scholarly journals Identification of ubiquitous high-molecular-mass, heat-stable microtubule-associated proteins (MAPs) that are related to the Drosophila 205-kDa MAP but are not related to the mammalian MAP-4.

1992 ◽  
Vol 89 (16) ◽  
pp. 7693-7697 ◽  
Author(s):  
M. Kimble ◽  
A. L. Khodjakov ◽  
R. Kuriyama
Keyword(s):  
Molecular Mass ◽  
High Molecular Mass ◽  
Microtubule Associated Proteins ◽  
Heat Stable ◽  
Associated Proteins

A high molecular mass phosphoprotein defined by a novel monoclonal antibody is closely associated with the intermicrotubule cross bridges in the Trypanosoma brucei cytoskeleton

1992 ◽  
Vol 103 (3) ◽  
pp. 665-675 ◽  
Author(s):  
A. Woods ◽  
A.J. Baines ◽  
K. Gull
Keyword(s):  
Molecular Mass ◽  
Trypanosoma Brucei ◽  
Minor Component ◽  
Relative Molecular Mass ◽  
Microtubule Associated Proteins ◽  
Heat Stable ◽  
Cytoplasmic Face ◽  
Associated Proteins ◽  
Cross Bridges ◽  
Main Component

The main component of the cell body cytoskeleton of Trypanosoma brucei is the highly organised array of stable, subpellicular microtubules on the cytoplasmic face of the plasma membrane. Although several microtubule associated proteins (MAPs) have been shown to be associated with this array, the mechanisms by which individual microtubules interact with one another and with the membrane are still largely undetermined. In this study we have used the T. brucei cytoskeleton as a complex immunogen for the production of monoclonal antibodies to define novel cytoskeletal antigens. Screening by immunofluorescence enabled the selection of an antibody, WCB-1, which detects an antigen associated specifically with the subpellicular microtubules and not with the flagellum microtubules. The antigen (WCB210) was shown to have a relative molecular mass of 210,000 by western blotting. Immunogold studies showed the epitope to be located on the membrane-facing side of the subpellicular cage; it appears to be closely associated with the cross-bridges lying between the microtubules. Unlike many MAPs this protein was shown not to be heat stable and is predicted to be a roughly globular monomer. Even though WCB210 is a very minor component of the cytoskeleton it is heavily phosphorylated. It is possible that this protein is involved in regulation of the subpellicular microtubule crossbridges by interaction with other proteins.

Unusual properties of a cold-labile fraction of Atlantic cod (Gadus morhua) brain microtubules

1989 ◽  
Vol 67 (11-12) ◽  
pp. 791-800 ◽  
Author(s):  
E. Strömberg ◽  
L. Serrano ◽  
J. Avila ◽  
M. Wallin
Keyword(s):  
Molecular Mass ◽  
Gadus Morhua ◽  
Atlantic Cod ◽  
Bovine Brain ◽  
Microtubule Assembly ◽  
Microtubule Associated Proteins ◽  
Heat Stable ◽  
Labile Fraction ◽  
Associated Proteins ◽  
Brain Microtubules

A cold-labile fraction of microtubules with unusual properties was isolated from the brain of the Atlantic cod (Gadus morhua). The yield was low, approximately six times lower than that for bovine brain microtubules. This was mainly caused by the presence of a large amount of cold-stable microtubules, which were not broken down during the disassembly step in the temperature-dependent assembly–disassembly isolation procedure and were therefore lost. The isolated cold-labile cod microtubules contained usually only a low amount of microtubule-associated proteins (MAPs). Three high molecular mass proteins were found, of which one was recognized as MAP2. Cod MAP2 differed from mammalian brain MAP2; it was not heat stable and had a slightly higher molecular mass. In contrast to mammalian MAPs, MAP1 was not found in the cold-labile fraction of microtubules. A new heat-labile MAP of higher molecular mass (400 kilodaltons) was however present, as well as a heat-stable protein of slightly lower molecular mass than MAP2. These MAPs showed similar tubulin-binding characteristics as bovine brain MAPs, since they coassembled with taxol-assembled bovine brain microtubules consisting of pure bovine tubulin. In spite of the fact that Ca2+ bound equally to cod and porcine tubulins, it did not inhibit cod microtubule assembly even at high concentrations (> 1 mM). In contrast, rings, spirals, and macrotubules were formed. The results show that there are major differences between this fraction of cod microtubules and microtubules from mammalian brain.Key words: microtubules, microtubule-associated proteins, calcium, cod.

