Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0009443 (cold)
 92,137 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A biochemical assay for taxol with sensitivity to 0.1 microM has been developed. Taxol-dependent formation of tubulin polymers occurs at 37 degrees C in 1.0 M glutamate in the absence of GTP. These polymers are cold-stable and hydrolyze GTP at 0 degrees C, whereas tubulin alone will not hydrolyze the nucleotide in the cold. This assay has been used to follow rabbit serum levels of taxol injected iv. Although the drug appears to be almost totally protein-bound, it is nevertheless rapidly cleared from serum. The apparent alpha-phase and beta-phase half-lives after iv bolus administration in one rabbit are 2.7 and 42 mins, respectively.
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PMID:Tubulin-dependent biochemical assay for the antineoplastic agent taxol and application to measurement of the drug in serum. 612 84

Dissociated bovine brain microtubule protein has been shown to reassemble at 0 degrees C in the presence of the drug taxol. Tubulin polymerization was monitored both by electron microscopy of the polymeric structures and by incorporation of tritiated GTP into filterable polymeric structures. Most of the labeled guanine nucleotide uptake into tubulin polymeric structures occurred in the first 30 minutes of incubation with the drug. The initial polymerization event results in the formation of protofilamentous tubulin ribbons. The first microtubules were noted after 1 hour of incubation with the drug. After 20 hours of incubation at 0 degrees C with taxol, the bulk of the polymerized tubulin appeared to be in the form of microtubules. Cold-stable tubulin rings with a mean diameter of 34 nm were present in the reaction mixture before the addition of taxol and throughout the 20-hour incubation. Most of the rings were apparently not involved in the taxol-induced microtubule assembly. The results are consistent with a model whereby taxol induces an initial formation of protofilamentous ribbon structures, mostly from free tubulin dimers, and a slower subsequent folding of the ribbon structures into microtubules.
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PMID:Taxol induces microtubule assembly at low temperature. 612 64

A crude membrane fraction was prepared from hamster brown adipose tissue. Extensive washing of the crude membranes was crucial for the appearance of specific beta-adrenergic receptor binding as assessed by (-)-[3H]dihydroalprenolol. Adrenergic agents competed for the specific binding sites with beta 1-specificity. Binding characteristics were very similar to those earlier found in intact cells, supporting our previous finding that a single (non-tumour) mammalian cell may contain as many as 60,000 beta-adrenergic receptors. Desensitization in situ (i.e. chronic norepinephrine stimulation due to cold acclimation) only marginally affected the number of beta 1-receptors and their affinity (Ki) for norepinephrine. Total (fluoride-stimulated) adenylate cyclase increased somewhat, but the Kact for norepinephrine slightly decreased. Thus the ratio Ki/Kact was rather unaffected by cold acclimation. However, the fraction of adenylate cyclase which could be stimulated by norepinephrine decreased drastically. GTP introduced a low-affinity form (for agonist) of the receptor. The form observed in isolated cells must primarily be the high-affinity form. The basis for desensitization must reside in a diminished ability to transfer the signal from the receptor to the cyclase. This change may be molecularly located in the N-protein or in its interaction with the receptor.
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PMID:The molecular basis for adrenergic desensitization in hamster brown adipose tissue: uncoupling of adenylate cyclase activation. 614 65

