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Biomedical subjects

J Avila

Publications and source records attributed to J Avila.

At least 235 records · Page 13Linked to original sources
PubMed1987

Potassium disturbances as a cause of metabolic neuromyopathy.

We report two cases of ascending muscular weakness progressing to areflexic quadriplegia caused by severe derangement of potassium homeostasis. The first patient presented with a 17-alpha-hydroxylase deficiency and severe hypokalemia. The second case had primary adrenocortical deficiency (Addison's disease) and extreme hyperkalemia. Complete recovery ensued after correction of the metabolic disorder in both cases. The role of potassium in the pathophysiology of neuromuscular excitation is discussed. We conclude that when neuromyopathy is present, metabolic causes should be considered and the serum potassium determined.

Addison Disease↗
PubMed1987

Centromeric proteins recognized by CREST sera and meiotic chromosome segregation.

Analysis of the peptides recognized by CREST sera was carried out in different mouse tissues and cells, including spermatozoa. In all cases, a polypeptide of Mr = 18,000 was recognized by the sera and occasionally two other proteins of Mr = 80,000 and Mr = 140,000 were observed after immunoblotting of nuclear proteins. In both early and late spermatids, centromeric staining was observed after incubation and immunofluorescence with CREST sera. After detergent treatment, it was even possible to detect centromeric staining in mature spermatozoa. In spermatid cells, the immunofluorescent pattern presented a binomial distribution of the number of fluorescent spots, with a mean value around half of the haploid number of chromosomes. Since this pattern is the result of chromosome segregation after meiosis II, our data suggest that this centromeric peptide is not directly implicated in the chromosome segregation process. On the other hand, the distribution of spots after immunofluorescence suggests a different organization of centromeric components in meiosis I and meiosis II.

Animals↗
PubMed1987

Tubulin phosphorylation by casein kinase II is similar to that found in vivo.

Purified brain tubulin subjected to an exhaustive phosphatase treatment can be rephosphorylated by casein kinase II. This phosphorylation takes place mainly on a serine residue, which has been located at the carboxy-terminal domain of the beta-subunit. Interestingly, tubulin phosphorylated by casein kinase II retains its ability to polymerize in accordance with descriptions by other authors of in vivo phosphorylated tubulin. Moreover, the V8 phosphopeptide patterns of both tubulin phosphorylated in vitro by casein kinase II and tubulin phosphorylated in vivo in N2A cells are quite similar, and different from that of tubulin phosphorylated in vitro by Ca/calmodulin-dependent kinase II. On the other hand, we have found an endogenous casein kinase II-like activity in purified brain microtubule protein that uses GTP and ATP as phosphate donors, is inhibited by heparin, and phosphorylates phosphatase-treated tubulin. Thus it appears that a casein kinase II-like activity should be considered a candidate for the observed phosphorylation of beta-tubulin in vivo in brain or neuroblastoma cells.

Animals↗
PubMed1987

In vitro conditions for the self-polymerization of the microtubule-associated protein, tau factor.

One of the microtubule associated proteins, tau factor, that appears associated to the paired helical filaments of Alzheimer's disease presents, by itself, after urea treatment, the ability of polymerizing in vitro as tested by immunoelectronmicroscopy. These polymers resemble in their width and appearance those of the paired helical filaments. The conditions required for this assembly have been studied and determined the protein concentration needed, the influence of salt concentration and pH as well as the possible modifications (deamination, acylation) which may be implied in such in vitro polymerization.

Alzheimer Disease↗
PubMed1987

Localization of the phosphorylation sites for different kinases in the microtubule-associated protein MAP2.

The phosphorylation of microtubule-associated protein 2 (MAP2) by four different kinases was studied in vitro to determine whether MAP2 is phosphorylated in its tubulin binding region or in the microtubule projection portion. Fragments corresponding to both regions of MAP2 were produced not only by chymotrypsin or trypsin digestion, but also using pepsin, a broad chain-specificity protease, a result supporting previous notions of the two-domain structure of MAP2. The position of these two functional domains was determined with respect to the carboxy terminal of the molecule, by labeling MAP2 exclusively at the carboxy terminal and subjecting it to pepsin digestion. The results suggested that the projection region of MAP2 contained the carboxy terminal of the protein. A phosphorylation map was constructed by subjecting phosphorylated MAP2 to enzymatic digestion using Staphylococcus aureus V8 protease or to chemical cleavage using N-chlorosuccinimide. The results indicated that all four kinases phosphorylated MAP2 in a 42-kilodalton peptide that contained the tubulin binding region but differed in the level and localization of the sites at which they phosphorylated the projection of MAP2.

