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Biomedical subjects

M Caplow

Publications and source records attributed to M Caplow.

At least 37 records · Page 2Linked to original sources
PubMed1983

Rate for nucleotide release from tubulin.

The lower limit for the first order rate constant for dissociation of GDP from the tubulin E-site has been determined to be 0.14 s-1; this corresponds to a reaction with a half-life of 5 s. Using this rate constant and the previously determined equilibrium constant for GDP dissociation, equal to 6.1 X 10(-8) M (Zeeberg, B., and Caplow, M. (1979) Biochemistry 18, 3880-3886), the calculated association rate constant is 2.2 X 10(6) M-1 s-1. The tubulin E-site is highly reactive and it is calculated that: the half-life is 5 s for quantitative displacement of E-site bound radioactive GDP, by added excess nonradioactive GDP; the half-life is about 260 ms for isotopic equilibration when a trace amount of radioactive GDP is added to 20 microM tubulin-GDP; the half-life is about 850 ms for re-establishing the equilibrium for GDP binding, when 20 microM tubulin is diluted 20-fold. Thus, tubulin-GDP nucleotide exchange is rapid, so that added radioactive guanine nucleotides can be used in studies of relatively rapid reactions involving the tubulin subunit.

Guanine Nucleotides↗
PubMed1982

Concerning the efficiency of the treadmilling phenomenon with microtubules.

Radioactive tubulin subunit incorporation into porcine and dogfish shark brain microtubules which are at steady state has been found to result primarily from a diffusional reaction, in which subunits are incorporated although there are an equal number of tubulin subunit additions to and losses from each of the two microtubule ends in a unit of time. Treadmilling is very inefficient, and the Wegner s-values are equal to 0.0005-0.001. At steady state there are approximately 5000 (pig brain) or 2500 (dogfish brain) tubulin subunits lost from the two microtubule ends/s; an equivalent number of subunit addition reactions maintains a constant microtubule mass. The rate constants for subunit loss and addition with porcine brain microtubules, determined from analysis of the steady state rate for radioactive subunit incorporation, are much larger than those measured previously, when the rate constants were determined from the disassembly rate following perturbation of the steady state by dilution (Zeeberg, B., Reid, R., and Caplow, M. (1980) J. Biol. Chem. 255, 9891-9899). To account for this discrepancy it is suggested that at steady state the microtubule is capped by a short finite length of tubulin-GTP subunits, which undergo extremely facile association and dissociation (2500-5000 subunits/microtubule/s). This cap would be rapidly lost following dilution so that the observed rate only measures the relatively slow loss of tubulin-GDP subunits (120 subunits/microtubule/s) which had been in the interior of the microtubule; this is not equal to the rate constant for subunit loss (and addition) from the ends at steady state. Because of this, previous estimates of the Wegner s-value for treadmilling which utilized dilution for determining the steady state molecular rate constants for subunit loss are believed to be too high.

Animals↗
PubMed1982

Dynamic properties of microtubules at steady state in the presence of taxol.

The dynamic properties of steady-state microtubules in the presence of the antitumor drug taxol and GTP, but in the absence of microtubule-associated proteins have been studied. The molecular rate constants for the loss or gain of subunits at steady state was found to be dramatically decreased as compared with that for microtubules formed in the presence of GTP and microtubule-associated proteins but in the absence of taxol [Zeeberg, B., Reid, R., and Caplow, M. (1980) J. Biol. Chem. 255, 9891-9899]. In light of this change it was surprising to find that the degrees of directionality for subunit flux into the microtubule at steady state are nearly identical within 1.5% of each other) in the two systems. One mechanism to account for this would be for taxol to cause a nearly identical decrease in the rate constants for subunit dissociation at both ends of the microtubule, with no effect on the rate constants for subunit addition. Similar results have previously been found in studies with an endogenous effector of the microtubule steady state, a protein kinase [Jameson, J. L. and Caplow, M. (1981) Proc. Natl Acad. Sci. USA, 78, 3413-3417]. In this case it was found that phosphorylation of microtubule-associated proteins altered the molecular rate constants for tubulin subunit addition and dissociation, but had no effect on the degree of directionality for subunit flux. It will be of interest to determine whether other exogenous or endogenous effectors also act in a manner such as to leave the directionality unaltered.

