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J Deinum

Publications and source records attributed to J Deinum.

At least 91 records · Page 5Linked to original sources
PubMed1984

Molecular weight dependency of heparin inhibition of microtubule assembly in vitro.

Low molar ratios of heparin inhibited in vitro assembly of bovine brain microtubule proteins and disassembled preformed microtubules. Addition of purified microtubule-associated proteins counteracted the assembly inhibition by heparin. Our results suggest that the polyanion heparin affects microtubule assembly by binding to the microtubule-associated proteins. This complex can not support nucleation or stabilize the microtubule structure although it still can associate with the tubulin polymer. In the presence of heparin, the critical concentration needed for microtubule assembly was increased. Furthermore, the absolute assembly difference induced by heparin, the delta A350, was only dependent on the concentration and the molecular weight of heparin, not of the total microtubule protein concentration, or the addition of microtubule-associated proteins. Commercial, standard heparin (Mr 6000-25 000) had an I50 of about 0.1/tubulin dimer. The heparin fraction(s) with a high molecular weight had a stronger effect than those with lower molecular weight. Substoichiometric amounts of taxol completely relieved the inhibition of assembly by heparin, although aberrant forms were present. These microtubules had a reduced amount of coassembled microtubule-associated proteins, and furthermore contained heparin.

Alkaloids↗
PubMed1983

The effect of S-100a and S-100b proteins and Zn2+ on the assembly of brain microtubule proteins in vitro.

The homologous proteins S-100a and S-100b affect the microtubule system in a distinctly different way in the presence of low molar ratios of Zn2+. Assembly of brain microtubule proteins can be almost completely inhibited and rapid disassembly can be induced by low molar amounts of S-100b in the presence of low molar ratios [2-4] of Zn2+. Higher molar ratios per S-100b (greater than 4) potentiate the general Zn2+ effect, promoting the formation of sheets of microtubules. However, the effect of S-100a is quite different, no inhibition of assembly can be observed and the presence of S-100a seems to protect the microtubule proteins against the effect of Zn2+ by chelating the Zn2+ and decreasing the free metal-ion concentration. S-100a or S-100b cannot bind to the microtubule polymer-form, either in the absence or in the presence of Zn2+.

Animals↗
PubMed1982

Effect of Cibacron blue on tubulin assembly in the absence and presence of microtubule-associated proteins.

Cibacron blue was found to inhibit assembly and increase the critical concentration of microtubule proteins. In the presence of 4 mol Cibacron blue/mol tubulin, assembly was completely inhibited and pre-formed microtubules disassembled. Addition of 8% (v/v) dimethylsulfoxide to Cibacron blue-inhibited samples induced assembly of normal microtubules in addition to sheets of protofilaments. Disassembly was induced upon addition of 1 mM colchicine or 2 mM Ca2+. No obvious difference was seen in the protein composition of these microtubules compared with controls. GTP exchange was not affected by the presence of Cibacron blue nor was GTP able to counteract its effect. This indicates that the exchangeable GTP site is not involved. The extent of assembly of phosphocellulose purified tubulin in the presence of 8% (v/v) dimethylsulfoxide was only slightly less in the presence of Cibacron blue, although the assembly rate was decreased. These results suggest that Cibacron blue might alter the binding of one or more of the associated proteins stimulating assembly.

Animals↗
PubMed1981

Spatial separation of the two essential thiol groups and the binding site of the exchangeable GTP in brain tubulin. A spin label study.

The assembly of microtubules from tubulin prepared without glycerol was inhibited by blocking the two most reactive sulfhydryl groups of the eight free sulfhydryl groups present per tubulin dimer. The assembly was also inhibited by Cu2+ ions in a redox-reaction with the two most reactive sulfhydryl groups. These two sulfhydryl groups had almost the same reactivity towards N-ethylmaleimide and p-chloromercuribenzoate, in spite of the fact that they are located on different subunits of tubulin. It was not possible to label just one single sulfhydryl group at a time by N-ethylmaleimide, and it was not possible to decide whether one or two free sulfhydryl group(s) are needed for assembly. The EPR technique based on the interaction of spin labels with transition metals was used for the study of the distance between the two most reactive sulfhydryl groups and the sites of exchangeable GTP and Mg2+, respectively. The sulfhydryl groups were spin labelled with a nitroxide derivative of N-ethylmaleimide. Cr(III)GTP was used as a paramagnetic substitute for GTP, and Mn2+ for Mg2+. It was found that: a. The exchange of GTP and the total content of GTP were not affected by modification of the sulfhydryl groups. b. The binding sites of the exchangeable GTP and Mg2+ are located 10 A, at least, from the two most reactive sulfhydryl groups. c. The distance between the spin labels introduced on the two most reactive sulfhydryl groups was larger than 17 A. The findings indicate that there is no direct interaction between exchangeable GTP and the two most reactive sulfhydryl groups.

Animals↗
PubMed1981

The effect of ruthenium red on the assembly and disassembly of microtubules and on rapid axonal transport.

