Search PubMed⌕ Search

Biomedical subjects

D L Purich

Publications and source records attributed to D L Purich.

At least 55 records · Page 3Linked to original sources
PubMed1991

Evidence against impaired brain microtubule protein polymerization at high glucose concentrations or during diabetes mellitus.

Previous studies suggest that brain microtubule protein exposed to high glucose levels or isolated from diabetic rats can become glucosylated and that this impairs GTP-induced microtubule polymerization. We set out to extend that investigation to define the mechanistic basis for inhibition of microtubule assembly during diabetes or on incubation at high glucose levels. Rat and bovine brain microtubule protein was purified by cycles of polymerization/depolymerization. When microtubules were incubated for 1 h in either buffer or buffer containing glucose (up to 165 mM), there was no difference in polymerization, a finding contrary to the earlier study. Other rats were injected with vehicle or streptozotocin (90 mg/kg) to induce diabetes as evidenced by serum glucose in excess of 300 mg%, and at 4 weeks, brain microtubule protein was isolated by the polymerization cycling method. Again, there was no difference in the amount or purity of isolated microtubule protein between control or diabetic rats. We also observed no increase in microtubule glucosylation, and GTP-induced polymerization in vitro was indistinguishable for protein derived from brains of normal rats and rats with diabetes as measured by turbidity or electron microscopy. Our results suggest that in vitro incubation with glucose or in vivo elevation of glucose during diabetes fails to impair microtubule polymerization, pointing to other mechanisms for the neuropathy associated with diabetes.

Animals↗
PubMed1990

Peptides corresponding to the second repeated sequence in MAP-2 inhibit binding of microtubule-associated proteins to microtubules.

Bovine brain high molecular weight microtubule-associated proteins (MAPs) can be displaced from assembled tubules by peptides corresponding to the second of three nonidentical repeated sequences in mouse MAP-2. The octadecapeptide m2 (VTSKCGSLKNIRHRPGGG) can release MAP-1b from MAP-containing microtubules, and the extended second-sequence peptide m2' (VTSKCGSLKNIRHRPGGGRVK) displaces MAP-1a and MAP-1b as well as MAP-2a and MAP-2b. Peptides m2 and m2' stimulate tubulin polymerization in the absence of MAPs or microtubule-stabilizing agents, and m2' acts as a competitive inhibitor of radiolabeled MAP-2 binding. The dissociation constant for MAP-2 binding to taxol-stabilized tubules was 3.4 microM in the absence of m2' and 14 microM in the presence of 1.5 mM of the m2' peptide. We estimate that the inhibition constant for peptide m2' is about 0.5 mM, about 100 times lower than for the Km of MAP-2. These observations suggest that the second repeated sequence in MAP-2 may represent an important recognition site for MAP binding to microtubules and that other structural features within MAP-2 may reinforce the strength of MAP-microtubule interactions.

Amino Acid Sequence↗
PubMed1990

Apparently irreversible GTP hydrolysis attends tubulin self-assembly.

The pathway of GTP hydrolysis associated with microtubule polymerization was investigated using an assay of intermediate 18O-exchange reactions. Under a variety of conditions influencing tubulin self-assembly, GTP was hydrolyzed without any evidence of multiple reversals characteristic of reversible phosphoanhydride-bond cleavage. These results also accord with published findings that ATP hydrolysis during actin polymerization fails to display intermediate exchange reactions [Carlier, M. F., Pantaloni, D., Evans, J. A., Lambooy, P. K., Korn, E. D. and Webb, M. R. (1988) FEBS Lett. 235, 211-214].

Energy Transfer↗
PubMed1989

The microtubule-binding fragment of microtubule-associated protein-2: location of the protease-accessible site and identification of an assembly-promoting peptide.

Thrombin cleavage of bovine brain microtubule-associated protein (MAP-2) yields two stable limit polypeptide fragments (28,000 and 240,000 Mr). The smaller cleavage product contains the microtubule-binding domain and is derived from the carboxyl terminus of MAP-2 while the 240,000 Mr fragment is derived from the amino terminus. The amino terminal sequence of the smaller cleavage product is homologous with the microtubule-binding fragment of tau in sequence and in a similar location relative to three imperfect octadecapeptide repeats implicated in microtubule binding. Peptides corresponding to the cleavage site and the three repeats of MAP-2 were synthesized. Only the second octadecapeptide repeat (VTSKCGSLKNIRHRPGGG) was capable of stimulating microtubule nucleation and elongation. Microtubules formed in the presence of this peptide displayed normal morphology and retained the inhibition properties of calcium ion, podophyllotoxin, and colchicine. Our result indicates that a region comprising only approximately 1% of the MAP-2 sequence can promote microtubule assembly.

Adenosine Triphosphate↗
PubMed1989

Use of the computer program EQUIL to estimate pH in model solutions and human urine.

