| 1. |
- Billger, Martin, et al.
(författare)
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Calpain processing of brain microtubules from the Atlantic cod, Gadus morhua.
- 1993
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Ingår i: Molecular and cellular biochemistry. - 0300-8177. ; 121:1, s. 85-92
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Tidskriftsartikel (refereegranskat)abstract
- Microtubules isolated from Atlantic cod (Gadus morhua) brains retained assembly competence and ultraculture, although treatment with rabbit calpain resulted in loss of MAPs. In addition, spirals and aberrant structures formed when calpain I was activated post assembly. No such effect was seen with calpain II. Soluble fractions from cod brain were found to contain proteolytic activity that could be blocked by exogenously added calpastatin. Calpain was also isolated from cod muscle tissue with 10 times less yield, compared to rabbit lung. On the basis of Ca(2+)-requirements for activation in the mM range, electrophoretic mobility, antigenicity and hydrophobicity, we conclude that the proteolytic activity was attributable to calpain II. There was no difference in effects of rabbit and cod calpain II on cod microtubule proteins, indicating that calpain is a conserved protein. Our results suggest that calpains might be involved in the Ca(2+)-dependent irreversible regulation of cod brain microtubules.
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| 2. |
- Billger, Martin, et al.
(författare)
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Microtubule-associated proteins-dependent colchicine stability of acetylated cold-labile brain microtubules from the Atlantic cod, Gadus morhua.
- 1991
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Ingår i: The Journal of cell biology. - 0021-9525. ; 113:2, s. 331-8
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Tidskriftsartikel (refereegranskat)abstract
- Assembly of brain microtubule proteins isolated from the Atlantic cod, Gadus morhua, was found to be much less sensitive to colchicine than assembly of bovine brain microtubules, which was completely inhibited by low colchicine concentrations (10 microM). The degree of disassembly by colchicine was also less for cod microtubules. The lack of colchicine effect was not caused by a lower affinity of colchicine to cod tubulin, as colchicine bound to cod tubulin with a dissociation constant, Kd, and a binding ratio close to that of bovine tubulin. Cod brain tubulin was highly acetylated and mainly detyrosinated, as opposed to bovine tubulin. When cod tubulin, purified by means of phosphocellulose chromatography, was assembled by addition of DMSO in the absence of microtubule-associated proteins (MAPs), the microtubules became sensitive to low concentrations of colchicine. They were, however, slightly more stable to disassembly, indicating that posttranslational modifications induce a somewhat increased stability to colchicine. The stability was mainly MAPs dependent, as it increased markedly in the presence of MAPs. The stability was not caused by an extremely large amount of cod MAPs, since there were slightly less MAPs in cod than in bovine microtubules. When "hybrid" microtubules were assembled from cod tubulin and bovine MAPs, these microtubules became less sensitive to colchicine. This was not a general effect of MAPs, since bovine MAPs did not induce a colchicine stability of microtubules assembled from bovine tubulin. We can therefore conclude that MAPs can induce colchicine stability of colchicine labile acetylated tubulin.
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| 3. |
- Fridén, B, et al.
(författare)
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Different assembly properties of cod, bovine, and rat brain microtubules.
- 1992
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Ingår i: Cell motility and the cytoskeleton. - : Wiley. - 0886-1544. ; 21:4, s. 305-12
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Tidskriftsartikel (refereegranskat)abstract
- Assembly properties of cod, bovine, and rat brain microtubules were compared. Estramustine phosphate, heparin, poly-L-aspartic acid, as well as NaCl, inhibited the assembly and disassembled both bovine and rat microtubules by inhibition of the binding between tubulin and MAPs. The assembly of cod brain microtubules was in contrast only marginally affected by these agents, in spite of a release of the MAPs. The results suggest that cod tubulin has a high intrinsic ability to assemble. This was confirmed by studies on phosphocellulose-purified cod tubulin, since the critical concentration for assembly was independent of the presence or absence of MAPs. The results show therefore that cod brain tubulin has, in contrast to bovine and rat brain tubulins, a high propensity to assembly under conditions which normally require the presence of MAPs. Even if cod MAPs, which have an unusual protein composition, were not needed for the assembly of cod microtubules, they were able to induce assembly of bovine brain tubulin. Both cod and bovine MAPs bound to cod microtubules, and bovine MAP1 and MAP2 bound to, and substituted at least the 400 kDa cod protein. This suggests that the tubulin-binding sites and the assembly-stimulatory ability of MAPs are common properties of MAPs from different species, independent of the tubulin assembly propensity.
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| 4. |
- Modig, C, et al.
(författare)
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Different stability of posttranslationally modified brain microtubules isolated from cold-temperate fish.
