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

A procedure employing streptolysin O to effect the selective permeabilization of either the apical or basolateral plasma membrane domains of MDCK cell monolayers grown on a filter support was developed which permeabilizes the entire monolayer, leaves the opposite cell surface domain intact, and does not abolish the integrity of the tight junctions. This procedure renders the cell interior accessible to exogenous macromolecules and impermeant reagents, permitting the examination of their effects on membrane protein transport to the intact surface. The last stages of the transport of the influenza virus hemagglutinin (HA) to the apical surface were studied in pulse-labeled, virus-infected MDCK cells that were incubated at 19.5 degrees C for 90 min to accumulate newly synthesized HA in the trans-Golgi network (TGN), before raising the temperature to 35 degrees C to allow synchronized transport to the plasma membrane. In cells permeabilized immediately after the cold block, 50% of the intracellular HA molecules were subsequently delivered to the apical surface. This transport was dependent on the presence of an exogenous ATP supply and was markedly inhibited by the addition of GTP-gamma-S at the time of permeabilization. On the other hand, the GTP analogue had no effect when it was added to cells that, after the cold block, were incubated for 15 min at 35 degrees C before permeabilization, even though at this time most HA molecules were still intracellular and their appearance at the cell surface was largely dependent on exogenous ATP. These findings indicate that GTP-binding proteins are involved in the constitutive process that effects vesicular transport from the TGN to the plasma membrane and that they are charged early in this process. Transport of HA to the cell surface could be made dependent on the addition of exogenous cytosol when, after permeabilization, cells were washed to remove endogenous cytosolic components. This opens the way towards the identification of cell components that mediate the sorting of apical and basolateral membrane components in the TGN and their polarized delivery to the cell surface.
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PMID:Transport of influenza HA from the trans-Golgi network to the apical surface of MDCK cells permeabilized in their basolateral plasma membranes: energy dependence and involvement of GTP-binding proteins. 212 1

Dampened oscillations of microtubule assembly can accompany polymerization at high tubulin subunit concentrations. This presumably results from a synchronization of dynamic instability behavior, which generates a large population of rapidly disassembling microtubules, that liberate tubulin-GDP oligomers. Subunits in oligomers cannot assemble until they dissociate, to allow GDP-GTP exchange. To determine whether rapidly disassembling microtubules generate oligomers directly, we measured the rate of dilution-induced disassembly of tubulin-GDP microtubules and the rate of dissociation of GDP from the so-formed tubulin-GDP subunits. The rate of GDP dissociation from liberated subunits was found to correspond to that of tubulin-GDP subunits (t1/2 = 5 s), rather than tubulin-GDP oligomers. This indicates that tubulin-GDP subunits are released from microtubules undergoing rapid disassembly. Oligomers apparently form in a side reaction from the high concentration of tubulin-GDP subunits liberated from the synchronously disassembling microtubule population. The rate of subunit dissociation is 0.11 s-1 with oligomers formed by concentrating tubulin-GDP subunits and 0.045 s-1 with oligomers formed by cold-induced microtubule disassembly. This difference provides evidence that the conformation of tubulin-GDP subunits released from rapidly disassembling microtubules differs from tubulin-GDP subunits that were not recently in the microtubule lattice.
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PMID:Mechanism for oscillatory assembly of microtubules. 229 38

Microtubules are capable of performing synchronized oscillations of assembly and disassembly which has been explained by reaction mechanisms involving tubulin subunits, oligomers, microtubules, and GTP. Here we address the question of how microtubule nucleation or their number concentration affects the oscillations. Assembly itself requires a critical protein concentration (Cc), but oscillations require in addition a critical microtubule number concentration (CMT). In spontaneous assembly this can be achieved with protein concentrations Cos well above the critical concentration Cc because this enhances the efficiency of nucleation. Seeding with microtubules can either generate oscillations or suppress them, depending on how the seeds alter the effective microtubule number concentration. The relative influence of microtubule number and total protein concentrations can be varied by the rate at which assembly conditions are induced (e.g. by a temperature rise): Fast T-jumps induce oscillations because of efficient nucleation, slow ones do not. Oscillations become damped for several reasons. One is the consumption of GTP, the second is a decrease in microtubule number, and the third is that the ratio of microtubules in the two phases (growth-competent and shrinkage-competent) approach a steady state value. This ratio can be perturbed, and the oscillations restarted, by a cold shock, addition of seeds, addition of GTP, or fragmentation. Each of these is equivalent to a change in the effective microtubule number concentration.
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PMID:Microtubule oscillations. Role of nucleation and microtubule number concentration. 230 70

