Ca(2+)-calmodulin regulated effectors of microtubule stability in bovine brain.
Stable microtubules (as defined by resistance to Ca2+, drug or cold temperature induced disassembly) form in abundance during tubulin assembly in brain crude extracts. We have previously shown that, in rat brain crude extracts, all microtubule stabilizing activity could be ascribed to a single Ca(2+)-calmodulin binding and Ca(2+)-calmodulin regulated protein, called "stable tubule only polypeptide", STOP145 [Pirollet, F., Rauch, C. T., Job, D., & Margolis, R. L. (1989) Biochemistry 28, 835-842]. We have now performed an exhaustive study of STOP-like effectors in bovine brain high-speed supernatants. All activity binds to cation exchangers and to Ca(2+)-calmodulin affinity columns. The activity can be resolved into two peaks on sizing columns. The first eluted peak contains a prominent 220-kDa protein. The second peak contains an apparently homogeneous 20-kDa polypeptide. A monoclonal antibody specific to rat brain STOP145 recognizes the 220-kDa protein, but not the 20-kDa species. The 220-kDa protein can be purified on a STOP antibody column and accounts for the bulk of stabilizing activity in the first peak. The 20-kDa protein does not bind to STOP antibody affinity columns. Sequence analysis of oligopeptide fragments of the 20-kDa protein shows 100% homology with bovine myelin basic protein (MBP). Anti-MBP antibodies recognize the 20-kDa, but not the 220-kDa species. We conclude that the 220-kDa protein is the bovine equivalent to rat brain STOP145 and that the 20-kDa species is MBP. Microtubule stabilization by MBP and STOP220 is abolished in the presence of Ca(2+)-calmodulin, and inhibition curves are similar for both proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
Pirollet
F
F
DBMS-CS, Inserm Unité 244, CENG, Grenoble, France.
Derancourt
J
J
Haiech
J
J
Job
D
D
Margolis
R L
RL
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000648:ultrastructure; D001923:Brain Chemistry; D002118:Calcium / Q000737:chemistry*; D002147:Calmodulin / Q000737:chemistry*; D002417:Cattle; D003080:Cold Temperature; D046911:Macromolecular Substances; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D008870:Microtubules / Q000737:chemistry*; D008969:Molecular Sequence Data; D004676:Myelin Basic Protein / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D011108:Polymers; D016415:Sequence Alignment; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism
D002147:Calmodulin; D046911:Macromolecular Substances; C113727:Map6 protein, rat; D008869:Microtubule-Associated Proteins; D004676:Myelin Basic Protein; D011108:Polymers; D014404:Tubulin; D002118:Calcium