Biomedical subjects

R H Himes

Publications and source records attributed to R H Himes.

At least 55 records · Page 3Linked to original sources

PubMed1991

Assembly properties of tubulin after carboxyl group modification.

By chemically modifying carboxyl groups we have investigated the role of the highly acidic COOH-terminal domains of alpha- and beta-tubulin in regulating microtubule assembly. Using a carbodiimide-promoted amidation reaction, as many as 25 carboxyl groups were modified by the addition of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and an amine nucleophile, [14C] glycine ethyl ester or [3H]methylamine, to assembled microtubules. Modification occurred primarily in the carboxyl-terminal region as demonstrated by limited proteolysis of modified tubulin by trypsin, chymotrypsin, subtilisin, and carboxypeptidase Y. Modified tubulin polymerized into microtubules with a critical concentration that was 15% of that for unmodified tubulin. Assembly of modified tubulin and microtubules formed from modified tubulin were less sensitive to Ca2+ and high ionic strength. Ca2+ binding studies under low ionic strength conditions indicated that modified tubulin does not contain the high affinity Ca2+ binding site. While assembly of unmodified tubulin was stimulated by Mg2+ up to 10 mM, assembly of the modified protein was inhibited by concentrations greater than 1 mM. When 24 residues were modified, polymerization was no longer stimulated by microtubule-associated proteins (MAPs) or polylysine and incorporation of high molecular weight MAPs into the polymers was reduced by about 70% compared to unmodified tubulin. These studies demonstrate that chemical modification of carboxyl groups in tubulin, most of which are localized in the COOH-terminal region, leads to an enhanced ability to polymerize and a decrease in interaction with MAPs and other positively charged species.

Animals↗

PubMed1991

Binding of guanine nucleotides and Mg2+ to tubulin with a nucleotide-depleted exchangeable site.

Binding of GTP and GDP to tubulin in the presence or absence of Mg2+ was measured following depletion of the exchangeable site--(E-site) nucleotide. The E-site nucleotide was displaced with a large molar excess of the nonhydrolyzable GTP analogue, GMPPCP, followed by the removal of the analogue. Using a micropartition assay, the equilibrium constant measured in 0.1 M 1.4-piperazinediethanesulfonic acid (Pipes), pH 6.9, 1 mM ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid, 1 mM dithiothreitol, and 1 mM MgSO4 at 4 degrees C was 9.1 x 10(6) M-1 for GTP and 4.4 x 10(6) M-1 for GDP. Removal of Mg2+ reduced the binding affinity of GTP by 160-fold while the affinity of GDP remained essentially unchanged. Similar values were obtained if 0.1 M Tris, pH 7.0, was used instead of Pipes. Binding of Mg2+ to tubulin containing GTP, GDP, or no nucleotide at the E-site was also examined by the micropartition method. Tubulin-GTP contained one high affinity Mg2+ site (K alpha = 1.2 x 10(6) M-1) in addition to the one occupied by Mg2+ as tubulin is isolated, while only weak Mg2+ binding to tubulin-GDP and to tubulin with a vacant E-site (K alpha = 10(3) M-1) was observed. It is suggested that Mg2+ binds to the beta and gamma phosphates of GTP, and only to the beta phosphate of GDP, as shown for the H. ras p21 protein.

Animals↗

PubMed1991

Interactions of the catharanthus (Vinca) alkaloids with tubulin and microtubules.

The dimeric Vinca alkaloids represent a group of important anti-tumor compounds whose intracellular target is tubulin, the protein monomer of microtubules. In this review data on the binding of these drugs to tubulin and microtubules in vitro are examined. The binding to tubulin is linked to a protein self-association reaction described by Na and Timasheff (1986a) as a ligand-induced plus ligand-mediated isodesmic self-association reaction. The simplest model which fits the binding data is one in which there is one intrinsic site which is linked to the self-association process. Effects of solution variables on the binding and self-association explain the wide variation of reported apparent binding constants for Vinca alkaloids to tubulin. The Vinca drugs also bind to microtubules via a low number of sites at the ends of microtubules with apparent high affinity and which are involved in the inhibition of tubulin dimer addition to the microtubule ends, and to sites along the microtubule wall with apparent low affinity which are involved in the disruption of the microtubules into spiraled protofilaments. This review also compares available binding data for different natural and semi-synthetic Vinca alkaloids.

