Isolation and partial characterization of a tubulin-like protein from human and swine synaptosomal membranes

Kornguth, Steven; Sunderland, E.

doi:10.1016/0005-2795(75)90220-2

Cited by 51 publications

(22 citation statements)

References 35 publications

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“…It is possible that some of the unaccounted-for enhanced labeling represents membrane-bound tubulin of post-crush retina. Reports that a substantial fraction of brain tubulin is particulate (21) and that some tubulin-like protein may be found in nerve endings (22) have recently been confirmed and extended (23,24), with tubulin tentatively being localized at the synaptic junction (25) and post-synaptic density (26).…”

Section: Discussionmentioning

confidence: 99%

Enhanced labeling of a retinal protein during regeneration of optic nerve in goldfish.

Heacock¹,

Agranoff²

1976

Proc. Natl. Acad. Sci. U.S.A.

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Goldfish retinas were examined for changes in the labeling pattern of protein during regeneration of retinal ganglion cell axons following unilateral optic nerve crush. At various times after optic nerve crush the normal retinas were incubated in vitro with [3H] Following intra-orbital crush of the optic nerve, in goldfish, the retinal ganglion cell must grow out a new axon several millimeters long. This process not only implies a substantial increase in the rate of protein synthesis but may also involve a selective increase in the synthesis of the proteins necessary for the renewal of axoplasm and replacement of structural elements of the axon. Formation of cell-specific markers involved in retinotectal recognition may be increased~during this period (10). There might, in addition, be an initial decrease in the synthesis of components necessary to maintain the functional integrity of the nerve ending until the growing nerve reaches the optic tectum (11).This report presents comparisons of the in vitro protein labeling pattern of normal goldfish retina with that of retinas removed at various times after optic nerve crush (postcrush retina). A double-labeling technique coupled with sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis (12) revealed an increased amino-acid incorporation into a protein fraction with properties of the microtubule subunit, tubulin. MATERIALS AND METHODSGoldfish (Carassius auratus), 6-7 cm in body length, were anesthetized with tricaine methane sulfonate prior to an intraorbital crush of the right optic nerve. The left optic nerve remained intact so that the left retina served as a control. Following surgery, the goldfish were stored in groups of 50 to 80 in 30 gallon tanks at 20-22°, and were fed daily.Fish were dark-adapted for 30 min prior to removal of retinas in order to facilitate separation of the retina from the pigment epithelium. After hemisection of the eye, the retina was floated free from the pigment layer with 0.86% saline and detached by cutting at the optic disk. The retinas were then rinsed and stored in ice-cold saline or incubation medium for up to 1 hr prior to incubation.In were evaporated to dryness under nitrogen immediately before use. After evaporation, each was resuspended in a small volume of incubation medium. The purity of each isotopic preparation was checked by paper chromatography. The incubation medium was that of Dunlop et al. (13): N-2-bydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes), 25 mM; MgSO4, 1.3 mM; CaCl2, 2.6 mM; K2HPO4, 1.2 mM; KCI, 5.9 mM; NaCl, 106.5 mM; glucose, 12 mM; and NaOH to pH 7.4.Retinas (in groups of 5 to 20) were pre-incubated for 2 min at 25°in 1.9 ml of incubation medium. Labeled precursor (0.1 ml) was then added (1.0-4.0 ,uCi per retina) and incubation was continued in air in a Dubnoff metabolic shaker at 250. All incubations using double-labeled methionine were carried out for 1 hr. The reaction was stopped by dilution with 2 volumes of ice-cold incubation medium containing 2 mM L-methionine. After c...

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Section: Discussionmentioning

confidence: 99%

Enhanced labeling of a retinal protein during regeneration of optic nerve in goldfish.

Heacock¹,

Agranoff²

1976

Proc. Natl. Acad. Sci. U.S.A.

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“…Brain tissue is composed primarily of membranous components, which contain many proteins that function as structural elements, enzymes, and receptors. The analysis of protein in CNS membrane fractions has provided evidence for the presence of tubulin in synaptic membranes (Feit and Barondes, 1970;Lagnado et al, 1971; Blitz and Fine, 1974; Kornguth and Sunderland, 1975; Walters and Matus, 1975; Wang and Mahler, 1976;Estridge, 1977; Kelly and Cotman, 1978;Rostas et al, 1979; Gozes and Littauer, 1979; Babitch and Benavides, 1979;Zisapel et al, 1980). In these studies tubulin was identified by various techniques, including colchicine binding, one-dimensional gel electrophoresis, peptide maps of proteins from one-dimensional gels, and two-dimensional gel electrophoresis (2DGE).…”

