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C M Lin

Publications and source records attributed to C M Lin.

At least 109 records · Page 6Linked to original sources
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↗
PubMed1986

Effects of pH on tubulin-nucleotide interactions.

Significant GTP-independent, temperature-dependent turbidity development occurs with purified tubulin stored in the absence of unbound nucleotide, and this can be minimized with a higher reaction pH. Since microtubule assembly is optimal at lower pH values, we examined pH effects on tubulin-nucleotide interactions. While the lowest concentration of GTP required for assembly changed little, GDP was more inhibitory at higher pH values. The amounts of exogenous GTP bound to tubulin at all pH values were similar, but the amounts of exogenous GDP bound and endogenous GDP (i.e., GDP originally bound in the exchangeable site) retained by tubulin rose as reaction pH increased. Endogenous GDP was more efficiently displaced by exogenous GTP than GDP at all pH values, but displacement by GTP was 10-15% greater at pH 6 than at pH 7. Dissociation constants for GDP and GTP were about 1.0 microM at pH 6 and 0.02 microM at pH 7. A small increase in the affinity of GDP relative to that of GTP occurs at pH 7 as compared to pH 6, together with a 50-fold absolute increase in the affinity of both nucleotides for tubulin at pH 7. The time courses of microtubule assembly and GTP hydrolysis were compared at pH 6 and pH 7. At pH 6, the two reactions were simultaneous in onset and initially stoichiometric. At pH 7, although the reactions began simultaneously, hydrolysis seemed to lag substantially behind assembly. Unhydrolyzed radiolabeled GTP was not incorporated into microtubules, however, indicating that GTP hydrolysis is actually closely coupled to assembly. The apparent lag in hydrolysis probably results from a methodological artifact rather than incorporation of GTP into the microtubule with delayed hydrolysis.

Animals↗
PubMed1986

Anticonvulsant activity of 2- and 3-aminobenzanilides.

A series of 2- and 3-aminobenzanilides derived from ring-alkylated anilines were prepared and evaluated for anticonvulsant activity. These benzanilides were prepared in the course of studies designed to determine the relationship between the benzamide structure and anticonvulsant effects. The compounds were tested in mice against seizures induced by maximal electroshock (MES) and pentylenetetrazole and in the rotorod assay for neurologic deficit. The 3-aminobenzanilide derived from 2,6-dimethylaniline, 21, was the most potent anti-MES compound, with an ED50 of 13.48 mg/kg and a protective index of 21.11 (PI = TD50/ED50). The activity profile for 21 compares favorably with that for phenobarbital and phenytoin.

Anilides↗
PubMed1985

Chloroplast promoters from higher plants.

This survey compiles 60 chloroplast promoter sequences from higher plants published to date and compares them with these sequences from procaryotic systems. The current evidence demonstrates that structurally defined chloroplast promoters are, in most cases, functionally active in initiating gene expression in chloroplasts.

Base Sequence↗
PubMed1985

Differential effects of magnesium on tubulin-nucleotide interactions.

Magnesium-depleted 2-(N-morpholino)ethanesulfonate (Mes), glutamate, tubulin and microtubule-associated proteins were prepared and used to study the effects of exogenously added MgCl2 on tubulin-nucleotide interactions in 0.1 M Mes with microtubule-associated proteins and in 1.0 M glutamate. Endogenous levels of Mg2+ in the systems studied were approximately stoichiometric with the tubulin concentrations and largely derived from the tubulin. We examined the effects of added Mg2+ on tubulin polymerization, GDP inhibition of polymerization, binding of GDP and GTP to tubulin, and GTP hydrolysis. Exogenously added Mg2+ had markedly different effects on these reactions. The order of their sensitivity for a requirement for added Mg2+ was as follows: GTP binding greater than GTP hydrolysis greater than polymerization greater than GDP binding. Inhibition of polymerization by GDP varied inversely with the Mg2+ concentration and was greatest in the absence of the cation. These results indicate that GDP and GDP-Mg2+ interact with similar affinity at the exchangeable site, while GTP-Mg2+ has a higher affinity for tubulin than does free GTP. Nevertheless, under appropriate conditions, free GTP can interact sufficiently well with tubulin to permit both nucleation and elongation reactions.

Animals↗
PubMed1985

Maytansine inhibits nucleotide binding at the exchangeable site of tubulin.