Animals↗

PubMed1990

Evidence that the tightly bound magnesium in tubulin is associated with the N-site GTP.

In an attempt to determine whether the tightly bound Mg2+ found in purified tubulin in associated with the N-site GTP or the E-site GDP or GTP, we removed the E-site nucleotide by several means: (i) alkaline phosphatase treatment; (ii) displacement using excess GMPPCP; and (iii) polymerizing tubulin in the presence of alkaline phosphatase and non-hydrolyzable analogues. The Mg2+ content remained equal to about 1 mol/mol tubulin under conditions where denaturation did not occur. Moreover, the Mg/GTP ratio always remained equal to 1. These results indicate that the Mg2+ is associated with the N-site GTP.

Animals↗

PubMed1990

Interaction of a new photosensitive derivative of vinblastine, NAPAVIN, with tubulin and microtubules in vitro.

We have synthesized a new photoreactive vinblastine derivative, 3-[[2-amino(4-azido-2-nitrophenyl)ethyl]-amino)-carbonyl)-O4-deceatyl -3-de (methoxycarbonyl)-vincaleukoblastine (NAPAVIN), which can be photoactivated with light in the 455-nm region as well as with ultraviolet irradiation. Previous studies had shown that photoactivated NAPAVIN is much more effective than vinblastine in inhibiting cell proliferation of multidrug resistant cell lines. The experiments reported here demonstrate that the unirradiated derivative is very similar to vinblastine in its interactions with brain tubulin and microtubules, regarding inhibition of in vitro assembly, binding, aggregation, and production of protofilament spirals. Irradiation of [3H]NAPAVIN in the presence of tubulin led to covalent binding of the drug to both subunits of the protein. Labeling also occurred when NAPAVIN was first irradiated, then incubated with tubulin in the dark, indicating the production of a fairly stable reactive species with a half-life of about 400 min. We conclude that labeling by this compound, under some conditions, occurs not by a nitrene but by an electrophilic photoproduct.

Animals↗

PubMed1990

Formylation of tetrahydrofolate by formyl phosphate.

Formyl phosphate is the putative intermediate in the formylation of tetrahydrofolate (THF) catalyzed by N10-formylTHF synthetase. In this study the non-enzymic reaction between formyl phosphate and THF was examined at 5 degrees C. 1H-NMR, HPLC and kinetic analysis of the proton-catalyzed conversion of the product to N5,10-methenylTHF were used to identify the product. In contrast to the enzyme reaction, which produces N10-formylTHF, N5-formylTHF was the only formylated THF derivative formed. The reaction was conducted at pH values of 3, 5, and 7, with the highest yield being obtained at pH 5 (64-85%, based on THF). The enzyme, therefore, changes the regioselectivity of this reaction by increasing the reactivity of the 10-nitrogen and either decreasing the reactivity of the 5-nitrogen or limiting its accessibility to formyl phosphate. 2-Mercaptoethanol, present in the reaction mixture to protect THF from O2, was also formylated by formyl phosphate, at the oxygen position.

Chemical Phenomena↗

PubMed1990

GTP analogues interact with the tubulin exchangeable site during assembly and upon binding.

