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Biomedical subjects

R H Himes

Publications and source records attributed to R H Himes.

At least 73 records · Page 4Linked to original sources
PubMed1986

Nucleotide stereochemistry in the formyltetrahydrofolate synthetase reaction.

In a recent study, we have shown that N10-formyltetrahydrofolate synthetase prefers (Sp)-MgATP beta S over the Rp isomer in the forward reaction. In this report the stereochemistry of ATP beta S produced from prochiral ADP beta S in the reverse reaction was determined. The ATP beta S product was purified and tested as a substrate for hexokinase (preference for the Rp isomer), adenylate kinase (preference for the Sp isomer) and N10-formyltetrahydrofolate synthetase. A comparison of kinetic constants for the product and the authentic Sp and Rp isomers shows that the product is the Sp diastereomer. 31P NMR was also used to identify the product as (Sp)-ATP beta S.

Adenosine Triphosphate↗
PubMed1986

Localization of the exchangeable nucleotide binding domain in beta-tubulin.

Limited proteolysis of tubulin by alpha-chymotrypsin cleaved the beta-subunit preferentially at Tyr 281, generating primarily 35 kD and 17 kD fragments which were located in the amino terminal and the carboxy terminal regions, respectively. A small amount of a 19 kD fragment from the C-terminal end was also produced. Alpha-Chymotrypsin-treated tubulin retained the ability to exchange GTP and covalently incorporate nucleotide by direct photoaffinity labeling. SDS-PAGE and autoradiography analysis of the [alpha-32P] GTP-labeled alpha-CT-treated tubulin showed that the 35 kD fragment was almost exclusively labeled, indicating that the exchangeable GTP binding domain resides in the amino terminal region of the beta-subunit.

Binding Sites↗
PubMed1986

Substrate and inhibitor activities of the screw sense isomers of metal-nucleotide complexes in the formyltetrahydrofolate synthetase reaction.

Phosphorothioate analogues of ATP and isomers of CrATP and CrADP were used to examine the nucleotide stereoselectivity of formyltetrahydrofolate synthetase from procaryotic and eucaryotic sources. Substrate activity of the thio-ATP analogues increased as the site of sulfur substitution was changed from the gamma to the alpha position. Thus, adenine nucleotide analogues substituted with sulfur at an alpha nonbridging position (ATP alpha S isomers) were the most active, and ATP gamma S was inactive. When Mg2+ was used as the divalent cation, both enzymes showed a clear preference (higher V/Km value) for the Sp isomer of ATP beta S although the magnitude of the preference was greater with the bacterial enzyme. With Cd2+ as the divalent cation the Rp isomer was preferred, but the difference was greater with the yeast enzyme. Both (Sp)-MgATP beta S and (Rp)-CdATP beta S have the delta or right-hand screw sense configuration of the metal chelate ring. The reversal of stereoselectivity when the cation was changed indicates that the metal ion is coordinated to the beta-phosphate group. No stereoselectivity was observed when ATP alpha S isomers were used in the presence of Mg2+ or Cd2+, suggesting that the metals are not coordinated to the alpha-phosphate. ATP beta S was also found to be a competitive inhibitor of MgATP and CdATP, and the lowest Ki values were obtained with the lambda screw sense isomers. The screw sense isomers of bidentate CrATP exhibited no detectable substrate activity but were competitive inhibitors of MgATP.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗
PubMed1986

Identification of a distinct class of vinblastine binding sites on microtubules.

Vinblastine, at concentrations above approximately 1 to 2 microM, causes depolymerization of steady-state bovine brain microtubules in vitro by a fraying of microtubule ends into protofilament-like spirals. Microtubule depolymerization is associated with the binding of vinblastine in approximately molar stoichiometry to tubulin in microtubules with apparent low affinity, as determined by binding experiments with radiolabeled vinblastine and by the ability of vinblastine to inhibit DEAE-dextran decoration of microtubule surfaces. Our data suggest that depolymerization occurs by a propagated mechanism, initially involving binding of vinblastine to a limited number of available sites on microtubule surfaces. This appears to cause loosening of protofilament associations which results in the exposure of new vinblastine-binding sites. Additional vinblastine binding in turn results in further loosening of protofilament associations. Such loosening, when it occurs at microtubule ends, results in protofilament-like splaying and end-wise depolymerization. Microtubule depolymerization appears mechanistically distinct from inhibition of microtubule polymerization by the drug, which is associated with the binding of vinblastine to small numbers of high-affinity binding sites on tubulin at one or both microtubule ends.

Animals↗
PubMed1985

Direct photoaffinity labeling of tubulin with guanosine 5'-triphosphate.