The antineoplastic drug maytansine inhibits the binding of exogenously added radiolabeled GDP and GTP to tubulin (50% inhibition at 9-10 microM drug at 0 degrees). Vinblastine was 1/10-th as inhibitory. Neither maytansine nor vinblastine displaced GDP from tubulin, and both drugs virtually eliminated dissociation of radiolabeled GDP from the exchangeable site. Maytansine also inhibits binding of nucleotides to a vacant exchangeable site. Maytansine thus prevents nucleotide exit and entry at the exchangeable site because of a direct physical obstruction or a conformational change in the tubulin molecule.

Binding Sites↗
PubMed1985

Anticonvulsant activity of some 4-aminobenzanilides.

A series of 4-aminobenzanilides derived from ring-alkylated anilines were prepared and evaluated for anticonvulsant activity. These benzanilides were prepared in the course of studies designed to determine the relationship between the benzamide structure and anticonvulsant effects. The compounds were tested in mice against seizures induced by electroshock and metrazole (pentylenetetrazole) and in the rotorod assay for neurologic deficit. All of the 4-aminobenzanilides showed activity at doses of 300 mg/kg against maximal electroshock seizures (MES). The 4-aminobenzanilide derived from 2,6-dimethylaniline (8) was the most potent anti-MES compound with an ED50 of 2.60 mg/kg and a protective index of 5.77 (PI = TD50/ED50). The activity profile for 8 compares quite favorably with that for phenobarbital and phenytoin in the same assays.

Anilides↗
PubMed1984

Deoxyguanosine nucleotide analogues: potent stimulators of microtubule nucleation with reduced affinity for the exchangeable nucleotide site of tubulin.

Four analogues of guanosine 5'-triphosphate (GTP) (dGTP, 3'-deoxy-GTP, arabinosyl-GTP, and 2',3'-dideoxy-GTP), which support more rapid and extensive microtubule assembly than GTP, were hydrolyzed more rapidly than GTP in reaction mixtures containing tubulin plus microtubule-associated proteins (MAPs). As with GTP, hydrolysis of the four analogues was initially closely coupled to the onset of polymerization and continued at a slower rate at the turbidity plateau. Relative to GTP, however, these analogues (and the cognate GDP analogues), particularly 3'-deoxy-GTP and 2',3'-dideoxy-GTP, bound poorly to tubulin and had a reduced ability to displace bound radiolabeled GDP under nonpolymerizing reaction conditions. Despite their reduced binding to the tubulin dimer, if polymerization occurred, all four analogues were incorporated into microtubules (as the diphosphates) in stoichiometric amounts comparable to the incorporation of GTP (in the form of GDP) with displacement of the GDP initially present in the exchangeable site. Microtubule nucleation was specifically enhanced in the presence of the analogues. With MAPs the analogues initiated microtubule assembly at temperatures 10-15 degrees C below that required by the GTP-supported reaction, and the average microtubule length was significantly reduced. In addition, MAP-independent polymerization occurred only with 2',3'-dideoxy-GTP with tubulin at 1.0 mg/mL, with the other three analogues at 2.0 mg/mL, and with GTP at 5.0 mg/mL. GTP inhibited analogue-supported polymerization at 20 degrees C with MAPs and at 37 degrees C without MAPs (tubulin, 3.5 mg/mL). Both 3'-deoxy-GTP and 2',3'-dideoxy-GTP were poor inhibitors of GTP binding and hydrolysis, but GTP potently inhibited the more vigorous hydrolysis of these analogues. We conclude that alteration of the ribose moiety reduces the affinity of a guanine nucleotide for the exchangeable site of tubulin but that a nucleotide's affinity for this site is not the major factor in its ability to support the nucleation of tubulin polymerization.

Animals↗
PubMed1984

Guanosine 5'-O-(3-thiotriphosphate), a potent nucleotide inhibitor of microtubule assembly.

Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and the two diastereoisomers of guanosine 5'-O-(2-thiotriphosphate) (GTP beta S) were prepared enzymatically, and their interactions with tubulin and microtubule-associated proteins (MAPs) in 0.1 M 2-(N-morpholino)ethanesulfonate, 0.5 mM MgCl2 were examined. GTP gamma S did not support microtubule assembly but instead inhibited the reaction. This analog was 1.5-2 times more potent than GDP in inhibiting both tubulin polymerization and GTP hydrolysis under conditions in which these reactions were dependent on MAPs. In contrast to the analog's inhibitory effects on polymerization and hydrolysis, however, radiolabeled GTP gamma S was only feebly bound by purified tubulin at 0 degrees C relative to the binding of GDP and GTP. There was a marked increase in the amount of GTP gamma S bound when the reaction temperature was raised to 37 degrees C or when MAPs were included in the reaction mixture. Only when both MAPs were present and the higher reaction temperature was used did the binding of GTP gamma S exceed that of GDP. Since substitution of sulfur for oxygen in a molecule should decrease its hydrophilic properties, these findings suggest that the exchangeable nucleotide binding site of tubulin becomes more hydrophobic at higher temperatures and in the presence of MAPs. The two isomers of GTP beta S were able to support MAP-dependent polymerization, although a 50-100-fold higher concentration of the analogs as compared to GTP was required. Neither isomer of GTP beta S had a significant inhibitory effect on GTP hydrolysis dependent on tubulin + MAPs.

