scholarly journals Intracellular localization of the high molecular weight microtubule accessory protein by indirect immunofluorescence.

1978 ◽  
Vol 76 (3) ◽  
pp. 781-786 ◽  
Author(s):  
J A Connolly ◽  
V I Kalnins ◽  
D W Cleveland ◽  
M W Kirschner
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Indirect Immunofluorescence ◽  
Tau Protein ◽  
Intracellular Localization ◽  
Double Diffusion ◽  
Specific Staining ◽  
Intact Cells ◽  
Dividing Cells ◽  
Brain Microtubules

Microtubule accessory proteins were isolated from porcine brain microtubules by phosphocellulose chromatography, and the high molecular weight protein (HMW protein), purified from this microtubule-associated fraction by electrophoretic elution from SDS gels, was used to raise antisera in rabbits. In agarose double diffusion tests, the antiserum obtained forms precipitin lines with purified HMW protein but not with tau protein or tubulin. When rat glial cells (strain C6) are examined by indirect immunofluorescence, this serum specifically stains a colchicine-sensitive filamentous cytoplasmic network in interphase cells, a network indistinguishable from that seen when cells are treated with antitubulin serum. In dividing cells, specific staining of the mitotic spindle and the stem body is observed with the antiserum to HMW protein. These studies indicate that HMW protein, like tau protein, is associated with microtubules in intact cells.

A probe for flagellar dynein in the mammalian mitotic apparatus

1981 ◽  
Vol 48 (1) ◽  
pp. 241-257
Author(s):  
G.W. Zieve ◽  
J.R. NcIntosh
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Indirect Immunofluorescence ◽  
Mitotic Spindle ◽  
Cultured Cells ◽  
Structural Studies ◽  
Mitotic Apparatus ◽  
Specific Staining ◽  
Bovine Sperm ◽  
Sperm Proteins

An anti-serum has been prepared in rabbits that precipitates high-molecular-weight bovine sperm proteins, including the dyneins. The activity of the serum against the dyneins is demonstrated by the recognition of dynein polypeptides in stained electrophoretic profiles of sperm proteins and in immunoprecipitates of radiolabelled sperm proteins. In addition, the serum stains the sperm flagella when used in indirect immunofluorescence and quantitatively inhibits the motility of demembranated sperm reactivated with ATP. However, the serum has additional anti-sperm activities besides those directed against the dyneins as demonstrated by the staining of the acrosome in indirect immunofluorescence. When used to immunoprecipitate proteins from extracts of cultured cells, the serum precipitates 2 polypeptides; one has a molecular weight higher than the flagellar dyneins, one lower. No specific staining of cultured cells is observed when an affinity-purified anti-dynein fraction IgG is used to stain a variety of cultured cells including bovine fibroblasts. We interpret these data to suggest that flagellar dynein is not a component of the mammalian mitotic spindle and discuss how this conclusion is consistent with recent genetic and structural studies on the mitotic spindle.

scholarly journals A theory relating the action of salts on bacterial respiration to their influence on the solubility of proteins

1947 ◽  
Vol 134 (875) ◽  
pp. 181-201 ◽  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Dehydrogenase Activity ◽  
Halophilic Bacteria ◽  
Convincing Evidence ◽  
Effect Of Temperature ◽  
Bacterial Respiration ◽  
Concentrated Solutions ◽  
Salting Out ◽  
Intact Cells

Evidence has been presented indicating that the action of concentrated solutions of salts on bacterial respiration may be partly explained in terms of salting-out. It has been suggested that the material upon which this action is exerted is probably one of the proteins concerned in respiration, perhaps a dehydrogenating enzyme. This theory provides satisfactory explanations for: ( a ) the relation between salt con­centration and rate of respiration or dehydrogenase activity; ( b ) the effect of temperature on this relation; and ( c ) the effect of pH on this relation, if it is further supposed that only the zwitterionic fraction of the protein is involved. The relative actions of various salts are in fair agreement with this suggestion, but provide no very convincing evidence either for or against it. The chief point of difficulty lies in the range of concentration over which the action is manifest. With halophilic bacteria, the evidence is consonant with the above view if the protein involved is one of high molecular weight. With normal organisms the salt concentra­tions are much lower than those causing salting-out. There is a little evidence that in normal organisms the dehydrogenating enzymes are less sensitive to salts than the intact cells, which may be the source of the discrepancy. No reason for this can yet be suggested, but the property must be absent from the enzymes of halophilic organisms, and whatever it is, its absence must be the foundation of the halophilic character.

scholarly journals Arrangement of high molecular weight associated proteins on purified mammalian brain microtubules.

