AMINO ACID COMPOSITIONS AND N-TERMINAL AMINO ACID OF ALL THE TRYPTIC PEPTIDES FROM THE α AND β POLYPEPTIDE CHAINS OF ADULT HEMOGLOBIN OF THE JAPANESE MONKEY (Macaca fuscata fuscata)

Globin prepared from adult hemoglobin of the Japanese monkey was separated into α and β polypeptide chains by the countercurrent distribution method. All the tryptic peptides obtained from these polypeptide chains were isolated and purified by column and paper chromatography. The amino acid compositions and the N-terminal amino acid of these tryptic peptides were analyzed. The results thus obtained suggested amino acid exchanges between Japanese monkey and human hemoglobin, that is, at four positions in α polypeptide chain and seven positions in β polypeptide chain. In comparison with the α and β polypeptide chains of adult hemoglobin from the rhesus monkey, the α chain is quite the same, and it is considered that there is one amino acid exchange in the β chains. The outline of this work was presented at Symposium 5 (Biochemical Polymorphisms in Man and Other Primates and their Anthropological Implications) of the 8th International Congress of Anthropological and Ethnological Science (3–10 September 1968, Tokyo     

SEQUENCE STUDIES ON THE TRYPTIC PEPTIDES AND THE CHYMOTRYPTIC PEPTIDES FROM THE α POLYPEPTIDE CHAIN OF All COMPONENT OF THE CHICKEN HEMOGLOBIN

Tryptic peptides and chymotryptic peptides from a polypeptide chain in All component of adult chicken hemoglobin were isolated and purified by using column chromatography and paper chromatography. Furthermore amino acid sequence analyses of these peptides were performed mainly by redigestion with enzyme, the PTC method and the DNP method. From the result of these experiments, the primary structure of this a polypeptide chain was determined. That is, a polypeptide chain in All component from adult chicken hemoglobin consisted of 141 amino acids. In comparison with the primary structure of a polypeptide chain from adult human hemoglobin, amino acid sequence exchanges were found at 35 positions, and with a polypeptide chain from horse hemoglobin, amino acid sequence exchanges were found at 40 positions.     

THE PRIMARY STRUCTURES OF α AND β CHAINS OF ADULT HEMOGLOBIN OF THE JAPANESE MONKEY (Macata fuscata fuscata)

Amino acid sequence of the tryptic peptides from the α and β chains of adult hemoglobin of the Japanese monkey were analyzed by partial hydrolyses with enzymes, the DNP method, PTC method and others after the isolation and purification of these peptides. Furthermore, the primary structures of the α and β chains of adult hemoglobin from the Japanese monkey were obtained by homology between these results and those of human adult hemoglobins. These primary structures show a high similarity to the primary structures of the α and β chains of adult hemoglobins from the human and rhesus monkey.     

THE SOLID-PHASE SYNTHESIS OF α-MELANOTROPIN HYDRAZIDE

The solid-phase synthesis of α-melanotropin hydrazide, a new analog of α-MSH, is described. Cleavage of the partially protected peptide from the solid support was accomplished with hydrazine in dimethylformamide. The protecting groups were removed with sodium in liquid ammonia and the product was purified by chromatography on carboxymethylcellulose to give a highly purified and melanotropically active tridecapeptide.     

Automated synthesis of 11C‐β‐hydroxybutyrate by enzymatic conversion of 11C‐acetoacetate using β‐hydroxybutyrate dehydrogenase

1‐[¹¹C]‐β‐hydroxybutyrate was produced by conversion from 1‐[¹¹C]‐acetoacetate using (D)‐β‐hydroxybutyrate dehydrogenase in the presence of nicotinamide adenine dinucleotide with purification by ion exchange column chromatography. Radiochemical yield at the end of the synthesis was 10% for a total synthesis time of 36 min. High‐performance liquid chromatography analysis showed ≤4% impurities, principally unconverted acetoacetate. Residual tetrahydrofuran (34±11 ppm) and ethanol (77±30 ppm) were well under the tolerable limits for human studies. Copyright © 2008 John Wiley & Sons, Ltd.     

SOLID PHASE SYNTHESIS OF THE PROTECTED 27 – 42 HEXADECAPEPTIDE OF THE HEAVY CHAIN FROM MYELOMA IMMUNOGLOBULIN M603 Elimination of Side Reactions Associated with Glycyl-2-oxypropionyl-resin

A fully protected 27–42 hexadecapeptide of the variable region of myeloma immunoglobulin M603 was synthesized on a 2-bromopropionyl-resin by the solid phase method. Side reactions due to cyclization of glycyl-2-oxypropionylresin were studied under different reaction conditions. The loss of peptide chains at the dipeptide and tripeptide stages due to diketopiperazine formation was also examined. These side reactions were circumvented by using a combination of fragment and stepwise coupling methods. The synthesized protected peptide was removed from the resin in 85% yield by photolysis, and purified by crystallization and by chromatography on a Sephadex LH-60 column.     