The question of whether nonhydrolyzable nucleotide analogues and other nucleoside triphosphates support tubulin assembly was addressed. Tubulin which contained residual GTP at the exchangeable site polymerized in the absence of added GTP in the presence of DMSO or glycerol. After maximum absorbance was reached, disassembly occurred at a slow rate. When 0.5 mM GMPPCP, GMPPNP, or ATP was included in the assembly reaction, disassembly did not occur, and about 0.1 mol of these nucleotides per mole of tubulin was incorporated into the protein. When 5 mM nucleotide was used or alkaline phosphatase was included in the case of the nonhydrolyzable analogues, a greater amount of assembly occurred and about 0.7-0.8 mol of analogue was incorporated. The products of the assembly reaction were cold-labile microtubules and protofilament ribbons. After cold-depolymerization of the microtubules and ribbons, a second cycle of assembly produced some microtubules, but cold-stable amorphous polymers were the major product. In addition, when GTP at the exchangeable site was first removed by a cycle of assembly, followed by depolymerization, assembly in the presence of GMPPCP, GMPPNP, or ATP produced a mixture of microtubules and cold-stable polymers, both of which contained bound analogue. Incorporation of GMPPCP, GMPPNP, or ATP into polymerized tubulin always occurred at the expense of GDP at the exchangeable site, the content of which decreased correspondingly. Incubation of tubulin with 5 mM GMPPCP, GMPPNP, or ATP under nonassembly conditions also displaced GDP.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗

PubMed1990

Stabilization of microtubules by GTP analogues.

We recently demonstrated that the nonhydrolyzable analogues of GTP (GMPPCP and GMPPNP) and ATP support the elongation phase of tubulin assembly and are incorporated into the E-site of polymerized tubulin. In this report we studied the stability of microtubules containing GTP analogues by examining length redistributions after shearing at polymer steady state. The mean length of a population of microtubules containing GMPPCP increased only by 37% over a 150 min time period after shearing. Microtubules which contained 70% ATP and 30% GDP at the E-site increased in length by 88%. In contrast, the mean length of microtubules assembled in the presence of GTP increased by 410% in the same time period. These results suggest that microtubules containing GMPPCP or ATP at their ends are stabilized from depolymerization.

Adenosine Triphosphate↗

PubMed1989

Tubulin dimer dissociation detected by fluorescence anisotropy.

We have demonstrated a concentration-dependent dissociation of bovine brain tubulin dimer covalently labeled with 5-[(4,6-dichlorotriazin-2-yl)amino]fluorescein (DTAF) or with fluorescein isothiocyanate (FITC) by fluorescence anisotropy and size-exclusion HPLC. The fluorescence anisotropy values decreased to a limiting value upon dilution of tubulin from 10(-5) to 8 x 10(-8) M. A dissociation constant in 0.1 M Pipes, pH 6.9, 1 mM EGTA, and 1 mM MgSO4 at 20 degrees C was estimated to be (8.4 x 10(-7) +/- (0.4 x 10(-7) M. Control experiments using monomeric and other dimeric proteins, urea-denatured tubulin, and DTAF-tubulin diluted into solutions of bovine serum albumin or unlabeled tubulin were consistent with the finding that the changes in anisotropy upon dilution are due to protein dissociation. These results were supported by size-exclusion HPLC data where an increase in the elution volume of DTAF-tubulin and FITC-tubulin was observed with decreasing protein concentrations. Reversibility of the dissociation process and the lack of denaturation at high dilution were shown by the ability of reconcentrated protein to assemble into microtubules to about the same extent as undiluted protein. Fluorescent lifetimes and limiting anisotropy values were found to be approximately identical at different tubulin concentrations, indicating that the anisotropy changes reflect changes in size or rotational correlation time of the protein. Studies on the effects of tubulin ligands and promoters or inhibitors of assembly demonstrated that their effects on tubulin dimer-monomer equilibria are small but reproducible.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Dynamics of Antarctic fish microtubules at low temperatures.