Alkaloids↗
PubMed1981

An isoenergetic exchange mechanism which accounts for tubulin-GDP stabilization of microtubules.

We have developed a coupled enzyme system composed of hexokinase, glucose, and nucleoside diphosphate kinase which is able to rapidly convert GTP at the exchangeable nucleotide binding site of tubulin to GDP. Using this method, we have studied the dynamic properties of microtubules and tubulin subunits in the presence of GDP. Conversion of GTP to GDP causes microtubules to change to a new, somewhat lower steady state level; dilution studies show that subunits disassemble from microtubules at steady state in the presence of GDP at a rate comparable to that in the presence of GTP; in reactions of existing microtubules with tubulin-GDP subunits it was found that tubulin-GDP subunits do not participate in net microtubule elongation. From these observations we conclude that in the presence of tubulin-GDP the microtubule steady state has an unusual property; in spite of the fact that the microtubule is continuously undergoing rapid subunit loss, but not subunit addition, a nearly constant steady state level of microtubule mass is maintained. This must mean that tubulin-GDP subunits, although unable to participate in a net addition, can participate in additions each of which compensates for the dissociation of a single subunit from the microtubule. This is equivalent to the existence of an isoenergetic exchange of a tubulin-GDP in solution for a subunit which had been lost from the end of a microtubule.

Adenosine Triphosphate↗
PubMed1981

Incorporation of radioactive tubulin into microtubules at steady state. Experimental and theoretical analysis of the effect of podophyllotoxin.

Addition of a substoichiometric amount of podophyllotoxin to microtubules which are at steady state decreases the rate of incorporation of radioactive subunits into the microtubules. The magnitude of this effect depends on the fraction of the total number of each of the microtubule ends which is capped by tubulin-podophyllotoxin subunits; these fractions depend on the concentration of podophyllotoxin used. A quantitative relationship is presented which correlates the extent of tubulin-podophyllotoxin capping of the microtubule ends with alterations in the rate for steady state subunit incorporation. An analysis based upon this relationship reveals that tubulin-podophyllotoxin caps both ends of the microtubule. Previously proposed mechanisms which involve tubulin-podophyllotoxin capping at only one end of the microtubule are analyzed.

Animals↗
PubMed1981

Modification of microtubule steady-state dynamics by phosphorylation of the microtubule-associated proteins.

Phosphorylation of purified microtubule-associated proteins (MAPs) inhibits the rate and extent of MAP-stimulated microtubule assembly. The extent of microtubule assembly is reduced as a result of a decrease in the fraction of tubulin polymerized, without a significant change in the critical protein concentration. The decreased rate of microtubule assembly using phosphorylated MAPs reflects a reduction in microtubule nucleation resulting in fewer, but 2-fold longer, microtubules at steady state. Analysis of microtubule (MT) dynamics at steady state reveals that the rate of directional incorporation of subunits (flux) is 22 subunits.MT-1.sec-1 with phosphorylated MAPs, compared to 10 subunits.MT-1.sec-1 with unphosphorylated MAPs. The initial rate of disassembly determined by isothermal dilution is 232 subunits.MT-1.sec-1 for microtubules assembled with phosphorylated MAPs, compared to 102 subunits.MT-1.sec-1 for microtubules assembled with unphosphorylated MAPs. By using these results, the directionality (the number of successful subunit additions relative to the total number of association events per unit time) for subunit addition is found to be 0.1 for microtubules assembled with either phosphorylated or unphosphorylated MAPs. These observations are interpreted in terms of a mechanism in which phosphorylation of MAPs increases the rate of steady-state subunit flux by an equivalent enhancement of the rates of subunit association and dissociation, such that the critical protein concentration and directionality remain unchanged.

Animals↗
PubMed1980

Exchange of tubulin dimer into rings in microtubule assembly--disassembly.

