Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Elongation factor Ts (EF-Ts) catalyzes the reaction EF-Tu X GDP + nucleotide diphosphate (NDP) reversible EF-Tu X NDP + GDP where NDP is GDP, IDP, GTP, or GMP X PCP. The EF-Ts-catalyzed exchange rates were measured at a series of concentrations of EF-Tu X [3H] GDP and free nucleotide. Plotting the rate data according to the Hanes method produced a series of lines intersecting on the ordinate, a characteristic of substituted enzyme mechanisms. GDP is a competitive inhibitor of IDP exchange, a result predicted for the substituted enzyme mechanism but inconsistent with ternary complex mechanisms that involve an intermediate complex containing EF-Ts and both substrates. The exchange of both GTP and the GTP analog GMP X PCP also follow the substituted enzyme mechanism. The maximal rates of exchange of GDP and GTP are the same, which indicates that the rates of dissociation of EF-Ts from EF-Tu X GDP and EF-Tu X GTP are the same. The steady-state maximal exchange rate is slower by a factor of 20 than the previously reported rate of dissociation of GDP from EF-Ts X EF-Tu. This is interpreted to mean that the rate-determining step in the exchange reaction is the dissociation of EF-Ts from EF-Tu X GDP.
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PMID:A study of the kinetic mechanism of elongation factor Ts. 404 68

The requirement of initiation factors F(1) (highly purified) and F(2) (electrophoretically homogeneous) for ribosomal binding of N-formylmethionyl transfer RNA (fMet approximately tRNA) at low Mg(2+) concentration (3.5 mM), with the trinucleoside diphosphate ApUpG as messenger, was studied under various experimental conditions with 30S + 50S ribosomes and with 30S subunits alone. The results were qualitatively the same in both cases but the amount of binding was two to three times higher when both 30S and 50S subunits were present. Although there was a virtually absolute requirement for F(2) in all cases, considerable binding occurred at 0 degrees in the absence of added F(1). F(1) addition stimulated binding up to twofold under these conditions. However, at 25 degrees , the temperature at which the reaction is usually carried out, there was very little binding with F(2) alone and addition of F(1) stimulated the reaction five- to sixfold. Contrary to current belief, the GTP analog 5'-guanylyldiphosphonate (GMP-PCP) cannot replace GTP in the binding reaction. In particular, there was but little stimulation of binding (about 1.5-fold) by addition of F(1) to F(2)-containing samples when GMP-PCP was used. In marked contrast, binding was stimulated up to sevenfold by addition of F(1) when GTP was substituted for the analog. Under these conditions, there was an ApUpG and F(1)-dependent hydrolysis of GTP. This is observable with 30S subunits alone and can hardly be related to the occurrence of translocation. The results may be interpreted to mean that a complex relatively stable at 0 degrees , but less stable at 25 degrees , is formed upon addition of F(2) alone. Conversion of the less stable to the more stable form of complex is made possible by addition of F(1). This is accompanied or mediated by cleavage of GTP.
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PMID:Polypeptide chain initiation in E. coli: studies on the function of initiation factor F1. 489 78

Mouse lymphoma cells were shown to be unresponsive to prostaglandin E1 in terms of cAMP production. The endogenous ATP concentration was shown to be low. Addition of ATP in the presence of IMBX into the incubation medium resulted in elevation of the intracellular pool of ATP and the cells responded to PGE1 by increasing cAMP production. The ATP effect is specific and cannot be substituted by GTP. ATP analogue (AMP-PCP) can, however, produce a similar effect to ATP. The intracellular ATP concentration can be lowered by incubation with iodoacetate and potassium cyanide. This caused a drastic decrease of the cAMP level. Ethionine on the other hand had no effect on the intracellular ATP level.
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PMID:ATP uptake by mouse lymphoma cells. 618 Jun 74

When purified muscle actin was mixed with microtubule-associated proteins (MAPs) prepared from brain microtubules assembled in vitro, actin filaments were organized into discrete bundles, 26 nm in diameter. MAP-2 was the principal protein necessary for the formation of the bundles. Analysis of MAP-actin bundle formation by sedimentation and electrophoresis revealed the bundles to be composed of approximately 20% MAP-2 and 80% actin by weight. Transverse striations were observed to occur at 28-nm intervals along negatively stained MAP-actin bundles, and short projections, approximately 12 nm long and spaced at 28-nm intervals, were resolved by high-resolution metal shadowing. The formation of MAP-actin bundles was inhibited by millimolar concentrations of ATP, AMP-PCP (beta, gamma-methylene-adenosine triphosphate), and pyrophosphate but not by AMP, ADP, or GTP. The addition of ATP to a solution containing MAP-actin bundles resulted in the dissociation of the bundles into individual actin filaments; discrete particles, presumably MAP-2, were periodically attached along the splayed filaments. These results demonstrate that MAPs can bind to actin filaments and can induce the reversible formation of actin filament bundles in vitro.
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PMID:Microtubule-associated proteins (MAPs) and the organization of actin filaments in vitro. 627 Jan 55