Complete replacement of the nucleotide on the exchangeable binding site of purified calf brain tubulin by the non-hydrolyzable GTP-analogue guanylyl-(beta,gamma-methylene)diphosphonate (GMPPCP) has been achieved by treatment of tubulin-GDP with phosphodiesterase-free alkaline phosphatase. GMPPCP binds to tubulin with a low affinity relative to GTP or GDP. Binding of the analogue is linked to magnesium ion concentration and, like the binding of other guanine nucleotides, is promoted by high concentrations of glycerol. The complex of pure tubulin and GMPPCP readily assembles at 37 degrees C into microtubules or curled ribbons of protofilaments, depending on buffer composition. Assemblies are cold-reversible at 0-2 degrees C, and multiple reversible assemblies can be observed during repeated heating/cooling cycles.
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PMID:Interactions of tubulin with guanylyl-(beta-gamma-methylene)diphosphonate. Formation and assembly of a stoichiometric complex. 233 45

Dolastatin 10, a cytostatic peptide containing several unique amino acid subunits, was isolated from the marine shell-less mollusk Dolabella auricularia (Pettit GR, Kamano Y, Herald CL, Tuinman AA, Boettner FE, Kizu H, Schmidt JM, Baczynskyj L, Tomer KB and Bontems RJ, J Am Chem Soc 109: 6883-6885, 1987). Since our preliminary studies demonstrated that dolastatin 10 inhibited tubulin polymerization and the binding of radiolabeled vinblastine to tubulin, an initial characterization of the properties of dolastatin 10 included a comparison to other antimitotic drugs interfering with vinca alkaloid binding to tubulin (vinblastine, maytansine, rhizoxin, and phomopsin A). Dolastatin 10 inhibited the growth of L1210 murine leukemia cells in culture, with a concordant rise in the mitotic index, and its IC50 value for cell growth was 0.5 nM. Comparable values for the other drugs were 0.5 nM for maytansine, 1 nM for rhizoxin, 20 nM for vinblastine, and 7 microM for phomopsin A. IC50 values were also obtained for the polymerization of purified tubulin in glutamate: 1.2 microM for dolastatin 10, 1.4 microM for phomopsin A, 1.5 microM for vinblastine, 3.5 microM for maytansine, and 6.8 microM for rhizoxin. Dolastatin 10 and vinblastine were comparable in their effects on microtubule assembly dependent on microtubule-associated proteins. Preliminary studies indicated that dolastatin 10, like vinblastine, causes formation of a cold-stable tubulin aggregate at higher drug concentrations. We confirmed that rhizoxin, phomopsin A, and maytansine also inhibit the binding of radiolabeled vinblastine and vincristine to tubulin. Dolastatin 10 and phomopsin A were the strongest inhibitors of these reactions, and rhizoxin the weakest. Dolastatin 10, phomopsin A, maytansine, vinblastine, and rhizoxin all inhibited tubulin-dependent GTP hydrolysis. The greatest inhibition of hydrolysis was observed with dolastatin 10 and phomopsin A, and the least inhibition with rhizoxin.
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PMID:Dolastatin 10, a powerful cytostatic peptide derived from a marine animal. Inhibition of tubulin polymerization mediated through the vinca alkaloid binding domain. 235 35

A factor with a molecular weight of 100 kDa, which markedly enhanced the guanine nucleotide exchange reaction of ras p21 proteins, was partially purified from bovine brain tissues. The factor was predominantly associated with the plasma membrane. When the partially purified factor and excess cold GTP were added to [3H]GDP.Gly12 p21 or Val12 in the presence of 2 mM MgCl2, the nucleotide exchange rate was stimulated up to 25-fold. The stimulation of the p21-nucleotide exchange reaction by the factor was completely blocked by the Y13-259 ras-neutralizing antibody. Taken together, these results suggest that the factor may control the rate limiting GDP/GTP exchange step in recycling of p21 in ras-mediated signal transduction.
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PMID:A novel membrane factor stimulates guanine nucleotide exchange reaction of ras proteins. 240 24

In order to evaluate the relationship between the calcium-calmodulin system and the adenylate cyclase activity of vascular smooth muscle, we examined the effects of several calcium effectors on the basal and stimulated adenylate cyclase activity. Thoracic aortae were removed from Wistar rats and the tissues were homogenated with cold homogenizing buffer containing 1 nM EDTA. Membrane protein fraction of the smooth muscle was prepared by centrifugation at 37,000 g. In this procedure, endogenous guanine nucleotides and contractile proteins remained. The protein fraction was incubated with 2 mM EGTA, 50 microM trifluoperazine, 0.1 microM A23187 or 25 microM calmodulin under basal and stimulated (50 microM isoproterenol, 100 microM GTP and 50 microM forskolin) conditions. The adenylate cyclase activity was determined by a method modified in our laboratory using double isotope counting. Trifluoperazine reduced the basal adenylate cyclase activity significantly (p less than 0.01) as well as the stimulated enzyme activities. A23187 did not affect the basal enzyme activity, but elevated the isoproterenol stimulated enzyme activity significantly (p less than 0.05). EGTA did not affect the basal and stimulated adenylate cyclase activities. Calmodulin elevated the basal enzyme activity significantly (p less than 0.02), but did not affect the stimulated enzyme activities. These results suggest that the calcium-calmodulin system is necessary for maintenance of the adenylate cyclase activity of vascular smooth muscle cells. The calmodulin acting site is considered to be the catalytic subunit, and stimulation of the enzyme is accelerated by calcium ion.
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PMID:Role of the calcium-calmodulin system in the adenylate cyclase activity of vascular smooth muscle. 250 33