An acetyl-coenzyme-A hydrolase from the supernatant fraction of rat liver is known to be rapidly inactivated at low temperature. Loss of catalytic activity is accompanied by apparent dissociation of tetrameric and dimeric forms of the enzyme into monomers. It was found that rewarming under appropriate conditions almost completely reversed the cold-induced inactivation and dissociation of the enzyme: At a protein concentration of 14 micrograms/ml, simple rewarming only partially restored the enzyme activity (less than 3% of the original activity), but at a higher concentration of the enzyme or in the presence of 1 mg/ml bovine serum albumin, the reactivation by warming was greater. Warming at 37 degrees C appeared to be optimal for reactivation; warming at 25 degrees C or at 43 degrees C was less effective. Longer exposure to cold did not affect reactivation on rewarming, but on repeated inactivation and reactivation the reactivation decreased to some extent, especially at lower concentrations of enzyme protein. Among various nucleotides tested, ATP greatly enhanced the restoration of the activity, while ITP, UTP and ADP were less effective and AMP, GTP, TTP and CTP had little effect. At an enzyme-protein concentration of 14 micrograms/ml, 2 mM ATP restored the enzyme activity to about 70% of that before cold treatment, while acetyl-CoA (0.5 mM) restored the activity about 50%. High concentrations of phosphate (0.92 M) and pyrophosphate (0.45 M) restored about 80% and 95%, respectively, of the original activity. Sucrose density gradient centrifugation of the active dimer at high enzyme concentration at 4 degrees C for 20 h produced a monomeric form without catalytic activity. Gel filtration showed that simple rewarming mostly converted the monomeric enzyme obtained in this way to the dimeric form, whereas on rewarming with ATP the monomer was mostly converted to a tetrameric form. The dimeric and tetrameric forms both had catalytic activity.
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PMID:A cold-labile acetyl-coenzyme-A hydrolase from the supernatant fraction of rat liver. Reactivation and reconstitution of the active species from the inactive monomer. 614 26

Interactions of both purified tubulin and microtubule protein (tubulin plus associated proteins) with two commonly used sulfonate buffers were examined. 1,4-Piperazineethanesulfonate (Pipes) and 4-morpholineethanesulfonate (Mes) at high concentrations induce the polymerization of purified tubulin in reactions requiring only buffer, tubulin and GTP. While both reactions were temperature-dependent, cold-reversible and inhibited by GDP, colchicine or Ca2+, there were significant differences between them. Substantially lower tubulin and buffer concentrations were required for Pipes-induced polymerization; and turbidity was much more intense in the Pipes-induced than in the Mes-induced reaction at the same protein concentration. Electron microscopy demonstrated that for the most part typical smooth-walled microtubules were formed in Mes, while aberrant forms were the predominant structures formed in Pipes. When the polymerization of microtubule protein was examined as a function of buffer concentration, biphasic patterns were observed with both Pipes and Mes: polymerization occurred at both low and high, but not intermediate, buffer concentrations. The turbidity observed at high concentrations of Pipes greatly exceeded that at low concentrations. With Mes, equivalent turbidity developed at both high and low buffer concentrations. Although associated proteins copolymerized with tubulin at low buffer concentrations, they were excluded from the polymerized material at high buffer concentrations. Pipes and Mes were compared to sodium phosphate, Tris/HCl and imidazole/HCl buffers at 0.1 M in several polymerization systems using both purified tubulin and microtubule protein. The sulfonate buffers were invariably associated with more vigorous reactions than the other buffers.
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PMID:Induction of polymerization of purified tubulin by sulfonate buffers. Marked differences between 4-morpholineethanesulfonate (Mes) and 1,4-piperazineethanesulfonate (Pipes). 627 66

We studied the characteristics of cytoplasmic microtubule reassembly from endogenous tubulin pools in situ using a Brij 58-lysed 3T3 cell system. Cells that were pretreated in vivo with colcemid retain endogenous tubulin in the depolymerized state after lysis. When lysed cells were removed from colcemid block and incubated in GTP-PIPES reassembly buffer at pH 6.9, microtubules repolymerized randomly throughout the cytoplasm, appeared to be free-ended and were generally not associated with the centrosomes. However, tubulin could be induced to polymerize in an organized manner from the centrosomes by increasing the pH to 7.6 in the presence of ATP and cAMP. Microtubules polymerized in ATP had significantly longer lengths than those assembled in GTP or UTP. When cells not treated with colcemid were lysed, the integrity of the cytoplasmic microtubule complex (CMTC) was maintained during subsequent incubation in reassembly buffer. However, in contrast to unlysed, living cells, microtubules of lysed cells were stable to colchicine. A significant fraction of the CMTC was stable to cold-induced disassembly whereas microtubules reassembled after lysis were extremely cold-sensitive. When cells not treated with colcemid were lysed and incubated in millimolar Ca++, microtubules depolymerized from their distal ends and a much reduced CMTC was observed. Ca++ reversal with EGTA rapidly resulted in a reformation of the CMTC apparently by elongation of Ca++ resistant microtubules.
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PMID:Cytoplasmic microtubule assembly-disassembly from endogenous tubulin in a Brij-lysed cell model. 630 13