The computer program EQUIL was designed to calculate relative supersaturations of solute components of common urinary stones. In an extended software version, quantitative consideration of charge balance for a priori or a posteriori pH estimation was added. The reliability of this computation was tested with hydrogen ion titration of buffer solutions containing HEPES [N-(2-hydroxyethyl)piperaizine-N'-ethanesulfonic acid] as well as samples of normal human urine. In the model solutions with HEPES, the difference between calculated pH values and the measured pH was smaller than 1.2% for any titration step within the buffer zone (pH 8.5-6.8). The pH values calculated for whole urine differed from the measured pH by 7% to 53%, and the calculated charge inbalance ranged from 2.6 to 9.6 mM. This net cation inbalance indicates that there is a need to account for other anionic components, including hippurate, amino acids, and isocitrate. In experimental solutions, charge balance calculations with EQUIL can be of great utility because they permit a priori estimation of pH or computation of the composition at a desired pH.

Buffers↗
PubMed1987

GTP regeneration influences interactions of microtubules, neurofilaments, and microtubule-associated proteins in vitro.

Interactions of microtubules, neurofilaments, and microtubule-associated proteins were investigated by turbidity and falling-ball viscometry measurements. We found evidence of endogenous GTPase activity in neurofilaments and microtubule-associated proteins (MAPs) in preparations that do not include urea or heat treatment, respectively. The absence or presence of either adenyl-5'-yl imidodiphosphonic acid or a GTP-regenerating system markedly influenced observed polymerization and gelation characteristics. Most significantly, the apparent viscosity of neurofilament and microtubule samples did not display a biphasic optimal MAP concentration profile when a GTP-regenerating system was operant. Likewise, GTP regeneration promoted the recovery of gelation following mechanical disruption of neurofilament/MAP/microtubule mixtures. These and other observations require some reassessment of proposed roles for microtubule-associated proteins in modulating neurofilament-microtubule interactions in vitro.

Animals↗
PubMed1987

The 28,000 Mr microtubule-binding domain of microtubule-associated protein-2 also contains a neurofilament-binding site.

We have developed a thrombin proteolytic cleavage procedure to obtain higher yields of the Mr 28,000 microtubule-binding and Mr 240,000 microtubule-projection components of MAP-2. The former is a highly basic component, whereas the latter and intact MAP-2 are acidic polypeptides. Most notably, our studies reveal that this Mr 28,000 fragment binds to neurofilaments, but the Mr 240,000 projection domain fails to interact. These data indicate that microtubules and neurofilaments share a common binding site on high-molecular-weight MAP-2.

Animals↗
PubMed1987

Modulation of microtubule assembly and stability by phosphatidylinositol action on microtubule-associated protein-2.

Exposure of elongating (or assembled) bovine brain microtubules to phosphatidylinositol leads to polymerization arrest (or disassembly). The efficiency of phosphatidylinositol far exceeds the action of related phospholipids including phosphatidylethanolamine, phosphatidylcholine, 1,2-diacylglycerol, phosphatidylserine, phosphatidylglycerol, or phosphatidic acid. Phosphatidylinositol increases the apparent critical concentration for assembly, and the inhibitory effect of phosphatidylinositol on polymerization is reversed at higher concentrations of microtubule-associated proteins (MAP)s. Taxol- and glycerol-treated microtubules are insensitive to the destabilizing action of phosphatidylinositol; centrifugation and subsequent gel electrophoresis of such samples revealed that both MAP-2a and MAP-2b were selectively desorbed. Likewise, the desorption of MAP-2 was visualized by indirect immunofluorescence microscopy using primary antibodies directed toward tubulin and MAP-2. The instability of microtubules exposed to phosphatidylinositol appears to be related to the MAP-2 content.

Alkaloids↗
PubMed1985

Ciliary dynein conformational changes as evidenced by the extrinsic fluorescent probe 8-anilino-1-naphthalenesulfonate.

The binding of 8-anilino-1-naphthalenesulfonate (ANS) to ciliary dynein ATPase leads to a marked increase in the dye's fluorescence intensity, accompanied by a blue shift in the observed fluorescence emission maximum. We found that dynein has 37 +/- 3 ANS binding sites and that experimentally applied ANS concentrations failed to alter enzyme activity. The fluorescence properties of the enzyme-dye complex were used to learn more about the binding characteristics of dynein substrates and effectors and to probe for possible conformational changes of the enzyme. The fluorescence of the dynein-ANS complex is increased by a number of substrates, including ATP, GTP, and UTP. The transfer of excitation energy from dynein chromophores to adsorbed ANS was also investigated. Our findings indicate that dynein appears to undergo a localized conformational change in its interaction with ATP. Native dynein was also found to be conformationally different from heat-activated or NEM-modified enzyme as evidenced by the emission and excitation spectra of the various enzyme-ANS complexes.