Irradiation of tubulin in the presence of [3H]GTP or [3H]GDP at 254 nm led to the covalent incorporation of nucleotide into the protein. The specific nature of the labeling was shown in the following manner: with tubulin depleted of exchangeable nucleotide, the amount of labeling increased to a plateau value as the [3H]GTP concentration was increased, with saturation being reached at a ratio of approximately 1.5; the same amount of labeling was obtained with GTP/tubulin ratios of 1 and 100; [3H]GMP was not incorporated into the dimer, nor did GMP inhibit the incorporation of [3H]GTP; [3H]ATP was not incorporated; [3H]GTP incorporation did not occur into denatured tubulin or into serum albumin. When [alpha-32P]GTP was used in the irradiation experiments, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the carboxymethylated protein demonstrated that the incorporated label was associated with the beta subunit. The radiation treatment did cause changes in the tubulin molecule resulting in a decrease in assembly competence and in sulfhydryl groups, but these effects were minimized when a large excess of GTP was present during irradiation. Labeling of tubulin in the assembled state was much less than that observed in the free state.

Adenosine Triphosphate↗
PubMed1985

Comparison of the effects of vinblastine, vincristine, vindesine, and vinepidine on microtubule dynamics and cell proliferation in vitro.

Vinepidine, a new derivative of vincristine, and three clinically used Catharanthus derivatives, vinblastine, vincristine, and vindesine, were examined for their abilities to inhibit net tubulin addition at the assembly ends of bovine brain microtubules at steady state. Although all four derivatives were generally similar in potency, their relative abilities to inhibit tubulin addition were distinguishable. Vinepidine and vincristine were the most potent derivatives (Ki, 0.079 +/- 0.018 (SD) microM and 0.085 +/- 0.013 microM, respectively), followed by vindesine (Ki, 0.110 +/- 0.007 microM) and vinblastine (Ki, 0.178 +/- 0.025 microM). In contrast to their relative abilities to inhibit microtubule assembly in vitro, vinblastine and its derivative, vindesine, were generally more potent than vincristine and vinepidine in inhibiting cell proliferation in culture. Vinblastine was nine times more potent than the weakest derivative, vinepidine, in B16 melanoma cells. In L-cells, vinblastine completely inhibited growth at 40 nM, whereas vincristine and vindesine caused about 25% inhibition, and vinepidine was inactive. When B16 melanoma cells were treated with drug before being injected into mice, retardation of tumor growth was best achieved with vindesine, one of the weaker of the four derivatives in vitro. The results demonstrate that chemical differences among the Catharanthus derivatives, which affect to small extents the abilities of the derivatives to inhibit microtubule assembly in vitro, result in significant differences in the order and the magnitude of the abilities of the drugs to inhibit cell growth.

Animals↗
PubMed1984

The influence of microtubule associated proteins on the production of polymorphic products from tubulin and microtubules.

Crude preparations of microtubule-associated proteins (MAPs), as well as purified MAP 2, influence the structure of products assembled from purified tubulin at low pH values. At pH 6.2, only 12% of the assembled products were microtubules (MTs) when assembly was conducted in 10% DMSO; 88% were large sheets of protofilaments. In the absence of DMSO, 28% of the structures were MTs. As the content of MAPs in the assembly reaction was increased, the proportion of MTs increased to 87% at a MAP/tubulin (w/w) ratio of 0.67 in the presence of DMSO and to 98% at a MAP/tubulin (w/w) ratio of 0.33 in the absence of DMSO. Purified MAP 2 was as effective as crude MAP preparations in promoting MT formation at pH 6.2. MTs formed from purified tubulin and MAP 2 were transformed into spirals of protofilaments upon the addition of Vinblastine (VLB). Spirals were also formed when VLB was added to a mixture of tubulin and MAP 2 at 4 degrees C. It thus appears that MAP 2 is a causative factor in initiating spiral formation in the presence of VLB.

Animals↗
PubMed1984

The action of two Vinca alkaloids on B16 melanoma in vitro.

The difference in the effectiveness of Vincristine and Vindesine against B16 melanoma proliferation was examined by treating the tumor tissue in vitro prior to subcutaneous implantation in adult mice and by treating cells in culture with the drugs. Vindesine caused greater inhibition of tumor growth than Vincristine when the tumor tissue was incubated with 50 microM drugs for 2 h prior to implantation. At nanomolar concentrations Vindesine retarded the proliferation of cells in culture to a greater extent than Vincristine. The greater effectiveness of Vindesine was also observed when cells were incubated with micromolar concentrations of the drugs followed by removal and recovery of the cell growth in vitro or in mice. The rate of drug uptake and amount of drug retained by either tumor pieces or cells in culture were similar for both Vinca alkaloids. It appears, therefore, that differences in drug uptake and retention by the tumor cells do not explain the greater effectiveness of Vindesine in inhibiting the proliferation of B16 melanoma cells.

