This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues.Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. were identified in L. muta and L. stenophrys venoms. In addition, both venoms contained a large number of bradykinin-potentiating peptides (BPP) and a C-type natriuretic peptide (C-NP). BPPs and C-NP comprised around 15% of the total venom proteins. In both species, the most abundant proteins were Zn 2+ -metalloproteinases (32-38%) and serine proteinases (25-31%), followed by PLA 2 s (9-12%), galactose-specific C-type lectin (4-8%), L-amino acid oxidase (LAO, 3-5%), CRISP (1.8%; found in L. muta but not in L. stenophrys), and NGF (0.6%).On the other hand, only six L. muta venom-secreted proteins matched any of the previously reported 11 partial or full-length venom gland transcripts, and venom proteome and transcriptome depart in their relative abundances of different toxin families. As expected from their close phylogenetic relationship, the venoms of L. muta and L. stenophrys share (or contain highly similar) proteins, in particular BPPs, serine proteinases, a galactose-specific C-type lectin, and LAO. However, they dramatically depart in their respective PLA 2 complement. Intraspecific quantitative and qualitative differences in the expression of PLA 2 molecules were found when the venoms of five L. muta specimens (3 from Bolivia and 2 from Peru) and the venom of the same species purchased from Sigma were compared.These observations indicate that these class of toxins represents a rapidly-evolving Author's personal copy gene family, and suggests that functional differences due to structural changes in PLA 2 s molecules among these snakes may have been a hallmark during speciation and adaptation of diverging snake populations to new ecological niches, or competition for resources in existing ones. Our data may contribute to a deeper understanding of the biology and ecology of these snakes, and may also serve as a starting point for studying structure-function correlations of individual toxins.

The study of the structure of the peptides arising from native, oxidized or reduced and maleinized bovine growth hormone on incubation with trypsin, chymotrypsin and pepsin is reported. The data obtained permitted the assembly of a unique sequence of amino acids for the poly‐peptide chain of the protein. Various corrections and one addition to the sequence previously communicated are made.

Further characterization of an aspartyl protease from Mucor bacilliformis with milk-clotting activity was performed. An extinction coefficient, epsilon 278 cm = 1.61 mL/mg/cm, a molecular mass of 35,400 Da and a pI of 5.2 were determined. Proteolytic activity and kinetic parameters were evaluated by using the hexapeptide Leu-Ser-pNO2-Phe-Nle-Ala-Leu-OMe as the substrate. The effect of pH and temperature on peptide cleavage, as well as protease heat stability, was determined. Such properties, taken as a whole, indicate that the M. bacilliformis protease can be considered a potential substitute for bovine chymosin in cheese manufacture.

Stimulation of full-grown amphibian oocytes with progesterone initiates a nontranscriptional signaling pathway that converges in the activation of Cdc2/cyclin B and reentry into meiosis. We observed that cholesterol depletion mediated by methyl-beta-cyclodextrin (MbetaCD) inhibited meiotic maturation, suggesting involvement of membrane rafts. In the present study, we further characterized caveolae-like membranes from Rhinella arenarum oocytes biochemically and functionally. The identification by mass spectrometry of a nonmuscle myosin heavy-chain associated with caveolar membranes showed evidence of direct involvement of the underlying cytoskeletal environment in the structure of oocyte rafts. Biophysical analysis using the fluorescent probe Laurdan revealed that MbetaCD-mediated cholesterol depletion affected membrane lipid order. In line with this finding, cholesterol removal also affected the localization of the raft marker lipid GM1. Results demonstrated that ceramide is an effective inducer of maturation that alters the distribution of the raft markers caveolin-1, SRC, and GM1, while progesterone seems not to affect membrane microdomain integrity. Cholesterol depletion had a greater effect on ceramide-induced maturation, thus suggesting that ceramide is an inducer more vulnerable to changes in the plasma membrane. MbetaCD treatment delayed tyrosine phosphorylation and MAPK activation in progesterone-induced maturation. Functional studies regarding tyrosine phosphorylation raise the possibility that the hormone receptor is located in the nonraft membrane in the absence of ligand and that it translocates to the caveola when it binds to progesterone. The presence of raft markers and the finding of signaling molecules from MAPK cascade functionally associated to oocyte light membranes suggest that this caveolae-rich fraction efficiently recreates, in part, maturation signaling.

No abstract

Monoclonal antibodies (MAb) to human GH (hGH) were used to correlate the antigenic topography of the hormone with its structure. Competition experiments performed in a solid phase RIA system allowed us to measure the reactivity toward the MAb of the following hGH derivatives: hGH 20K (a natural variant lacking residues 32-46), hGH selectively modified in His or Met residues, hGH with the C and/or N-terminal disulfide bond reduced and carbamidomethylated, and hGH cleaved between residues 142-143. Results indicated that fragment 32-46 participates in the structure of epitopes EB1/EB3 and that the C-terminal bridge is located in epitope 10D6, whereas opening of both disulfide bridges alters the entire hGH antigenic surface. His-151 and Met-170 were placed in epitopes NA71 and AC8, respectively, whereas His-18 and Met-14 would be involved in the hGH antigenic domain formed by overlapping epitopes 3C11, 10C1, and HG3. MAb AE5, AE12, and AC3 define a flexible hGH region related to sequence 134-150; the respective epitopes show high conformational mobility induced by modifications in other regions of the molecule. Binding of the different hGH derivatives to lactogenic receptors from female rat liver gave some insights on the localization of the hormone-binding site. Epitopes EB1/EB3 and 10D6 were discarded because there was not a direct correlation between their drastic immunological alterations and the binding properties of the respective hGH derivatives. In the same way, epitopes AE5, AE12, and AC3 were excluded from the hGH-binding domain because a disruption in those sites did not affect the hGH interaction with receptors. We conclude that the hGH structure defined by epitopes 3C11, 10C1, and HG3 is probably related to the binding properties of the hormone.

No abstract

Among the abnormalities in erythrocyte porphyrin metabolism already described in patients with chronic renal failure on hemodialysis, a decrease in blood aminolevulinate dehydratase activity has been reported, suggesting the presence in uremic plasma of an inhibitor of the enzyme. The aim of this work has been to isolate and characterize such an inhibitor. Blood samples from 105 patients with chronic uremia were collected; plasma was applied to Sephadex G-100 columns and the fraction with the highest inhibiting capacity was identified and purified by subsequent SDS-polyacrylamide gel electrophoresis, followed by electroelution and electroblotting. It was demonstrated that the factor present in plasma of uremic patients inhibited blood aminolevulinate dehydratase in a concentration-dependent manner; its inhibitory properties were abolished after heat, trypsin and TCA treatment indicating its peptidic nature. The purified inhibitor has an apparent molecular mass of 56.2 kD, it inhibits blood aminolevulinate dehydratase in a competitive way and the K_i value is 12×10^–6 M. The amino acid composition of the inhibitor has been determined and it has been found that its N-terminal amino acid is blocked. The isolated peptide may play a role in heme biosynthesis disturbances and in the pathogenesis of uremic anemia.