Adenosine Triphosphatases↗
PubMed1985

Taxol stabilization of microtubules in vitro: dynamics of tubulin addition and loss at opposite microtubule ends.

We have investigated the effects of taxol on steady-state tubulin flux and on the apparent molecular rate constants for tubulin addition and loss at the two ends of bovine brain microtubules in vitro. These microtubules, which consist of a mixture of 70% tubulin and 30% microtubule-associated proteins (MAPs), undergo a net addition of tubulin at one end of each microtubule (A end) and a precisely balanced net loss of tubulin at the opposite end (D end) at steady state in vitro. They do not exhibit to a detectable extent the "dynamic instability" behavior described recently for MAP-free microtubules, which would be evident as an increase in the mean microtubule length and a decrease in the number of microtubules in the suspensions [Mitchison, T., & Kirschner, M. (1984) Nature (London) 312, 237-242]. We used a double-label procedure in which microtubules were labeled with tritium and carbon-14 at A ends and carbon-14 at D ends to distinguish the two ends, combined with a microtubule collection procedure that permitted rapid and accurate analysis of retention of the two labels in the microtubules. We found that taxol slowed the flux of tubulin in a concentration-dependent manner, with 50% inhibition occurring between 5 and 7 microM drug. The effects of taxol on the apparent molecular rate constants for tubulin addition and loss at the two microtubule ends were determined by dilution analysis at an intermediate taxol concentration. The results indicated that taxol decreased the magnitudes of the dissociation rate constants at the two ends to similar extents, while exerting little effect on the association rate constants.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaloids↗
PubMed1985

Microtubule-associated proteins (MAPs): a monoclonal antibody to MAP 1 decorates microtubules in vitro but stains stress fibers and not microtubules in vivo.

A monoclonal antibody (mAb 7-1.1) was produced against a bovine brain microtubule-associated protein (MAP) preparation that had been separated from tubulin after initial purification by cycles of microtubule assembly and disassembly in vitro. The antibody reacted specifically with two high molecular weight polypeptides of the MAP 1 class, designated MAP 1.1 and MAP 1.2, and also with the surfaces of MAP 1-containing microtubules that had been assembled in vitro. Double immunofluorescence microscopy using mAb 7-1.1 and a well-characterized rabbit anti-tubulin antibody revealed that mAb 7-1.1 stained stress fibers in fixed and permeabilized cultured mammalian cells rather than microtubules. The antibody also stained cell nuclei in a punctate fashion. mAb 7-1.1 is one of a number of monoclonal antibodies that react with presumptive MAP 1 polypeptides. Some of the MAP 1 antibodies have been found to bind specifically to microtubules in fixed and permeabilized cells, while others have been reported to react with nonmicrotubule structures. Our results, together with the results of other investigations, indicate that "MAP 1" may be a family of several high molecular weight polypeptides that adventitiously behave as MAPs by the criterion of in vitro coassembly with tubulin through cycles of polymerization and depolymerization but whose cellular distributions, and perhaps functions, are varied.

Animals↗
PubMed1983

Concerning the equilibrium exchange kinetics and mechanism of the sheep brain glutamine synthetase reaction.

Isotope exchange measurements of sheep brain glutamine synthetase have yielded conflicting experimental findings and interpretation, leaving in doubt the question of whether the enzyme operates via ordered or random pathways of enzyme-substrate interactions. We now report new experimental evidence that demonstrates the earlier discrepant results may be attributed to the choice of reaction conditions used to achieve equilibration of the chemical reaction prior to addition of isotopic tracer. A random kinetic mechanism, perhaps not of the rapid-equilibrium type, is most compatible with the exchange data. We also discuss other potential time-dependent processes that may compromise the equilibration of reaction systems and affect the outcome of exchange experiments, and criteria for equilibration are suggested for the aid of other workers.

Adenosine Diphosphate↗
PubMed1982

A reinvestigation of dynein ATPase kinetics and the inhibitory action of vanadate.

The kinetic properties of sea urchin flagellar dynein ATPase have been reinvestigated using a continuous assay which regenerates ATP and contains P1,P5-di(adenosine-5')pentaphosphate, a potent adenylate kinase inhibitor. Earlier studies (Shimizu, T. (1981) Biochemistry 20, 4347-4354) revealed complex, highly cooperative kinetics with respect to MgATP2- concentration in the absence of this inhibitor. With Ap5A, the kinetics are characteristic of classical Michaelis-Menten enzymes. Isolated 21 S and 14S enzyme forms were also examined, and their kinetic parameters are presented Vanadate inhibition patterns in the presence of P1,P5-di(adenosine-5')pentaphosphate lose their nonlinear character, and we observe linear noncompetitive inhibition of the "mixed" type.

Adenosine Triphosphatases↗