Modulation of microtubule dynamic instability in vivo by brain microtubule associated proteins

1995 ◽  
Vol 108 (4) ◽  
pp. 1679-1689 ◽  
Author(s):  
R. Dhamodharan ◽  
P. Wadsworth
Keyword(s):  
Dynamic Behavior ◽  
Dynamic Instability ◽  
Average Rate ◽  
Stable Maps ◽  
Microtubule Dynamic ◽  
Unit Distance ◽  
Microtubule Associated Proteins ◽  
Heat Stable ◽  
Associated Proteins

Heat-stable brain microtubule associated proteins (MAPs) and purified microtubule associated protein 2 (MAP-2) were microinjected into cultured BSC-1 cells which had been previously injected with rhodamine-labeled tubulin. The dynamic instability behavior of individual microtubules was then examined using low-light-level fluorescence microscopy and quantitative microtubule tracking methods. Both MAP preparations suppressed microtubule dynamics in vivo, by reducing the average rate and extent of both growing and shortening events. The average duration of growing events was not affected. When measured as events/unit time, heat-stable MAPs and MAP-2 did not significantly alter the frequency of rescue; the frequency of catastrophe was decreased approximately two-fold by heat-stable MAPs and MAP-2. When transition frequencies were calculated as events/unit distance, both MAP preparations increased the frequency of rescue, without altering the frequency of catastrophe. The percentage of total time spent in the phases of growth, shrink and pause was determined. Both MAP-2 and heat-stable MAPs decreased the percentage of time spent shortening, increased the percentage of time spent paused, and had no effect on percentage of time spent growing. Heat-stable MAPs increased the average pause duration, decreased the average number of events per minute per microtubule and increased the probability that a paused microtubule would switch to growing rather than shortening. The results demonstrate that addition of MAPs to living cells reduces the dynamic behavior of individual microtubules primarily by suppressing the magnitude of dynamic events and increasing the time spent in pause, where no change in the microtubule length can be detected. The results further suggest that the expression of MAPs directly contributes to cell type-specific microtubule dynamic behavior.

Immunoreactive forms of erythrocyte spectrin and ankyrin in brain

1982 ◽  
Vol 299 (1095) ◽  
pp. 301-312 ◽  
Keyword(s):  
Molecular Mass ◽  
Erythrocyte Membranes ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Molecular Masses ◽  
Low Ionic Strength ◽  
Polypeptide Chains ◽  
The Brain ◽  
Total Membrane ◽  
Rotary Shadowing

Polypeptides immunologically related to erythrocyte spectrin and ankyrin have been detected in brain. The cross-reacting proteins include soluble as well as membrane-associated forms. A class of soluble cross-reacting polypeptides have been identified as high molecular mass microtubule-associated proteins (MAPS). MAP1, a group of polypeptides of molecular mass ca . 370 kDa contains a component that cross-reacts with anti-ankyrin IgG. MAP2, a polypeptide of molecular mass 300 kDa cross-reacts with anti-spectrin IgG, with the shared antigenic sites localized to the α chain of spectrin. The functional basis for structural homology between MAP1 and ankyrin may involve association with tubulin, since erythrocyte ankyrin binds to microtubules polymerized from pure brain tubulin. Spectrin did not associate with microtubules, but does have in common with MAP2 the ability to bind to actin (Brenner & Korn 1979; Sattilaro et al . 1981) and the shape of a flexible rod as visualized by rotary shadowing (Shotton et al . 1979; Voter & Erickson 1981). Immunoreactive forms of spectrin and ankyrin are also present in membrane fractions. A homologue of spectrin which constitutes 3% of the total membrane protein has been purified from low ionic strength extracts of membranes. This protein contains two non-identical polypeptide chains of molecular masses of 260 and 265 kDa, binds to F-actin, and displaces binding of erythrocyte spectrin to erythrocyte membranes. The brain protein pas been visualized by rotary shadowing as an extended rod-like molecule 195 nm in length. These studies indicate that the organization of proteins in the membrane-cytoskeleton complex of erythrocytes has direct relevance to other types of cells, and suggest the existence of families of proteins related to spectrin and ankyrin.