- 1994
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Ingår i: Molecular and cellular biochemistry. - 0300-8177. ; 130:2, s. 137-47
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Tidskriftsartikel (refereegranskat)abstract
- Microtubule proteins were isolated by a temperature-dependent assembly-disassembly method from brain tissue of for cold-temperature fish; one fresh water fish (Oncorhynchus mykiss), and three marine fish (Labrus berggylta, Zoarces viviparus and Gadus morhua). The alpha-tubulins from all four fish species were acetylated. The alpha-tubulins from the marine fish were composed of a mixture of tyrosinated and detyrosinated tubulin, while the fresh water fish tubulin only reacted with an antibody against detyrosinated tubulin. The isolated microtubules had a similar MAP composition. A 400 kD protein and a MAP2-like protein were found, but MAP1 was missing. All microtubules disassembled upon cooling to 0 degrees C. In spite of these common characteristics, the assembly of microtubules from Labrus berggylta was inhibited by colchicine and calcium, in contrast to the assembly of microtubules from Oncorhynchus mykiss and Zoarces viviparus. For the latter, colchicine was not completely inhibitory even at a concentration as high as 1 mM, and calcium induced the formation of both loosely and densely coiled ribbons. The effects of calcium and colchicine on microtubules from Oncorhynchus mykiss and Zoarces viviparus were modulated by either fish or cow MAPs, indicating that the effects are due to intrinsic properties of the fish tubulins and not the MAPs. In view of these findings, our results suggest that there is no correlation between colchicine sensitivity, inability of calcium to inhibit microtubule assembly, and acetylation and detyrosination.
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| 5. |
- Strömberg, E, et al.
(författare)
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Differences in the effect of Ca2+ on isolated microtubules from cod and cow brain.
- 1994
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Ingår i: Cell motility and the cytoskeleton. - : Wiley. - 0886-1544. ; 28:1, s. 59-68
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Tidskriftsartikel (refereegranskat)abstract
- Isolated microtubules from cod and cow brains were compared with respect to their response to calcium ions. The effect of Ca2+ on cod microtubules was found to be temperature dependent. In contrast to cow microtubules, cod microtubules assembled at 18 degrees C. At this temperature the assembly was inhibited by Ca2+ concentrations of 2 mM and higher. This was also found for cow microtubules at 37 degrees C. However, at 30 degrees C there was no effect of 2 mM Ca2+ of the amount of assembly or disassembly of cod microtubules consisting of only tubulin or of tubulin and microtubule-associated proteins (MAPs). The morphology was affected though, since some coiled ribbons formed from tubulin and MAPs. The calcium-binding calmodulin did not alter the effect of calcium on cod microtubules markedly. At higher Ca2+ concentrations (> 4 mM), coiled ribbons were formed from cod tubulin and MAPs, but mainly amorphous aggregates and very few coiled ribbons were formed from cod tubulin alone, indicating that the Ca2+ effect is modulated by cod MAPs. The modulatory effect of cod MAPs was however not species specific, since both cod and cow MAPs had the same effect on cod microtubules, in spite of a different protein composition. A MAP-dependent effect of Ca2+ was also found for cow microtubule proteins. The assembly of pure cow tubulin, as well as that of cow tubulin and MAPs, was inhibited by 2 mM Ca2+. In the presence of 10 and 20 mM Ca2+, pure cow tubulin formed amorphous aggregates, rings, and even paracrystals, while the assembly of cow tubulin and MAPs was inhibited. Our results suggest therefore that the effect of Ca2+ can be moderated by MAPs, but depends on intrinsic properties of the different tubulins.
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| 6. |
- Wallin, Margareta, 1952, et al.
(författare)
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Assembly of Atlantic cod (Gadus morhua) brain microtubules at different temperatures: dependency of microtubule-associated proteins is relative to temperature.
- 1993
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Ingår i: Archives of biochemistry and biophysics. - : Elsevier BV. - 0003-9861. ; 307:1, s. 200-5
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Tidskriftsartikel (refereegranskat)abstract
- Isolated cod (Gadus morhua) brain microtubules were found to have a broad temperature interval for assembly. In contrast to mammalian microtubules they assembled even at as low temperatures as 14 degrees C. Evidence was found that temperature alters the dependency of microtubule-associated proteins (MAPs) for assembly. The assembly was MAPs-dependent at low, but not at higher temperatures. Assembly at +18 degrees C was inhibited by both NaCl and estramustine phosphate. These compounds are well known to inhibit the binding of MAPs to tubulin. At higher temperatures there was no MAPs dependency for assembly, despite that MAPs bound to the microtubules. Cow MAPs had the same effect as cod MAPs, suggesting that despite differences in MAP composition, the effect is not caused by the unusual composition of cod MAPs. The results therefore suggest that these differences in MAPs dependency are due to intrinsic properties of cod tubulin or tubulin-to-tubulin interactions. Small temperature-induced conformational changes of tubulin and a slight enrichment of acetylated and detyrosinated tubulin in microtubules assembled at +30 degrees C as compared to +15 degrees C, were observed. The ability to alter the assembly stimulating effect of MAPs may be important for the cell to regulate microtubule dynamics and stability. In addition, changes in tubulin conformation and composition of tubulin isoforms may reflect adaptations for microtubule assembly at low temperatures.
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