Isoforms of soluble alpha-tubulin in oocytes and brain of the frog (genus Rana): changes during oocyte maturation.

Abstract

Rana oocytes have previously been shown to contain much more soluble tubulin than does the brain, suggesting different assembly and disassembly dynamics of frog oocyte tubulin compared to that in brain. By using centrifugation, SDS-PAGE, two-dimensional gel electrophoresis and Western blots, probed with anti-alpha-tubulin monoclonal antibodies, polymorphic alpha-tubulins (isoforms) were compared in brains and follicle-enclosed oocytes of northern (Rana pipiens) and southern (R. berlandieri) frogs. Oocyte tubulin in both species had isoforms with greater ranges of isoelectric point (pI) than those of brain tubulins; in particular, the oocyte tubulin pIs ranged further into the acidic region of the isoelectric-focusing gels than corresponding brain tubulin. This difference may, in part, be responsible for the previously reported assembly differences between oocyte tubulin (undetectable assembly) and brain tubulin (high assembly). Isoforms of alpha-tubulin with relatively acidic pI were more abundant in northern frog brain and oocyte soluble extracts than in analogous extracts from southern frogs. Furthermore, additional acidic alpha-tubulin isoforms were found in progesterone-treated oocytes (i.e., eggs), indicating increased heterogeneity of acidic alpha-tubulin isoforms during oocyte meiotic maturation. Among northern frog oocyte soluble components fractionated on Superose-6b columns, tubulin complexes with apparent molecular mass of about 1800 kDa were found to contain acidic alpha-tubulin isoforms while the putative oligomeric tubulins with an apparent molecular mass of about 250 kDa contained an additional relatively basic alpha-tubulin isoform. The acidic alpha-tubulin isoforms, therefore, are proposed to be associated with cold-adaptable cells of brain and oocytes, and may also be involved in stabilization of large soluble tubulin complexes in oocytes of the northern frog.