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E Hamel

Publications and source records attributed to E Hamel.

At least 199 records · Page 11Linked to original sources
PubMed1988

Transforming growth factor beta as a potent promoter in two-stage BALB/c 3T3 cell transformation.

We have tested transforming growth factor beta (TGF beta) in the two-stage BALB/c 3T3 cell transformation assay for possible tumor-promoting activity, since it has several effects similar to those of tumor-promoting phorbol esters. After initiation of BALB/c 3T3 cells with 3-methylchol-anthrene, treatment with TGF beta at 1 ng/ml alone or in combination with epidermal growth factor (EGF) for 4 weeks enhanced the number of transformed foci by 5- to 6-fold in comparison with uninitiated cells. Initiation treatment alone induced no or very few transformed foci in several assays. Treatment with phorbol-12,13-didecanoate (PDD) at 100 ng/ml for 4 weeks enhanced the number of transformed foci in initiated BALB/c 3T3 cells by 4- to 5-fold in comparison with uninitiated cells. Thus, TGF beta at 1 ng/ml is as potent as PDD at 100 ng/ml for tumor-promoting activity in the two-stage BALB/c 3T3 cell transformation assay. The enhancing effect of TGF beta was dose-related in the dose range tested (0.03-1 ng/ml) and was not reversible. Some of the foci induced by combined MCA-TGF beta-EGF treatment were cloned, and eight out of nine clones tested produced tumors in nude mice. TGF beta (1 ng/ml) plus EGF (2 ng/ml) increased the saturation density to a similar extent as PDD (100 ng/ml) but did not affect the growth of BALB/c 3T3 cells. We observed no change in junctional intercellular communication, as measured by the dye transfer method, when cells were treated with TGF beta during the two-stage BALB/c 3T3 cell transformation assay. Nevertheless, there was selective communication between transformed and surrounding nontransformed cells; MCA-TGF beta transformed cells intercommunicated among themselves but not with surrounding nontransformed cells. Our results indicate that TGF beta has potent tumor-promoting activity in vitro, but that this activity is not mediated by a complete blockage of intercellular communication, as is suggested for phorbol ester tumor promoters.

Animals↗
PubMed1988

CSF-endorphines in acute and chronic brain lesions.

In order to answer the question of an opioid influence on consciousness, a radio-immuno-assay (n = 852) of beta-endorphin and beta-LPH (beta-lipotropic hormone) in both ventricular CSF and blood plasma was carried out in 101 neurosurgical patients. The following results were obtained: I) beta-END and beta-LPH levels were found to be lower in the CSF than in blood plasma. II) beta-END and beta-LPH in the CSF was the same in both sexes. III) beta-END levels in the CSF decreased with age. IV) beta-END and beta-LPH levels showed a diurnal rhythm with a maximum in the late a. m. hours. V) beta-END levels in the ventricular CSF tend to decrease parallel to a drop in conciousness as well as with longlasting comatous states. VI) beta-END in ventricular CSF becomes higher with increasing systolic arterial blood pressure. VII) beta-END and beta-LPH levels in ventricular CSF are not correlated with the type of the disease, CSF pressure, body temperature or respiratory changes.

Adult↗
PubMed1988

Heterogeneous vasomotor responses of anatomically distinct feline cerebral arteries.

