Some Researches on Histones

The histone extracted from calf thymus glands is a complex system of proteins, which can be fractionated by chromatography on carboxymethyl cellulose columns into three principal fractions (1) very lysine-rich, (2) moderately lysine-rich, (3) arginine-rich. When examined by starch gel chromatography each of these gives more than one band. Methods have been devised for further separation of the components in some cases. The components show characteristic differences in end groups and certain amino acids as well as in their basic character. Histones extracted from various rat tissues can be separated into similar fractions, of which the amino acid analyses are similar to those derived from calf thymus, within the experimental error. To this extent, no species or tissue specificity of the fractionated histones was observed. Although all the histone fractions contain approximately one basic amino acid to three non-basic amino acids their structure is not regular, as Phillips has shown that in certain fractions the number of non-basic groups between two basic groups may vary from 0 to seven or more. The possible functions of histones are discussed.     

Neuropeptide-processing carboxypeptidases

Neuropeptides are generally produced from precursor proteins by selective cleavage at specific sites, usually involving basic amino acids. Enzymes such as the prohormone convertases and carboxypeptidase E are highly specific for these basic amino acid-containing sites. In addition to this "traditional" pathway, several neuropeptides are known to be cleaved at non-basic sites, and the enzymes responsible for these cleavages have not been conclusively identified. In a recent search for novel members of the metallocarboxypeptidase family, we found three human genes. One of these, named "CPA-5," has a specificity for C-terminal hydrophobic amino acids and mRNA expression in brain, pituitary, and testis. To test whether CPA-5 protein has a distribution pattern in pituitary that is consistent with a role for this enzyme in the non-basic processing of proopiomelanocortin-derived peptides such as beta-endorphin and adrenocorticotropin, we examined the distribution of CPA-5 using immunocytochemistry. In the pituitary, CPA-5 is detected in the neurointermediate lobe and in scattered cells in the anterior lobe. In the AtT-20 corticotroph cell line, CPA-5 has a perinuclear distribution. Taken together, these results are consistent with a role for CPA-5 in the intracellular processing of proopiomelanocortin-derived peptides at non-basic sites.     

Carbohydrate and protein histochemistry during oogenesis inHalobatrachus didactylus (Schneider, 1801) from the Bay of Cadiz (Spain)

Summary Histological and histochemical characteristics were studied inHalobatrachus didactylus (Schneider, 1801) during oogenesis. Three phases could be differentiated: previtellogenesis (oogonia and basophilic oocytes), vitellogenesis (yolk synthesis) and maturation-spawning. Glycogen, glycoproteins and proteins rich in certain amino acids were present in the previtellogenic as well as in the vitellogenic cytoplasm oocytes. No acid mucosubstances were detected. Three types of yolk (vesicles, vacuoles and granules) contained different types of organic reserves; granules were essentially proteic whereas globules were lipidic. Carbohydrates and proteins were present in vesicles.     

Identification of Basic Nuclear Proteins by their Boronate Complex

A new histochemical reaction for the identification of histone type basic proteins has been developed. Carbonyldiimidazol is used to activate the basic proteins of TCA-extracted nuclei, their m-aminophenylboronic acid complex is prepared, and the DNA-free, histone-containing nuclei are stained with toluidine blue at pH 5.5.     

Identification of Basic Nuclear Proteins by their Boronate Complex

A new histochemical reaction for the identification of histone type basic proteins has been developed. Carbonyldiimidazol is used to activate the basic proteins of TCA-extracted nuclei, their m-aminophenylboronic acid complex is prepared, and the DNA-free, histone-containing nuclei are stained with toluidine blue at pH 5.5.     

Proteins, nucleic acids and genetic codes.

