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Query: UMLS:C0009443 (cold)
 92,137 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Treatment of isolated factor F1 by 1% dimethylsuberimidate in the presence of 50 mM (NH4)2SO4 leads to the formation of four different types of cross-linked dimers of the subunits, on average one dimer per molecule of the enzyme. This treatment results in 60-70% inactivation of factor F1. Factor F1 treated with dimethylsuberimidate does not show a change in the sedimentation coefficient and is not inactivated in the cold; it is not inactivated in the presence of Mg2+ either, nor is it activated by anions. Incubation of the cross-linked factor F1 with ADP does not lead to inactivation, although the ability to tightly bind ADP is retained. The total quantity of tightly bound ADP reaches 5 mol per mol of the cross-linked factor F1. Cross-linking of factor F1 also prevents the slow inactivation of the enzyme coupled with the hydrolysis of Mg-ATP and Mg-GTP. The dependence of the inactivation rate constant on the concentration of Mg-ATP and Mg-GTP at substrate concentrations of 0.05-2 mM is characterized by the same values of Km,app as those of the ATPase and GTPase activities of factor F1. The probability of the inactivation of factor F1 per turnover remains constant for all the concentrations of the substrates studied and is 2 . 10(-6) per turnover for the ATPase reaction and 2 . 10(-5) per turnover for the GTPase reaction. Moderate hydrostatic pressure (up to 150 atmospheres) greatly accelerates ATP-induced inactivation of factor F1. The activation volume (delta V*) of the inactivation process is equal to 5.1 . 10(-4) cm3/g, which is evidence of considerable changes in the extent of protein hydration during inactivation. Inactivation of the enzyme under pressure is accompanied by dissociation into subunits. Dimethyladipimidate, which does not cause intersubunit cross-linking in the molecule of factor F1, does not alter the properties of the native enzyme. It is suggested that the formation of one intersubunit cross-link in the molecule of factor F1 by dimethylsuberimidate affects the ability of the enzyme to undergo co-operative rearrangements of the quaternary structure under the influence of Mg2+, ADP, ATP, anions, and low temperature. The rate constants of ATP binding to the active site of factor F2 (k+1) = 2 . 10(8) M-1 . min-1), of ATP release from the active site (k-1 = 2 . 10(-2) min-1), and of ADP and Pi release from the active site (k2 = 5 . 10(3) min-1) have been determined. The results obtained confirm the correctness of Boyer's idea, according to which ATP is formed in the active site of mitochondrial ATPase without any external source of energy. Energy is used at the stage of the release of synthesized ATP from the active site of ATPase in the solution.
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PMID:Structural rearrangements in soluble mitochondrial ATPase. 645 13

The effect of kainate, an heterocyclic analogue of glutamate on tubulin polymerization was studied. We demonstrate that kainate induces a dose-dependent aggregation of rat brain tubulin either purified by DEAE-Sephadex A-50 or in the presence of MAPs into a mesh-like structure. Such polymer is cold-, CaCl2- and colchicine-insensitive. Removal of kainate from the incubation medium yields free tubulin competent for polymerizing into normal microtubules in the presence of GTP.
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PMID:Kainate reversibly aggregates brain tubulin in vitro. 647 71

Microtubule assembly has been studied turbidometrically in supernatant fluids prepared from rat brain by high-speed centrifugation. It was confirmed that assembly occurred in the absence of added GTP. Zinc ions (500 microM, but in the presence of 1 mM EGTA) stimulated assembly under these conditions. Zinc-stimulated assembly produced microtubules with normal characteristics, as judged by electron microscopy, SDS-polyacrylamide gel electrophoresis and inhibition of assembly by fructose-6-phosphate or colchicine. However, microtubules formed in the presence of such zinc concentrations were more stable to cold than controls, although the rate constant for the disassembly reaction was unchanged. Neither the stimulation of assembly by zinc nor the effect on cold stability was affected by trifluoperazine suggesting that a calmodulin-related mechanism is not involved. Microtubule "seeds" had little effect in the presence of zinc, suggesting that it may be acting on the nucleation phase of the assembly reaction. This was supported by the findings that zinc reduced the critical concentration of brain supernatant necessary for assembly and that zinc did not affect the rate constant for assembly. The results suggest zinc can in some way stabilize microtubules; possible mechanisms are discussed.
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PMID:Microtubule assembly in rat brain extracts. Further characterization of the effects of zinc on assembly and cold stability. 653 18