Animals↗
PubMed1984

Separation of active tubulin and microtubule-associated proteins by ultracentrifugation and isolation of a component causing the formation of microtubule bundles.

A new method for separating microtubule-associated proteins (MAPs) and tubulin, appropriate for relatively large-scale preparations, was developed. Most of the active tubulin was separated from the MAPs by centrifugation after selective polymerization of the tubulin was induced with 1.6 M 2-(N-morpholino)ethanesulfonate (Mes) and GTP. The MAPs-enriched supernatant was concentrated and subsequently clarified by prolonged centrifugation. The supernatant (total soluble MAPs) contained almost no tubulin, most of the nucleosidediphosphate kinase activity of the microtubule protein, good activity in promoting microtubule assembly in 0.1 M Mes, and proteins with the electrophoretic mobility of MAP-1, MAP-2, and tau factor. The pellet, inactive in supporting microtubule assembly, contained denatured tubulin, most of the ATPase activity of the microtubule protein, and significant amounts of protein with the electrophoretic mobility of MAP-2. Insoluble material at this and all previous stages, including the preparation of the microtubule protein, could be heat extracted to yield soluble protein active in promoting microtubule assembly and containing MAP-2 as a major constituent. The total soluble MAPs were further purified by DEAE-cellulose chromatography into bound and unbound components, both of which induced microtubule assembly. The bound component (DEAE-MAPs) contained proteins with the electrophoretic mobility of MAP-1, MAP-2, and tau factor. The polymerization reaction induced by the unbound component (flow-through MAPs) produced very high turbidity readings. This was caused by the formation of bundles of microtubules. Although the flow-through MAPs contained significantly more ATPase, tubulin-independent GTPase, and, especially, nucleosidediphosphate kinase activity than the DEAE-MAPs, preparation of a MAPs fraction without these enzymes required heat treatment.

Animals↗
PubMed1984

Stability of tubulin polymers formed with dideoxyguanosine nucleotides in the presence and absence of microtubule-associated proteins.

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.

Animals↗
PubMed1984

Inhibition of tubulin polymerization with ribose-modified analogs of GDP and GTP. Reduced inhibition with microtubule-associated proteins and magnesium.

Inhibitory effects of ribose-modified GDP and GTP analogs on tubulin polymerization were examined to explore nucleotide structural requirements at the exchangeable GTP binding site. With microtubule-associated proteins and Mg2+, GTP-supported polymerization was only modestly inhibited by GDP, and still weaker inhibitory activity was found with two analogs, dGDP and 9-beta-D-arabinofuranosylguanine-5'-diphosphate (araGDP). Omission of Mg2+ significantly enhanced the inhibitory effects of GDP, dGDP and araGDP and resulted in weak inhibition of the reaction by several other GDP analogs. The relative inhibitory activity of the GDP analogs had no discernible relationship to the relative activity of cognate GTP analogs in supporting microtubule-associated protein-dependent polymerization. One GTP analog, 2',3'-dideoxyguanosine 5'-triphosphate (ddGTP), supports polymerization both with and without microtubule-associated proteins. The inhibitory activity of GDP and GDP analogs in ddGTP-supported polymerization was much greater in the absence of microtubule-associated proteins than in their presence; and both reactions were more readily inhibited than was microtubule-associated protein-dependent, GTP-supported polymerization. Microtubule-associated protein-independent, ddGTP-supported polymerization was also potently inhibited by GTP and a number of GTP analogs. GTP was in fact twice as inhibitory as GDP. The relative inhibitory activity of the GTP analogs was comparable to the relative inhibitory activity of the cognate GDP analogs and very different from their relative activity in supporting polymerization.

Animals↗
PubMed1983

Microtubule assembly with the guanosine 5'-diphosphate analogue 2',3'-dideoxyguanosine 5'-diphosphate.

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.

Animals↗