Michael L. Shelanski

Vinblastine-Induced Precipitation of Microtubule Protein

Vinblastine quantitatively precipitates a protein from supernatants obtained from high-speed centrifugation of homogenates of HeLa cells and of pig brain. This protein migrates as a single band on gel electrophoresis, has a mobility identical to that of purified microtubule protein, and-like microtubule protein-binds colchicine. The precipitation is partially inhibited by 0.9 percent NaCl.

Isolation of Filaments from Brain

A method is presented for the isolation of filaments of 90-angstrom diameter from the white matter of bovine brain by first floating the myelinated axons in a centrifugal field and then fractionating the axons on a series of density gradients. This results in a fraction that contains two types of bundles of filaments but few other constituents. The filaments are stable over a wide range of temperatures and at both low and high ionic strength. Their density and their resistance to digestion by ribonuclease and deoxyribonuclease indicate that they are primarily protein. The molecular weight of the subunit is approximately 60,000. The protein does not comigrate with microtubule protein and does not bind cholcicine or nucleotides.

Induction in vitro of Microtubular Crystals by Vinca Alkaloids

The addition of vinblastine or vincristine to solutions of pure microtubule protein or to supernatants from high-speed centrifugation of rabbit-brain homogenates results in the formation of a fine precipitate. Examination of this precipitate by electron microscopy reveals ordered structures with areas of ladder-like configuration.

Is tubulin a glycoprotein

[14C]Glucosamine is incorporated in vivo in mouse brain into the major protein species present in purified tubulin preparations when analyzed both by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by isoelectric focusing. The radioactivity incorporated into tubulin can be recovered as a mixture of glucosamine and galactosamine.

Observations on axoplasmic transport in rabbits with aluminum-induced neurofibrillary tangles

We have studied axoplasmic transport in rabbits as it is affected by aluminum-induced neurofibrillary degeneration. The analyses were performed 8–15 days after AlCl3 administration except for one animal in which 30 days elapsed. Morphological changes were examined parallel to transport measurement. The anterograde fast axoplasmic component in the appropriate nerve was measured using [3H]leucine injected in the anterior horn of the L-7 level of the spinal cord and/or spinal ganglion at the same level. In both aluminum-treated and control animals, the rate of axoplasmic transport was similar at about 440 mm/day, and there was no correlation between this rate and the intensity of morphologic alteration. The effect of aluminum-induced neurofibrillary degeneration on retrograde axon transport was studied using Evans blue-albumin injected into the tongue. In all the controls and in one-half of the aluminum-treated rabbits, there was an accumulation of fluorescent granules in the perikarya of the XII cranial nucleus. In the rest of the experimental animals, fluorescent granules did not appear in the relevant perikarya.

Some features of the vinblastine-induced assembly of porcine tubulin.

Abstract Porcine tubulin precipitated by 10 −3 , m vinblastine (VLB) contains approximately 0.50 molecule of VLB bound per 110,000-molecular-weight tubulin dimer. The amount of precipitate, followed by turbidity, is a linear function of the initial tubulin concentration. The rate of precipitation is roughly first order in protein concentration. Vindoline and velbanamine halves of VLB are ineffective separately or together in producing the tubular aggregates observed for VLB precipitates by electron microscopy. At 10 −3 , m concentrations no turbidity is observed nor is there any competition with VLB-induced turbidity. Removal of GTP from tubulin by dialysis or incubation of tubulin in the absence of added GTP blocks VLB-induced assembly. Readdition of GTP at room temperature or above restores sensitivity to VLB precipitation. The β,γ methylene analog of GTP cannot substitute for GTP in this process. About 0.7 mol of added GTP is found bound per mole of tubulin dimer. During the course of VLB-induced assembly, roughly half of this GTP is displaced. These results show interesting similarities and differences in the VLB-induced assembly of tubulin and the normal in vitro assembly of microtubules. Further comparisons between both assembly processes should be useful.

Some Biochemical Aspects of Neurotubule and Neurofilament Proteins

In the few years since the isolation of the subunit protein of microtubules (1, 2, 23, 26) work on its chemistry, localization and metabolism has proceeded at a feverish pace. In their initial studies Taylor and his co-workers noted exceptionally high levels of colchicine-binding activity in brain homogenates and in axoplasm. Weisenberg, Borisy and Taylor (31) developed a simple procedure for the purification of the microtubule subunits (tubulin) from brain enabling investigators to use large quantities of highly purified protein for biochemical studies. Studies on metabolism and distribution of tubulin in brain were aided by the observation that vinblastine quantitatively and selectively precipitated tubulin from 100,000 g supernatants of brain homogenates (17, 18, 32).