Common and distinct tubulin binding sites for microtubule-associated proteins.

A specific binding assay was developed that monitors the interaction of 125I-labeled microtubule-associated proteins (MAPs) with tubulin or its fragments bound to nitrocellulose membrane. To identify the tubulin-binding domains for MAPs we have examined the binding of rat brain 125I-labeled MAP2 or 125I-labeled tau factors to 60 peptides derived from porcine alpha- and beta-tubulin. MAP2 and tau factors specifically interacted with two peptides derived from the carboxyl-terminal region of beta-tubulin, which are located between positions 392-445 and 416-445. In addition, there is a distinct tau-binding site at the amino-terminal region of alpha-tubulin. tau factors but not MAP2 displayed strong interaction with a peptide derived from the amino-terminal domain of alpha-tubulin between positions 1 and 75. To narrow down the location of the beta-tubulin binding site that is common to MAP2 and tau factors, we have synthesized five peptides that are homologous to the corresponding sequence from the porcine or rat carboxyl-terminal region. Binding studies with the synthetic peptides suggest that amino acid residues 434-440 of beta-tubulin are crucial for the interaction of MAP2 and tau factors.

Modulation of mRNA for microtubule-associated proteins during brain development.

The heterogeneity of tau microtubule-associated proteins from rat brain is developmentally determined. Newborn rat brain contains two tau polypeptides (tau 0) with somewhat different molecular weights than the five tau components associated with microtubules from 12-day-old brain (tau 12). tau 0 and tau 12 are immunologically related and crossreact with antibodies against tau 12 proteins. Enrichment of the tau mRNA was achieved by prior hybridization of unfractionated poly(A)-containing mRNA to cDNA preparations containing tubulin and actin sequences. The remaining unhybridized mRNA was further fractionated by electrophoresis on methylmercury hydroxide agarose gels. Experiments involving cell-free translation of mRNA indicated that the major differences in the composition of tau proteins from newborn and developing brain are controlled at the mRNA level. The mRNA from newborn rat brain directed the synthesis of five tau proteins, two of which are specific for newborn brain, whereas the other three forms are characteristic of the developing brain. Thus, the appearance in newborn brain of mRNA species specific for three tau 12 forms precedes the phase of the synthesis of these proteins in the cell. By contrast, mRNA from 12-day brain directed the synthesis of four tau proteins specific for the developing brain, one of which is not synthesized by mRNA from newborn brain. None of the newborn tau 0 forms were synthesized with mRNA isolated from 12-day brain.

Functionally impaired tRNA from ethionine treated rats as detected in injected Xenopus oocytes.

Treatment of rats with ethionine was found to cause severe impairment in the aminoacylation capacity of tRNA. This effect was only observed when assayed in injected oocytes, while invitro assays of aminoacylation failed to detect differences between normal tRNA and tRNA from ethionine treated animals. The effect of ethionine on the tRNA population was not uniform and differed for various amino acid specific tRNAs. Thus liver tRNA from ethionine treated rats showed a decreased capacity for phenylalanine aminoacylation, while no change was found in the case of leucine. On the other hand, the level of histidine aminoacylation was higher for tRNA from ethionine treated animals. An even more complex response was observed with methionine aminoacylation where tRNA from ethionine treated animals showed an initially faster rate than control tRNA. With more prolonged incubation periods, the methionyl-tRNA from ethionine treated animals was deacylated at an accelerated rate while the level of normal methionyl-tRNA remained almost constant. In addition to the aminoacylation reaction, the participation of aminoacyl-tRNA in protein synthesis was severely impaired. In this case, both the injected oocyte system and the cell-free wheat germ assay revealed these differences which were manifested with various mRNA and viral RNA preparations.

Abundance of tRNAPhe lacking the peroxy Y-base in mouse neuroblastoma.

Affinity chromatography on anti-Y (Y is a tricyclic imidazopurine to which is attached a complex four-carbon side chain) antibody immobilized to Sepharose was used to determine the proportion of rat liver tRNAPhe species containing the peroxy Y-nucleoside. Unfractionated Unfractionated mammalian tRNA was aminoacylated with labeled phenylalanine. The phenylalanyl-tRNA was then chemically acetylated to yield N-acetylphenylalanyl-tRNA. When this preparation was applied to the antibody column, between 6-10% of the radioactivity was not bound to the column, indicating a deficiency of peroxy Y-nuceloside in a minor isoaccepting tRNAPhe species. In contrast to normal tissues (including embryonic tissue), about 85% of the tRNAPhe from mouse neuroblastoma C-1300 or N-18 tumors lack the peroxy Y-base, a property which is not affected by tumor age. Rat liver labeled N-acetylphenylalanyl-tRNA preparations were resolved on Plaskon chromatography (RPC-5) into two minor peaks closely followed by a mojor component. A high proportion of the two minor tRNAPhe species was unable to bind to anti-Y antibodies. Upon mild acid treatment, the minor and major tRNAPhe species eluted simultaneously from Plaskon columns, at a much reduced salt concentration. These results would indicate that the two minor tRNAPhe species from rat liver as well as the major component contain a tricyclic imidazopurine base that differs from each other in its side chain. About 85% of the N-acetylphenylalanyl-tRNA from neuroblastoma was resolved by Plaskon chromatography as an early eluting peak. The position of this major neuroblastoma tRNAPhe species was not altered by mild acid treatment, and its elution position from the column almost coincides with that of acid-treated normal rat liver tRNAPhe. The latter results would suggest that most of the tRNAPhe chains from neuroblastoma lack the tricyclic imidazopurine of normal rat liver tRNAPhe, but are very close if not identical in primary nucleotide sequence.