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
Long chain glycols augment in size and birefringence the in vivo mitotic apparatus (MA) oE marine eggs. Dinitrophenol and caffeine antagonize the effect but they can be balanced by glycols. Caffeine inhibits the phosphodiesterase for cyclic AMP (CAMP) and CAMP levels increase in its presence. However, added dlbutyryl CAMP does not affect MAS o r cleavage, but is taken up by eggs. Oxygen uptake studies show that caffeine depresses oxidative metabolism but does not affect ATP levels. Action through the pentosephosphate shunt is suggested.Glycols influence the assembly of tubulin. Optical ultracentrifuge patterns of tubulin polymerized without glycol show a 6s and 30s peak. Similar patterns of tubulin polymerized at pH 6.4 in glycol and depolymerized in its absence show 6S, 8-18S, and 30S, peaks. The 8-18s peak appears in equilibrium with the 6s peak. If glycol is added t o cold tubulin polymerized without glycol, only 6s and 30s peaks occur. Preparations with no 30s peak d o not show 450 A rings in the EM.Calcium depolymerizes microtubules. In the absence of glycols 450 a rings are seen.In the presence of glycol, much higher concentrations of calcium are necessary for depolymerization, and few 450 8, rings occur.We suggest that glycols prevent formation of the stable 30s peak, favor an intermediate structure in equilibrium with the 6s peak, and antagonize calcium depolymerization. Their in vivo effects may arise from these interactions.
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