1. The vasomotor reactivity to a number of neurotransmitters and blood-borne substances was evaluated in several anatomically distinct arteries of the cat cerebral circulation. Few regional differences were observed in their vasoconstrictor responses to noradrenaline, dopamine, 5-hydroxytryptamine and prostaglandin F2 alpha. Only the anterior cerebral artery reacted strongly to all vasoconstrictor agents. 2. Adenosine, acetylcholine and histamine induced pronounced relaxation in the vast majority of the major cerebral arteries. The relaxation elicited by adenosine showed a slight degree of heterogeneity between the arteries and the overall response accounted for 81 +/- 6% of the pharmacologically-induced tone. On the other hand, the dilatation induced by acetylcholine and histamine varied as a function of the anatomical localization of the cerebral arteries. The acetylcholine-induced vasodilatation was significantly more pronounced in the middle cerebral, anterior communicating and anterior cerebellar arteries, with respective responses of 72, 66 and 83% of the induced tone as compared to 43% in the other vessels. However, all arteries were equally sensitive to acetylcholine with an overall mean pD2 value of 7.47 +/- 0.06. The most heterogeneous results were obtained with histamine and applied both to the magnitude of the maximal response and the sensitivity of the various arteries to this amine. The intensity of the relaxation varied from 20% (anterior communicating artery) to 118% (posterior cerebellar artery). 3. Among the neuropeptides studied, substance P and bradykinin were considerably less potent than vasoactive intestinal peptide on all the cerebral arteries. The least responsive vessel to bradykinin was the anterior cerebral artery with a maximal response of 22 +/- 5% of the induced-tone and a pD2 value of 7.56 +/- 0.24. All vessels responded weakly to substance P and those from the vertebrobasilar circulation were significantly less sensitive to this neuropeptide with pD2 values around 8.07 as compared to 9.82 in the more rostral arteries. Although all vessels were equally sensitive to vasoactive intestinal peptide, the dilator responses were significantly less pronounced in the middle cerebral and basilar arteries (maximal response of 86 +/- 5% and 69 +/- 6% of the induced-tone, respectively, as compared to 110 +/- 9% in the other vessels). 4. The vertebrobasilar arteries were as reactive, if not more reactive, to vasoconstrictors than the vessels originating from the carotid circulation. In contrast, the dilator responses were less marked in most caudal arteries. Such dichotomies may be important in the regulation of local cerebral blood flow. 5. The results emphasize the considerable heterogeneity in the vasomotor responses to a given substance among the various cerebral arteries. Further, they suggest the presence of multiple receptor populations which mediate opposite effects and which are distributed in different proportions among the cephalic arteries.

Animals↗
PubMed1988

Interactions of tubulin with potent natural and synthetic analogs of the antimitotic agent combretastatin: a structure-activity study.

Combretastatin, an antineoplastic and antimitotic agent, was isolated from the bark of Combretum caffrum [Can. J. Chem. 60: 1374-1376 (1982); Biochem. Pharmacol. 32:3864-3867 (1983)]. Structurally, combretastatin consists of two substituted benzene rings linked by a saturated, hydroxy-substituted two-carbon bridge. A large number of combretastatin analogs have now been synthesized or obtained from C. caffrum. These vary in substituents on the phenyl rings or bridge carbons, bridge length, unsaturation of the bridge (i.e., stilbene derivatives, with the two phenyl rings oriented either cis or trans), and in precise ring structure (two major variants, with the bridge incorporated into a third six-member ring to form a phenanthrene structure or a methyl group eliminated from vicinal methoxy substituents to form a benzodioxole ring). Available analogs (17 natural products and 22 synthetic agents) were examined for antimitotic and cytotoxic activity and for effects on tubulin polymerization and colchicine binding. Nineteen compounds inhibited cell growth by 50% or more at concentrations of 1 microM or less, and 14 inhibited tubulin polymerization by at least 50% at stoichiometric drug concentrations. The most potent cytotoxic agents generally strongly inhibited both tubulin polymerization and the binding of colchicine to tubulin. The most promising compound is the (cis)-stilbene derivative (cis)-1-(3,4,5-trimethoxyphenyl)-2-(3'-hydroxy-4'-methoxyphenyl)ethene, which has been named combretastatin A-4. This compound inhibited cell growth by 50% at 7 nM, inhibited tubulin polymerization by 50% at 2.5 microM (1/4 molar equivalent), and competitively inhibited colchicine binding with an apparent Ki of 0.14 microM.

Animals↗
PubMed1987

Neutral endopeptidase-24.11, mu and delta opioid receptors after selective brain lesions: an autoradiographic study.

