José A. Santomé

Sequence of thirteen amino acids in the C-terminal end of bovine growth hormone and localization of a disulflde bridge☆

Abstract The carbamidomethyl derivative of reduced bovine growth hormone (BGH) was extensively attacked by carboxypeptidases A and B, and approximately 19 moles of various amino acids was liberated. The correct sequence of 13 amino acids could be established by selective cleavage of the methionyl bonds in the molecule and isolation of a C-terminal dodecapeptide by filtration through Sephadex G-25. The structure of this peptide was studied by carboxypeptidase A attack, Edman degradation, and further study of the fragments obtained by chymotryptic digestion. A disulfide bond connecting the two half-cystines in the dodecapeptide was discovered by the comparative study of the peptides released by the action of cyanogen bromide on native or reduced and blocked hormone. The structure of the C-terminal end in BGH is:

Binding properties of Echinococcus granulosus fatty acid binding protein

EgFABP1 is a developmentally regulated intracellular fatty acid binding protein characterized in the larval stage of parasitic platyhelminth Echinococcus granulosus. It is structurally related to the heart group of fatty acid binding proteins (H-FABPs). Binding properties and ligand affinity of recombinant EgFABP1 were determined by fluorescence spectroscopy using cis- and trans-parinaric acid. Two binding sites for cis- and trans-parinaric acid were found (K(d(1)) 24+/-4 nM, K(d(2)) 510+/-60 nM for cis-parinaric acid and K(d(1)) 32+/-4 nM, K(d(2)) 364+/-75 nM for trans-parinaric). A putative third site for both fatty acids is discussed. Binding preferences were determined using displacement assays. Arachidonic and oleic acids presented the highest displacement percentages for EgFABP1. The Echinococcus FABP is the unique member of the H-FABP group able to bind two long chain fatty acid molecules with high affinity. Structure-function relationships and putative roles for EgFABP1 in E. granulosus metabolism are discussed.

Isolation, characterization and binding properties of two rat liver fatty acid-binding protein isoforms

Mammalian liver has only one fatty acid-binding protein (L-FABP) while the liver of non-mammalian vertebrates expresses a liver basic FABP (Lb-FABP) in addition to other members of the FABP family. We explore the possibility that L-FABP isoforms accomplish, in the liver of mammals, the metabolic functions corresponding to the different FABPs present in the liver of non-mammalian vertebrates. We have isolated isoforms I and II which have a different residue 105, Asn in the former and Asp in the latter. We made a conformational comparison of the apo-isoforms by intrinsic fluorescence emission and fourth-derivative spectroscopy, native-state proteolysis and unfolding curves. Ligand affinity was studied by measuring cis-parinaric acid displacement by different ligands. They have differences in their molecular conformation, including the environment of the binding site. Isoform II has probably a more open conformation than isoform I, thus allowing the binding of a greater variety of ligands. The affinity of isoform II for lysophospholipids, prostaglandins, retinoids, bilirubin and bile salts is greater than that of isoform I. These characteristics of rat L-FABP isoforms I and II suggest that they may accomplish different functions as happens with those of the different FABP types in non-mammalian species.

Presence and subcellular localization of tyrosine aminotransferase and p-hydroxyphenyllactate dehydrogenase in epimastigotes of Trypanosoma cruzi

Cell-free extracts of epimastigotes of Trypanosoma cruzi contain tyrosine aminotransferase (TAT) and p-hydroxyphenyllactate dehydrogenase (pHPLDH). The TAT activity could be separated from aspartate aminotransferase (ASAT) by polyacrylamide gel electrophoresis or DEAE-cellulose chromatography; the latter procedure also allowed complete separation of pHPLDH. The subcellular localization of both T. cruzi enzymes, as determined by digitonin extraction, subcellular fractionation by differential centrifugation, and isopycnic ultracentrifugation in sucrose gradients, was mainly cytosolic, with low mitochondrial activities.

Influence of some chemical treatments on the immunological properties of various mammalian growth hormones

Abstract The antigenic reactivity of some mammalian growth hormones in their native, oxidized, reduced and nitrated conditions were studied in a hemagglutination system using antisera elicited in rabbits injected with the native hormones. With the limitation imposed by the exclusive use of rabbit antisera, the following conclusions were arrived at: (a) the determinant areas are of small size in all cases; (b) the modification of the cystine, methionine, tryptophan and tyrosine residues does not greatly modify the immunological reactivity of human and horse growth hormones; (c) bovine growth hormone is very sensitive to any modification of the amino acids mentioned before with the only exception of the tyrosines.