scholarly journals Mechanical properties of brain tubulin and microtubules.

1988 ◽  
Vol 106 (4) ◽  
pp. 1205-1211 ◽  
Author(s):  
M Sato ◽  
W H Schwartz ◽  
S C Selden ◽  
T D Pollard
Keyword(s):  
Mechanical Properties ◽  
Viscoelastic Properties ◽  
Microtubule Assembly ◽  
Cross Link ◽  
Microtubule Associated Proteins ◽  
Heat Stable ◽  
Microtubule Protein ◽  
Associated Proteins ◽  
Steady Shearing ◽  
Brain Tubulin

We measured the elasticity and viscosity of brain tubulin solutions under various conditions with a cone and plate rheometer using both oscillatory and steady shearing modes. Microtubules composed of purified tubulin, purified tubulin with taxol and 3x cycled microtubule protein from pig, cow, and chicken behaved as mechanically indistinguishable viscoelastic materials. Microtubules composed of pure tubulin and heat stable microtubule-associated proteins were also similar but did not recover their mechanical properties after shearing like other samples, even after 60 min. All of the other microtubule samples were more rigid after flow orientation, suggesting that the mechanical properties of anisotropic arrays of microtubules may be substantially greater than those of randomly arranged microtubules. These experiments confirm that MAPs do not cross link microtubules. Surprisingly, under conditions where microtubule assembly is strongly inhibited (either 5 degrees or at 37 degrees C with colchicine or Ca++) tubulin was mechanically indistinguishable from microtubules at 10-20 microM concentration. By electron microscopy and ultracentrifugation these samples were devoid of microtubules or other obvious structures. However, these mechanical data are strong evidence that tubulin will spontaneously assemble into alternate structures (aggregates) in nonpolymerizing conditions. Because unpolymerized tubulin is found in significant quantities in the cytoplasm, it may contribute significantly to the viscoelastic properties of cytoplasm, especially at low deformation rates.

Haemophilus paragallinarumsecretes metalloproteases

2005 ◽  
Vol 51 (10) ◽  
pp. 893-896 ◽  
Author(s):  
P C Rivero-García ◽  
C Vázquez Cruz ◽  
P Sánchez Alonso ◽  
S Vaca ◽  
E Negrete-Abascal
Keyword(s):  
Molecular Mass ◽  
Proteolytic Activity ◽  
Culture Media ◽  
High Molecular Mass ◽  
Secreted Proteins ◽  
Extracellular Proteases ◽  
Heat Stable ◽  
Infectious Coryza ◽  
Ph Range ◽  
Polyclonal Serum

Haemophilus paragallinarum secretes metalloproteases into different culture media lacking serum. Secreted proteins, concentrated by precipitation with 70% ammonium sulphate ((NH4)2SO4) or methanol, displayed proteolytic activity at >100 kDa molecular mass in 10% polyacrylamide gels co-polymerized with porcine gelatin (0.1%). They were active in a broad pH range (4–9); pH 7.5 being the optimum. Protease activity was inhibited by 20 mmol EDTA/L and reactivated by calcium. The proteolytic activity was heat-stable at 40, 50, and 60 °C, but its activity diminished at 70 °C or higher. Secreted proteins partially degraded chicken immunoglobulin G (IgG) and cross-reacted with a polyclonal serum against a high molecular mass protease secreted by Actinobacillus pleuropneumoniae. Extracellular proteases could play a role in infectious coryza caused by H. paragallinarum.Key words: pathogenicity, secreted protein, infectious coryza.

scholarly journals MAP 1C is a microtubule-activated ATPase which translocates microtubules in vitro and has dynein-like properties.