Opioid receptor bindings of four different ligands, dihydromorphine (DHM), D-Ala2-D-Leu5-enkephalin (DADLE), ethylketocyclazocine (EKC) and phencyclidine (PCP), were investigated with the treatment of 5,5'-dithiobis-(2-nitrobenzoic acid), DTNB, and 5,5'-dithiobis-(2-nitro-N-2'-hydroxyethylbenzamide), DTNHEB; a relative positive charged analog of DTNB. DTNB and DTNHEB effectively inhibited the binding of DHM and DADLE. Despite the presence of maximally effective concentrations of DTNB for DHM and DADLE, the receptor binding of EKC decreased intermediately, like effect of a partial agonist. DTNHEB inactivated the binding of EKC in a similar fashion to that of DHM. DTNB did not alter the intensity of the decrease of EKC binding by DTNHEB, even given concurrently. It suggests that an anionic center of the receptor has multiple active sulfhydryl sites. The ability of GTP to inhibit DADLE binding to the receptor disappeared by the pre-treatment of DTNB, and DTNB-induced inactivation of opioid agonist binding was potentiated in the presence of NaCl. DTNB-sensitive site may couple a mechanism of ligand binding that GTP regulated. The receptor binding of PCP was not influenced by DTNB and/or DTNHEB.
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PMID:Regulation of opioid receptor binding; possible mechanisms of sulfhydryl groups in the binding site. 631 63

Poliovirus replicase- and host factor-catalyzed copying of 3'-terminal polyadenylic acid [poly(A)] of poliovirion RNA was studied. Host factor-stimulated synthesis of polyuridylic acid [poly(U)] by the replicase required ATP in addition to UTP. ATP was not required for the oligouridylic acid-primed copying of 3'-terminal poly(A) of virion RNA. GTP, CTP, and AMP-PCP (5'-adenylyl beta-gamma methylenediphosphate, an ATP analog) could not replace ATP in host factor-stimulated synthesis of poly(U). Antibodies to poliovirus genome-linked protein (VPg) specifically precipitated in vitro-synthesized poly(U) from a host factor-stimulated reaction. The poly(U) synthesized in a host factor-stimulated reaction was shown to be attached to VPg precursor polypeptide(s) via a tyrosine-phosphate bond as found in poliovirion VPg-RNA.
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PMID:ATP is required for initiation of poliovirus RNA synthesis in vitro: demonstration of tyrosine-phosphate linkage between in vitro-synthesized RNA and genome-linked protein. 632 50

Evidence in this and other reports from this laboratory suggest that adrenergic nerves in rat heart ventricle slices incubated in a Na+-deprived (choline+) medium containing Ca++ (Ch+--Ca++), transport (by a cocaine-sensitive mechanism) 3H-norepinephrine outwardly from synaptic vesicles attached or fused to the plasma membrane. The 3H-amine secretion was not inhibited by probenecid, an anion transport inhibitor which may prevent exocytosis. The 3H-amine release was rapidly inhibited by exogenous nucleotides ATP, UTP, and GTP greater than ADP greater than AMP greater than the nucleoside adenosine. Magnesium++ tended to increase and reserpine to decrease the effect of ATP. Neither increasing the [Ca++] nor [Mg++] (to compete with Ca++ for ATP) decreased the effect of 3 mM ATP. After secretion began, lowering the Ca++ concentration by ommission, or by the inclusion of either a low concentration of EDTA or the Ca++-binding, but non-energy-conserving synthetic analogs of ATP: AMP--PCP and AMP--PNP, gradually lowered the rates of secretion. By comparison, the rapid effects of the energy-conserving nucleotides suggested that their effects were at least partially independent of chelation, and were energy dependent. ATP, unlike cocaine, did not inhibit the uptake of NE in a Krebs HCO3 medium. Inhibition of (Na+ + K+)-ATPase by ouabain neither inhibited the release by Ch+--Ca++, nor antagonizes the release inhibiting effect of ATP. Hence, ATP did not increase apparent retention of NE by stimulating the uptake of released NE. The ATP-inhibited secretion was not increased by theophylline.
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PMID:The effect of exogenous adenosinetriphosphate on the choline-calcium stimulated release of 3H-norepinephrine in rat heart ventricle slices. 668 77