Crystal structure of axolotl (Ambystoma mexicanum) liver bile acid‐binding protein bound to cholic and oleic acid

The family of the liver bile acid‐binding proteins (L‐BABPs), formerly called liver basic fatty acid‐binding proteins (Lb‐FABPs) shares fold and sequence similarity with the paralogous liver fatty acid‐binding proteins (L‐FABPs) but has a different stoichiometry and specificity of ligand binding. This article describes the first X‐ray structure of a member of the L‐BABP family, axolotl (Ambystoma mexicanum) L‐BABP, bound to two different ligands: cholic and oleic acid. The protein binds one molecule of oleic acid in a position that is significantly different from that of either of the two molecules that bind to rat liver FABP. The stoichiometry of binding of cholate is of two ligands per protein molecule, as observed in chicken L‐BABP. The cholate molecule that binds buried most deeply into the internal cavity overlaps well with the analogous bound to chicken L‐BABP, whereas the second molecule, which interacts with the first only through hydrophobic contacts, is more external and exposed to the solvent. Proteins 2006.

Trinitrophenylation of equine growth hormone

Abstract The reactivity of the amino groups in equine growth hormone towards trinitrophenylation with picryl sulfonic acid was investigated, and the localization in the molecule of the various kinetically distinguishable amino groups was achieved. The N-terminal residue provides the most reactive amino group followed by the ϵ-amino groups of lysines 179 and 156 and lysines 63, 143, and 165 in decreasing order. Total trinitrophenylation of equine growth hormone brings about a complete loss of the growth-promoting capacity of the protein, but half-maximal potency is still present when there is more than 80% substitution in lysine 179, about 50% in lysine 156, and approximately 20% in each of lysines 63, 143, and 165 besides 100% reaction of the N-terminal group. On the other hand, the immunological properties, as measured in this work, are practically unmodified, even in completely trinitrophenylated equine growth hormone.

Purification and partial characterization of a fatty acidbinding protein from the yeast, Yarrowia lipolytica

A low‐molecular‐mass fatty acid‐binding protein was isolated from the cytosol of the yeast Yarrowia lipolytica. Purification was achieved by a two‐step procedure involving size‐exclusion and cation‐exchange chromatography. The isolated protein exists as a monomer of 15 kDa, is basic and has a blocked N‐terminus. Internal amino acid sequencing suggests that this protein may belong to a novel class of fatty acid‐binding proteins.

Complex assembly of calgranulins A and B, two S100-like calcium-binding proteins from pig granulocytes

Calgranulin A (CAGA) and calgranulin B (CAGB) are two S100-like calcium-binding proteins that in human, bovine and mouse granulocytes are associated into a heterocomplex. We have previously identified in pig granulocytes the porcine homologue of CAGA and a novel S100-like protein which was named calgranulin C (CAGC). As pig CAGA is not associated with CAGC, we herein investigate its possible association with other proteins. CAGA was purified from pig granulocytes by gel filtration followed by Mono Q chromatography. The purified fractions were analysed by SDS-polyacrylamide gel electrophoresis, isoelectric focusing, mass spectrometry, chemical cross-linking and hydrophobic interaction chromatography. The CAGA-associated protein was further characterized by amino acid sequencing. Two CAGA-containing fractions were isolated. One of them was identified as a CAGA homodimer. The other fraction consists of a heterocomplex containing CAGA and a pI 7.0 calcium-binding protein; this protein has a molecular mass of 15,877.9 +/- 3.8 Da (mean +/- SD) whereas it migrates on 10 and 16% polyacrylamide gels as a 24- and 20-kDa protein, respectively. The pI 7.0 protein was identified by internal amino acid sequencing as the porcine homologue of CAGB. The stoichiometry of the heterocomplex was estimated to be 1:1. Both the CAGA homodimer and CAGA/CAGB were found to be non-covalently associated. Unlike the homodimer, CAGA/CAGB was bound to a Phenyl Superose column in a calcium-dependent manner. Our results suggest that pig granulocytes contain, in addition to CAGC, a CAGA homodimer and a CAGA/CAGB heterodimer. It is proposed that CAGB/CAGB and the CAGA homodimer may play different roles in vivo.

Presence of a Fatty Acid-Binding Protein and Lipid Stores in Flight Muscles of Dipetalogaster maximus (Hemiptera: Reduviidae)

Abstract A fatty acid-binding protein (FABP) from the cytosolic fraction of the triatomine Dipetalogaster maximus (Uhler) flight muscles was purified by a procedure based on gel filtration, reverse-phase high performance liquid chromatography, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein has an apparent molecular mass of 14 kDa, and its N-terminus is unblocked. Its N-terminal sequence was obtained by submitting an SDS-PAGE band blotted onto a polyvinylidene difluoride membrane to Edman degradation. The sequence obtained indicates that this FABP belongs to the heart type. This is the first time that a fatty acid-binding protein has been reported for a triatomine. The presence of said FABP, abundant mitochondria, and lipid stores in the flight muscles of D. maximus suggests that beta oxidation of fatty acids is used by the triatomine thoracic muscle as an energy source, and could be related to its dispersal capacity.