Animals↗
PubMed1983

Possible treadmilling in zinc(II)-induced sheet polymers of bovine brain microtubule protein.

We have examined the ability of zinc(II)-induced sheet polymers, formed from thrice-cycled bovine brain microtubule protein prepared in the absence of glycerol, to exchange with tubulin subunits at steady state. By a rapid filtration assay in which labeled GTP was used as a marker for tubulin addition and loss, we found that steady-state sheet polymers, formed in 0.5 mM-ZnCl2, 1 mM-dithiothreitol, and 100 mM-2-(N-morpholino)ethanesulfonic acid (pH 6.75) in the presence of a GTP-regenerating system at 37 degrees C, incorporated the label in a time-dependent manner to a maximum level. The steady-state uptake of label was inhibited by colchicine, podophyllotoxin and vinblastine. In pulse-chase experiments, we observed that label added onto sheet polymers in a short pulse was retained for a period equal to that required by the polymers to become fully labeled in a continuous pulse; thereafter, the label was lost gradually to a baseline level. An average of 82% of the label was retained in the sheet polymers after a "cold" chase of equal duration to the time of the pulse. Sheet polymers assembled from microtubule protein prepared in the presence of glycerol gave similar results. Using a double-labeling procedure to analyze tubulin addition and loss simultaneously, we found that the rates of steady-state addition and loss were similar. Sheet polymers retained their structural integrity throughout these experiments, as determined by electron microscopy. We believe that the data are consistent with a "treadmilling" mechanism of polymerization and depolymerization, analogous to that documented to occur in steady-state microtubules in vitro. Such a mechanism is discussed in the context of recent findings from structural studies, and a model consistent with established structural data is offered.

Animals↗
PubMed1983

On the nonlinear relationship between the initial rates of dilution-induced microtubule disassembly and the initial free subunit concentration.

We have examined the dilution-induced in vitro disassembly kinetics of bovine brain microtubules, initially at steady state, using a wider range of dilutions (2-100-fold) than previously employed. In contrast to earlier results, as well as to the simple nucleation-condensation model for microtubule formation, the initial rate of dimer loss from microtubule ends was not a linear function of the initial concentration of unpolymerized tubulin. Over a 2-20-fold dilution range, plots of the initial rate of dimer loss versus the initial unpolymerized tubulin concentration were approximately linear. However, at greater dilutions, rates of microtubule depolymerization increased nonlinearly. For example, between a 10-fold dilution and a 100-fold dilution, the initial rate of dimer loss for microtubule-associated protein-containing microtubules increased by 300%, rather than a maximum of 11% expected on the basis of a linear rate plot. The nonlinear response was observed for dimer loss from opposite microtubule ends separately and with microtubules containing and lacking associated proteins. Qualitatively similar results were obtained using a wide range of experimental protocols, from which we can reasonably exclude methodological artifact as a basis for the data. We can also reasonably exclude the dissociation of the high molecular weight microtubule-associated proteins 1 and 2 from the microtubules as an explanation for the nonlinearity of the rate plots. The nonlinearity of the rate plots indicates that kinetic constants obtained under nonsteady state conditions of extreme microtubule dilution may not describe the steady state condition accurately.

Animals↗
PubMed1983

Nuclear magnetic resonance studies of formyltetrahydrofolate synthetase interactions with formate and methylammonium ion.

Using nuclear magnetic resonance techniques, we have measured the internuclear distances separating the nucleotide-bound metal from the carbon and hydrogen nuclei of formate as well as the carbon of methylammonium cation when bound to formyltetrahydrofolate synthetase. Measurements were made of the paramagnetic effect on the spin-lattice relaxation rates (1/T1) of 13C and 1H nuclei arising from the replacement of Mg2+ with Mn2+, which binds to the enzyme in the form of a metal-nucleotide complex. Distances from Mn2+ to the formate carbon and proton were found to be 6.3 and 7.4 A, respectively, in the E . ATP . Mn2+ . formate complex and 6.0 and 7.1 A, respectively, in the E . ADP . Mn2+ . formate complex. When tetrahydrofolate was added to the latter complex, the exchange of formate was greatly reduced and became rate limiting for relaxation. These results are consistent with substantial conformational effects produced by the binding of the cofactor. The distance from Mn2+ to the methylammonium carbon in the E . ADP . Mn2+ . CH3NH+3, E . ADP . Mn2+ . formate . CH3NH3+, and E . ADP . Mn2+ . tetrahydrofolate . CH3NH3+ complexes was estimated to be in the range of 7.4-12 A. However, in the E . ADP . Mn2+ formate . tetrahydrofolate . CH3NH3+ complex, the data suggest that exchange of cation contributes significantly to relaxation. These results, combined with other known features of the enzyme, suggest that there may be a monovalent cation site within the active site of the enzyme.