The cellular localization of the rat brain neutral endopeptidase (NEP, EC 3.4.24.11) was investigated by quantitative autoradiography of the enzyme inhibitor [3H]N-[(2RS)-3-hydroxyaminocarbonyl-2-benzyl-1-oxopropyl]glycine ([3H]HACBO-Gly) after lesions of the striatum, nigrostriatal and corticostriatal pathways. The effect of these lesions on NEP levels was compared with that on delta and mu opioid receptors, selectively labeled with [3H]Tyr-D-Thr-Gly-Leu-Thr ([3H]DTLET) and [3H]Tyr-D-Ala-Gly-MePhe-Glycinol ([3H]DAGO), respectively. Twenty-one days after injection of kainate in the caudate putamen (CP), the NEP level was locally decreased (52%) but the time course of this decrease was different from that of mu and delta opioid receptors: [3H]DAGO binding was diminished by 40% from day 2 whereas that of [3H]DTLET was reduced by 51% from day 7. Kainic acid injection in the CP induced in the globus pallidus (GP) and substantia nigra (SN) a distant reduction of the 3 opioid markers. Likewise after injection of colchicine in the CP, [3H]HACBO-Gly binding was decreased in the GP (60%) and SN (58%), [3H]DTLET binding was reduced by 54 and 55% in the GP and SN, respectively and [3H]DAGO labeling was diminished by 49% in the GP, and 58% in the SN. Finally, lesion of the nigrostriatal dopamine pathway by 6-hydroxydopamine did not induce any change of NEP level in the CP and GP whereas delta and mu opioid receptor levels were diminished respectively by 25 and 29% in the CP, and 45 and 39% in the GP, a new finding of the present study. Taken together these data suggest that NEP is in part associated with striatal intrinsic neurons. In the GP and SN, a large part of NEP seems to be presynaptically associated with nerve terminals endowed with mu and delta opioid receptors, which originate from efferent striatal neurons. In contrast to opioid receptors in the CP, the NEP appears not to be associated with dopaminergic nerve terminals originating from the SN. Cortical ablation did not affect any of the opioid markers.

Animals↗
PubMed1987

A simple method for the rapid determination of the stereospecificity of NAD-dependent dehydrogenases applied to mammalian IMP dehydrogenase and bacterial NADH peroxidase.

The stereospecificity of IMP dehydrogenase (IMP:NAD+ oxidoreductase, EC 1.1.1.205) from two different sources was determined. The enzyme preparations were obtained from murine lymphoblasts and from Escherichia coli. Both enzymes transferred the 2-3H of IMP to the pro-S position of carbon atom C-4 of the nicotinamide ring in NAD. Thus, B-sided stereospecificity is common to the enzyme from two very different species. In addition, the studies described here demonstrate that alcohol dehydrogenase and NADH peroxidase, used as auxiliary enzymes, in combination with a microdistillation procedure, should permit rapid determination of the stereospecificity of any NAD-dependent dehydrogenase for which the appropriate tritiated substrate is available.

Alcohol Dehydrogenase↗
PubMed1987

Interrelationships of tubulin-GDP and tubulin-GTP in microtubule assembly.

We previously reported that direct incorporation of GDP (i.e., without an initial hydrolysis of GTP) into microtubules occurs throughout an assembly cycle in a constant proportion. The exact proportion varied with reaction conditions, becoming greater under all conditions in which tubulin-GDP increased relative to tubulin-GTP (low Mg2+ and GTP concentrations, high tubulin concentrations, and in the presence of exogenous GDP). These findings led us to explore further interrelationships of tubulin-GDP and tubulin-GTP in microtubule assembly. We have now determined the minimum amount of tubulin-GTP required for the initiation of microtubule assembly and the relative efficiency with which tubulin-GDP participates in microtubule elongation. When GTP, GDP, and tubulin concentrations were varied at a constant Mg2+ concentration (0.2 mM), initiation of assembly required that 35% of the nucleotide-bearing tubulin be in the form of tubulin-GTP, and incorporation of tubulin-GDP into microtubules during elongation was only 60% as efficient as would be predicted on the basis of its proportional concentration in the reaction mixtures. Very different results were obtained when the Mg2+ concentration was varied. Even though Mg2+ enhances the binding of GTP to tubulin (the equilibrium constant for the exchange of GTP for GDP was 0.2 in the absence of exogenous Mg2+, 3 with 0.2 mM Mg2+, 5 with 0.5 mM Mg2+, and 11 with 2 and 4 mM Mg2+), as Mg2+ was increased the proportion of tubulin-GTP required for the initiation of microtubule assembly rose greatly, and the direct incorporation of tubulin-GDP into microtubules during elongation became progressively more efficient. In the absence of exogenous Mg2+, only 20% tubulin-GTP was required for initiation, and tubulin-GDP was directly incorporated into microtubules half as efficiently as would be predicted on the basis of its concentration in the reaction mixture. At the highest Mg2+ concentration examined (4 mM), 80% tubulin-GTP was required for initiation of assembly, and tubulin-GDP was incorporated into microtubules as efficiently as tubulin-GTP.

