. Other nucleoside triphosphates also activated the cells and the potency orders of the nucleotides were ATP greater than UTP greater than ITP greater than CTP greater than GTP for all the responses. 3. Pretreatment of the cells with UTP desensitized the response to ATP and the pretreatment of ATP desensitized the response to UTP. 4. The responses to ATP and UTP were inhibited by neither pertussis nor cholera toxin. 5. The receptor for UTP, however, may be a distinct pyrimidinoceptor different from the purinoceptor of the cells for ATP, because the 50% effective concentration of UDP was much larger than that of UTP, while ATP and ADP were essentially equipotent ligands to the endothelial cells.  相似文献   

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