1977 ◽  
Vol 72 (3) ◽  
pp. 642-654 ◽  
Author(s):  
L A Amos
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Mammalian Brain ◽  
Lateral Interactions ◽  
High Molecular Weight Protein ◽  
Protein Molecules ◽  
Associated Proteins ◽  
Weight Protein ◽  
High Molecular Weight Proteins ◽  
Brain Microtubules

The arrangement of the high molecular weight proteins associated with the walls of reconstituted mammalian brain microtubules has been investigated by electron microscopy of negatively stained preparations. The images are found to be consistent with an arrangement whereby the high molecular weight molecules are spaced 12 tubulin dimers apart, i.e., 960 A, along each protofilament of the microtubule, in agreement with the relative stoichiometry of tubulin and high molecular weight protein. Molecules on neighbouring protofilaments seem to be staggered so that they give rise to a helical superlattice, which can be superimposed on the underlying tubulin lattice. In micrographs of disintegrating tubules there is some indication of lateral interactions between neighbouring high molecular weight molecules. When the microtubules are depolymerized into a mixture of short spirals and rings, the high molecular weight proteins appear to remain attached to their respective protofilaments.

scholarly journals Isolation and characterization of a large, neurite-associated glycoconjugate from neuroblastoma cells

1983 ◽  
Vol 96 (3) ◽  
pp. 661-668 ◽  
Author(s):  
EAG Chernoff ◽  
GA Maresh ◽  
LA Culp
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cells ◽  
Cell Fraction ◽  
Serum Starvation ◽  
Affinity Column ◽  
Human Neuroblastoma ◽  
Intact Cells ◽  
Isolation And Characterization

A high molecular weight glycoconjugate has been isolated from neurite-producing neuronal tumor cells in culture and has been designated as I(0) based on its elution characteristics in gel filtration chromatography. This molecule cannot be found in a variety of nonneuronal cells. I(0) is found in the substratum-attached material or cell fraction of neurite-producing neuroblastoma cells, depending upon culture conditions. It is found in the substratum-bound fraction of B104 rat neuroblastoma cells during serum starvation and in the EGTA-detached cell fraction of B104 cells grown in chemically defined N2 medium. It occurs only in the cell fraction of the human neuroblastoma line Platt. Examination of behavioral variants of the B104 rat line further strengthens the association of I(0) with neurite production; the constitutive neurite-producing E(R)B9 variant contains I(0) while the non-neurite-producing E(R)A11 variant does not. I(0) is large, eluting in the void volume of sepharose-CL2B columns. Radioiodination of intact cells with lactoperoxidase shows I(0) to be a cell surface component. Metabolic radiolabeling studies show that it contains a high proportion of polysaccharide to protein, does not contain mannose, and is unsulfated. Alkaline borohydride reduction release two size classes of large polysaccharide chain. The alkaline reduction results, along with the mannose incorporation studies, show the presence of O-glycosidic linkages and few, if any, N-linkages. Resistance to nitrous acid deamination, insensitivity to glycosaminoglycan lyases, and the absence of sulfation, indicate that I(0) does not contain the glycosaminoglycans hyaluronic acid, chondroitin-, dermatan-, or heparin- sulfates. Affinity column chromatography reveals high binding affinity of I(0) to polyornithine and no binding to gelatin (collagen) or the glycosaminoglycans hyaluronate and heparin. These studies describe a unique high molecular weight glycoconjugate on the surface of neurite-producing neuroblastoma cell lines from two species.

scholarly journals ISOLATION AND PARTIAL CHARACTERIZATION OF MACRO- AND MICRONUCLEI FROM PARAMECIUM AURELIA

1972 ◽  
Vol 53 (1) ◽  
pp. 105-115 ◽  
Author(s):  
Donald J. Cummings
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Calcium Ions ◽  
Gel Electrophoresis ◽  
Partial Characterization ◽  
Intact Cells ◽  
Molecular Weights ◽  
High Molecular Weight Species ◽  
Nonionic Detergents

