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B Bhattacharyya

Publications and source records attributed to B Bhattacharyya.

90 records · Page 5Linked to original sources
PubMed1977

Podophyllotoxin as a probe for the colchicine binding site of tubulin.

The binding of [3H]podophyllotoxin to tubulin, measured by a DEAE-cellulose filter paper method, occurs with an affinity constant of 1.8 X 10(6) M-1 (37 degrees at pH 6.7). Like colchicine, approximately 0.8 mol of podophyllotixin are bound per mol of tubulin dimer, and the reaction is entropy-driven (43 cal deg-1 mol-1). At 37 degrees the association rate constant for podophyllotoxin binding is 3.8 X 10(6) M-1 h-1, approximtaely 10 times higher than for colchicine; this is reflected in the activation energies for binding which are 14.7 kcal/mol for podophyllotoxin and 20.3 kcal/mol for colchicine. The dissociation rate constant for the tubulin-podophyllotoxin complex is 1.9 h-1, and the affinity constant calculated from the ratio of the rates is close to that obtained by equilibrium measurements. Podophyllotxin and colchicine are mutually competitive inhibitors. This can be ascribed to the fact that both compounds have a trimethoxyphenyl ring and analogues of either compound with bulky substituents in their trimethoxyphenyl moiety are unable to inhibit the the binding of either of the two ligands. Tropolone, which inhibits colchicine binding competitively, has no effect on the podophyllotoxin/tubulin reaction. Conversely, podophyllotoxin does not influence tropolone binding. Moreover, the tropolone binding site of tubulin does not show the temperature and pH lability of the colchicine and podophyllotoxin domains, hence this lability can be ascribed to the trimethoxyphenyl binding region of tubulin. Since podophyllotoxin analogues with a modified B ring do not bind, it is concluded that both podophyllotoxin and colchicine each have at least two points of attachment to tubulin and that they share one of them, the binding region of the trimethoxyphenyl moiety.

Animals↗
PubMed1976

Anion-induced increases in the rate of colchicine binding to tubulin.

The rate of binding of colchicine to tubulin to tubulin is enhanced by certain anions. Among the inorganic anions tested, only sulfate was effective. The organic anions include mostly dicarboxylic acids, among which tartrate was the most effective. This effect occurs onlt at low concentrations of colchicine (less than 0.6 X 10(-5) M). The rate increase dor sulfate and L-(+)-tartrate is ca. 2.5-fold at 1.0 mM and plateaus at a limiting value of ca. 4-fold at 100mM. The overall dissociation rate of the colchicine from the complex, which includes both the true rate of dissociation and the rate of irreversible denaturation of tubulin, is not influenced by 1.0 mM tartrate. The affinity constants for colchicine determined from the rate constants are 8.7 X 10(6) and 2.1 X 10(7) M-1 in the absence and the presence of 1.0 mM L-(+)-tartrate. The limiting value is 3.2 X 10(7) M-1. The affinity constant calculated from steady-state measurements is 3.2 X 10(6) M-1 with or without anions. The binding of other ligands like podophyllotoxin, vinblastine, and 1 -anilino-8-naphthalenesulfonate to tubulin is not affected by tartrate. No major conformational changes resulting from anion treatment could be detected by circular dichroism or intrinsic fluorescence. However, the ability of tubulin to polymerize is inhibited by L-(+)-tartrate at concentrations that increase the rate of colchicine binding. We conclude that anions must have a local effect at or near the binding site which enhances the binding rate of colchicine and which may be related to inhibition of polymerization.

Anilino Naphthalenesulfonates↗
PubMed1976

Tubulin aggregation and disaggregation: mediation by two distinct vinblastine-binding sites.

Rat brain tubulin possesses two distinct binding sites for vinblastine per molecule: a high-affinity site with an affinity constant of 6.2 x 10(6) M-1 and a low-affinity site with an affinity constant of 8 x 10(4) M-1. The high-affinity site is labile, with a t1/237 degrees of 3.5 hr, is protected by colchicine, and is unaffected by salt, whereas the low-affinity site is stable but is inhibited by salt. Binding to both sites is rapid. The high-affinity binding constant of vinblastine to tubulin (6.2 x 10(6) M-1) corresponds to the half-maximal concentration of vinblastine needed to prevent polymerization of tubulin in vitro, whereas the low-affinity binding constant (8 x 10(4) M-1) corresponds to the half-maximal concentration of vinblastine required to aggregate tubulin. We conclude that vinblastine binding to the high- and low-affinity sites, respectively, accounts for the depolymerization and aggregation behavior of tubulin.

Animals↗
PubMed1975

Membrane-bound tubulin in brain and thyroid tissue.