Analysis of γβ, βγ, γγ, ββ continuous turns in proteins

Abstract: We report the observation of continuous turns in proteins which comprise individual γ‐turns or β‐turns or both that are situated immediately one after the other along the polypeptide chain. The continuous turns were identified from a representative data set of three‐dimensional protein crystal structures. The γβ/βγ, γγ and ββ continuous turns represent peptides of varying amino acid residue lengths and conformations. The continuous turns frequently observed in proteins were: γβ, between a coil and a strand; βγ, between a helix and a strand; γγ, between coils; and ββ, either between a strand and a coil or between strands or coils. We determined the statistically significant amino acid residue preferences at individual positions in the turn, calculated amino acid positional potentials and analyzed main chain hydrogen bonds and side‐chain interactions likely to stabilize the continuous turns. The data on continuous turns have been integrated in the database of structural motifs in proteins (DSMP) on our web server at ( http://www.cdfd.org.in/dsmp.html ). This is useful to make queries on sequences compatible with different continuous turns.     

Properties of a Resiniferatoxin-stimulated,Calcium Inhibited but Phosphatidylserine-dependent Kinase,which is Distinct from Protein Kinase C Isotypes α, β1γ, δ, ε and η

We have separated a resiniferatoxin-stimulated histone-kinase activity from human neutrophils, elicited mouse macrophages and murine alveolar macrophages by hydroxyapatite chromatography. The assay conditions for resiniferatoxin kinase were optimized as part of this study and in the presence of phosphatidylserine but absence of Ca²⁺ the K_a for histone IIIs phosphorylation by resiniferatoxin was calculated as 16 nm . Using a phosphate gradient of 20–500 mm , peaks of protein kinase C activity could be washed from the hydroxyapatite column in 300 nm phosphate and resiniferatoxin kinase recovered in 500 mm phosphate. At the optimum concentration of 160 nm , the ability of resiniferatoxin to induce enzyme activity was compared with a range of phorbol esters all at the same concentration. These related compounds failed to activate resiniferatoxin kinase although they have previously been shown to activate protein kinase C isotypes. Similarly sn-1,2,-dioleoylglycerol and the potent irritant capsaicin at 30 μm failed to activate the kinase. A Scatchard analysis of [³H] phorbol dibutyrate binding produced a linear plot (K_d 41·6 nm ; B_max 11·6 fmol unit^?1) and binding was inhibited by resiniferatoxin and 12-O-tetradecanoylphorbol-13-acetate (TPA), with resiniferatoxin 700 times more potent than TPA in this respect. A radiolabeled resiniferatoxin binding assay was also used to demonstrate specific binding of [³H]resiniferatoxin which could be inhibited by unlabelled compound. Resiniferatoxin kinase activity was shown to be distinct from the protein kinase C isotypes α, β₁, γ δ and ε by means of immunological analysis and from the η isotype, because that isotype was not stimulated by resiniferatoxin but was stimulated by TPA when a pseudosubstrate was used. In addition the resiniferatoxin-stimulated activity was inhibited in-vitro by the addition of Ca²⁺ (K_i 0·1-0·5 nm free Ca²⁺). Further purification of resiniferatoxin kinase by Superose chromatography indicated a major activity fraction of about 70–90 kDa. Thus resiniferatoxin kinase, isolated from human and mouse inflammatory cells is distinct from the known isotypes of protein kinase C and is a major resiniferatoxin receptor.     

QUANTITATION OF THE β-ELIMINATION REACTION AS USED ON GLYCOPROTEINS

The carbohydrate side chains of mucus-type glycoproteins are O-glycosidic bonds between N-acetylgalactosamine to the hydroxyl groups of serine and threonine in the protein core. The alkaline catalyzed β-elimination reaction, in the presence of sodium borohydride, is used for determining the number of side chains. The present paper presents a study of the quantitativeness of the alkaline borohydride procedure, using four parameters: the loss of seryl and threonyl residues, the formation of alanine and 2-aminobutanoic acid; the decrease in N-acetylhexosamine and the recovery of the amino sugar alcohols. Bovine, ovine and porcine submandibular glycoproteins were studied. Evidence is presented for the existence of N-acetylglucosamine involvement in O-glycosidic linkages to serine and threonine. Results for the relative rates of β-elimination indicate that serine-linked glycosides are released more rapidly than threonine-linked glycosides.     

