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Biomedical subjects

M Wallin

Publications and source records attributed to M Wallin.

At least 91 records · Page 5Linked to original sources
PubMed1985

Effect of estramustine phosphate on the assembly of isolated bovine brain microtubules and fast axonal transport in the frog sciatic nerve.

Estramustine phosphate (0.01 to 0.5 mM), an estradiol mustard derivative used in the therapy of prostatic carcinoma, inhibited the assembly of brain microtubules proteins in vitro and disassembled preformed microtubules. In the presence of estramustine phosphate, the minimum microtubule-protein concentration sufficient for the assembly of microtubules was increased. Low concentrations of taxol (20 microM) completely reversed the inhibition of assembly by estramustine phosphate. The effects were specific to estramustine phosphate since neither estradiol 17 beta-phosphate, the hormonal moiety of the drug, nor nornitrogen mustard, the alkylating moiety, had any effect on assembly. Estramustine phosphate (0.1 to 0.5 mM) was also found to reversibly inhibit fast axonal transport in the frog sciatic nerve. The nerve content of adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate was not significantly affected by estramustine phosphate. Our results suggest that the cytotoxic action of estramustine phosphate could be dependent partially on an interaction with microtubules, probably via the microtubule-associated proteins.

Animals↗
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↗
PubMed1983

Characterization of acid and alkaline phosphatase activity in preparations of tubulin and microtubule-associated proteins.

Acid and alkaline phosphatase activity, determined by the hydrolysis of p-nitrophenyl phosphate, was found in preparations of microtubules purified from bovine brain by temperature-dependent assembly-disassembly and ion-exchange chromatography. Phosphocellulose-purified tubulin contained an associated acid phosphatase activity, stimulated by Mg2+ and by Zn2+. Alkaline phosphatase activity with a pH optimum of 10.4 was measured in a fraction of microtubule-associated proteins (MAPs). Kinetics and the effects of sodium fluoride, sodium tartrate, sulfhydryl-blocking agents, EDTA and Zn2+ are reported.

Acid Phosphatase↗
PubMed1983

The ATPase activity in brain microtubule preparations is membrane-associated.

Microtubule protein prepared from bovine brain by a temperature-dependent assembly-disassembly procedure contained Mg2+- or CA2+-stimulated ATPase activity. However, activity decreased with successive cycles of assembly-disassembly such tht 15% of the Mg2+-stimulated and 31% of the Ca2+-stimulated activity of the second-cycle material remained after seven cycles. Microtubule preparations purified by three cycles of assembly-disassembly contained many membrane fragments and vesicles which were absent in microtubule preparations cycled eight times. Histochemistry and electron microscopy revealed that much of the activity is associated with the vesicles. Vesicles with an accumulation of led phosphate deposits (indication of ATPase activity) were observed in high-speed pellets (150,000 g, 60 min) of microtubule-associated proteins. Most of the activity in the microtubule-associated protein preparations, but only a fraction of the total protein is pelleted. 53-78% of the ATPase activity, but only 6% of the total protein, is recovered in a microtubule-associated protein fraction eluted from phosphocellulose with 0.17 M NaCl. Polypeptides resolved on SDS polyacrylamide gradient gels have estimated molecular weights of 30,000-76,000. Electron micrographs of this material revealed short filaments, vesicles, and small ring-like structures. None of the inhibitors of possible contaminating ATPases affected the ATPase activity.

Adenosine Triphosphatases↗
PubMed1983

Characterization of microtubules isolated from dogfish (Squalus acanthias and Scyliorhinus canicula) brain in the absence of glycerol.

Microtubules were isolated by two cycles of assembly-disassembly from dogfish brain in the absence of glycerol. The microtubule protein preparation consist mainly of tubulin as characterized by SDS-polyacrylamide gel electrophoresis, but tau and high molecular weight microtubule associated proteins (MAPs) were also identified, in addition to several other MAPs. The microtubules were cold-sensitive and had an assembly temperature optimum at 21-25 degrees C. Ca2+, at a concentration of 1 mM or higher, induced spirals of several protofilaments, sheets and "macrotubules". In the presence of colchicine (0.1-1.0 mM) spirals as well as microtubules were seen. These structures were often found to be clustered.

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↗
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↗
PubMed1980

Ca2+- or Mg2+-dependent enzymatic ATP hydrolysis associated with the microsomal fraction of frog sciatic nerves.

The microsomal fraction of frog sciatic nerves was found to contain Ca2+- or Mg2+-dependent hydrolytic activity toward different nucleoside di- and triphosphates. In the presence of Ca2+ substrate specificity was in the order CTP > UTP > GTP > ATP. When Mg2+ was used, the triphosphates were approximately equally good substrates. ATP hydrolytic activity was very similar with Ca2+ or Mg2+ as the cofactor, whereas Ca2+ was the more potent activator of hydrolysis of the other triphosphates tested. The preparation showed some activity toward the nucleoside diphosphates but none toward the monophosphates or p-nitrophenylphosphate. The enzymic properties of ATP hydrolysis were more closely studied. The hydrolysis was optimal at 18--24 degrees C in the presence of 1 mM-Ca2+ or 1 mM-Mg2+. Ca2+- and Mg2+-ATP hydrolysis displayed pH maxima around 8.0--8.5 and 7.4--8.0, respectively. Vmax values for Ca2+- and Mg2+-ATP hydrolysis similar: approx. 12 mumol Pi per h per mg protein with a Km value of approx. 0.05 mM. The ATP hydrolysis activity was inhibited by NaF but unaffected by ouabain, vanadate, cytochalasin B, and various drugs known to influence ATPase activity of mitochondria. Zn2+ stimulated the ATP hydrolysis activity at low concentrations (10(-6)-10(-5) M) and inhibited it at higher concentrations. The possibility that these observations account for stimulation and inhibition of axonal transport in frog sciatic nerves exposed to similar concentrations of Zn2+ is discussed.

Adenosine Triphosphate↗
PubMed1979

Differences in microtubule stability to colchicine in extracts of guinea pig, rat and rabbit brain.

1. Microtubules (MT) from a guinea pig brain 25,000 g supernatant are not depolymerized by colchicine in contrast to MT from similar preparations of rat and rabbit. 2. The colchicine-stability was lost if the guinea pig brain homogenate was centrifuged at a higher g-level, further purified or if only the grey matter was used. 3. The association constant of colchicine to tubulin did not differ between a stable and a labile guinea pig brain preparation. 4. The GTP-hydrolysis was higher in the guinea pig preparation containing stable MT, than in the preparation containing labile MT. Additional GTP added to the polymerized MT before colchicine exposure, labilized the MT. Preincubation with NaF decreased the GTP-hydrolysis and caused a colchicine depolymerization. 5. The results indicate species differences in colchicine sensitivity of in vitro polymerized MT, probably depending on differences in GTP-hydrolysis.

Animals↗
PubMed1979

Inhibition of fast axonal transport and microtubule polymerization in vitro by colchicine and colchiceine.

The effects of colchicine and colchiceine on fast axonal transport in frog sciatic nerves were studied in vitro. Colchiceine inhibited the transport to about the same extent as colchicine. Preincubation at low temperature potentiated the inhibitory effect of either drug. The polymerization of purified brain tubulin was inhibited by colchiceine at 5-10 times higher concentrations than colchicine. The similarity of the effects obtained with colchicine and colchiceine indicates that both drugs arrest axonal transport by interfering with microtubule function. Colchicine and colchiceine did not affect the levels of high energy phosphates (ATP and CrP) in frog nerves indicating that a reduced energy supply was not responsible for the arrested transport.

Adenosine Triphosphate↗