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confidence: 99%

The Characterization of Tubulin in CNS Membrane Fractions

Strocchi

Brown

Young

et al. 1981

Journal of Neurochemistry

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Rough endoplasmic reticulum (RER), smooth endoplasmic reticulum (SER), and a plasma membrane (PM) fraction enriched in synaptic membranes were isolated from rat forebrain. The proteins in these membrane fractions were analyzed by two-dimensional gel electrophoresis (2DGE) in the isoelectric range of 5.1 to 6.0 by a modification of the O'Farrell procedure. Proteins were detected by Coomassie Brilliant Blue staining of the electrophoretograms. The results of these analyses were compared with 2DGE analysis of cytosol proteins, with particular attention given to tubulin subunits and actin. The RER contained one major protein (53K 5.4) in the beta-tubulin region with a molecular weight of 53,000 and an isoelectric point of 5.4. The SER contained at least two major proteins in the alpha-tubulin region; one with a migration identical to 53K 5.4 and other proteins with slightly higher apparent molecular weights and more acidic isoelectric points (54K, 5.4 to 5.3), identical to cytoplasmic beta-tubulin. The PM fraction also contained multiple overlapping proteins (54K, 5.4 to 5.3) in the beta-tubulin area and a trace amount of the 53K 5.4 protein. The proteins in the beta-tubulin region were removed from the 2DGE electrophoretogram and digested by Staphylococcus aureus V8 protease, and the peptides separated on one-dimensional polyacrylamide gels. The peptide patterns of 53K 5.4 protein from RER and SER were almost identical and differed significantly from the cytoplasmic beta-tubulin pattern; however, the peptide maps of the PM and SER beta-tubulin region were identical to the cytoplasmic beta-tubulin. The 2DGE analysis of RER did not contain proteins in the region of cytoplasmic alpha-tubulin. SER and PM contained proteins in the alpha-tubulin region with a similar, but not identical, peptide analysis to cytoplasmic alpha-tubulin. Significant amounts of actin were detected in 2DGE analysis of SER and PM, and the peptide analysis of the actin was identical to the cytoplasmic actin analysis. The RER fraction contained only trace amounts of actin. The cytosol and all membrane fractions contained a protein (68K 5.6) found among microtubule-associated proteins, as judged by molecular weight and isoelectric point. Several proteins present in all membrane fractions (61K 5.1 and 58K 5.1) bound to concanavalin A agarose.

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“…On the other hand, there is increasing evidence for a direct association of tubulin (the major subunit component of microtubules) with membranes. For example, there is specific colchicine binding to isolated membrane fractions from brain (6, 10); and tubulin has been identified in synaptic vesicles by polyacrylamide gel electrophoresis (7, 11,19), by reaction with specific antibodies (33), and recently by analysis on two-dimensional electropheretograms and by tryptic peptide mapping (17). Stephens' (30) analyses of membranes of the scallop have also supported the existence of membrane-bound tubulin: it was clearly shown that tubulin is associated with ciliary but not flageUar membranes.…”

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Interaction of microtubule proteins with phospholipid vesicles.

Caron¹,

Berlin²

1979

The Journal of Cell Biology

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We have examined the interaction of unilamellar dimyristoyl phosphatidylcholine liposomes with the high-speed supernate of brain homogenate and with tubulin purified through one or two cycles of microtubule assembly-disassembly. Tubulin and certain high molecular weight proteins are selectively adsorbed from these mixtures onto liposomes. The composition of adsorbed proteins is similar to that obtained during corresponding cycles of microtubule assembly, suggesting the equivalency of these processes. Adsorption induces stacking and/or fusion of liposomes into multilamellar structures indicating strong protein-lipid interaction. In addition, liposome-adsorbed tubulin forms extensive intermolecular disulfide bridges that are inert to reducing agents in the aqueous medium. The observations form a basis for further study of the distribution, function, and properties of membrane-bound tubulin. KEY WORDS tubulin membrane disulfides phospholipid microtubules liposomesPharmacological evidence has implicated microtubules in the control of a wide range of surface phenomena. For example, microtubule disassembly by colchicine enables the movement of surfacebound concanavalin A into caps in leukocytes (1, 35) and prevents the segregation of membrane proteins (26, 32) and probably lipids (4) that normally accompanies phagocytosis in neutrophils. In addition, studies ofneutrophils from mice with the Chedak-Higashi syndrome show that failure of microtubule assembly causes colchicine-like effects but in the absence of drugs (25). These results indicate that microtubule assembly is required to control and direct the distribution of membrane components.We have suggested that microtubule regulation of surface events may occur indirectly through control of the microfflament-contractile system (5). On the other hand, there is increasing evidence for a direct association of tubulin (the major subunit component of microtubules) with membranes. For example, there is specific colchicine binding to isolated membrane fractions from brain (6, 10); and tubulin has been identified in synaptic vesicles by polyacrylamide gel electrophoresis (7, 11,19), by reaction with specific antibodies (33), and recently by analysis on two-dimensional electropheretograms and by tryptic peptide mapping (17). Stephens' (30) analyses of membranes of the scallop have also supported the existence of membrane-bound tubulin: it was clearly shown that tubulin is associated with ciliary but not flageUar membranes. Finally, previous in vitro studies in our laboratory demonstrated fluorescence resonance energy transfer between fluorescein-labeled neutrophil membranes and rhodamine-labeled brain tubulin (2). At 37~ a high degree of transfer indicated close approximation of the membrane and tubulin. At 0~ transfer did not occur.While these studies indicate the presence of tubulin in isolated membrane fractions, they indicate neither the molecular nor functional basis of the tubulin-membrane interaction. In particular, a major difficulty in establishing the molecu...

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Isolation and partial characterization of a tubulin-like protein from human and swine synaptosomal membranes

Cited by 51 publications

References 35 publications

Enhanced labeling of a retinal protein during regeneration of optic nerve in goldfish.

Enhanced labeling of a retinal protein during regeneration of optic nerve in goldfish.

The Characterization of Tubulin in CNS Membrane Fractions

Interaction of microtubule proteins with phospholipid vesicles.

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