Animals↗
PubMed1987

Neuronal versus endothelial origin of vasoactive acetylcholine in pial vessels.

Functional pial vessels denuded in situ of the endothelial cell layer exhibit a markedly decreased choline uptake capacity (-53%) but integrally preserved choline acetyltransferase (ChAT) activity and acetylcholine (ACh) release mechanisms. These studies demonstrate that endothelial cells possess a specific choline uptake system. However, the unimpaired ChAT activity in denuded pial vessels implies that the endothelial pool of choline is not significantly metabolized into ACh. In spite of possible differences in the mechanisms that govern release processes in endothelial and neuronal elements, taken together the findings of the present study suggest that the ACh released following depolarization of pial blood vessels originates predominantly from cholinergic perivascular nerve terminals.

Acetylcholine↗
PubMed1987

Nucleotide interconversions in microtubule protein preparations, a significant complication for accurate measurement of GTP hydrolysis in the presence of adenosine 5'-(beta, gamma-imidotriphosphate).

Pursuing the observation of Carlier and Pantaloni [Carlier, M.-F., & Pantaloni, D. (1982) Biochemistry 21, 1215-1224] that adenosine 5'-(beta, gamma-imidotriphosphate) (pNHppA) strongly inhibited tubulin-independent phosphatases in microtubule protein preparations, we observed with a number of commercial preparations of pNHppA that a major proportion of the terminal phosphate of [gamma-32P]GTP added to microtubule protein preparations was rapidly converted into ATP. Initially postulating degradation of pNHppA to AMP followed by stepwise conversion of AMP to ATP, we isolated two nucleoside monophosphate kinase activities from microtubule protein capable of generating ATP from AMP + GTP. The amounts of these enzymes in microtubule protein preparations, however, are probably too low to account for rapid ATP formation. Instead, ATP formation most likely is caused by nucleoside diphosphate kinase acting on ADP contaminating commercial pNHppA preparations. Such ADP contamination was demonstrated by high-performance liquid chromatography, with the amount of ATP formed with different pNHppA preparations proportional to the amount of ADP contamination. Repurification of commercial pNHppA until it was free of contaminating ADP also resulted in the elimination of ATP formation. The repurified pNHppA potently inhibited GTP hydrolysis in microtubule protein preparations. In addition, especially when supplemented with equimolar Mg2+, the repurified pNHppA strongly inhibited GTP hydrolysis and microtubule assembly in reaction mixtures containing purified tubulin and heat-treated microtubule-associated proteins (which contain negligible amounts of tubulin-independent phosphatase activity). We conclude that studies of microtubule-dependent GTP hydrolysis which make use of pNHppA must be interpreted with extreme caution.

Adenosine Triphosphate↗
PubMed1987

Opioid receptors in rat neostriatum: radioautographic distribution at the electron microscopic level.