On the basis of the previous article (Morchio and Traverso [1999]), we discuss the possible interactions between the first proteic fragments developed in the hydrophobic layer made of hydrocarbons, which would have covered the surface of the primitive seas, and the nitrogenous bases, particularly the pyrimidinic ones, which would have found in such hydrophobic layer favourable conditions to their prebiotic synthesis. These interactions would have presumably brought, on the basis of the physicochemical laws, at the moment the only ones at work, to the linkage of various bases and so to the construction of the first nucleic acid chains (most likely RNA). Interestingly enough this result would have been obtained by inserting two more bases between those hydrogen bound to the amino acids and this might have been the ground for the future "triplets". These interactions might have been particularly significant because of two important consequences: the birth of a rough genetic code and the starting of interactions of the co-operative type between bases and amino acids that would have made the growth of both proteic and nucleic acid fragments easier and faster. We conclude that the development of the genetic code was neither a "frozen accident" nor an occurrence directed by any information flow.     

Single-column amino acid analysis of connective tissue proteins and related materials.

A single-column procedure is described which, in general, is useful for the amino acid analysis of any simple or complex protein, but in particular the procedure is useful for the separation and quantitation of the amino acids and amino sugars, if present, in collagen, elastin and related materials from a variety of connective tissues. In addition to the amino acids commonly found in proteins, the system resolves hydroxyproline, hydroxylysine, desmosine, isodesmosine, glucosamine, galactosamine and also a number of other ninhydrin-positive compounds ordinarily not found in acid hydrolysates of proteins. Chromatograms of the analysis of a synthetic mixture of amino acid standards and also collagen and elastin hydrolysates indicate the multiple use of the procedure and the nearbaseline separation of all the amino acids. The basic amino acids are well spread, thus providing flexibility for the isolation and identification of additional ninhydrin-positive components. The analysis, which requires approximately 2 hr and four sodium citrate buffers was performed with a Durrum (D-500) amino acid analyzer.     

Functional properties of whey,whey components,and essential amino acids: mechanisms underlying health benefits for active people (review)

Whey proteins and amino acid supplements have a strong position in the sports nutrition market based on the purported quality of proteins and amino acids they provide. Recent studies employing stable isotope methodology demonstrate the ability of whey proteins or amino acid mixtures of similar composition to promote whole body and muscle protein synthesis. Other developing avenues of research explore health benefits of whey that extend beyond protein and basic nutrition. Many bioactive components derived from whey are under study for their ability to offer specific health benefits. These functions are being investigated predominantly in tissue culture systems and animal models. The capacity of these compounds to modulate adiposity, and to enhance immune function and anti-oxidant activity presents new applications potentially suited to the needs of those individuals with active lifestyles. This paper will review the recent literature that describes functional properties of essential amino acids, whey proteins, whey-derived minerals and other compounds and the mechanisms by which they may confer benefits to active people in the context that exercise is a form of metabolic stress. The response to this stress can be positive, as with the accretion of more muscle and improved functionality or greater strength. However, overall benefits may be compromised if immune function or general health is challenged in response to the stress. From a mechanistic standpoint, whey proteins, their composite amino acids, and/or associated compounds may be able to provide substrate and bioactive components to extend the overall benefits of physical activity.     

Schistosoma mansoni: histochemical analysis of the postacetabular gland secretion of cercariae

Contents of the funduses and ducts of the postacetabular glands of Schistosoma mansoni cercariae, the secreted deposits, and the surface film were compared by their histochemical reactions. Techniques for carbohydrate-containing substances, neutral and acid mucosubstance, proteins and amino acids, and enzymes were used. The secretion reacted differently before (within the glands) and after (in secreted deposits) emission.Before emission, the postacetabular gland contents reacted as a neutral mucosubstance containing periodate-engendered and periodate-reactive aldehydes rich in vic-glycols or their substituted amines, probably hexoses other than glucose, such as fucose or galactose. No reactions of significance were observed for acid groups or for glycogen or lipids. In this state, the secretion is termed mucigen.After emission, the secretion stained not only as mucigen, but also as acid mucosubstance, apparently sialomucin. After emission, it is termed mucin.It is probable that acid radicals were present in mucigen but were masked stearically by the presence of adjacent neutral radicals or basic proteins. The surface film reacted as both a neutral and an acid mucosubstance. Evidence suggested that the film itself was neutral and that the reaction for acid mucosubstance was from an overlay of mucin secreted from the postacetabular glands.Proteins and amino acids, especially arginine, and some tyrosine and tryptophan were indicated in mucigen and in mucin by the histochemical tests. There was no histochemical evidence of enzymes. Secretion of the postacetabular glands is concluded from histochemical reactions, as from earlier chromatographic data (Stirewalt and Evans 1960), to be a carbohydrate-protein-lipid complex.     