We have prepared native radioactive tubulin dimer from two species: [35S]tubulin dimer, by in vivo labeling of rat brain, and porcine [3H]ethyltubulin, as previously described [Zeeberg, B., Cheek, J., & Caplow, M. (1980) Anal. Biochem. 104, 321--327]. After microtubule assembly with radioactive tubulin dimer and nonradioactive dimer and rings, the tubulin in the rings and the dimer obtained upon disassembly have approximately equal specific activities. Therefore, during the reaction sequence dimer + rings leads to 37 degrees C microtubules leads to 0 degrees C dimer + rings the tubulin initially in rings becomes indistinguishable from tubulin initially in dimer. Under nonpolymerizing conditions (0 degrees C) radioactive tubulin dimer and radioactive guanine nucleotide are incorporated into rings at approximately equal rates. This indicates that there is a pathway for nucleotide incorporation into rings under nonpolymerizing conditions which involves the incorporation of dimer-bound nucleotide. We also report results on the lack of the mirror image equilibrium during the disassembly process, using porcine [3H]-ethyltubulin dimer, rat [35S]tubulin dimer, and a [3H]-GDP.porcine tubulin dimer complex. In all three cases there is no significant disassembly-dependent incorporatioin of radioactivity into rings when microtubules are disassembled in the presence of radioactive dimer. These results demonstrate that, for rat and porcine tubulin, rings are formed during microtubule disassembly by direct cleavage of intact rings, without a tubulin dimer intermediate.

Animals↗
PubMed1980

Incorporation of radioactive tubulin into microtubules at steady state. Experimental and theoretical analyses of diffusional and directional flux.

Subunit flux into porcine brain microtubules at steady state has been studied using both radioactive guanine nucleotide and a radioactive tubulin dimer, [3H]ethyltubulin, which is obtained by a reductive ethylation (Zeeberg, B., Cheek, J., and Caplow, M., (1980) Anal. Biochem. 104, 321--327). We have also determined the molecular rate constants for steady state dimer loss from and addition to the microtubule. This is the first study where the rate for flux of radioactive subunits at steady state has been correlated with the measured molecular rate constants. Also, we derive a theoretical analysis of the observed flux of subunits into a polymer at steady state in pulse and chase experiments; this is used to relate the flux and molecular rate constant to a steady state model. Analysis of our results shows that, at steady state, there is only a small difference in the number of assembly and disassembly events at a given microtubule end (as compared with the total number of events at both ends) in a unit of time; predominant opposite end assembly-disassembly (Margolis, R. L., and Wilson, L. (1978) Cell 13, 1--8) is, therefore, ruled out. The almost complete absence of opposite end assembly-disassembly in the steady state provides a means for very efficient regulation of the microtubule network in vivo, since a small modulation in the relative magnitudes of the molecular rate constants can increase the subunit flux dramatically.

Animals↗
PubMed1980

Uptake of the components of phenylalanylphenylalanine and maltose by intestinal epithelium.

The observed rate of phenylalanine absorption into rat intestinal rings with 0.5 or 5.0 mM phenylalanine is greater than that for absorption of phenylalanine from 0.25 or 2.5 mM Phe-Phe, respectively. With the amino acid phenylalanine, V for absorption is the same whether Na+ is present (149 mM) or absent, but the concentration at which the half-maximal transport rate occurred (Kt) is greater in the absence of Na+. For Phe-Phe, the V decreases in the absence of Na+ whilst Kt is not influenced by the Na+ concentration. The different effect of Na+ on Phe and Phe-Phe transport indicates that the absorptive mechanism for Phe-Phe is different from that for phenylalanine. Absorption of a mixture of [U-14C]Phe-[he and Phe-[G-3H]Phe showed identical rates of uptake of the carboxyl and amino terminal amino acids. Studies of transport of radioactive maltose showed that the rates of uptake of the reducing and non-reducing glucosyl moieties are identical. Radioactive maltose absorption is not inhibited by glucose oxidase. These results provide evidence that in intestinal epithelium, hydrolysis of Phe-Phe and maltose does not occur on the cell surface with release of the hydrolyzed products to the medium. Rather, hydrolysis and release of the reaction products occur at a point on the cytosol side of a diffusion barrier located in the brush border membrane.

Animals↗
PubMed1980

Inhibition of microtubule assembly by phosphorylation of microtubule-associated proteins.