[14,15-3H]14,15-Dihydroforskolin [( 3H]DHF) has been used as a radioactive ligand to identify forskolin binding sites in rat brain membranes. The binding was saturable and reversible. The binding sites showed positive cooperative properties as evident from an upward convex Scatchard plot and a Hill coefficient of 1.6. The equilibrium dissociation constants (KD) were in the range between 10 microM and 10 nM as estimated from the limiting slopes of the curved Scatchard plot. Half-maximal saturation of the binding sites was observed at a ligand concentration of 225 nM. The binding kinetics were very rapid: Binding equilibrium was reached in less than 2 min and a large excess of cold forskolin displaced 80% of the radioligand within 2 min. The dissociation reaction was not first order, characterized by a decreasing dissociation rate constant. Bound [3H]DHF could be displaced with forskolin (IC50 0.3 microM), 14,15-dihydroforskolin (IC50 0.8 microM) and 7-desacetylforskolin (IC50 3 microM). However, nucleotides (ATP, GTP) and other receptor ligands (adenosine, isoproterenol) had no effect on the binding. Although the density of the forskolin binding sites (3.2 pmole/mg protein) is similar to those of other adenylate cyclase linked receptors, discrepancies between the KD and the ED50 obtained in adenylate cyclase studies and the finding that activation of the enzyme by forskolin is negative cooperative makes it difficult to clearly relate the binding sites to adenylate cyclase.
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PMID:Characterization of forskolin binding sites in rat brain membranes using [14,15-3H]14,15-dihydroforskolin as a ligand. 653 36

We have examined the effects of dilution, Ca2+, reduced temperature, and triphosphate depletion on microtubules formed from purified tubulin, heat-treated microtubule-associated proteins (MAPs), and either GTP, 2',3'-dideoxyguanosine 5'-diphosphate (ddGDP), or 2',3'-dideoxyguanosine 5'-triphosphate (ddGTP). The stability of the polymer formed with tubulin plus ddGTP without MAPs was also examined. In all cases dilution resulted in rapid depolymerization of polymer until a new turbidity plateau was established. These experiments yielded estimates of the critical concentration of tubulin of 0.09 mg/ml with GTP plus MAPs, 0.04 mg/ml with either ddGDP or ddGTP plus MAPs, and 0.07 mg/ml with ddGTP minus MAPs. Addition of CaCl2 to polymer resulted in depolymerization of microtubules formed with either GTP or ddGDP plus MAPs; but both with and without MAPs the polymer formed with ddGTP was stable to Ca2+. The polymer formed with ddGTP minus MAPs was the most cold-labile, major depolymerization occurring at 25 degrees C. With MAPs, microtubules were progressively less cold-labile when formed with GTP, ddGDP, or ddGTP. Depolymerization with GTP was virtually complete at 15 degrees C, with ddGDP at 5 degrees C, and with ddGTP at 0 degrees C. Rapid triphosphate depletion was achieved with phosphofructokinase. GTP-formed tubules were rapidly and completely depolymerized at all GTP concentrations after the enzyme was added to the reaction mixture. Both with and without MAPs polymer formed with ddGTP was progressively more stable upon enzyme addition the higher the initial ddGTP concentration. At specific ddGTP concentrations, however, less depolymerization was observed following enzyme addition if MAPs were present. Microtubules formed with ddGDP plus MAPs were unaffected by phosphofructokinase addition. This comparison of the properties of microtubules formed with MAPs and either ddGDP or ddGTP demonstrates that their stability is enhanced rather than reduced following nucleotide hydrolysis. The greater stability of microtubules formed with ddGTP plus MAPs than of the polymer formed with ddGTP minus MAPs similarly implies substantial enhancement of microtubule stability by the MAPs.
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PMID:Stability of tubulin polymers formed with dideoxyguanosine nucleotides in the presence and absence of microtubule-associated proteins. 669 77

Interaction between porcine brain tubulin and neurofilaments was investigated with reference to network formation in vitro. When a mixture of tubulin and neurofilaments was incubated at 33--34 degrees C, its low-shear viscosity measured with a falling ball viscometer, was far above the sum of viscosities of the separate components and gelation was observed upon increasing the concentrations of both tubulin and neurofilaments above a certain level. Gelation that required Mg2+ and GTP was inhibited by Ca2+,Ca2+-calmodulin or ATP. Antimitotic drugs suppressed gelation at substoichiometric concentrations and cold treatment destroyed the formed gel, which indicated that gelation occurred in conjunction with tubulin polymerization. Assaying by centrifugation revealed that the amount of tubulin co-sedimented with neurofilaments was evidently larger in the presence of neurofilaments than in their absence. Furthermore, electron micrographs showed that a large number of microtubules which were shorter than usual were formed in the presence of neurofilaments. Interestingly, measurements with an Ostwald-type viscometer demonstrated that neurofilaments elevated the viscosity of the tubulin solution in a concentration-dependent fashion. In other words, neurofilaments had the ability to stimulate polymerization of tubulin. We conclude that polymerization of tubulin with neurofilaments produces three-dimensional networks in vitro.
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PMID:Interaction of tubulin with neurofilaments: formation of networks by neurofilament-dependent tubulin polymerization. 689 May 51

The adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1)-stimulating factor from rat osteosarcoma cytosol was purified 600-fold by ion-exchange chromatography. The factor has an apparent Mr of 20000, is cold-labile, but retains activity at -20 degrees C in 10% glycerol. The factor enhanced parathyroid hormone stimulation of adenylate cyclase and restored hormone responsiveness to membranes washed with 0.5 M NaCl. These 'GTP-like' effects were not inhibited by 100 microM GDP-beta-S, which completely abolished the GTP enhancement of both basal and hormone-stimulated adenylate cyclase. Adenylate cyclase activity in the presence of the stimulating factor was linear with time, and showed hyperbolic dependence on factor concentration. The factor also linearized (in double reciprocal plots) the downward-concave Mg2+-dependence of adenylate cyclase, increasing the apparent affinity of the enzyme for Mg2+. The presence of the factor in two clonal osteosarcoma cell lines correlated with parathyroid hormone-stimulatable adenylate cyclase. Factor stimulation was absent while GTP stimulation was retained in the hormone-nonresponsive clone. Factor and hormone sensitivity were restored by in vivo passage. This factor thus may represent a guanyl nucleotide-independent path for cellular regulation of hormone response.
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PMID:Osteosarcoma cytosol factor promotes parathyroid hormone stimulation of adenylate cyclase independent of GTP. 693 11

The self-assembly of tubulin devoid of microtubule-associated proteins (MAPs) has been studied using a MES buffer containing dimethyl sulfoxide (Me2SO). Between 6% and 12% v/v Me2SO, the tubulin forms polymers which resemble microtubules in their morphology and chemical properties. These Me2SO microtubules, like normal microtubules, require GTP for assembly and are sensitive to cold, calcium ions, colchicine, and hydrostatic pressure. The polymerization shows a critical concentration which is dependent on the concentration of Me2SO. 8% Me2SO was found to be the optimum concentration for microtubule assembly. In these conditions, a linear Van t'Hoff plot is obtained, with delta H0/kJ . mol-1 = 26.5 over the range of 10-35 degrees C, and delta S0/J . K-1 . mol-1 = 186, in contrast to the assembly with MAPS or glycerol. The kinetics of polymerization shows that the apparent stoichiometry coefficient of nucleation has the value of 2. Ultracentrifugation analysis shows that there are no oligomers present at low temperatures in the absence of free nucleotide, while in identical conditions, tubulin with MAPs does form oligomers. Although the solvent conditions used supported propagation of assembly, nucleation was found to be very dependent on the transiently locally high Me2SO concentrations formed when Me2SO was added to initiate assembly. It is concluded that Me2SO preferentially stabilizes the lateral interactions.
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PMID:Tubulin polymerization in dimethyl sulfoxide. 706 96

The rate of depolymerization of microtubules upon lowering the temperature was found to depend on the amount of time elapsed since the beginning of the assembly process. In the first minutes following self-assembly at 37 degrees C, microtubules are more cold sensitive and depolymerize faster than later at the steady state. In the meanwhile, no change occurred in the average length nor in the shape of the distribution of microtubules. On the other hand, the evolution with time of the apparent dissociation rate constant of tubulin from microtubules was in good correlation with the GTP content of microtubules following assembly, showing that GTP hydrolysis modifies the tubulin-tubulin interactions. Microtubule-bound GTP was not exchangeable for GDP, but steady-state GTP hydrolysis was inhibited by GDP. This result indicates that GDP and GTP exhibit different affinities for tubulin in the body and at the ends of microtubules. It is proposed that GTP-tubulin dissociates faster from microtubules than GDP-tubulin. In other words GTP hydrolysis contributes to the stabilization of microtubules.
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PMID:Mechanism of tubulin assembly: guanosine 5'-triphosphate hydrolysis decreases the rate of microtubule depolymerization. 707 50

A brain preparation, consisting of nuclei and perikarya, was able to bind tritium-labeled diguanosine tetraphosphate ([3H]Gp4G). The binding was linear with both time and amount of extract and apparently presented two dissociation constant values (KD) of 0.16 and 0.6 microM, respectively, as determined by equilibrium binding experiments. Inhibition of [3H]Gp4G specific binding by 50% at equilibrium was accomplished by cold Gp4G, guanosine 5'-tetraphosphate, diguanosine triphosphate, and GTP at 0.7, 2.8, 3.1, and 10 microM concentrations, respectively. Diadenosine tetraphosphate (Ap4A) at concentrations up to 100 microM did not affect the observed binding of [3H]Gp4G to brain. These results suggest that this binding is specific and requires the existence of 4 phosphates plus at least 1 guanosine residue in the molecule. The binding of Gp4G to brain increased with time of development reaching a plateau at about 20 days after birth. The data are discussed in relation to previous results on the binding of Ap4A to brain and to DNA polymerase-alpha (Grummt, F., Waltl, G., Jantzen, H-M., Hamprecht, K., Huebscher, U., and Kuenzle, C. C. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 6081-6085; Rapaport, E., Zamecnik, P. C., and Baril, E. F. (1981) Proct. Natl. Acad. Sci. U. S. A. 78, 838-842).
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PMID:Binding of P1,P4-bis(5'-guanosyl)tetraphosphate to brain. 711 13


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