Brain and thyroid tissue contain membrane-bound colchicine-binding activity that is not due to contamination by loosely bound cytoplasmic tubulin. This activity can be solubilized to the extent of 80 to 90% by treatment with 0.2% Nonidet P-40 with retention of colchicine binding. Extracts so obtained contain a prominent protein band in disc gel electrophoresis that co-migrates with tubulin. Membranes, and the solubilized protein therefrom, exhibit ligand binding properties like tubulin; for colchicine the KA is approximately 1 X 10(6) M-1 in brain and approximately 0.6 X 10(6) M-1 in thyroid; for vinblastine the KA is approximately 8 X 10(6) M-1 for both tissues; and for podophyllotoxin the Ki is approximately 2 X 10(-6) M for both tissues. Displacement by analogues of colchicine is of the same order as for soluble tubulin. Although membrane-bound colchicine-binding activity shows greater thermal stability and a higher optimum binding temperature (54 degrees versus 37 degrees) than soluble tubulin, this appears to be the result of the membrane environment since the solubilized binding activity behaves like the soluble tubulin. Antibody against soluble brain tubulin reacts with membranes and solubulized colchicine-binding activity from both brain and thyroid gland. We conclude that brain and thyroid membrane preparations contain firmly bound tubulin or a very similar protein.

Adenylyl Cyclases↗
PubMed1974

Promotion of fluorescence upon binding of colchicine to tubulin.

Colchicine, which does not fluoresce in aqueous media and organic solvents, exhibits marked fluorescence on combination with brain tubulin, with a corrected excitation maximum at 362 nm, an emission maximum at 435 nm, and a quantum yield of about 0.03. From fluorescence measurements it was found that rat brain tubulin binds 0.83 moles of colchicine per dimer (molecular weight 110,000) with an association constant of 3.2 muM(-1) at pH 7.0 and 37 degrees . These results are in excellent agreement with those obtained with the binding of [(3)H]-colchicine. The enthalpy of binding is 10 kcal/mole, with an entropy change of 62 entropy units. The fluorescence can be ascribed to the tropolone moiety. However, the A ring of colchicine is also involved in binding. Denaturing agents abolish fluorescence, whereas podophyllotoxin, another antimitotic agent, decreases fluorescence competitively. Fluorescence is a convenient method for determining the binding of colchicine to tubulin that does not require the separation of free colchicine from bound colchicine and yields values for physical and biochemical parameters that are in excellent agreement with those obtained from the binding of [(3)H]colchicine.

Animals↗
PubMedDate not supplied

Mechanism for antagonism of paraoxon by hemicholinium-3 analogues.

In a newly synthesized series of DMAE analogues (bis-diethyl analogue of hemicholinium-3), selected chemicals (TL-402 = NAM-242 greater than JGC-VII-110) showed significant protection of mouse lethality after acute toxic doses of paraoxon (in vivo). DMAE and NAM-250 (like hemicholinium-3) showed minimal or no antagonism against paraoxon-induced toxicity in mice. Studies with DMAE analogues demonstrate weak anticholinesterase activity. The pattern for the neuromuscular inhibition of TL-402, NAM-242 and JGC-VII-110 is different from that of hemicholinium-3. LD50 studies identified compounds with less inherent toxicity (TL-402, NAM-242 and JGC-VII-110) and showed significant antagonism in contrast to DMAE and NAM-250. These chemicals (DMAE and NAM-250) are as toxic as the parent compound hemicholinium-3. All compounds in this series showed potent antinicotinic activity in different nicotinic-receptor preparations. The antinicotinic activity correlates with their action on the acetylcholine receptor-ion channel complex at frog neuromuscular junctions (in vitro). Electrophysiological studies demonstrate that the antinicotinic agents significantly depressed both the end plate current (EPC) amplitude and the time constant of decay (tau EPC) at the end plate of frog. In presence of paraoxon, voltage- and concentration-dependent shortening of tau EPC is observed which is more prominent than the decrease of the amplitude of EPC. The antinicotinic agents which showed significant antagonism of paraoxon both in vivo and in vitro (TL-402, NAM-242 and JGC-VII-110) also produced profound tetanic rundown after neurally or ionophoretically evoked EPC. These effects are voltage-dependent. The marked shortening of tau EPC, linear relationship between 1/tau vs DMAE analogue concentrations and potential-dependent tetanic rundown suggest that these analogues produce antagonism of paraoxon primarily by reducing end plate permeability by blocking nicotinic ACh-R associated ion channels in their open form. The antinicotinic activity of these agents is related to acetal or corresponding ether substitution.

Animals↗