32P labeling of microtubular protein by endogenous protein kinase activity is shown to result from a net increase in protein-bound phosphate and is not the result of a phosphate exchange reaction between ATP and phosphoprotein. Protein phosphorylation is maximal in the presence of 0.5 mM Mg2+ and 0.25 mM ATP, resulting in approximately 2.8 nmol of phosphate/mg of protein. However, phosphorylation can be increased two-to threefold by cAMP. The protein substrates for phosphorylation either the absence or presence of cAMP are the microtubule-associated proteins which copurify with tubulin and promote microtubule assembly. Phosphorylation of microtubule-associated proteins inhibits both the rate and extent of microtubule assembly when the protein is exposed to conditions which result in dissociation of rings. These results are taken to indicate that phosphorylation modifies MAPs so that they have a reduced ability to form an assembly-competent complex with tubulin.

Animals↗
PubMed1979

Determination of free and bound microtubular protein and guanine nucleotide under equilibrium conditions.

The dissociation constant for GDP binding to the E site of tubulin isolated by chromatography on Sepharose 6B is equal to 6.1 X 10(-8) M, as determined by the Hummel-Dryer procedure. This is smaller than any previously reported value, and the discrepancy with earlir results is analyzed. By use of a recently described column centrifugation procedure [Penefsky, H. S. (1977) J. Biol. Chem. 252, 2891-2899], it was established that GDP and GTP bind to the same site. GTP is bound 2.8-fold tighter than GDP, and the dissociation constant is 2.2 X 10(-8) M. A new method for the determination of dissociation constants for a protein-bound ligand, based on a quantitative analysis of the loss of ligand during exclusion chromatography, is presented. This has been used to determine that the dissociation constant for GDP bound to tubulin is equal to 5.5 X 10(-8) M, in excellent agreement with that determined independently from the Hummel-Dryer method. A previous theoretical treatment [Dixon, H. B. F. (1976) Biochem. J. 159, 161-162] of ligand loss during exclusion chromatography is discussed.

Animals↗
PubMed1978

Reactions of tubulin-associated guanine nucleotides.

Only exchangeably bound nucleotide (E-site) is involved in the reaction of the transplhosphorylase activity in microtubular protein. Contrary to earlier reports, we find that the nonexchangeable nucleotide (N-site) is not a substrate. This conclusion is based upon comparison of: (a) rates of hydrolysis of endogenous tubulin-associated GTP and added [32p]GTP: (b) hydrolysis rates for added [32p]GTP and [3h]GTP; (c) the 32P/3H ratio in bound and free GTP after reaction with [3h, 32p]gTP. During the course of the above studies we have made the unusual observation of a time dependent augmentation in the expected amount of GTP relative to GDP at the E-site; there is either a net conversion of E-site GDP to E-site GTP, or a means for providing additional E-site GTP from another source.

Glycoproteins↗
PubMed1977

Microtubular protein catalytic interactions with nucleotides.

Purified tubulin prepared from pig brain in the absence of added guanine nucleotides contains 1 mol each of GDP and GTP/mol of tubulin. Incubation of a tubulin preparation with inorganic [32P]phosphate results in the incorporation of 32P into tubulin-associated GDP and GTP. Typically, in a 5-h incubation, 0.45 mM 32Pi reacts with 22 muM tubulin to form 3.3 muM [32P]GDP, and 0.3 muM [32P]GTP. The yield of labeled nucleotide is decreased as the result of a hydrolase activity associated with the preparation. The [32P]GTP is exclusively beta-labeled and is therefore formed by phosphorylation of [32P]GDP by a phosphate donor other than inorganic phosphate, most likely by nonradioactive GTP. Added GDP significantly decreases the yield of labeled GTP, but increases the yield of labeled GDP in a manner that is consistent with a partial protection against [32P]GDP hydrolysis, but not consistent with significant additional [32P]GDP formation. Added GMP is an inhibitor of both labeling activities associated with the preparation, although it has no effect on the hydrolase activity. Added ADP (0.4 MM) does not form labeled ADP or ATP and does not influence the labeling of GDP or GTP. The formation of [32P]GDP was shown to occur by an exchange mechanism rather than through net synthesis from GMP and Pi. These results provide evidence for a reversible guanidylation of the protein. The labeling activity is always specifically associated with highly purified tubulin preparations. Microtubular protein preparations are found to catalyze an exchange of oxygen from H218O into inorganic phosphate. Thus, there are two distinct catalytic properties associated with tubulin.

Animals↗