1987 ◽  
Vol 105 (3) ◽  
pp. 1273-1282 ◽  
Author(s):  
B M Paschal ◽  
H S Shpetner ◽  
R B Vallee
Keyword(s):  
Atpase Activity ◽  
Heavy Chain ◽  
Sedimentation Coefficient ◽  
Minor Component ◽  
High Molecular Mass ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Axonemal Dynein ◽  
A Minor

We observe that one of the high molecular mass microtubule-associated proteins (MAPs) from brain exhibits nucleotide-dependent binding to microtubules. We identify the protein as MAP IC, which was previously described in this laboratory as a minor component of standard microtubule preparations (Bloom, G.S., T. Schoenfeld, and R.B. Vallee, 1984, J. Cell Biol., 98:320-330). We find that MAP 1C is enriched in microtubules prepared in the absence of nucleotide. Kinesin is also found in these preparations, but can be specifically extracted with GTP. A fraction highly enriched in MAP 1C can be prepared by subsequent extraction of the microtubules with ATP. Two activities cofractionate with MAP 1C upon further purification, a microtubule-activated ATPase activity and a microtubule-translocating activity. These activities indicate a role for the protein in cytoplasmic motility. MAP 1C coelectrophoreses with the beta heavy chain of Chlamydomonas flagellar dynein, and has a sedimentation coefficient of 20S. Exposure to ultraviolet light in the presence of vanadate and ATP results in the production of two large fragments of MAP 1C. These characteristics suggest that MAP 1C may be a cytoplasmic analogue of axonemal dynein.

scholarly journals The epididymal soluble prion protein forms a high-molecular-mass complex in association with hydrophobic proteins

2005 ◽  
Vol 392 (1) ◽  
pp. 211-219 ◽  
Author(s):  
Heath Ecroyd ◽  
Maya Belghazi ◽  
Jean-Louis Dacheux ◽  
Jean-Luc Gatti
Keyword(s):  
Molecular Mass ◽  
Prion Protein ◽  
Hydrophobic Interactions ◽  
Protein Complexes ◽  
Biochemical Properties ◽  
High Molecular Mass ◽  
Soluble Form ◽  
Disulphide Bonds ◽  
Apolipoprotein J ◽  
Associated Proteins

We have shown previously that a ‘soluble’ form of PrP (prion protein), not associated with membranous vesicles, exists in the male reproductive fluid [Ecroyd, Sarradin, Dacheux and Gatti (2004) Biol. Reprod. 71, 993–1001]. Attempts to purify this ‘soluble’ PrP indicated that it behaves like a high-molecular-mass complex of more than 350 kDa and always co-purified with the same set of proteins. The main associated proteins were sequenced by MS and were found to match to clusterin (apolipoprotein J), BPI (bacterial permeability-increasing protein), carboxylesterase-like urinary excreted protein (cauxin), β-mannosidase and β-galactosidase. Immunoblotting and enzymatic assay confirmed the presence of clusterin and a cauxin-like protein and showed that a 17 kDa hydrophobic epididymal protein was also associated with this complex. These associated proteins were not separated by a high ionic strength treatment but were by 2-mercaptoethanol, probably due to its action on reducing disulphide bonds that maintain the interaction of components of the complex. Our results suggest that the associated PrP retains its GPI (glycosylphosphatidylinositol) anchor, in contrast with brain-derived PrP, and that it is resistant to cleavage by phosphatidylinositol-specific phospholipase C. Based on these results, the identity of the associated proteins and the overall biochemical properties of this protein ensemble, we suggest that ‘soluble’ PrP can form protein complexes that are maintained by hydrophobic interactions, in a similar manner to lipoprotein vesicles or micellar complexes.

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