The tubulins of Antarctic fishes, purified from brain tissue and depleted of microtubule-associated proteins (MAPs), polymerized efficiently in vitro to yield microtubules at near-physiological and supraphysiological temperatures (5, 10, and 20 degrees C). The dynamics of the microtubules at these temperatures were examined through the use of labeled guanosine 5'-triphosphate (GTP) as a marker for the incorporation, retention, and loss of tubulin dimers. Following attainment of a steady state in microtubule mass at 20 degrees C, the rate of incorporation of [3H]GTP (i.e., tubulin dimers) during pulses of constant duration decreased asymptotically toward a constant, nonzero value as the interval prior to label addition to the microtubule solution increased. Concomitant with the decreasing rate of label incorporation, the average length of the microtubules increased, and the number concentration of microtubules decreased. Thus, redistribution of microtubule lengths (probably via dynamic instability and/or microtubule annealing) appears to be responsible for the time-dependent decrease in the rate of tubulin uptake. When the microtubules had attained both a steady state in mass and a constant length distribution, linear incorporation of labeled tubulin dimers over time occurred at rates of 1.45 s-1 at 5 degrees C, 0.48 s-1 at 10 degrees C, and 0.18 s-1 at 20 degrees C. Thus, the microtubules displayed greater rates of subunit flux, or treadmilling, at lower, near-physiological temperatures. At each temperature, most of the incorporated label was retained by the microtubules during a subsequent chase with excess unlabeled GTP.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Formation and utilization of formyl phosphate by N10-formyltetrahydrofolate synthetase: evidence for formyl phosphate as an intermediate in the reaction.

N10-Formyltetrahydrofolate synthetase from bacteria and yeast catalyzes a slow formate-dependent ADP formation in the absence of H4folate. The synthesis of formyl phosphate by the enzyme was detected by trapping the intermediate as formyl hydroxamate. That the "formate kinase" activity was part of the catalytic center of N10-formyltetrahydrofolate synthetase was shown by demonstrating coordinate inactivation of the "kinase" and synthetase activities by heat and a sulfhydryl reagent, similar effects of monovalent cations, similar Km values for substrates, and similar Ki values for the inhibitor phosphonoacetaldehyde for both activities. The relative rates of the kinase activities for the bacterial and yeast enzymes are about 10(-4) and 4 x 10(-6) of their respective synthetase activities. These slow rates for the kinase reaction can be explained by the slow dissociation of ADP and formyl phosphate from the enzyme. This conclusion is supported by rapid-quench studies where a "burst" of ADP formation (6.4 s-1) was observed that is considerably faster than the steady-state rate (0.024 s-1). The demonstration of enzyme-bound products by a micropartition assay and the lack of a significant formate-stimulated exchange between ADP and ATP provide further evidence for the slow release of the products from the enzyme. The synthesis of N10-CHO-H4folate when H4folate was added to the E-formyl phosphate-ADP complex is also characterized by a "burst" of product formation. The rate of this burst phase at 5 degrees C occurs with a rate constant of 18 s-1 compared to 14 s-1 for the overall reaction at the same temperature. These results provide further evidence for formyl phosphate as an intermediate in the reaction and are consistent with the sequential mechanism of the normal catalytic pathway. Positional isotope exchange experiments using [beta,gamma-18O]ATP showed no evidence for exchange during turnover experiments in the presence of either H4folate or the competitive inhibitor pteroyltriglutamate. The absence of scrambling of the 18O label as observed by 31P NMR suggests that the central complex may impose restraints to limit free rotation of the P beta oxygens of the product ADP.

Adenosine Diphosphate↗

PubMed1989

Binding of vinblastine to stabilized microtubules.

Addition of 2-200 microM vinblastine to microtubules at steady state in vitro causes the microtubules to depolymerize, with the formation of protofilament spirals and other aggregated forms of microtubule protein. The presence of such spirals and protein aggregates, which are difficult to separate from microtubules, has complicated attempts to measure the binding of vinblastine to microtubules. We have found that stabilizing bovine brain microtubules in vitro with dimethyl sulfoxide, taxol, or a combination of dimethyl sulfoxide and taxol prevents or greatly retards the formation of protofilamentous spirals, thus permitting us to measure the binding of vinblastine to intact microtubules. Reciprocal plots of binding data indicate the presence of 1.4-1.7 vinblastine binding sites/mol of tubulin in the microtubule, with a Ka of approximately 3-4 x 10(3) M-1. The Ka value obtained is within 1 order of magnitude of the apparent intrinsic binding constant for the binding of vinblastine to tubulin dimers. The results support the idea that depolymerization of microtubules by intermediate and high concentrations of vinblastine occurs by stoichiometric binding of vinblastine to tubulin along the microtubule surface.

