Biomedical subjects
Publications and source records attributed to L Serrano.
In the transition state for unfolding of barnase, the hydrophobic core between the major alpha-helix and beta-sheet is somewhat weakened, the C terminus of the major helix is largely intact but its N terminus is exposed and a major loop has been invaded by solvent.
A magnesium-dependent heparin-inhibited protein kinase activity associated with brain microtubule preparations has been identified as casein kinase II using a monospecific polyclonal antibody. This enzyme appears enriched in cold-stable microtubule fractions. By immunofluorescence microscopy using an antiserum against casein kinase II, the in situ immunolabeling of some microtubule assays has been observed. Thus, mitotic spindles are stained by the anti-casein kinase II antibody in fibroblast cells. In neuroblastoma cells induced to differentiate, the labeling of microtubule arrays inside developing axon-like processes is also seen. These results support the view that casein kinase II can modulate cytoskeletal assembly and dynamics through phosphorylation of microtubule proteins.
Eleven patients with distal subungal onychomycosis of the fingers by Trichophyton rubrum infection were treated with daily doses of oral terbinafine, a new allylamine derivative. A 125-mg capsule was given twice daily. Each patient's nailplate was marked appropriately following the Zaias and Drachman method for the determination of antifungal efficacy in onychomycosis. Patients were monitored monthly for the antifungal effect of the drug, as well as side-effects, including laboratory values for liver and kidney function tests and a full blood count. At the end of 6 months, all 11 patients were clinically and mycologically normal. It can be concluded that 250 mg (125 mg b.i.d.) oral terbinafine is the most appropriate drug for the treatment of finger onychomycosis. No side-effects of any kind were noted in this 6-month period. Onychomycosis is a common disease of the toenails. No topical treatments have yet been found to be satisfactory. In a very motivated patient-doctor relationship, the use of topically applied thiabendazole has proved effective, particularly after nailplate avulsion. Systemic antifungal treatment is possible with griseofulvin and ketoconazole. Griseofulvin may cause enzyme induction in the liver, which requires a higher dose intake to achieve results. This has been demonstrated using the Zaias and Drachman method of judging successful antifungal clinical effects in onychomycosis. Ketoconazole is a very effective drug which must be carefully monitored. Its difficulties are with liver function, anti-androgenic dysfunction in males, and adrenal dysfunction. There is a great need for an effective and safe oral systemic drug. Terbinafine is an allylamine derivative which is highly antifungal.(ABSTRACT TRUNCATED AT 250 WORDS)
Taxol-stabilised erythrocyte microtubules assembled less readily than similarly prepared brain microtubules on adding 10(-4) M-10(-3) M concentrations of calcium at 2 degrees C. Scatchard plot analyses of the high affinity calcium binding sites showed that the erythrocyte tubulin contained only 0.9 high affinity binding sites per dimer compared to 1.4 binding sites per dimer for brain tubulin. Association constants, however, for calcium binding to both erythrocyte and brain tubulin were similar (3.0 x 10(-6) M and 2.1 x 10(-6) M). The beta-tubulin subunit appeared to be responsible for the lower calcium binding ability of erythrocyte tubulin as shown by a gel overlay assay with 45Ca. Strains-all, a dye that stains many calcium binding proteins blue, did not stain erythrocyte beta-tubulin or its chymotryptic C-terminal fragment blue as was the case for brain beta-tubulin and its chymotryptic C-terminal fragment. We suggest that the lower calcium binding ability of erythrocyte beta-tubulin may be implicated in the differential behaviour of erythrocyte microtubules.
