Subunit Protein Of Microtubules Research Articles

Identification of tubulin associated with rat brain myelin

During development, axons in both the central and peripheral nervous systems become ensheathed with myelin [ 11. Biochemical manifestations of myelination include rapid accumulation of the myelin membrane proteins [2,3]. These can be divided into three classes; the proteolipid proteins, the basic proteins and the Wolfgram proteins [3-61. The Wolfgram proteins are characterized as the high molecular weight proteins of the myelin fraction [4]. The Wolfgram proteins can be resolved electrophoretically into several polypeptides [2,3,7-91, two of which, WI and W2, have been characterized immunochemically [IO]. The_ myelin membrane is principally derived from oligodendroglia plasma membrane [5,6]. A major protein component which is synthesized by glial and neuronal cells is tubulin [ 1 l131, the subunit protein of microtubules [ 14,151. Tubulin can be associated with membranes [ 16181, and was shown to accumulate in the particulate fraction of brain with maturation [ 131, concomitantly with myelin formation [ 19 1. Together, these observations suggest the possibility that tubulin may be a component of the myelin membrane. By the criteria of SDS-polyacrylamide gel electrophoresis for molecular weight determination, peptide mapping and colchicine binding, we have now demonstrated that tubulin is a constituent of the high molecular weight portion of the myelin fraction.

Tubulin microheterogeneity increases with rat brain maturation.

MICROTUBULES are present in all eukaryotic cells and have been found to have a variety of structural and dynamic roles in cell shape, division, motility, transport and secretion1. In nervous tissue neurite outgrowth and axoplasmic transport are also thought to depend on microtubule integrity2. The micro-tubule subunit protein, tubulin, is a heterodimer composed of two polypeptides α and β (refs 3,4). The α and β subunits show microheterogeneity and both have been resolved into two or three components5–8. The question therefore arises as to whether changes occur in the relative proportions of the multiple forms of tubulin upon assumption of different roles within the nerve cell. We show here that cytoplasmic rat brain tubulin, as resolved by isoelectric focusing, is highly heterogeneous. Moreover, tubulin microheterogeneity seems to be developmentally determined, increasing from seven to nine distinct components during early postnatal rat brain maturation. However, extensive tubulin microheterogeneity is prominent in the brain, as tubulin from other organs is less heterogeneous.

The Subcellular Distribution of [3H]-Colchicine-Binding Activity and Tubulin in Pig Blood Platelets

The subcellular distribution of the [3H]-colchicine-binding protein, believed to be tubulin, the subunit protein of microtubules, has been investigated in mammalian blood platelets. Studies on a soluble extract from pig platelets and two particulate fractions (viz. membrane-rich and granule-rich fractions) have shown that about 98% of the colchicine-binding activity in a platelet homogenate is located in the soluble phase. This result is in agreement with polyacrylamide gel electrophoresis experiments which show that the soluble fraction contains a substantial amount of 55,000 MW tubulin, whereas the membrane and granule-rich fractions contain very little of this component. The [3H]-colchicine-binding activity of the platelet soluble phase is largely precipitated by 40-50% ammonium sulphate and also by vinblastine sulphate in millimolar concentrations. Moreover the colchicine-binding protein in the platelet soluble fraction has a sedimentation coefficient of 5.9 S, is eluted in the void volume of a Sephadex G-100 column, and binds to DEAE-Sephadex at low ionic strength and is eluted from this ion-exchanger at an ionic strength of 0.47 M-KCl. In addition, most of the colchicine-binding activity of the platelet soluble phase is associated with protein which will undergo temperature-dependent polymerization in vitro and which has a molecular weight on SDS-polyacrylamide gels of 55,000. All these experimental findings suggest that the colchicine-binding activity of pig platelet homogenates is due to the presence of the microtubule protein, tubulin, which is largely found in the soluble compartment of the cells.

The isolation and characterisation of platelet microtubule proteins

Tubulin, the microtubule subunit protein, has been isolated from a soluble extract of pig platelets, by an in vitro polymerisation process. Several physicochemical properties of platelet tubulin have been investigated and compared with those of mammalian brain tubulins. The molecular weight of the tubulin monomeric subunits was found to be 55 000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and the platelet protein co-migrated with rat, rabbit, guinea-pig, mouse and calf brain tubulins. The [ 3H]colchicine-binding dimer form of platelet tubulin sedimented in a 5% to 20% linear sucrose gradient with a sedimentation coefficient of 5.7 S, compared with that of 5.9 S for rat brain tubulin. The amino acid compositions of platelet and mammalian brain tubulins were found to be very similar, with glutamate and asparate as the predominant residues. Moreover, the one and two dimensional fingerprints of 125I-labelled tryptic peptides of platelet and brain tubulins showed considerable similarity. Platelet tubulin could be separated into two non-identical (α and β) subunits by Tris/glycine discontinuous alkaline sodium dodecyl sulphate-polyacrylamide gel electrophoresis and the β-tubulin had two or three 125I labelled peptides not seen in the α-tubulin. Two, or perhaps three, high-molecular-weight proteins were always present in the platelet tubulin samples, prepared by in vitro polymerisation and these had molecular weights greater than 200 000 and were probably analogous to the high-molecular weight proteins reported to be present in brain microtubule preparations. Platelet tubulin with its associated high molecular weight proteins will assemble in vitro into tubular structures which in electron micrographs resemble brain microtubules and the microtubules seen in whole platelets. It is believed that this is one of the first mammalian microtubule systems other than that of nervous tissue which has been isolated and studied in detail at the subunit level.

