Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.16.2 (PCP)
 3,761 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

(1-Aminoethyl)boronic acid (Ala-B), an analogue of alanine in which a boronic acid group replaces the carboxyl group, has been synthesized and found to inhibit the first two enzymes, alanine racemase (from Bacillus stearothermophilus, EC 5.1.1.1) and D-alanine:D-alanine ligase (ADP-forming) (from Salmonella typhimurium, EC 6.3.2.4), of the D-alanine branch of bacterial peptidoglycan biosynthesis. In both cases, time-dependent, slow binding inhibition is observed due to the generation of long-lived, slowly dissociating complexes. Ala-B inhibits alanine racemase with a Ki of 20 mM and a kappa inact of 0.15-0.35 min-1. Time-dependent loss of activity is paralleled by conversion of the 420-nm chromophore of initial bound PLP aldimine to a 324-nm absorbing species. On dilution of Ala-B, racemase activity is regained with a t1/2 of ca. 1 h. The D-Ala-D-Ala ligase also shows progressive inhibition by Ala-B provided ATP (but not AMP-PNP or AMP-PCP) is present. The presence of D-alanine along with ATP also leads to Ala-B-induced inactivation. Kinetic analysis suggests Ala-B can compete with D-alanine at either of the two D-alanine binding sites, and on inactivation with Ala-B, labeled D-alanine, and labeled ATP, the inactive enzyme has stoichiometric amounts of D-alanine, ADP, Pi, and Ala-B bound. The half-life of inactive enzyme complexes varied from approximately 2 h (without D-alanine) to 4.5 days (with D-alanine). No D-Ala-D-Ala-B dipeptide was detected.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:(1-Aminoethyl)boronic acid: a novel inhibitor for Bacillus stearothermophilus alanine racemase and Salmonella typhimurium D-alanine:D-alanine ligase (ADP-forming). 266 72

The patch-clamp open-cell recording configuration has been used to investigate the effects of non-hydrolyzable analogues of ATP on the diazoxide-activation of KATP channels in the insulin-secreting cell line RINm5F. K+ channels inhibited by 0.1, 0.5 and 1.0 mM ATP were consistently activated by 200 microM diazoxide. During sustained activation of channels, exchange of ATP for either AMP-PNP, AMP-PCP or ATP gamma S abolished the effects of diazoxide. If diazoxide was added to the membrane in the continued presence of AMP-PNP, AMP-PCP or ATP gamma S either no effects were observed or alternatively a small transient activation of channels occurred. This study suggests that protein phosphorylation is necessary for diazoxide to activate ATP-sensitive potassium channels in insulin-secreting cells.
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PMID:Protein phosphorylation is required for diazoxide to open ATP-sensitive potassium channels in insulin (RINm5F) secreting cells. 266 56

1. The inside-out configuration of the patch-clamp method was used to study the effects of MgATP, free ATP and Mg2+ on single ATP-sensitive K+ channel currents in rat pancreatic beta-cells. 2. Magnesium ions caused a marked reduction of channel activity: 5 mM-free Mg2+ produced a 50% reduction in the activity of inward currents recorded at -60 mV in symmetrical K+ concentrations. 3. Inhibition of channel activity by MgATP does not involve phosphorylation as both free ATP (i.e. ATP in the absence of divalent cations) and non-hydrolysable ATP analogues were effective inhibitors. 4. Magnesium ions produced a striking reduction in the ability of ATP (total) to inhibit channel activity. When channel activity was plotted as a function of the total ATP concentration, the Ki for channel inhibition was 4 microM in Mg2(+)-free solution, compared to a Ki of 26 microM in the presence of 2 mM-Mg2+. The shape of the relationship between channel activity and the total ATP concentration was not changed by Mg2+. When channel activity was plotted as a function of the free ATP concentration, however, Mg2+ had little effect on Ki. This suggests that free ATP is the more potent inhibitor of channel activity and that MgATP has little inhibitory effect. 5. ATP analogues that dissociate only as far as the tribasic form were also able to inhibit channel activity. This suggests that both ATP4- and ATPH3- can block the channel. 6. Like ATP, ADP was more effective at inhibiting channel activity in the absence of Mg2+, that is as the free base. The non-hydrolysable ATP analogues AMP-PNP and AMP-PCP, however, were more effective in the presence of Mg2+. 7. It is suggested that (1) the potency of inhibition is related to the amount of negative charge carried by the ion and (2) the intracellular concentration of free ATP will be an important modulator of channel activity in the intact beta-cell.
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PMID:ATP-sensitive K+ channels in rat pancreatic beta-cells: modulation by ATP and Mg2+ ions. 269 45