The distribution of mu-opioid receptors, selectively labeled in vitro with a monoiodinated Met-enkephalin analog [( 125I]FK 33-824), was analyzed by light and electron microscopic radioautography in sections from the neostriatum of the rat. In the light microscope, patches of high receptor densities were detected amidst a moderately labeled matrix. The number of silver grains, as counted in 1-micron thick plastic-embedded sections, was 3 times greater inside the patches than in the intervening matrix. In both compartments, the proportion of labeled binding sites associated with the neuropil was significantly higher (greater than 70%) than that associated with nerve cell bodies or myelinated fascicles. Quantitative analyses of electron microscopic radioautographs revealed that the majority of silver grains corresponding to specifically bound [125I]FK molecules originated from radioactive sources associated with apposed neuronal membranes. Of the total number of specific binding sites, 53% was associated with axodendritic, 18% with axoaxonic and 3% with axosomatic interfaces. The occurrence of multiple labeled foci along the plasma membrane of certain perikarya and dendrites suggested that some of the binding sites might be associated with somato/dendritic elements. The high incidence of labeling along axoaxonic interfaces indicated that others were linked to the membrane of axons and/or axon terminals. A major finding of the present study was that only a small proportion of specific FK binding sites (7% of total) was associated with synaptic junctions. Labeled synapses were primarily of the asymmetric type and were found predominantly on dendritic branches and spines. A few were observed on nerve cell bodies. Labeled symmetric synapses were rare and encountered exclusively on dendritic branches. The high frequency with which specifically labeled binding sites were found to be associated with neuronal interfaces involving axonal processes strongly suggests that even if non-junctional these binding sites correspond to functional receptors. Whether these receptors are activated by endogenous ligand molecules released by the labeled terminals themselves or from terminals located at a distance from the labeled interfaces remains to be determined.

Animals↗
PubMed1987

Photoaffinity labeling of tubulin subunits with a photoactive analogue of vinblastine.

A photoactive, radioactive analogue of vinblastine, N-(p-azido[3,5-3H]benzoyl)-N'-(beta-amino-ethyl)vindesine ([ 3H]NABV), was used to localize the Vinca alkaloid binding site(s) on calf brain tubulin after establishing that its in vitro interactions with tubulin were comparable to those of vinblastine. Microtubule assembly was inhibited by 50% with 2 microM NABV or vinblastine. At higher drug concentrations, NABV and vinblastine both induced tubulin aggregation, and both drugs inhibited tubulin-dependent GTP hydrolysis. Vinblastine and NABV inhibited each other's binding to tubulin, but the binding of neither drug was inhibited by colchicine. Two classes of binding sites for NABV and vinblastine were found on calf brain tubulin. High-affinity sites had apparent KD values of 4.2 and 0.54 microM for NABV and vinblastine, respectively, whereas the low-affinity binding sites showed apparent KD values of 26 and 14 microM for NABV and vinblastine, respectively. Mixtures of tubulin and [3H]NABV were irradiated at 302 nm and analyzed for incorporation of radioactivity into protein. Photolabeling of both the alpha- and beta-subunits of tubulin with increasing concentrations of [3H]NABV exhibited a biphasic pattern characteristic of specific and nonspecific reactions. Nonspecific labeling was determined in the presence of excess vinblastine. Saturable specific covalent incorporation into both subunits of tubulin was observed, with an alpha:beta ratio of 3:2 and maximum saturable incorporation of 0.086 and 0.056 mol of [3H]NABV/mol of alpha-tubulin and beta-tubulin, respectively. Such photolabeling of the tubulin subunits will permit precise localization of Vinca alkaloid binding sites, including identification of the amino acid residues involved, an essential requirement for understanding the interactions of these drugs with tubulin.

Affinity Labels↗
PubMed1987

Effect of oral morphine and naloxone on pituitary-adrenal response in man induced by human corticotropin-releasing hormone.

To further investigate the role of opioids in the regulation of the pituitary-adrenal axis we studied the effect of morphine and naloxone on human corticotropin-releasing hormone (hCRH)-induced ACTH, immunoreactive (ir) beta-endorphin, and cortisol release in normal subjects. Protocols: 1. 30 mg of a slow-release preparation of morphine or placebo was given orally 3 h prior to administration of hCRH (0.1 mg iv) (N = 7). 2. Naloxone (4 mg as bolus iv) or placebo was given 5 min prior to hCRH (N = 7). 3. Naloxone (4 mg iv as bolus followed by a continuous infusion of 6 mg over 75 min) or placebo was started 15 min prior to hCRH (N = 6). hCRH was injected at 11.00 h (protocol 1, 2) or at 17.00 h (protocol 3). Oral morphine not only suppressed basal hormone levels (P less than 0.02), but also the peak response to hCRH compared with placebo (cortisol: 270 +/- 50 vs 559 +/- 80 nmol/l; ACTH: 5.1 +/- 1.5 vs 13.1 +/- 2.7 pmol/l; ir beta-endorphin: 48.5 +/- 8.7 vs 88 +/- 14 pmol/l; mean +/- SEM, P less than 0.02). Similarly, the maximum incremental changes and the area under the curve were significantly reduced for all three hormones compared with placebo (P less than 0.05). After 4 mg of naloxone in the morning, no significant hormonal changes in response to hCRH were observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Oral↗
PubMed1987