Phosphoryl Group Transfer by a Fraction of the Soluble Proteins of Catecholamine Storage Vesicles

Abstract: The terminal phosphate group of ATP was transferred to ADP by an enzyme present in the soluble core proteins of adrenal medulla catecholamine storage vesicles. It was purified 10–30-fold by DEAE Sephadex chromatography (Fraction I). The enzyme required divalent metal ions for activation; Mn²⁺ was almost as effective as Mg²⁺, but Ca²⁺ was only a weak activator. Activation by Mg²⁺ took place over a very narrow concentration range (0.5–3 m m ). The specificity of the enzyme activity to nucleoside triphosphates was broad, to the nucleoside diphosphates narrow, favouring adenosine diphosphate. In dependence on the pH the activity increased from pH 4 to pH 7 and remained constantly high between pH 7 and 9. The Arrhenius plot was linear between 5 and 70°C, with an activation energy of 11.1 kcal/mol. The phosphoryl group transfer reaction depended on the function of thiol groups; p -hydroxymercuribenzoate inhibited 50% of the enzyme activity; dithioerythritol reactivated it completely. Gel electrophoresis revealed that in Fraction I, a protein of molecular weight about 45,000, was enriched compared with the total soluble proteins. The enzyme-enriched Fraction I differed significantly in its relative amino acid composition from that of the total soluble proteins; in general, the acidic amino acids were reduced and the more basic acids enhanced.     

Monoclonal Antibodies for Detection of 4-Hydroxynonenal Modified Proteins

A promising approach to study lipid peroxidation pathology is antibodies recognizing aldehydes which react with and became bound to amino acid side chains of proteins. We present in this study the characterization of several monoclonal antibodies which recognize 4-hydroxynonenal (HNE) modified proteins. Six out of 20 antibodies recognizing HNE modified BSA were able to detect HNE-protein adducts in peroxidized liver microsomes. Two of these antibodies were selected and characterized. Both antibodies could also detect HNE-protein adducts in oxidized low density lipoprotein. They exhibit no detectable cross reaction with proteins modified by malonaldehyde, nonanal, nonenal and 4-hydroxyhexenal. Protein bound 4-hydroxyoctenal and 4-hydroxydecenal were recognized to some extent. Further characterization revealed that the two antibodies are highly selective for HNE bound to histidine with only some cross reaction to HNE bound to lysine and cysteine. Preliminary quantitative ELISA-analysis showed that oxidized microsomes and oxidized LDL contain 12 nmol and 3 nmol HNE-histidine per mg protein respectively.     

Amino Acid Residues in the ω-Minus Region Participate in Cellular Localization of Yeast Glycosylphosphatidylinositol-Attached Proteins

The final destination of glycosylphosphatidylinositol (GPI)-attached proteins in Saccharomyces cerevisiae is the plasma membrane or the cell wall. Two kinds of signals have been proposed for their cellular localization: (i) the specific amino acid residues V, I, or L at the site 4 or 5 amino acids upstream of the GPI attachment site (the omega site) and Y or N at the site 2 amino acids upstream of the omega site for cell wall localization and (ii) dibasic residues in the region upstream of the omega site (the omega-minus region) for plasma membrane localization. The relationships between these amino acid residues and efficiencies of cell wall incorporation were examined by constructing fusion reporter proteins from open reading frames encoding putative GPI-attached proteins. The levels of incorporation were high in the constructs containing the specific amino acid residues and quite low in those containing two basic amino acid residues in the omega-minus region. With constructs that contained neither specific residues nor two basic residues, levels of incorporation were moderate. These correlations clearly suggest that GPI-attached proteins have two different signals which act positively or negatively in cell wall incorporation for their cellular localization.     