Beef heart mitochondrial ATPase (F1) catalyzes the hydrolysis of the ATP analog adenyl-5-yl imidodiphosphate (AMP-PNP). The reaction products are inorganic phosphate and adenyl-5-yl phosphoramidate (AMP-PN) as determined by HPLC analysis. The hydrolysis occurs in both the presence and absence of added divalent metal ions and is stimulated by potassium. The kinetic properties of the hydrolytic reaction depend markedly on the identity of the added divalent metal. GMP-PNP and AMP-CPP are also hydrolyzed, while AMP-PCP is not. Adenyl-5-yl phosphoramidate is a potent effect of beef heart mitochondrial ATPase activity. Based on these data, a reinterpretation of work based on the assumption that AMP-PNP is not hydrolyzed is presented.
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PMID:Hydrolysis of adenyl-5-yl imidodiphosphate by beef heart mitochondrial ATPase. 286 12

Glutamine synthetase of plants is the physiological target of tabtoxinine-beta-lactam, a toxin produced by several disease-causing pathovars of Pseudomonas syringae. This toxin, a unique amino acid, is an active site-directed, irreversible inhibitor of glutamine synthetase from pea. ATP is required for inactivation. Neither ADP, AMP, nor adenosine 5'-(beta,gamma-methylene)triphosphate (AMP-PCP) supports inactivation. Adenyl-5'-yl imidophosphate (AMP-PNP) is slowly hydrolyzed by glutamine synthetase to produce adenyl-5'-yl phosphoramidate (AMP-PN) and inorganic phosphate as identified by 31P NMR spectroscopic analysis. AMP-PNP also supports a slow inactivation of glutamine synthetase by tabtoxinine-beta-lactam. These data are consistent with gamma-phosphate transfer being involved in the inactivation. Completely inactivated glutamine synthetase has 0.9 mumol of toxin bound/mumol of subunit. One mumol of ATP is bound per mumol of subunit of glutamine synthetase in the absence of either the toxin or another active site-directed inhibitor, methionine sulfoximine; whereas, a 2nd mumol of either [alpha- or gamma-32P]ATP is bound per mumol of subunit when glutamine synthetase is incubated in the presence of either toxin or methionine sulfoximine until all enzyme activity is lost. These data suggest that the gamma-phosphate hydrolyzed from ATP during inactivation remains with the enzyme-inhibitor complex, as well as the ADP. The open chain form, tabtoxinine, was neither a reversible nor an irreversible inhibitor of glutamine synthetase, suggesting that the beta-lactam ring is necessary for inhibition. The inactivation of glutamine synthetase with tabtoxinine-beta-lactam is pseudo-first-order when done in buffer containing 15% (v/v) ethylene glycol. The rate constant for this reaction is 3 X 10(-2) S-1, and the Ki for the toxin is 1 mM. Removal of the ethylene glycol from the buffer allows the reaction to proceed in a non-first-order manner with the apparent rate constant decreasing with time. As the enzyme is inactivated in these conditions, the binding affinity for the toxin appears to decrease, while the Km observed for glutamate does not change.
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PMID:Inactivation of pea seed glutamine synthetase by the toxin, tabtoxinine-beta-lactam. 287 40

1. Adult female Culex pipiens and Culiseta inornata have purinergic receptors that respond to extracellular ADP and related compounds. Stimulation of these receptors caused ingestion of artificial diets. Addition of bicarbonate to the saline solvent enhanced the phagostimulatory effect. Saline-bicarbonate was as effective a solvent as blood plasma for Cx. pipiens, and was used in the dose-effect determinations. Ranking of the potencies was: ADP greater than AMP-PNP greater than ATP = AMP greater than AMP-PCP much greater than 2'dAMP greater than 2'dADP greater than 2'dATP. At 1 mM concentration, ITP, GTP, CTP, UTP, c-AMP, 2'AMP, 3'AMP, DPG, or GSH + glucose caused fewer than 50% of the insects to gorge, as did 2'3'dd-ATP, A tetra P, and AMP-CPP at 100 microM. 2. The potency ranking for Cu. inornata was: ADP greater than AMP-PNP greater than ATP greater than AMP-PCP much greater than AMP much greater than AMP-S. The concentrations required to produce the ED50 response (inducing 50% of the test insects to gorge) were much higher than those required for Cx. pipiens; however, saline, not saline-bicarbonate, was used as the solvent. With the exception of the very low potency of AMP for Cu. inornata, the ADP potency index values for the other chemicals tested on both species are similar.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Purinergic reception by culicine mosquitoes. 290 19