A method was developed for the isolation of macro- and micronuclei from Paramecium aurelia. This method utilized ionic and nonionic detergents to rupture the intact cells, calcium ions and spermidine were employed to protect the nuclei, and the nuclei were purified by centrifugation. Macronuclei consisted of 22% DNA, 10% RNA, and 68% protein. Micronuclei were composed of 9% DNA, 11% RNA, and 80% protein. DNA from both macro- and micronuclei had a density of 1.687 g/cc in CsCl and 1.417 g/cc in Cs2SO4. These values corresponded to G + C content of about 23%. The RNA of macronuclei was examined by gel electrophoresis, and two high molecular weight species were identified having molecular Weights of 1.3 x 106 and 2.8 x 106 daltons. Three syngens were studied, and in each case the conditions for isolation of the nuclei were the same and no differences were observed in the properties of the nuclei.

scholarly journals Effect of tau on the vinblastine-induced aggregation of tubulin.

1981 ◽  
Vol 89 (3) ◽  
pp. 680-683 ◽  
Author(s):  
R F Ludueña ◽  
A Fellous ◽  
J Francon ◽  
J Nunez ◽  
L McManus
Keyword(s):  
Molecular Weight ◽  
Rat Brain ◽  
High Molecular Weight ◽  
Microtubule Assembly ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Spiral Structures ◽  
The Individual ◽  
Brain Microtubules ◽  
Induced Aggregation

Two microtubule-associated proteins, tau and the high molecular weight microtubule-associated protein 2 (MAP 2), were purified from rat brain microtubules. Addition of either protein to pure tubulin caused microtubule assembly. In the presence of tau and 10 microM vinblastine, tubulin aggregated into spiral structures. If tau was absent, or replaced by MAP 2, little aggregation occurred in the presence of vinblastine. Thus, vinblastine may be a useful probe in elucidating the individual roles of tau and MAP 2 in microtubule assembly.

High molecular weight tau: preferential localization in the peripheral nervous system

1991 ◽  
Vol 100 (1) ◽  
pp. 55-60 ◽  
Author(s):  
I.S. Georgieff ◽  
R.K. Liem ◽  
W. Mellado ◽  
J. Nunez ◽  
M.L. Shelanski
Keyword(s):  
Spinal Cord ◽  
Molecular Weight ◽  
Nervous System ◽  
High Molecular Weight ◽  
Peripheral Nervous System ◽  
Tau Protein ◽  
Protein A ◽  
Amino Terminal ◽  
Neuronal Tissues ◽  
Associated Proteins

Using epitope mapping we have demonstrated that a high molecular weight protein (Mr approximately 115 × 10(3)) present in brain and spinal cord is a member of the tau family of microtubule-associated proteins. Antibodies directed against the amino-terminal, middle and carboxyl-terminal portions of tau recognize this protein. A limited survey of neuronal tissues has shown that this high molecular weight tau protein is present in brain, spinal cord, dorsal root ganglia, dorsal and ventral roots and peripheral nerves. High molecular weight tau protein is expressed at higher levels in spinal cord than in brain and is the only form of tau detected in the adult peripheral nervous system.

scholarly journals Ultrastructural localization of the high molecular weight proteins associated with in vitro-assembled brain microtubules

1975 ◽  
Vol 65 (1) ◽  
pp. 237-241 ◽  
Author(s):  
WL Dentler ◽  
S Granett ◽  
JL Rosenbaum
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Ultrastructural Localization ◽  
Thin Sections ◽  
Microtubule Associated Proteins ◽  
Brain Extracts ◽  
Associated Proteins ◽  
High Molecular Weight Proteins ◽  
Brain Microtubules

Microtubules isolated from brain extracts by in vitro assembly (1, 19, 23) are composed principally of two tubulins and two high molecular weight proteins (microtubule-associated proteins [MAPS] 1 and 2) (2,5,7,20). Recently, it was demonstrated that in vitro-assembled brain microtubules (neurotubules) are coated with filaments (5, 7) which are similar to the filaments attached to neurotubules in situ (4, 15, 21, 24, 25), and it was suggested that the filaments are composed of the higher molecular weight MAPs (5, 7, 12). In this study, microtubules were assembled in the presence and absence of the MAPs, and thin sections of the microtubules were examined by electron microscopy. The results show that the filaments only occur on microtubules assembled in the presence of the MAPs and it is therefore concluded that the filaments are composed of the high molecular weight MAP's.

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