Activation of the oncogenic potential of ras oncogenes occurs by point mutations at codons 12, 13, 59, 61, and 63 of the sequences that codify for its product, a 21-kDa protein designated as p21. This activation has been postulated by computer models as modifiers of the structure of the protein, which may alter its biochemical and biological activities. We have expressed in bacteria the normal ras p21 and five mutated p21 proteins with mutations at positions 12, 59, 61, 12 plus 59, and 12 plus 61. Purification was carried out by solubilization from bacterial pellets in 7 M urea and chromatography through a Sephadex G-100 column to obtain greater than 95% purified proteins. Circular dichroic (CD) spectra showed that the normal protein and that activated by substitution of Ala59 to Thr59 are very similar in their overall structure. By contrast, point mutations affecting either 12 or 61 residues substantially altered the structure of the proteins. When the parameters of Chen et al. [Biochemistry II, 4120-4131 (1972)] were applied to the CD spectra, both normal and thr59-mutated ras proteins showed a less organized structure than mutated proteins at position 12 or 61. Since the Thr59 mutant has more similar transforming activity than other activated proteins, but a GTPase activity similar to that of the normal protein, our results support the hypothesis that there is more than one mechanism of activation of the ras p21 protein. One of these mechanisms involves important structural alterations by point mutations at position 12 or 61 which reduce the GTPase activity of the protein. Another mechanism will be that induced by a substitution of Ala59 to Thr59 which does not substantially alter the protein conformation. A putative alternative mechanism for the activation of this mutant is discussed.
The capacity for self-polymerization and shape of the tubulin polymers assembled after digestion with trypsin, Pronase, chymotrypsin, subtilisin, Staphylococcus aureus proteinase V8 and proteinase K were investigated. Digestion with trypsin, Pronase or chymotrypsin resulted in a decrease in the ability of tubulin for self-assembly, whereas limited proteolysis with subtilisin, S. aureus proteinase V8 or proteinase K resulted in an increase in such ability. The shape of the assembled polymers varied from typical microtubules (after the treatment with trypsin or Pronase) to sheets (after the treatment with chymotrypsin) and from hooked microtubules with a constant polarity (after the treatment with subtilisin) to the disappearance of a defined polarity of such polymers (after the treatment with S. aureus V8 proteinase or proteinase K). These results indicate that the tubulin C-terminal regions are involved in the regulation of microtubule polymerization, shape, directional growth and lateral interactions between tubulin protofilaments.
Calcium has been suggested to be an important element in the regulation of microtubule dynamics 'in vivo'. In this report we have analyzed the possibility that microtubule-associated protein 2 (MAP2) binds calcium. MAP2 was blue-stained with the cationic carbocyanine dye 'stains-all' in a similar way to that of calcium-binding proteins and bound 45Ca as estimated from dot-blotting experiments. The calcium-binding characteristics of MAP2, determined by equilibrium dialysis, indicated that MAP2 bound about 3 mol (n = 2.9 +/- 0.4) of calcium per mol of protein (Kd = (0.9 +/- 0.2).10(-5) M). Analysis of the Scatchard plots from equilibrium dialysis and dot-blot assays indicated that MAP2 also presented low-affinity calcium-binding sites (Kd = (0.3 +/- 0.2).10(-4) M). Incubation of nitrocellulose blots of proteolytically digested MAP2 with 45Ca indicated that the calcium-binding sites were located in the region that is not involved in the interaction with tubulin (projection region).
Purified brain microtubule protein is phosphorylated by endogenous protein kinase activities in the presence of [gamma-32P] ATP or [gamma-32P] GTP. Here we show that certain microtubule-associated proteins are phosphorylated differently by GTP or ATP as direct phosphoryl donors, suggesting the presence of distinct kinase activities, with different specificities, associated with microtubule protein.
The discovery that certain proteins may require zinc for their activity, and the fact that several of them cannot be purified in large amounts, has led us to develop a rapid, sensitive method to detect these proteins in samples. This method is based on the fractionation of the proteins by gel electrophoresis, blotting onto nitrocellulose paper, and overlaying with 65Zn. We have tested the procedure with well-characterized zinc-binding proteins. In the case of tubulin, we have used this method to localize its zinc-binding site. It was found that zinc binds to the first 150 amino acids of both alpha- and beta-tubulin subunits.