We recently reported the partial purification of a cAMP-independent and Ca2+-calmodulin-independent glycogen synthase kinase from porcine renal cortex (Schlender, K. K., Beebe, S. J., and Reimann, E. M. (1981) Cold Spring Harbor Conf. Cell Proliferation, 389-400). Subsequent purification indicated that the enzyme preparation consisted of at least three forms of glycogen synthase kinase which could be resolved by ATP gradient elution from aminoethylphosphate-agarose (AEP-agarose). The predominant form of glycogen synthase kinase, which eluted from AEP-agarose between 2 and 6 mM ATP, was purified approximately 800-fold and is designated GSK-A1. It had a molecular weight of 45,000-50,000 as determined by gel filtration and sucrose density gradient centrifugation. It catalyzed the transfer of 1 mol of 32P/mol of synthase subunit into a low molecular weight (10,000) CNBr peptide which was tentatively identified as Ser-7 (site 2) by high performance liquid chromatography. This phosphorylation decreased the activity ratio (activity in the absence of glucose-6-P divided by activity in the presence of 7.2 mM glucose-6-P) from 0.95 to about 0.55. GSK-A1 appeared to be specific for and had low s0.5 values for both substrates, ATP (13 microM) and glycogen synthase (0.3-0.4 microM). The enzyme could not use GTP as the phosphate donor. GSK-A1 was not affected by the protein kinase inhibitor, cAMP, cGMP, Ca2+-calmodulin, EGTA, or trifluoperazine and had a broad pH optimum (pH 7.0-8.5). A second form, GSK-A2, was eluted from AEP-agarose between 7 and 9 mM ATP. GSK-A2 could transfer a 2nd mol of 32P/mol of synthase subunit and decreased the activity ratio to 0.30. The interrelation among these multiple forms is not clear, but the data suggest that multiple kinases are required to form the highly inactivated glycogen synthase in renal tissues.
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PMID:Purification and characterization of a cAMP- and Ca2+-calmodulin-independent glycogen synthase kinase from porcine renal cortex. 631 98

Our experiments have delineated the flow of information in the cyclic nucleotide cascade of vision of ROS. A single, photoexcited rhodopsin molecule activates several hundred phosphodiesterase molecules in two stages. First, photoexcited rhodopsin (R*) interacts with transducin (T), a peripheral membrane protein consisting of alpha- (39 kD), beta- (36 kD), and gamma- (approximately 10 kD) subunits. R* catalyzes the exchange of GTP for GDP bound to the subunit of transducin. About 500 T alpha- GTPs are produced per photoexcited rhodopsin at low light levels. T alpha-GTP, released from the beta- and gamma-subunits of transducin, then interacts with the phosphodiesterase to relieve the inhibitory constraint imposed by its gamma-subunit. Hydrolysis of GTP bound to T alpha serves to restore the system to the dark state. Transducin is the amplified signal carrier in this light-triggered cascade. The formation of hundreds of T alpha- GTPs is likely to be the first stage of amplification in visual excitation. The photoactivation of the phosphodiesterase in ROS closely resembles the activation of adenylate cyclase in hormone-sensitive cells. Our cholera toxin labeling studies have shown that transducin is akin to the signal-coupling G protein of the adenylate cyclase system. Cholera toxin specifically ADP- ribosylates and inactivates the GTPase activity of T alpha, just as it does with Gs. The action of pertussis toxin on ROS further underscores the homology of the photoreceptor and hormone-responsive systems. It seems likely that transducin, the stimulatory G protein, and the inhibitory G protein are members of the same family of signal-amplifying proteins. The study of the cyclic nucleotide cascade of vision is proving to be rewarding in affording a view of a recurring motif of signal amplification in nature in addition to providing insight into the mechanism of vision.
Cold Spring Harb Symp Quant Biol 1983
PMID:Transducin and the cyclic GMP phosphodiesterase: amplifier proteins in vision. 632 79