The assembly of microtubules was found to decrease in proportion to the mount of added ruthenium red, indicating a high affinity of ruthenium red for the microtubule system. An equimolar amount of ruthenium red per tubulin dimer inhibited the microtubule assembly completely and disassembled existing microtubules. Binding of ruthenium red to tubulin is accompanied by a shift in the absorption maximum fro 535 to 538 nm. The binding is very strong, as shown by the finding that ruthenium red could not be displaced from tubulin by gel chromatography on Sephadex G-25, or by the addition of Ca2+ or Mg2+. The binding of ruthenium red to tubulin did not affect the single colchicine site, nor the Mg2+ site(s), as shown by use of Mn2+ as an EPr probe. Ruthenium red also interfered with microtubules in an intact cell system, as it inhibited rapid axonal transport in the frog sciatic nerve, measured by the accumulation of [3H]leucine-labelled proteins in front of a ligature.

Animals↗
PubMed1981

Synthesis and binding to tubulin of an allocolchicine spin probe.

A spin probe for the colchicine binding site on tubulin has been synthesized from allocolchicine. The probe is competitive to colchicine with an apparent inhibition constant of 11 microM while allocolchicine has an inhibition constant of 2 microM. Microtubule assembly is inhibited to 50% by 7 microM of the spin probe. As a mitotic poison the spin probe is less potent than colchicine. These data suggest that the probe binds to the same site on tubulin as colchicine and in spite of the somewhat lower efficiency, it seems to be a valuable tool for the study of the microtubule system.

Animals↗
PubMed1980

Metal analysis by energy dispersive x-ray fluorescence of bovine brain tubulin and microtubule-associated proteins prepared by phosphocellulose chromatography.

It has been shown by trace metal analysis that tubulin isolated from bovine brain does not contain strongly bound transition metal ions. The traces of zinc and iron found in the fraction of microtubule-associated proteins might originate from previously reported phosphatase activity (Larsson, H., Wallin, M. and Edström, A. (1979) J. Neurochem. 32, 155--161).

Animals↗
PubMed1978

The reaction of mercaptans with tyrosinases and hemocyanins.

1. Titration of Neurospora tyrosinase with 2-mercaptoethanol shows that the increase of absorbance at 700 nm is directly correlated to the loss of enzymatic activity. Approximately 2 mol of 2-mercaptoethanol per mole of protein are needed for full development of the green, enzymatically inactive complex. The increase of absorbance at 700 nm is also proportional to the intensity of the EPR signal and the amount of non-covalently bound 2-[35S] mercaptoethanol to the enzyme. The maximal EPR intensity reaches 70% of the protein concentration and at most 0.7--0.8 mol of 2-[35S] mercaptoethanol is bound per mol of enzyme. 2. Stopped-flow measurements show that in the reaction between 2-mercaptoethanol and Neurospora tyrosinase a raction intermediate with a strong absorption band at 360 nm is formed in an apparent second-order reaction. This intermediate displays no EPR-detectable signals. The intermediate decays in a similar complex fashion as the absorption band at 700 nm is formed. 3. The reaction of Neurospora tyrosinase with a variety of sulfhydryl compounds was also investigated. In most cases green coloured, enzymatically inactive complexes are formed displaying slightly different EPR signals. However, with cysteine and cysteamine violet coloured, enzymatically inactive complexes are formed which show rather different EPR signals. The integrated EPR intensities amount to 40--70% of the protein concentration. Based on simulations of 9 and 35 GHz spectra all observed EPR spectra can be represented as true S = 1/2 systems. The cysteamine complex can be interpreted as arising from a mixed valence Cu2+ . Cu+ complex. The 2-mercaptoethanol spectra can, however, arise from sulphur radicals. 4. Treatment of Agaricus bispora tyrosinase and Cancer pagures hemocyanin with 2-mercaptoethanol results in green-coloured, EPR detectable complexes similar to the one found with Neurospora tyrosinase. No such complexes are formed when hemocyanins from Helix pomatia and Panulirus interruptus were treated with this reagent.

Basidiomycota↗
PubMed1978

The state of copper in Neurospora laccase.

1. Neurospora crassa laccase has been prepared from the growth medium and studied by optical absorption, circular dichroism and electron paramagnetic resonance (EPR) spectroscopy. The molecular weight, the copper content and the amino acid composition have also been determined. 2. The molecular weight as determined by gel filtration in 6 M guanidine hydrochloride and by sodium dodecyl sulfate gel electrophoresis is found to be 64 000. The enzyme contains 3.8 copper ions per 64 000. 3. The visible and the near ultraviolet difference absorption spectrum shows two maxima, at 330 and 595 nm, and a shoulder at about 720 nm. The circular dichroism spectrum between 300 and 760 nm contains five bands in the oxidized enzyme. After reduction of the enzyme with ascorbate there remains only a band at 305 nm. 4. EPR measurements show that 52% of the total copper in the protein is paramagnetic. Two EPR signals of equal intensity with different hyperfine splitting constants, of 9 and 18.5 mT, are present, which are assigned to Type 1 Cu2+ and Type 2 Cu2+, respectively, as found in other blue copper-containing oxidases.

Amino Acids↗