A neuroblastoma protein related to the brain microtubule-associated protein, MAP-1B, as determined by immunoprecipitation and coassembly with brain microtubules, becomes phosphorylated when N2A mouse neuroblastoma cells are induced to generate microtubule-containing neurites. To characterize the protein kinases that may be involved in this in vivo phosphorylation of MAP-1B, we have studied its in vitro phosphorylation. In brain microtubule protein, MAP-1B appears to be phosphorylated in vitro by an endogenous casein kinase II-like activity which also phosphorylates the related protein MAP-1A but scarcely phosphorylates MAP-2. A similar kinase activity has been detected in cell-free extracts of differentiating N2A cells. Using brain MAP preparations devoid of endogenous kinase activities and different purified protein kinases, we have found that MAP-1B is barely phosphorylated by cAMP-dependent protein kinase, Ca/calmodulin-dependent protein kinase, or Ca/phospholipid-dependent protein kinase whereas MAP-1B is one of the preferred substrates, together with MAP-1A, for casein kinase II. Brain MAP-1B phosphorylated in vitro by casein kinase II efficiently coassembles with microtubule proteins in the same way as in vivo phosphorylated MAP-1B from neuroblastoma cells. Furthermore, the phosphopeptide patterns of brain MAP-1B phosphorylated in vitro by either purified casein kinase II or an extract obtained from differentiating neuroblastoma cells are identical to each other and similar to that of in vivo phosphorylated neuroblastoma MAP-1B. Thus, we suggest that the observed phosphorylation of a protein identified as MAP-1B during neurite outgrowth is mainly due to the activation of a casein kinase II-related activity in differentiating neuroblastoma cells. This kinase activity, previously implicated in beta-tubulin phosphorylation (Serrano, L., J. Díaz-Nido, F. Wandosell, and J. Avila, 1987. J. Cell Biol. 105: 1731-1739), may consequently have an important role in posttranslational modifications of microtubule proteins required for neuronal differentiation.
Insulin receptor kinase phosphorylated tubulin in an insulin-dependent fashion. Two different populations of phosphotubulin were found. In tubulin dimers containing tyrosine at the carboxyl-terminal of their alpha subunit, phosphate was incorporated in that residue, and the phosphorylated protein did not assemble into polymers. In tubulin dimers lacking this tyrosine residue, phosphate was incorporated into different tyrosine residues located in other parts of the molecule, and the phosphoprotein retained its capacity to polymerize.
Limited proteolysis of tubulin with subtilisin results in the cleavage of both tubulin subunits yielding S-tubulin heterodimer and 4 kDa peptide fragments containing the carboxyl-terminal domains of alpha- and beta-polypeptide chains. S-tubulin binds colchicine and the characterization of the binding of colchicine to S-tubulin molecules showed a decreased rate of decay of colchicine binding activity as compared to that of undigested tubulin. However, S-tubulin exhibited a lower colchicine binding constant than tubulin. Peptide fragments resulting from the controlled tryptic proteolysis of both pure tubulin and S-tubulin were purified by filtration chromatography and presented a strong colchicine binding activity with association constants of 4.5 X 10(6) and 2.7 X 10(6) M-1, respectively. Furthermore, these studies support our initial findings on the localization of the tubulin site for colchicine (Serrano L, Avila J, Maccioni RB: J Biol Chem 259:6607-6611, 1984) and define the colchicine binding domain in a domain of alpha-subunit from the point of limited tryptic cleavage to the site of subtilisin controlled proteolysis of that tubulin subunit. On the basis of these alterations in the interaction of colchicine upon removal of the C-terminal moiety of tubulin and since no change in the number of binding sites was found after subtilisin digestion, we suggest that the carboxyl-terminal region of tubulin subunits modulates the binding of colchicine.
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.
Microtubule associated protein tau factor self-assembles into filamentous structures resembling the paired helical filaments found in Alzheimer disease. Tau polymerization requires of a previous modification; conversion of glutamine into glutamic acid by deamination.
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.
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.
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.
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.