Alkaloids↗

PubMed1988

GTP hydrolysis by tubulin occurs with water oxygen incorporation into inorganic phosphate.

Tubulin assembly was conducted in [18O]H2O and the resulting mixture of GTP, GDP and Pi was examined by 31P-NMR. Two Pi signals, separated by about 0.02 ppm, were observed. By combining this mixture with a solution of Pi containing all five possible 16O and 18O isotopomers of Pi, it was shown that the two signals were due to [16O4]- and [16O3, 18O]Pi. The amount of 18O incorporated into the Pi was that expected if the hydrolysis of GTP during tubulin assembly occurs with cleavage of the gamma-phosphorus-bridge oxygen bond.

Animals↗

PubMed1988

Effect of solution variables on the binding of vinblastine to tubulin.

Vinblastine binding to tublin was measured in different buffers using tubulin prepared by two different methods and three different binding assay methods. In 100 mM 1,4-piperazinediethanesulfonic acid (Pipes) buffer containing 1 mM MgSO4 and 1 mM ethylene glycol bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), the data appeared to be consistent with one site with a Ka value of 3.4 X 10(6) M-1 and another site with a Ka value of 2.8 X 10(5) M-1. However, in buffers of lower ionic strengths and without Mg2+ the Ka values were lower. The lowest value (2 X 10(4) M-1) was obtained in 10 mM phosphate buffer, in which only one site was evident under the conditions used. Neither the binding assay used nor the method for tubulin preparation affected the Ka value. Using HPLC, aggregation induced by vinblastine was evident in buffers which gave the largest Ka values. Tubulin aggregation in the presence of vinblastine was also confirmed by analytical ultracentrifugation. The results support the proposal of Na and Timasheff [Biochemistry 25, 6214 (1986)] that the apparent Ka value is influenced by the degree of aggregation induced by vinblastine and that the intrinsic binding constant to the dimer is represented by the lowest value, about 2 X 10(4) M-1.

Animals↗

PubMed1988

The effects of dimethyl sulfoxide on the kinetics of tubulin assembly.

The kinetics of tubulin assembly were examined in the absence and presence of dimethyl sulfoxide at 37 degrees C. Inclusion of 1.4 M (10%) dimethyl sulfoxide lowered the critical protein concentration about 8-10-fold, from 9.4 microM in the absence of the organic solvent to 1.1 microM in its presence. This decrease was due solely to an effect on k-, the off rate constant. The on rate constant k+, was essentially unaffected. Another effect of dimethyl sulfoxide was in the nucleation process. The pseudo-first-order rate constant of elongation, kapp (k+[m]), was greatly increased by inclusion of dimethyl sulfoxide. This was due to an increase in the microtubule number concentration, [m]. The microtubules formed in the presence of dimethyl sulfoxide were much shorter than those formed in its absence, accounting for the higher number concentration. The nucleation number, n, was calculated by plots of ln kapp vs. ln c0 or ln t10% vs. ln c0, and the value appeared to be about 4 to 5, although some variability was found. It was shown that a plot of kapp vs. c0 to determine n, is not appropriate because of the inability to distinguish between linear and curved plots in the range of tubulin concentration used in assembly studies.

Animals↗

PubMed1988

Nuclear magnetic resonance relaxation studies of the interaction of ligands with the monomer and tetramer forms of formyltetrahydrofolate synthetase.