The GDP analogue 2',2'-dideoxyguanosine 5'-diphosphate (ddGDP) supports efficient tubulin polymerization. Microtubule-associated protein (MAP) dependent microtubule assembly occurs in 0.1 M 2-(N-morpholino)-ethanesulfonate, and sheets of parallel protofilaments are formed in 1.0 M glutamate without MAPs. The nucleotide is bound to tubulin in the course of polymerization, presumably in the exchangeable GTP site. The ddGDP is not hydrolyzed, however, and is completely stable in the reaction mixture. Nor was the nonexchangeable GTP bound to tubulin hydrolyzed in ddGDP-supported polymerization: equivalent amounts of GTP remained associated with polymerized tubulin after polymerization with either ddGDP or GTP. Higher concentrations of ddGDP than GTP were required under all conditions. Nevertheless, under optimum conditions for the ddGDP-supported reaction, polymerization began with a shorter lag period and both the rate and extent of polymerization were greater with ddGDP than with GTP. The MAP-dependent reaction with ddGDP is temperature dependent, cold reversible, and inhibited by calcium and antimitotic drugs. It differs from the GTP-supported reaction in being most vigorous at minimal Mg2+ concentrations and exquisitely sensitive to GDP inhibition.
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PMID:Microtubule assembly with the guanosine 5'-diphosphate analogue 2',3'-dideoxyguanosine 5'-diphosphate. 635 8

The effect of GTP analogs and Mg on the structure of Ns and Ni, the stimulatory and inhibitory regulatory components of adenylyl cyclase, were studied in a comparative manner. Both N proteins, which are alpha beta gamma heterotrimers that differ in their alpha subunits, when exposed to GTP analogs underwent a Mg-dependent conformational change that was not dependent on subunit dissociation. This was seen both as change in sedimentation behavior at 4 degrees C from 4 S to about 3 S and by a property of the new conformation to retain guanine nucleotide tightly bound to it. Warming to 32 degrees C promoted subunit dissociation, each protein giving a mixture of 2 S alpha G and 2 S beta gamma complexes. For both Ns and Ni, these reactions were reversible: 2 S complexes of Ns and Ni associated to 3 S forms on cooling to 4 degrees C, provided the Mg concentration was at or below 10 mM and detergent concentration was below 1%, and the 3 S complexes of these proteins reverted to 4 S forms and released the nucleotide in the cold on chelation of free Mg with EDTA. Reconstitution assays with Ns-deficient membranes from cyc- S49 lymphoma cells revealed that the 3 S form of Ns is a "pre-active" form of the protein. The scheme below summarizes these findings, where G represents a guanine nucleotide. (Formula: see text) Ns and Ni differ in that more Mg is necessary to promote the 4 S to 3 S conversion of Ns than of Ni, and in that both the 2 S to 3 S to 4 S conversions proceeded more readily with Ni than with of Ns. Mg could not be shown to promote subunit dissociation. The above scheme is suggested as a plausible description of the reaction sequence leading from an unactivated to an activated N protein.
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PMID:Effects of guanine nucleotides and Mg on human erythrocyte Ni and Ns, the regulatory components of adenylyl cyclase. 638 98


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