Polypeptides similar to the alpha and beta subunits of tubulin are exposed on the neuronal surface.

THE finding by Borisy and Taylor1 that nervous tissue was the source of large amounts of soluble tubulin, the subunit protein of microtubules, has greatly facilitated the biochemical study of microtubular structure. From 10% to 20% of the total protein of developing brain is in the form of soluble tubulin2,3 and most studies have focused on this form of the protein. In 1970, however, Feit and Barondes demonstrated a large amount of colchicine binding activity, a term which has become associated with the presence of tubulin, in particulate fractions of brain4—fractions expected to be free of microtubule-derived tubulin. Recent studies indicate that polypeptides with the same size and drug-binding properties as soluble tubulin are present in synaptosomal plasma membranes5–7. These studies suggest a membrane-associated form of the protein which is insoluble in non-chaotropic buffers. I here report experiments which provide additional evidence for membrane-associated tubulin-like proteins and also suggest that a portion of this polypeptide(s) may be exposed on the external surface of the cell.

Immunochemical determination of tubulin

Tubulin, the subunit protein of microtubules is found in all eukaryotic cells. Microtubules are functionally important for a wide variety of cellular activities such as mitosis, cell shaping, secretion and motility. They are also abundant in the nervous tissue where neurite outgrowth as well as axoplasmic transport are thought to be dependent on microtubules integrity [l] . Detection and quantitation of tubulin is, thus, of importance in trying to understand differentiation processes. It has recently been shown that during postnatal development of the rat brain, there is a decline in the rate of tubulin synthesis as compared to that of the total protein (2-5). The decrease in the relative amounts of this protein occurs in the soluble fraction and is accompanied by a comparable decline in the percentage of its mRNA (3-5). The relative amounts of rat brain [2,5] or chick brain [6] iubulin have been determined by colchicine binding as well as by electrophoretic resolution on polyacrylamide gels followed by densitometric scanning of the stained gels. An alternative method is the detection and quantitation of tubulin by specific anti-tubulin antibodies, which allows a more sensitive assay as tubulin determinations can be carried out also on membrane bound protein not necessarily in its functional form. Specific tubulin antibodies have been used mainly for visualization of the mitotic spindle, as well as of the microtubular network in the cell [7,8] by immunofluorescence

Regulation of Microtubules in Tetrahymena

The coupled resorption and redifferentiation of oral structures which occurs in Tetrahymena pyriformis under conditions of amino acid deprivation has been studied by transmission electron microscopy. Two patterns of ciliary resorption have been found, ( a ) in situ , and ( b ) after withdrawal into the cytoplasm. No autophagic vacuoles containing cilia or ciliary axonemes have been seen. Stomatogenic field basal bodies arise by a process of rapid sequential nucleation, with new ones always appearing next to more mature ones, even though the latter may not be fully differentiated. Accessory radial ribbons of microtubules develop immediately adjacent to oral field basal bodies as a late step in their maturation. It can be seen that the formation of basal bodies and their orientation within the oral complex are separate processes. This is true for about 130 of the approximately 170 oral basal bodies; the remaining 40 or so form within the patterned groups of ciliary units as a later event. Clusters of randomly oriented thin-walled microtubules are found surrounding oral basal bodies at all times during stomatogenesis. They may either represent stores of microtubule subunit protein, or serve as effectors of basal body movement during their orientation into pattern.

A new colchicine binding assay for tubulin

A simple and sensitive procedure is described for assaying tubulin, the subunit protein of microtubules. Tubulin is equilibrated with [ 3H]colchicine after which the remaining free colchicine is removed by adsorption to activated charcoal. Values for the amount of tubulin in various rat tissues have been determined by both the charcoal method and by gel filtration on Sephadex G-100, and in all cases the values determined by the two methods correspond closely.

Regulation of microtubules in Tetrahymena. I. Electron microscopy of oral replacement.