Intracellular ATP-dependent Ca2+-sequestration mechanisms were studied in isolated dispersed rat pancreatic acini following treatment with saponin or digitonin to disrupt their plasma membranes. In the presence of 45Ca2+ concentrations less than 10(-6) mol/liter, addition of 5 mmol/liter ATP caused a rapid increase in 45Ca2+ uptake exceeding the control by fivefold. ADP mimicked the ATP effect by 50 to 60%, whereas other nucleotides such as AMP-PNP, AMP-PCP, CTP, UTP, ITP, GTP, cAMP and cGMP did not. Maximal ATP-promoted Ca2+ uptake was obtained at 10(-5) mol/liter Ca2+. Inhibition of Ca2+ uptake by mitochondrial inhibitors was dependent on the Ca2+ concentration, indicating the presence of different Ca2+ storage systems. Whereas the apparent half-saturation constant found for mitochondrial Ca2+ uptake was approximately 4.5 X 10(-7) mol/liter, in the presence of antimycin and oligomycin (nonmitochondrial uptake) it was approximately 1.4 X 10(-8) mol/liter. In the absence of Mg2+ both ATP- and ADP-promoted Ca2+ uptake was nearly abolished. The Ca2+ ionophore and mersalyl blocked Ca2+ uptake, Electron microscopy showed electron-dense precipitates in the rough endoplasmic reticulum of saponin-treated cells in the presence of Ca2+, oxalate and ATP, which were absent in intact cells and in saponin-cells without ATP or pretreated with A23187. The data suggest the presence of mitochondrial and nonmitochondrial ATP-dependent C2+ storage systems in pancreatic acini. The latter is likely to be located in the rough endoplasmic reticulum.
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PMID:Calcium uptake into acini from rat pancreas: evidence for intracellular ATP-dependent calcium sequestration. 680 Dec 63

The translocation of the mRNA in relation to the ribosome during peptide synthesis represents an example for a mechanochemical reaction in which the chemical bond energy of GTP is transformed into coordinated motion. We demonstrate here that translocation can be explained simply by binding equilibria between the tRNA, the mRNA, and their binding sites on the ribosome. The presence of two cognate tRNAs shifts the association constant for the 70 S ribosome . AUGU3 complex from 6.8 x 10(5) to 2.2 x 10(8) M-1. The elongation factor G and GTP or guanosine-5'-(beta,gamma-methylene)triphosphate GMP-PCP) displace the methionine tRNAs which can be formylated (tRNAfMet) from the quaternary complex 70 S . AUGU3 . tRNAfMet . tRNAPhe. Only the ternary complex Phe-tRNAPhe . elongation factor Tu . GMP-PCP shows an absolute preference for the aminoacyl-tRNA binding site (A site) (K a = 6.6 x 10(6) M-1). AcPhe-tRNAPhe, (N alpha-acetylphenylalanyl-tRNA) an analogue of a peptidyl-tRNA exhibits a 20-fold higher affinity to the peptidyl-tRNA binding site (P site) (K a = 3.5 x 10(6) M-1) as against the A site (K a = 1.8 x 10(6) M-1) at 8 mM Mg2+. Compared to aminoacyl-tRNA and tRNA, peptidyl-tRNA shows a 3- to 15-fold higher affinity toward complementary oligonucleotides both in the binary complex and in the presence of 70 S ribosomes (UUCA . AcPhe-tRNAPhe: K a = 1.9 x 10(5) M-1), UUCA . tRNAPhe:K a = 3.2 x 10(4) M-1). This indicates a stabilization of the peptidyl-tRNA . mRNA complex during translocation. Our data support a concept of mRNA translocation in which the removal of the deacylated tRNA from the P site requires GTP energy and a peptidyl-tRNA . mRNA complex diffuses from its low affinity site (A) to its high affinity binding site (P).
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PMID:Mechanism of translocation. Binding equilibria between the ribosome, mRNA analogues, and cognate tRNAs. 703 57

The double-stranded RNA bacteriophage phi 6 contains a virion-associated RNA-dependent RNA polymerase complex. Removal of the virus envelope and the nucleocapsid surface protein, P8, reveals a nucleocapsid core particle (proteins P1, P2, P4, P7) which is the viral polymerase complex, capable of synthesizing RNA strands of positive polarity. The in vitro plus strand synthesis (transcription) reaction of the particle obtained from the mature virion was optimized and its activation and inactivation were investigated. Purine nucleoside triphosphates (NTPs), binding to a low-affinity binding site in the polymerase complex, activated plus strand synthesis. GTP was the preferred NTP, but dGTP, ddGTP, and the noncleavable analog GMP-PCP could also switch on transcription. This NTP-binding site is probably different from that of the unspecific viral NTPase found in protein P4 and also from that of the rNTP-specific RNA polymerase active site. Binding of purine NTPs was sufficient for the switch-on; hydrolysis of the NTP was not required. Besides nucleotides, divalent cations had an effect on phi 6 in vitro plus strand synthesis. Magnesium ions are required for the activity but calcium ions inhibit the reaction. Manganese ions are shown to dissipate the effect of magnesium and calcium ions, leading to uncontrolled, exceptionally high level plus strand synthesis.
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PMID:In vitro transcription of the double-stranded RNA bacteriophage phi 6 is influenced by purine NTPs and calcium. 788 44


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