Search PubMed⌕ Search

Biomedical subjects

C I Rivas

Publications and source records attributed to C I Rivas.

24 records · Page 2Linked to original sources
PubMed1988

Purification, amino terminal analysis, and peptide mapping of proteins after in situ postelectrophoretic fluorescent labeling.

Proteins fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were stained in situ with either 5-(dimethylamino)-1-naphthalene sulfonyl chloride (dansyl chloride) or fluorescein isothiocyanate. This staining procedure can be carried out in less than 30 min without previous fixation of the proteins. It is not dependent on such factors as charge or molecular weight of the proteins and can detect 50 ng of protein in a 10-mm-wide gel slot. Fluorescent staining with dansyl chloride was used to localize proteins after electrophoresis for subsequent electroelution, amino terminal analysis, and peptide mapping. The electroelution can be carried out in less than 3 h with yields approaching 100%. The staining of only one strip of a preparative gel allowed the electroelution of proteins without covalent modification. For amino terminal analysis, identical results were obtained when the hydrolysis step was carried out after electroelution or directly in the gel pieces. The peptide mapping can be carried out with the proteins in solution (after electroelution) or directly in the gel pieces. The amino terminal and peptide mapping analysis of each protein in a mixture can be completed within 30 h from the beginning of the electrophoretic fractionation. The method appears to be applicable to a wide range of proteins showing very different biochemical properties.

Amino Acids↗
PubMed1988

Anti-idiotypic antibodies that react with microtubule-associated proteins are present in the sera of rabbits immunized with synthetic peptides from tubulin's regulatory domain.

A fundamental question in microtubule research is how the interactions of tubulin subunits with microtubule-associated proteins (MAPs) are controlled. The answer should provide insight into the regulation of the cellular processes in which microtubules are implicated. Previous work demonstrated the interaction of MAPs with a 4-kDa C-terminal domain of tubulin alpha and beta subunits. Synthetic peptides from the variable region of the 4-kDa C-terminal moiety of tubulin subunits, alpha-(430-441) and beta-(422-434), bind to MAP-2 and to the MAP tau, and a preferential interaction of the beta peptide is observed. To define the regulatory significance of the substructure of the C-terminal tubulin domain, we produced rabbit antisera against these MAP-interacting peptides. We found that these antisera contained not only antibodies to the original synthetic peptides but also antibodies to MAPs. Here, we report that these antibodies, which react with MAP-1, MAP-2, and tau, appear to be a population of anti-idiotypic antibodies directed to the anti-peptide antibodies. They can inhibit MAP-induced tubulin assembly into microtubules in vitro, and the addition of MAPs overcomes the inhibition. The recognition by these anti-idiotypic antibodies of the tubulin-binding domain on MAPs provides unequivocal evidence that the tubulin region defined by the synthetic peptides is directly involved in the interaction with MAPs.

Animals↗
PubMed1988

Antibodies to synthetic peptides from the tubulin regulatory domain interact with tubulin and microtubules.

The carboxyl-terminal region of tubulin alpha and beta subunits plays a major role in regulating its assembly into microtubules and constitutes an essential domain for the selective interaction of microtubule-associated proteins (MAPs). With the goal of understanding the structural basis of the regulatory function of the carboxyl-terminal domains of tubulin subunits, we have produced rabbit antisera against two MAP-interacting peptides Lys-Asp-Tyr-Glu-Glu-Val-Gly-Val-Asp-Ser-Val-Glu of alpha-tubulin and Tyr-Gln-Gln-Tyr-Gln-Asp-Ala-Thr-Ala-Asp-Glu-Gln-Gly of beta subunit. The affinity-purified alpha and beta anti-peptide antibodies interacted specifically with tubulin and with the respective peptide antigens but did not interact with MAPs. Substoichiometric amounts of both antibodies showed the capacity to inhibit in vitro MAP-induced tubulin assembly and to promote a fast depolymerization of preassembled microtubules. Taxol-promoted assembly of pure tubulin was not inhibited by the antibodies. In the presence of MAP-2 and taxol, the antibodies decreased the MAP-2 content of taxol-promoted microtubules. The interaction with microtubules was corroborated by immunofluorescence experiments in HeLa and NE-18 lung carcinoma cells. The epitopes recognized by the alpha and beta anti-peptide antibodies appear to be located in the outer surface of the microtubular structure.

Alkaloids↗