Previous work using n.m.r. spectroscopy to investigate the binding between formyltetrahydrofolate synthetase and its ligands was done using the catalytically active tetrameric form of the enzyme. By removal of specific monovalent cations the tetramer dissociates to four identical, catalytically inactive monomers, which are capable of binding nucleotides with affinities similar to those obtained with the tetramer. In the studies reported here, we examined the interaction of metal-nucleotide, formate and monovalent cations with the monomer using n.m.r. relaxation measurements. We were able to demonstrate that formate binds to the monomer. The spin-lattice relaxation rate (1/T1) of the formate carbon in the monomer.M2+.ADP.formate complex is enhanced when Mg2+ is replaced by Mn2+. By assuming that the exchange of formate is not rate-limiting and that tau c of the monomer is the same as that of the tetramer, the distance between the Mn2+ and the formate carbon was calculated and found to be similar in the monomer and tetramer complexes. The spin-lattice relaxation rates of [13C]trimethylammonium ion (an inactive monovalent cation), [13C]methylammonium and [15N]ammonium ions (both active monovalent cations), were measured in the presence of tetramer, MnADP and formate. The relaxation rates of methylammonium and ammonium ions were enhanced under these conditions whereas the relaxation rate of trimethylammonium ion was not. The results indicate that the active monovalent cations bind near the MnADP binding site. A distance from the Mn2+ to the ammonium nitrogen of between 0.5 and 0.6 nm was calculated.

Ammonia↗

PubMed1987

Substrate activity of synthetic formyl phosphate in the reaction catalyzed by formyltetrahydrofolate synthetase.

Formyl phosphate, a putative enzyme-bound intermediate in the reaction catalyzed by formyltetrahydrofolate synthetase (EC 6.3.4.3), was synthesized from formyl fluoride and inorganic phosphate [Jaenicke, L. v., & Koch, J. (1963) Justus Liebigs Ann. Chem. 663, 50-58], and the product was characterized by 31P, 1H, and 13C nuclear magnetic resonance (NMR). Measurement of hydrolysis rates by 31P NMR indicates that formyl phosphate is particularly labile, with a half-life of 48 min in a buffered neutral solution at 20 degrees C. At pH 7, hydrolysis occurs with P-O bond cleavage, as demonstrated by 18O incorporation from H2(18)O into Pi, while at pH 1 and pH 13 hydrolysis occurs with C-O bond cleavage. The substrate activity of formyl phosphate was tested in the reaction catalyzed by formyltetrahydrofolate synthetase isolated from Clostridium cylindrosporum. Formyl phosphate supports the reaction in both the forward and reverse directions. Thus, N10-formyltetrahydrofolate is produced from tetrahydrofolate and formyl phosphate in a reaction mixture that contains enzyme, Mg(II), and ADP, and ATP is produced from formyl phosphate and ADP with enzyme, Mg(II), and tetrahydrofolate present. The requirements for ADP and for tetrahydrofolate as cofactors in these reactions are consistent with previous steady-state kinetic and isotope exchange studies, which demonstrated that all substrate subsites must be occupied prior to catalysis. The k cat values for both the forward and reverse directions, with formyl phosphate as the substrate, are much lower than those for the normal forward and reverse reactions. Kinetic analysis of the formyl phosphate supported reactions indicates that the low steady-state rates observed for the synthetic intermediate are most likely due to the sequential nature of the normal reaction.

Adenosine Diphosphate↗

PubMed1987

Polymorphic assembly of subtilisin-cleaved tubulin.

Limited proteolysis of tubulin with subtilisin results in cleavage of both the alpha and beta subunits, releasing small peptides from the C-terminal ends. At 37 degrees C the digested tubulin assembles into polymorphic structures: microtubules with attached ribbons in the presence of GTP, rings in the presence of GDP, and protofilament spirals in the presence of vinblastine. Undigested tubulin does not assemble under these conditions. Rings and Vinca-induced spiral structures are assembled from undigested tubulin only when microtubule-associated proteins, high Mg2+ concentrations, or polycations are present. Thus, cleavage with subtilisin affects assembly in a manner similar to the addition of these agents. It appears that binding of positively charged substances may act by neutralizing the charge on the highly acidic C-terminal regions of the alpha- and beta-subunits, while cleavage with subtilisin produces the same effect by removing these peptides. Undigested and subtilisin-digested tubulin form sheets of protofilaments in the presence of Zn2+, which indicates that the binding sites for the 2-3 Zn2+ ions necessary to induce sheet formation do not reside in the C-terminal regions of the monomers.

Animals↗