Animals↗
PubMed1986

A Trypanosoma cruzi monoclonal antibody that recognizes a superficial tubulin-like antigen.

A monoclonal antibody (MAB 10), obtained from mice infected with Trypanosoma cruzi, was found to recognize a superficial antigen in living or fixed parasites. It reacted more strongly with T. cruzi than with related parasites such as T. brucei and Leishmania. In immunoblots it recognized a single trypanosoma polypeptide and also brain tubulin, both of which had the same electrophoretic mobility. Further analysis suggested that the alpha-tubulin subunit contained the epitope recognized by MAB 10. These results suggest that a surface tubulin-like protein is present is T. cruzi.

Animals↗
PubMed1986

Phosphorylation of tubulin by a calmodulin-dependent protein kinase.

Calmodulin-dependent protein kinase was purified from porcine brain cytosol through sequential steps involving acid precipitation, DEAE-chromatography, and calmodulin-Sepharose chromatography. The purified enzyme contained a major Mr 50,000 and a minor Mr 60,000 peptide. Porcine brain tubulin was a major substrate for this kinase. Under optimal conditions 2.6 mol of phosphate were incorporated per mol of tubulin. The kinase phosphorylated both tubulin subunits at their carboxyl-terminal region. Limited proteolysis, using trypsin and chymotrypsin, of phosphorylated and unphosphorylated tubulins resulted in different cleavage patterns as determined by peptide mapping. Phosphorylated tubulin was unable to bind to microtubule-associated protein or to polymerize, but regained its assembly capacity after phosphatase treatment.

Animals↗
PubMed1986

Localization of the high affinity calcium-binding site on tubulin molecule.

Tubulin is a calcium-binding protein. Two different modes of interaction of calcium with tubulin have been described: a high affinity interaction to one or two binding sites and lower affinity interactions to several other binding sites. In the present study, we have used limited proteolysis of tubulin with trypsin, chymotrypsin, and subtilisin to localize the high affinity calcium-binding sites. Our results indicate that two sites are located in the carboxyl-terminal region of both tubulin subunits, and that tubulin deprived of its carboxyl-terminal region is able to polymerize in the presence of 0.5 mM calcium.

Amino Acid Sequence↗
PubMed1986

A calcium binding protein from Drosophila melanogaster which activates cAMP phosphodiesterase: comparison of this protein with porcine brain calmodulin.

A calcium binding protein from Drosophila melanogaster has been isolated and characterized. This protein shows several analogies with pig brain calmodulin in its molecular weight, isoelectric point, peptide maps, calcium binding properties, and ability to activate cyclic AMP phosphodiesterase. However, some differences were observed; the most remarkable one is the presence of tryptophan, an amino acid which is absent from all the calmodulins analyzed previously.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1986

Quantitation and characterization of tau factor in porcine tissues.

Using a monospecific antibody against brain tau factor purified by affinity chromatography, we have studied the distribution of tau factor or related polypeptides in different cells. The presence of tau in all cell types tested was demonstrated by a radioimmunoassay. Tau factor-related proteins were found in liver, spleen, pancreas, kidney and lung, although at much lower levels than that found in neural cells. In all cases, they copolymerized with tubulin and were heat-resistant. When the distribution of tau factor-related proteins was studied by Western blotting, tau factor antiserum reacted against peptides with an electrophoretic mobility that was similar to those of brain tau factor peptides. Immunofluorescence studies have also been performed with the same antibody to determine the distribution of tau factor-related peptides in PK15 cells. Our results indicated that these peptides were associated to the microtubule network.

Animals↗
PubMed1986

Characterization and structural aspects of the enhanced assembly of tubulin after removal of its carboxyl-terminal domain.