The coupled resorption and redifferentiation of oral structures which occurs in Tetrahymena pyriformis under conditions of amino acid deprivation has been studied by transmission electron microscopy. Two patterns of ciliary resorption have been found, (a) in situ, and (b) after withdrawal into the cytoplasm. No autophagic vacuoles containing cilia or ciliary axonemes have been seen. Stomatogenic field basal bodies arise by a process of rapid sequential nucleation, with new ones always appearing next to more mature ones, even though the latter may not be fully differentiated. Accessory radial ribbons of microtubules develop immediately adjacent to oral field basal bodies as a late step in their maturation. It can be seen that the formation of basal bodies and their orientation within the oral complex are separate processes. This is true for about 130 of the approximately 170 oral basal bodies; the remaining 40 or so form within the patterned groups of ciliary units as a later event. Clusters of randomly oriented thin-walled microtubules are found surrounding oral basal bodies at all times during stomatogenesis. They may either represent stores of microtubule subunit protein, or serve as effectors of basal body movement during their orientation into pattern.

Colchicine and the mitotic spindle of the aquatic phycomycete Allomyces.

Exposure of germlings of Allomyces neo-moniliformis to colchicine for 0 to 5 min after zoospore encystment was found to block 30% of germlings derived from flagellated zoospores and 55% of germlings derived from deflagellated zoospores in C-metaphase configurations at the first mitotic division. The zoospore lacks a pool of colchicine binding protein, and protein synthesis is absent during the time when colchicine first becomes effective in inducing C-metaphase. From these observations it is concluded that the microtubule subunit protein of the spindle apparatus of the first mitotic division to a large extent is derived from the depolymerization of the cytoplasmic microtubules of the zoospore. GTP, Mg2+, and ATP were observed to be antagonistic to the action of colchicine in vivo. It is suggested that these compounds may compete with colchicine for binding to the subunit protein in vivo. Germlings derived from flagellated zoospores are appreciably less subject to the action of colchicine in the presence of the antagonistic compounds than are germlings derived from deflagellated zoospores. This differential sensitivity to colchicine is interpreted as reflecting a difference in the quantity of microtubule subunit protein present at the time of exposure to colchicine.

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).KeywordsSynaptic VesicleSodium BorohydrideCyanogen BromideTubulin SubunitColchicine BindingThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

MICROTUBULE PROTEIN

The subunit protein of microtubules, tubulin, has been demonstrated to be present in isolated nerve endings by gel electrophoresis, amino acid composition, and peptide mapping. The tubulin constitutes approximately 28% of the soluble protein of the nerve endings. The transport of tubulin to the nerve endings has been demonstrated and its relationship to slow transport is discussed.

Heterogeneity of tubulin subunits.

Tubulin, the subunit protein of microtubules, is a dimer that sediments at 6 S and has a molecular weight of 110,000. Using high resolution polyacrylamide gel electrophoresis, we have demonstrated the presence of two peptide chains, of molecular weight 56,000 and 53,000, in tubulin purified from brain. Two peptide chains of similar molecular weight were identified in each of the A- and B-tubulins isolated from flagella of sea urchin sperm. In all cases, the protein concentrations in the bands were equal. Each of the subunits ran as a single band when eluted from the gel and electrophoresed again in the same type of gel. Chromatography of purified brain tubulin on DEAE-Sephadex columns gave only a single peak containing both subunits in equal amounts. Cyanogen bromide peptides were prepared from each of the bands after elution from polyacrylamide gel. While certain of the peptides appear to be common to both subunits, substantial differences exist between them. The tubulin dimer is composed of two nonidentical subunits.

Microfilaments from amoeba proteins

Summary Bundles of microfilaments have been obtained from a fraction of homogenised amoebae cells after the addition of EDTA. These bundles are made up of many single microfilaments 30–40 A in diameter. An attempt has been made to obtain the protein subunit of these microfilaments. In the detergent Sarkosyl, a subunit of sedimentation constant 2.0×10−13 and mol. wt approx. 42 500 has been found. The amino acid composition of this protein is similar to that of muscle actin, plasmodium actin and microtubule subunit protein. On removing the Sarkosyl, the protein reaggregates into bundles of microfilaments like those originally formed with EDTA.

Ultrastructural changes in skeletal muscle induced by colchicine.

COLCHICINE arrests mitotic division of many plant and animal cells by interfering with the formation of the mitotic spindle. 1-7 Cellular division is arrested at metaphase, bizarre and abnormal nuclear configurations occur, and the cell often dies. Colchicine also has a profound effect on nonmitotic differentiating cells. It induces the formation of cytoplasmic filaments in the interphase HeLa cells in vitro 8 and neurofibrillary tangles in tissue cultures of rat neurons. 9 Neurofibrillary change has recently been noted in the brainstem and spinal cord of rabbits after intracisternal injection of colchicine. 10 The variable effects of colchicine apparently depend on its binding to the subunit protein of microtubules. 11 Although colchicine consistently causes severe ascending paralysis in experimental animals, a thorough morphological study of skeletal muscle has not been undertaken. Ferguson 12 reported delayed paralysis of the hindlimbs in acute toxicity experiments in rats and prominent muscle atrophy in