Calcium sensitivity of sea urchin tubulin in in vitro assembly and the effects of calcium-dependent regulator (CDR) proteins isolated from sea urchin eggs and porcine brains

Abstract

Tubulin was purified from unfertilized eggs or embryos of the sea urchin. In vitro assembly of sea urchin tubulin into microtubules (MTs) was highly sensitive to Ca2+ ions. At low ionic strength, the self-assembly was inhibited by 5 x 10(-7) M free Ca2+, and MT elongation which was initiated by mixing sea urchin tubulin with Tetrahymena cilia outer fiber fragments was inhibited by 10(-6)--10(-5) M free Ca2+. Increase in the ionic strength of the assembly medium lowered the Ca2+ sensitivity, in contrast to brain MT assembly. Brain microtubule-associated proteins (MAPs), which stimulated the sea urchin MT assembly, lowered the Ca2+ sensitivity. Calcium-dependent regulator (CDR) protein was purified from unfertilized eggs of the sea urchin by the pruification procedures used for brain CDR. The sea urchin CDR associated with brain tubulin in a Ca2+-dependent manner indistinguishable from that of brain CDR when assayed by the ammonium sulfate fractionation method. Moreover, both CDRs had Ca2+-dependent inhibitory effects on brain MT assembly. However, neither procine brain CDR nor sea urchin CDR showed a Ca2+-dependent inhibitory effect on sea urchin MT assembly. Sea urchin tubulin assembly initiated by brain MAPs was not inhibited by these CDRs with Ca2+, either. These results suggest that CDRs are not species-specific, but that tubulins respond in a highly specific manner.