Clostridium↗
PubMed1983

Tubulin-zinc interactions: binding and polymerization studies.

The binding of Zn2+ to tubulin and the ability of this cation to promote the polymorphic assembly of the protein were examined. Equilibrium binding showed the existence of more than 60 potential Zn2+ binding sites on the dimer, including a number of high-affinity sites. The number of high-affinity sites, estimated by using a standard amount of phosphocellulose to remove more weakly bound Zn2+, reached a maximum of 6-7.5 with increasing levels of Zn2+ in the incubation solution. The number also increased with time of incubation at a single Zn2+ concentration. It is suggested that tubulin is slowly denatured in the presence of Zn2+, exposing more binding sites. Cu+ and Cd2+ were effective inhibitors of Zn2+ binding; Mg2+, Mn2+, and Co2+ were much less effective, and Ca2+ was without effect. Zn2+ does not replace the tightly bound Mg2+. GTP reduces the amount of Zn2+ binding under equilibrium conditions and the amount bound to high-affinity sites. Zinc-induced protofilament sheets are produced at a Zn2+/tubulin ratio of 5 in the presence of 0.5 mM GTP, conditions where about two to three Zn2+ ions would be bound to the dimer. At higher GTP concentrations, less assembly occurred, and the products were narrower sheets and microtubules. Zn2+-tubulin, isolated from phosphocellulose, will not assemble unless Mg2+ and dimethyl sulfoxide (Me2SO) or more Zn2+ is added. Broad protofilament sheets, formed from Zn2+-tubulin in the presence of Mg2+ and Me2SO, contain slightly more than one Zn2+ per dimer. It is concluded that Zn2+ stimulates tubulin assembly by binding directly to the protein, not via a ZnGTP complex.

Binding Sites↗
PubMed1982

Assembly of formyltetrahydrofolate synthetase monomers. Kinetic evidence for a temperature-induced conformational change.

The assembly of Clostridium acidi-urici formyltetrahydrofolate synthetase (formate: tetrahydrofolate ligase (ADP-forming), EC 6.3.4.3) monomers induced by low concentrations of CsCl to form the catalytically active tetramer shows a pronounced lag phase in the time-course progress curve. Preincubation of the monomer for 20 min at 20 degree C in a medium of either high ionic strength, 10% dimethylsulfoxide or 1 M glycerol removes the lag period. Shorter preincubation periods produce the same effect if the medium contains both high ionic strength and dimethylsulfoxide or glycerol. Preincubation at 0 degree C in the presence of these agents has little effect. The reassociation reaction is first-order with respect to monomer concentration, before preincubation at 20 degree C, but second-order after. It is proposed that two conformers of the monomer exist in equilibrium in a temperature-dependent reaction and that the conformer able to reassociate is more favored at 20 degree C than at 0 degree C. High ionic strength or the presence of the organic solvents shift the equilibrium far in the direction of the conformer capable of reassociation, changing the rate-limiting step from a first-order conformational step to a second-order monomer association step.

Clostridium↗
PubMed1981

Inhibition of tubulin self-assembly in vitro by fluorodinitrobenzene.

The self-assembly of bovine brain tubulin into microtubules is inhibited by low molar ratios of 1-fluoro-2,4-dinitrobezene (FDNB). Binding studies using [14C]FDNB indicate that the incorporation of between one and two dinitrophenyl groups is sufficient to inhibit assembly completely, although more dinitrophenyl groups can be incorporated using higher FDNB/tubulin ratios. Dinitrophenyltubulin, under assembly conditions, tends to assemble into amorphous aggregates. Thiolysis by 2-mercaptoethanol removes the dinitrophenyl moieties. Paper chromatography and high-voltage electrophoresis of acid-hydrolyzed modified tubulin containing one dinitrophenyl group identified the residue as S-dinitrophenylcysteine. The beta-monomer of tubulin is preferentially modified at low FDNB/tubulin ratios. The reaction with FDNB is greatly reduced when tubulin is in polymerized form. FDNB reduces the colchicine binding activity but does not affect the Mg(II) content of the protein.

Animals↗
PubMed1981

A simple procedure for the isolation of brain actin.

Actin has been isolated from bovine brain by forming an actin-containing gel from acetone powders. Solubilization of the gel and chromatography on Sephadex G150 produces actin in good yield. Acetone powders of crude pellets of brain homogenates obtained in the isolation of tubulin produces about 80% of the actin isolated from acetone powder of whole brain. The procedure therefore can be used to isolate both proteins from the same brain.

Actins↗