Sulfhydryls and the in vitro polymerization of tubulin.

Abstract

The free sulfhydryls of brain tubulin prepared by cyclic polymerization procedures both with and without glycerol have been examined. The average free sulfhydryl titer of tubulin prepared with glycerol (7.0 sulfhydryls/55,000 mol wt) is greater than that of tubulin prepared without glycerol (4.0 sulfhydryls/55,000 mol wt). Diamide, a sulfhydryl-oxidizing agent, inhibits the polymerization of tubulin. Diamide also disperses the 20S and 30S oligomers of tubulin seen in analytical ultracentrifuge patterns of tubulin solutions and, depending on the temperature at which diamide is added, converts all or part of the oligomeric material to 6S dimers. Electron microscopy demonstrates that diamide also destroys the 450-/~, ring structures characteristic of tubulin solutions. All diamide effects are reversible by the addition of 10 mM dithioerythreitoi, a sulfhydryl-reducing agent. That diamide interacts with sulfhydryls on tubulin is directly demonstrated by a 50% decrease in the free sulfhydryl titer of tubulin measured after diamide treatment. Concentrations of CaC12 which inhibit polymerization also decrease the free sulfhydryl titer of tubulin.Since the in vivo assembly and disassembly of microtubules is a basic cellular process about which little is known, potential regulatory mechanisms involving modifications of tubulin such as phosphorylation (4), tyrosylation (1), and the presence or absence of carbohydrate (7) are of considerable interest. Modifications of the reactivity of sulfhydryl groups on tubulin similarly suggest themselves as interesting possibilities for control. Previous work on tubulin isolated from mammalian brain by ion exchange procedures has demonstrated that there are 8-11 half-cystines/55,000 monomer (4, 16) and that most of the half-cystine residues exist as easily titratable sulfhydryls in the native tubulin molecule. Eipper (4) has reported that in rat brain tubulin there are 11 half-cystine residues all accounted for by easily titratable sulfhydryls. However, Lee et al. (16) have shown in calf brain that while 8 of the 10 sulfhydryls detected by amino acid analysis exist as available sulfhydryls, the remaining two sulfurs are involved in an interchain disulfide bond. The differences in these two reports may result from differences in the techniques used to isolate tubulin.With the introduction of the in vitro polymerization conditions developed by Weisenberg (25), we can now investigate the sulfhydryls of polymerizable tubulin. We have determined average sulfhydryi titers of tubulin prepared with and without glycerol and we have also investigated the effects of diamide, a sulfhydryl-oxidizing agent, on tubulin polymerization. Diamide is a substituted diazene introduced by Kosower (10, 11) which stoichiometricaily oxidizes low molecular weight thiols to disulfides according to reactions 1 and 2 (10) and, in particular, rapidly converts reduced glutathione to oxidized glutathione. Diamide has recently been shown to inhibit cell division in sea

Mechanism of Destruction of Microtubule Structures by 4-Hydroxy-2-Nonenal

“…Tubulin has 20 cysteine and 24 histidine residues per dimer. Studies on residue-selective alkylation or oxidative modification have demonstrated that some of these residues are important for polymerization (Hosono et al, 2005;Kuriyama and Sakai, 1974;Mellon and Rebhun, 1976;Oliver et al, 1976;Rai and Wolff, 1998).…”

Microtubule assembly and conanavalin A capping in lymphocytes: reappraisal using normal and abnormal human peripheral blood cells.

“…(We assume, of course, that lymphocytes and PMN are equivalently permeable to colchicine.) In contrast with colchicine, microtubule disassembly caused by diamide is independent of the assembly/disassembly equilibrium (5,11,12) and so occurs with equal rapidity in both lymphocytes and PMN.…”

“…As described (5,8), the majority of PMN were also readily capped after exposure to 100uM diamide for 10 min. This glutathione-oxidizing agent is thought to cause microtubule disassembly and to inhibit microtubule assembly by causing oxidation of critical tubulin-SH residues (11,12).…”

Activation‐independent nuclear translocation of mitogen activated protein kinase ERK1 mediated by thiol‐modifying chemicals

“…4B). It is known that thiol-reactive compounds can inhibit microtubule polymerization in vitro by directly modifying tubulin at cysteins [29]. The actin cytoskeleton on the other hand was affected neither by PAO nor by mitogens (data not shown).…”