[Criteria of brain death: 3d generation. Interpretation of criteria established by the Federal Chamber of Physicians in determining brain death].

The criteria of brain death published by the Federal Chamber of Physicians in 1982 in their second generation have stood the test of time and remain valid. The most important amendments in issuing a third generation are the following: In primary cerebral lesions supratentorial lesions have to be distinguished from infratentorial processes. In primarily the infratentorial processes EEG activities of the cortex max outlast apneic brain stem areflexia. Therefore in infratentorial processes controls of the EEG are indispensable. The extinction of evoked potentials, certain conditions being observed, may prove the loss of brain stem function and therefore may allow to shorten observation time, and may offer an alternative to isoelectricity in the EEG or cerebral perfusion stop as a diagnostic procedure. Additional tests for brain death are commented on. Special considerations are necessary in brain death in children.

Brain Damage, Chronic↗
PubMed1986

Neuronal localization of the neutral endopeptidase 'enkephalinase' in rat brain revealed by lesions and autoradiography.

The cellular localization of rat brain enkephalinase was studied after induction of selective unilateral lesions using in vitro quantitative autoradiography of the specific binding of the enzyme inhibitor [3H]-N-[(2RS)-3-hydroxyaminocarbonyl-2-benzyl-1-oxopropyl]glycine ([3H]HACBO-Gly). Twenty-one days following injection of kainic acid in the caudate-putamen (CP) [3H]HACBO-Gly binding was locally decreased by 52% with a concomitant reduction of 67 and 78% in the ipsilateral substantia nigra (SN) and globus pallidus (GP), respectively. Inhibition of axonal transport in the CP by unilateral stereotaxic injection of colchicine induced a large (30-60%) and progressive decrease in enkephalinase labelling within the ipsilateral GP and SN. Taken together these results strongly suggest that in the CP a large fraction of enkephalinase is localized on intrinsic striatal neurones, and that the enzyme present both in the GP and the SN is partly localized on nerve terminals originating from neurones in the CP. No change in [3H]HACBO-Gly binding was observed in the CP following injection of 6-hydroxydopamine in the nigrostriatal bundle, contrasting with the 30% depletion in opioid receptors. This would indicate that enkephalinase is present in only very low amounts, if at all, on striatal dopaminergic nerve terminals.

Animals↗
PubMed1986

Morpholino derivatives of benzyl-benzodioxole, a study of structural requirements for drug interactions at the colchicine/podophyllotoxin binding site of tubulin.

We performed a structure-activity evaluation of the effects of methoxy substituents in the benzyl moiety of a series of morpholinyl Mannich base derivatives of 6-benzyl-1,3-benzodioxol-5-ol ("morpholino compounds") on the ability of these compounds to inhibit tubulin polymerization in vitro. Structurally these agents are most similar to the natural product podophyllotoxin and, like podophyllotoxin, they inhibited in vitro tubulin polymerization, tubulin-dependent GTP hydrolysis, and the binding of colchicine to tubulin. The benzyl ring (C ring) of these compounds appeared to be analogous to the trimethoxybenzene ring (E ring) of podophyllotoxin (with its methoxy substituents at the 3', 4' and 5' positions), but the morpholino compound superficially most similar to podophyllotoxin (with 3', 4' and 5' methoxy substituents) was the least active in the series. The most potent methoxy-substituted morpholino compounds bear these substituents either at the 2' and 4' positions (NSC 370277) or at the 2', 4' and 6' positions (NSC 381577). NSC 370277 and NSC 381577 were essentially identical in their inhibitory effects on tubulin polymerization, but the latter compound was considerably more effective as an inhibitor of the binding of colchicine to tubulin. The most active of the monomethoxy substituted compounds bore this group at position 4'. A number of compounds with alternative substituents at this position (in particular, alkyl-substituted amines) also had significant in vitro inhibitory effects on tubulin polymerization. Although the morpholino compounds appear to possess only limited cytotoxicity, these findings suggest possible modifications of the antimitotic benzyl-benzodioxole compounds described previously [Batra et al., Molec. Pharmac. 27, 94 (1985)] to enhance their antineoplastic activity.