Amino-Acid Composition of Wood Proteins

The ammo-acid compositions of proteins present in the sapwoodand heartwood from three species of Eucalyptus, in normal, reaction,and opposite wood from Pinus radiata seedlings, and in reactionand opposite wood from Eucalyptus goniocalyx, are listed. Thequestion of the location of protein within wood cell walls isdiscussed. The amounts of tyrosine found in the woods from P.radiata seedlings are incompatible both with the belief thatit determines the intensity of staining of reaction wood cellwalls with Millon's reagent, and that it is the source of thep-hydroxybenzaldehyde obtained when milled wood lignin is oxidizedwith nitrobenzene. Loss of amino acids in the presence of milledwood lignin, -cellulose, hemicellulose, glucose, mannose, galactose,xylose, arabinose, glucuronic, and galacturonic acids is demonstrated.The presence of numerous peaks which are not indentifiable withknown amino acids in the chromatograms of wood hydrolysatesis believed to be due largely to the products of interactionof amino acids and sugars. Some retention of nitrogen by theresidues of wood hydrolysates occurs. The significance of theresults obtained with the wood samples listed above is discussedagainst this background.     

Microheterogeneous Cytosolic High-Mobility Group Proteins from Broccoli Co-Purify with and Are Phosphorylated by Casein Kinase II

A group of low molecular weight protein substrates was found to co-purify with casein kinase II from broccoli (Brassica oleracea var italica). These substrates showed very high affinity toward casein kinase II and were efficiently phosphorylated even in the presence of an excess of exogenous substrates. The broccoli substrates were purified from cytosolic extracts as a double band of related proteins migrating at 18.7 and 20 kD. Further microheterogeneity was revealed by anion-exchange high-performance liquid chromatography and mass spectroscopy. The actual molecular masses of the three major components identified by mass spectroscopy were determined to be 12,691, 13,256, and 14,128 D. The substrates showed characteristic amino acid composition with a high content of polar amino acids, including about 20% each of acidic and basic amino acids. They were soluble in 2% trichloroacetic acid. The substrates cross-reacted with an antibody against wheat high-mobility group protein d (HMGd) but not HMGa. The isolated broccoli HMGs showed general DNA-binding activity without preference for AT-rich DNA. The presence of these HMG proteins in the cytosolic fraction is similar to the distribution characteristics of the animal HMG-1 subgroup. On the basis of amino acid composition and DNA-binding specificity, the isolated broccoli HMGs resemble other plant HMGs homologous to the HMG-1 subgroup.     

Some of the amino acid chemistry going on in the Laboratory of Amino Acids, Peptides and Proteins

Some of the chemistry of amino acids going on in our laboratory (Laboratoire des Amino acides Peptides et Protéines) is described as well as some mass spectrometry methodology for their characterization particularly on solid supports. Several aspects are presented including: (i) the stereoselective synthesis of natural and unnatural amino acids using 2-hydroxypinan-3-one as chiral auxiliary; (ii) the stereoselective synthesis of natural and unnatural amino acids by deracemization of alpha-amino acids via their ketene derivatives; (iii) the synthesis of alpha-aryl-alpha-amino acids via reaction of organometallics with a glycine cation; (iv) the diastereoselective synthesis of glycosyl-alpha-amino acids; (v) the synthesis of beta-amino acids using alpha-aminopyrrolidinopiperazinediones as chiral templates; (vi) the reactivity of urethane-N-protected N-carboxyanhydrides. To characterize natural and non natural amino acids through their immonium ions by mass spectrometry, some methodology is also described.     