Limited subtilisin cleavage of tubulin results in formation of S-tubulin heterodimer and a 4-kDa carboxyl-terminal peptide fragment. This carboxyl-terminal domain constitutes an essential site for MAPs interaction and plays a role in modulating the interactions responsible for tubulin self-assembly into microtubules [Serrano et al. (1984) Proc. Natl Acad. Sci. USA 81, 5989; and Biochemistry 23, 4675]. In the present communication it is shown that addition of the 4-kDa peptide fragment from porcine tubulin to porcine S-tubulin in a molar ratio of about 2:1 does not affect the assembly of the latter. On the other hand, consistent with previous findings on the binding of the 4-kDa peptide by MAP-2, the peptide inhibited MAP-2-induced tubulin assembly (molar ratio of peptide to tubulin, about 2:1; peptide to MAP-2, about 30:1). Comparison of the amino acid composition of the 4-kDa peptide fragment and the C-terminal amino acid residues of S-tubulin with the amino acid sequence of tubulin indicated the subtilisin cleavage site on the tubulin molecule to be between residues Glu417 and Phe418 of the alpha-subunit sequence and between Glu407 and Phe408 of the beta-subunit sequence. The circular dichroism of the 4-kDa fragment in water as solvent is indicative of a molecule with an unordered structure, but when the solvent is changed to a water-trifluoroethanol mixture, the fragment becomes more highly structured. The critical concentration for S-tubulin assembly is not affected by MAPs nor by polylysine, but is decreased by either taxol of dimethylsulfoxide. S-tubulin, with its greater propensity for self-association, has a different conformation from tubulin as shown by a 50% decrease in alpha-helical content, a more hydrophobic environment of at least some of the tryptophan residues as judged from fluorimetry, and a greater compaction indicated by f/f0 = 1.3, as compared to 1.4 for tubulin. The latter point is supported by the observation that the value of the sedimentation coefficient, s20,w = 5.7 S, of the 92-kDa S-tubulin molecule is not significantly different from that of the 100-kDa tubulin, s20,w = 5.8S.

Amino Acids↗
PubMed1986

Physicochemical characterization of the heat-stable microtubule-associated protein MAP2.

MAP2 purified without heat treatment was compared to MAP2 prepared with the usual boiling step. No significant differences were found by isoelectric focusing, peptide mapping, fluorescence spectroscopy and circular dichroism. Hydrodynamically, MAP2 is a monomer with s degree 20,w = 3.5 +/- 0.1 S and Mr = 220 000. The molecular mass was measured by sedimentation equilibrium and verified by gel chromatography in denaturing solvent and dodecyl sulfate gel electrophoresis at different acrylamide concentrations. The high frictional ratio, f/fmin = 3.7, indicated that MAP2 was clearly not globular but had either a very elongated shape or an unordered expanded structure. Circular dichroic results were consistent with a predominantly unordered structure independently of the preparation procedure. This supports the notion that MAP2, in solution, is a very flexible and non-compact protein. Examination of the circular dichroism of MAP2 as a function of temperature indicated that the thermal stability of this protein was probably due to its mostly unordered structure and the fact that the little ordered structure present was resistant to thermal denaturation.

Amino Acids↗
PubMed1986

Characterization of a membrane-specific tubulin isoform by peptide mapping.

A membrane-specific tubulin-like protein, found in preparations of synaptic plasma membranes and brain mitochondria, was analyzed by chemical and proteolytic peptide mapping to determine which part of the molecule was different from cytoplasmic tubulin. The membrane polypeptide was identical to alpha tubulin in the first two-thirds of the molecule containing the amino terminal, as found by peptide mapping. However, some differences were observed in the peptide maps of the carboxy terminal one third of the molecule which includes a domain that is important in the regulation of tubulin self-assembly.

Animals↗
PubMed1986

Location of the regions recognized by five commercial antibodies on the tubulin molecule.

Through limited proteolysis of the tubulin molecule with trypsin, chymotrypsin, subtilisin, or pronase we have mapped the regions recognized by five commercial antibodies. Two of them recognized a sequence between amino acids 340 and 400 near the C terminal of the alpha or beta subunits, one recognized a sequence between amino acids 120 and 150 present in both subunits, and another one probably recognized a conformational epitope. Simultaneously we have confirmed the results obtained for other antibodies which recognized a region previously mapped on the tubulin molecule.

Amino Acid Sequence↗
PubMed1986

The removal of the carboxy-terminal region of tubulin favors its vinblastine-induced aggregation into spiral-like structures.

Vinblastine induces brain tubulin to assemble into spirals. This process is stimulated by microtubule-associated proteins (MAPs) which copolymerize with brain microtubules assembled in vitro. When the carboxy terminal of tubulin is removed by subtilisin digestion, vinblastine readily induces the aggregation of tubulin into spiral-like or circular structures, even in the absence of MAPs. These results suggest that in the absence of MAPs, the carboxy-terminal domain of tubulin may inhibit vinblastine-induced polymerization of tubulin into spiral-like structures.

Biopolymers↗
PubMed1986

The combined use of limited proteolysis and differential peptide staining for protein characterization.

A method to characterize a protein by peptide mapping is described. It involves a combination of limited proteolysis and differential staining with the dye 'stains all' which allows the identification of the proteolytic fragments by their size and colour. When this procedure was used for tubulin, specific acidic fragments were identified and localized in the molecule.

Electrophoresis, Polyacrylamide Gel↗