Golgi apparatus was prepared from rat liver, and enzymatic properties and the subunit structure of the H+-ATPase were characterized. GTP (and also ITP) was found to drive H+-transport with about 20% of the initial velocity as that of ATP. Bafilomycin, a specific inhibitor for vacuolar H+-ATPase, inhibited the activity at 2.5 nM. The H+-ATPase was completely inhibited in the cold in the presence of MgATP (5 mM) and NaNO3 (0.1 M). The cold inactivation of the H+-ATPase resulted in release of a set of polypeptides from Golgi membrane, with molecular masses almost identical to that of the hydrophilic sector of chromaffin granule H+-ATPase (72, 57, 41, 34, and 33 kDa). Three of these polypeptides (72, 57, and 34 kDa), cross-reacted with antibodies against the corresponding subunits of the chromaffin granule H+-ATPase. A counterpart of the 39-kDa hydrophobic component of chromaffin granule H+-ATPase was identified in the membrane, but no 115-kDa component was found. Hence, the Golgi H+-ATPase shows typical features of vacuolar H+-ATPase, in relatively low substrate specificity, its response to inhibitors, inactivation by cold treatment in the presence of MgATP, and subunit composition judged by antibody cross-reactivity.
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PMID:H+-translocating ATPase in Golgi apparatus. Characterization as vacuolar H+-ATPase and its subunit structures. 253 Feb 22

The inhibition of microtubule assembly by Ruthenium red (Deinum, J., Wallin, M., Kanje, M. and Lagercrantz, C. (1981) Biochim. Biophys. Acta 675, 209-213) could be counteracted by either taxol or dimethyl sulfoxide. Ruthenium red remained bound to the assembled microtubules. Microtubules assembled in the presence of Ruthenium red and taxol showed the typical taxol-dependent stability. The dimethyl sulfoxide-induced microtubules showed normal assembly characteristics, e.g., were GTP dependent, could be disassembled by cold, colchicine and Ca2+ and had no alterations in ultrastructure. The absolute disassembly induced by Ca2+ in the presence of dimethyl sulfoxide and Ruthenium red was dependent on the microtubule protein concentration, but independent in the absence of Ruthenium red. Ruthenium red was strongly bound to purified tubulin also in the presence of 8% (v/v) dimethyl sulfoxide. The dimethyl sulfoxide-induced assembly of purified tubulin in the presence of Ruthenium red was slightly stimulated, although the critical protein concentration was the same. It was found by resonance Raman spectroscopy with a flow technique that Ruthenium red did not bind to a specific calcium binding site on tubulin, although binding to a GTP binding site cannot be excluded. The wavenumbers of the lines in the region 375-500 cm-1 differ from those found for Ruthenium red bound to typical calcium-binding proteins such as calmodulin. Although Ruthenium red binds to serum albumin as well, the spectrum with albumin resembled that of the free dye.
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PMID:The binding of Ruthenium red to tubulin. 257 24

The cell body of Trypanosomatidae is enclosed in densely packed, crosslinked, subpellicular microtubules closely underlying the plasma membrane. We isolated the subpellicular microtubules from bloodstream Trypanosoma brucei parasites by use of a zwitterion detergent. These cold stable structures were solubilized by a high ionic strength salt solution, and the soluble proteins that contained tubulin along with several other proteins were further fractionated by Mono S cation exchange column chromatography. Two distinct peaks were eluted containing one protein each, which had an apparent molecular weight of 52 kDa and 53 kDa. (Mr was determined by SDS-gel electrophoresis). Only the 52 kDa protein showed specific tubulin binding properties, which were demonstrated by exposure of nitrocellulose-bound trypanosome proteins to brain tubulin. When this protein was added to brain tubulin in the presence of taxol and GTP, microtubule bundles were formed with regular crosslinks between the parallel closely packed microtubules. The crosslinks were about 7.2 nm apart (center to center). Under the same conditions, but with the 53 kDA protein or without trypanosome derived proteins, brain tubulin polymerized to single microtubules. It is thus suggested that the unique structural organization of the subpellicular microtubules is dictated by specific parasite proteins and is not an inherent property of the polymerizing tubulin. The in vitro reconstituted microtubule bundles are strikingly similar to the subpellicular microtubule network of the parasite.
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PMID:Isolation of a subpellicular microtubule protein from Trypanosoma brucei that mediates crosslinking of microtubules. 258 98


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