Photodynamic effects of protoporphyrin on human erythrocytes. Nature of the cross-linking of membrane proteins

Protoporphyrin-sensitized photooxidation in human red blood cell membranes leads to severe deterioration of membrane structure and function. The membrane damage is caused by direct oxidation of amino acid residues, with subsequent cross-linking of membrane proteins. The chemical nature of these cross-links was studied in model systems, isolated spectrin and red cell ghosts. Cysteine and methionine are not involved in the cross-linking reaction. Further it could be shown that dityrosine formation, the crucial mechanism in oxidative cross-linking of proteins by peroxidase-H2O2 treatment, plays no role in photodynamic cross-linking. Experimental evidence indicated that a secondary reaction between free amino groups and a photooxidation product of histidine, tyrosine or tryptophan is involved in photodynamic cross-linking. This was deduced from the reaction observed between compounds containing a free amino group and photooxidation products of these amino acids, both in model systems, isolated spectrin and erythrocyte ghosts. In accordance, succinylation of free amino groups of membrane proteins or addition of compounds with free amino groups protected against cross-linking. Quantitative data and consideration of the reaction mechanisms of photodynamic oxidation of amino acids make it highly probable that an oxidation product of histidine rather than of tyrosine or tryptophan is involved in the cross-linking reaction, via a nucleophilic addition by free amino groups.     

Mutual Amino Acid Catalysis in Salt-Induced Peptide Formation Supports this Mechanism's Role in Prebiotic Peptide Evolution

The presence of some amino acids and dipeptides under the conditions of the salt-induced peptide formation reaction (aqueous solution at 85 °C, Cu(II) and NaCl) has been found to catalyze the formation of homopeptides of other amino acids, which are otherwise produced only in traces or not at all by this reaction. The condensation of Val, Leu and Lys to form their homodipeptides can occur to a considerable extent due to catalytic effects of other amino acids and related compounds, among which glycine, histidine, diglycine and diketopiperazine exhibit the most remarkable activity. These findings also lead to a modification of the table of amino acid sequences preferentially formed by the salt-induced peptide formation (SIPF) reaction, previously used for a comparison with the sequence preferences in membrane proteins of primitive organisms     

Characterization of Transport Proteins for Aromatic Compounds Derived from Lignin: Benzoate Derivative Binding Proteins

In vitro growth experiments have demonstrated that aromatic compounds derived from lignin can be metabolized and represent a major carbon resource for many soil bacteria. However, the proteins that mediate the movement of these metabolites across the cell membrane have not been thoroughly characterized. To address this deficiency, we used a library representative of lignin degradation products and a thermal stability screen to determine ligand specificity for a set of solute-binding proteins (SBPs) from ATP-binding cassette (ABC) transporters. The ligand mapping process identified a set of proteins from Alphaproteobacteria that recognize various benzoate derivatives. Seven high-resolution crystal structures of these proteins in complex with four different aromatic compounds were obtained. The protein-ligand complexes provide details of molecular recognition that can be used to infer binding specificity. This structure-function characterization provides new insight for the biological roles of these ABC transporters and their SBPs, which had been previously annotated as branched-chain amino‐acid-binding proteins. The knowledge derived from the crystal structures provides a foundation for development of sequence-based methods to predict the ligand specificity of other uncharacterized transporters. These results also demonstrate that Alphaproteobacteria possess a diverse set of transport capabilities for lignin-derived compounds. Characterization of this new class of transporters improves genomic annotation projects and provides insight into the metabolic potential of soil bacteria.     

From selective substrate analogue factor Xa inhibitors to dual inhibitors of thrombin and factor Xa. Part 3

Highly potent and selective substrate analogue factor Xa inhibitors were obtained by incorporation of non-basic or modestly basic P1 residues known from the development of thrombin inhibitors. The modification of the P2 and P3 amino acids strongly influenced the selectivity and provided potent dual factor Xa and thrombin inhibitors without affecting the fibrinolytic enzymes. Several inhibitors demonstrated excellent anticoagulant efficacy in standard clotting assays in human plasma.     

Obtaining a protein concentrate from integral defatted sunflower flour

Proteins which are found in integral defatted sunflower flour (27% of protein in dry weight) allow us to produce a protein concentrate by means of extraction of proteins with a basic pH solution, followed by their precipitation with an acid pH solution. Once the suitable conditions for pH and temperature were fixed in order to carry out these processes, a solid proteic concentrate (71% of protein in dry weight) was obtained which was rich in glutamic and aspartic acids, with a liquid supernatant very rich in phosphorus and potassium, which might be used as an agricultural fertilizer.