The possible involvement of adenosine diphosphate (ADP) in haemostatic platelet aggregation was investigated by determining the duration of primary haemorrhage as standardized bleeding times from punctures of small mesenteric arteries in anaesthetized rats. The bleeding times were highly significantly increased by infusing into the mesenteric arterial blood flowing towards the punctures either the nucleotide-dephosphorylating enzyme apyrase or the ADP-receptor antagonists ATP, adenosine 5'-(beta,gamma-methylene)triphosphonate (AMP-PCP) or 2-methylthioadenosine 5'-(beta,gamma-methylene)triphosphonate (2-MeS-AMP-PCP). The increases in bleeding times could not be accounted for by local vasodilator effects of the agents. It is concluded that the presence of ADP through local release and/or formation at sites of vascular injury contributes significantly to haemostasis, presumably by accelerating platelet aggregation.
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PMID:Evidence for the dependence of arterial haemostasis on ADP. 290 27

The dynein arms that power ciliary motility are normally permanently attached by one end exclusively to subfiber A of each axonemal doublet (N) while the other (head) end transiently attaches to the subfiber B of the adjacent doublet (N + 1) to produce sliding of the doublets. In Tetrahymena axonemes, sliding of contiguous groups of doublets is induced by ATP suggesting that, in the absence of exogenous protease, there may be sets of potentially active and potentially inactive or refractory arms in a single axoneme. In the presence of a non-hydrolyzable analog of ATP, beta,gamma-methylene adenosine 5'-triphosphate (AMP-PCP), about half the doublets in an axonemal preparation retain all arms bound to subfiber A, but half the doublets show long regions where some arms are pulled away from subfiber A of doublet N and attached to subfiber B of doublet N + 1 by their head ends. In AMP-PCP-induced splaying, positional information regarding arm state is retained. Analysis reveals that throughout regions where B subfiber attachment is found, small groups of about four subfiber B attached arms alternate with groups of about four arms that remain attached to subfiber A. This unique pattern of attachment suggests that arms function co-operatively in groups of four. Further, the repetition of the pattern is reminiscent of metachronal activity seen at higher levels of biological organization. This suggests that in these regions we have instantaneously preserved groups of arms capable of attaching to and detaching from doublet N + 1 in rapid succession. This appearance could be used to delineate the potentially active sets of arm, primed for mechanochemical activity, within an axoneme.
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PMID:Dynein arm attachment probed with a non-hydrolyzable ATP analog. Structural evidence for patterns of activity. 296 33

The dynein arms of ciliary doublet microtubules cause adjacent axonemal doublets to slide apart with fixed polarity. This suggests that there is a unique mechanochemistry to the dynein arm with unidirectional force generation in all active arms and also that not all arms are active at once during a ciliary beat. Negative stain and thin-section images of arms in axonemes treated with beta, gamma methylene adenosine triphosphate (AMP-PCP) show a consistent subunit construction where the globular head of the arm interacts with subfiber B of doublet N+1. This interpretation differs from that provided by freeze etch and STEM interpretations of in situ arm construction and has implications for the mechanochemical cycle of the arm. A computer model of the arms in relation to other axonemal structures has been constructed to test these interpretations. Attachment of the head of the arm subfiber B is directly demonstrable in splayed axonemes in AMP-PCP. About half of the doublets in an axoneme show such attachments, while half do not. This might imply that about half the doublets in an axoneme are active at any given instant and can be identified as such. This information may be useful in probing questions of how active arms differ biochemically from inactive arms and of how microtubule translocators in general become active.
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PMID:Dynein as a microtubule translocator in ciliary motility: current studies of arm structure and activity pattern. 297

Spin-labeled derivatives of AMP-PCP, ATP, and 2'-deoxy-ATP, with a nitroxide moiety attached to the ribose ring [3'-O-(1-oxy-2,2,5,5-tetramethylpyrroline-3-carbonyl)nucleotide], are used to study the nucleotide binding site stoichiometry of sarcoplasmic reticulum (SR) ATPase. With all derivatives, a maximal binding of 4.5 nmol/mg of SR protein is found, a value close to the number of phosphorylation sites obtained with ATP. The spin-labeled nucleotides cannot be utilized by the enzyme as substrates. Binding of spin-labeled nucleotides is inhibited by labeling the ATPase with fluorescein 5'-isothiocyanate, indicating that all the labeled nucleotide is located at the catalytic site. Additions of spin-labeled ATP to vesicle suspensions during steady turnover demonstrate competitive inhibition of both catalysis and the regulatory effect normally exhibited by ATP. As secondary binding of spin-labeled ATP is not detected at pertinent concentrations, it is suggested that both functions of ATP may be effected through a single site.
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PMID:Interaction of spin-labeled nucleotides with sarcoplasmic reticulum adenosinetriphosphatase. 297 48


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