Benzyl Compounds↗
PubMed1986

Direct incorporation of guanosine 5'-diphosphate into microtubules without guanosine 5'-triphosphate hydrolysis.

Using highly purified calf brain tubulin bearing [8-14C]guanosine 5'-diphosphate (GDP) in the exchangeable nucleotide site and heat-treated microtubule-associated proteins (both components containing negligible amounts of nucleoside diphosphate kinase and nonspecific phosphatase activities), we have found that a significant proportion of exchangeable-site GDP in microtubules can be incorporated directly during guanosine 5'-triphosphate (GTP) dependent polymerization of tubulin, without an initial exchange of GDP for GTP and subsequent GTP hydrolysis during assembly. The precise amount of GDP incorporated directly into microtubules is highly dependent on specific reaction conditions, being favored by high tubulin concentrations, low GTP and Mg2+ concentrations, and exogenous GDP in the reaction mixture. Minimum effects were observed with changes in reaction pH or temperature, changes in concentration of microtubule-associated proteins, alteration of the sulfonate buffer, or the presence of a calcium chelator in the reaction mixture. Under conditions most favorable for direct GDP incorporation, about one-third of the GDP in microtubules is incorporated directly (without GTP hydrolysis) and two-thirds is incorporated hydrolytically (as a consequence of GTP hydrolysis). Direct incorporation of GDP occurs in a constant proportion throughout elongation, and the amount of direct incorporation probably reflects the rapid equilibration of GDP and GTP at the exchangeable site that occurs before the onset of assembly.

Animals↗
PubMed1986

Tubulin polymerization with ATP is mediated through the exchangeable GTP site.

Glycerol-induced tubulin polymerization supported by non-guanine nucleotides was examined. The electrophoretically homogeneous tubulin was devoid of nucleoside diphosphate kinase activity and 95% saturated with exchangeable GDP and nonexchangeable GTP. All purine ribonucleoside 5'-triphosphates were active but no polymerization occurred with CTP or UTP. All polymerization reactions, as a function of nucleotide concentration, were similar: above a minimum (threshold) concentration, as the amount of nucleotide increased the reaction became progressively more rapid and extensive with a progressively shorter nucleation period. Threshold concentrations of ATP, XTP, ITP and GTP were 0.6 mM, 0.3 mM, 30 microM and 7 microM, respectively. Most ribose- and polyphosphate-modified ATP analogs also supported polymerization at high concentrations, but the activity of these analogs relative to ATP was very similar to the activity of cognate GTP analogs relative to GTP. Polymerization with ATP was associated with an ATPase reaction. ATP hydrolysis was potently inhibited by GDP and GTP and altered by antimitotic drugs in parallel with the effects of these agents on GTP hydrolysis. Substantial amounts of [8-14C]GDP bound in the exchangeable site of tubulin were displaced during polymerization with GTP or ATP, but much higher concentrations of ATP were required for equivalent displacement of the tubulin-bound GDP. Polymerization with GTP or ATP was inhibited in a qualitatively similar manner by GDP, with increasing concentrations of GDP causing a progressive prolongation of the nucleation period and reduction in reaction rate and extent. However, complete inhibition of polymerization required that GDP:GTP much greater than 1, but that GDP:ATP much less than 1. Inhibition appeared to be primarily competitive, since with higher triphosphate concentrations higher GDP concentrations were required for comparable inhibition. We conclude that ATP effects on tubulin polymerization are mediated through a feeble interaction at the exchangeable GTP site.

Adenosine Triphosphate↗