Reversed phase thin-layer chromatography for the separation of β-endorphin, β-lipotropin and enkephalins

A novel, simple technique using reversed phase thin-layer chromatography has been developed to separate β-endorphin, β-lipotropin and Met-and Leuenkephalins. This method is useful for the rapid determination of the purity of these opioid peptides. The effectiveness of several solvent systems has been assessed. The resolution between β-endorphin and β-lipotropin achieved by this method exceeds that reported using high performance liquid chromatography or a variety of column techniques. This system also eliminates the problem of unrecognized loss of peptide due to binding on HPLC or other types of columns since the entire stationary phase is visualized using fluorescamine detection. Efficient, inexpensive and reliable simultaneous separation with visualization of several opioid peptides, their peptide precursors and their degradation products is now possible using the new technique. Such separations are required for many purposes including preparation of samples for radioimmunoassay of opioid peptides.     

ISOLATION AND CHARACTERIZATION OF β-LIPOTROPIN FROM THE PITUITARY GLAND OF THE OSTRICH,STRUTHIO CAMELUS

Avian β-lipotropin (β-LPH) was purified from adenohypophyseal glands of the ostrich Struthio camelus by a procedure involving acid/acetone extraction, NaCl fractionation, CM-cellulose chromatography, Sephadex G-75 chromatography and paper electrophoresis (pH 6.7). The 90-amino acid peptide behaved as a single substance during polyacrylamide-gel electrophoresis, isoelectric focusing (pI of 6.0) and N-terminal analysis, the N-terminal amino acid being alanine. Ostrich β-LPH exhibited lipolytic activity corresponding to an average minimal effective dose of 0.088 μg in rabbit adipose tissue.     

Comparison of the binding of α-helical and β-sheet peptides to a hydrophobic surface

The induction and stabilisation of secondary structure for a series of amphipathic α-helical and β-sheet peptides upon their binding to lipid-like surfaces has been characterised by reversed phase high-performance liquid chromatography (RP-HPLC). In addition, a series of peptides which have been shown to switch from β-sheet to α-helical conformation upon transfer from a polar to a non-polar solution environment also have been studied. Binding parameters related to the hydrophobic contact area and affinity for immobilised C18 chains were determined at temperatures that ranged from 5 to 85°C, allowing conformational transitions for the peptides during surface adsorption to be monitored. The results demonstrated that all peptides which adopt secondary structure in solution also exhibited large changes in their interactive properties. Overall, this study demonstrates that the hydrophobic face of each amphipathic peptide dominates the binding process and that hydrophobic interactions are a major factor controlling the surface induction of secondary structure.     

Prediction of γ‐turns from amino acid sequences

Abstract: We predicted γ‐turns from amino acid sequences using the first‐order Markov chain theory and enlarged representative data sets corresponding to protein chains selected from the Protein Data Bank (PDB). The following data sets were used for training and deriving the probability values: (1) an initial data set containing 315 protein chains comprising 904 γ‐turns and (2) a later data set in order to include new entries in the PDB, containing 434 protein chains and comprising 1053 γ‐turns. By excluding 93 protein chains that were common to these two training data sets, we generated two mutually exclusive data sets containing 222 and 341 protein chains for testing our predictions. Applying amino acid probability values derived from training data sets on to testing data sets yielded overall prediction accuracies in the range 54–57%. We recommend the use of probability values derived from the data set comprising 315 protein chains that represents more γ‐turns and also provides better predictions.     

Integrin α5β1: a new purification strategy based on immobilized peptides

Abstract: Novel efficient and robust affinity chromatography material: There are several strategies known for the purification of integrins by affinity chromatography, but the disadvantages of common strategies like insufficient selectivity or compelling conditions for the elution still require alternatives. A new strategy, based on the immobilized C‐terminally modified peptide Ac‐Gly‐Ala‐c‐(Cys^SS‐Arg‐Arg‐Glu‐Thr‐Ala‐Trp‐Ala‐Cys^SS)‐Gly‐Ala‐O(CH₂CH₂O)₂CH₂CH₂‐NH₂ allows for the affinity purification of the integrin α₅β₁. While RGD peptides have been proven in the past to be inappropriate for selective purification of integrins by affinity chromatography, the new peptide can be efficiently used for selective enrichment of the integrin α₅β₁. It is a specific ligand of the target protein, but does not contain an RGD sequence. The application of well‐characterized affinity chromatography material with a site‐specifically immobilized peptide allows to obtain integrin α₅β₁ in a single chromatography step without contamination by other integrins. This process combines the advantages of a selective and monospecific protein‐ligand recognition with mild elution conditions and a low sensitivity of the immobilized ligand with respect to column regeneration.     

THE PH DEPENDENCE OF BINDING OF α, α‘-DIBROMO-P-XYLENESULFONIC ACID TO LYSOZYME

The chemical modification of lysozyme (I) has been accomplished with α,α'-dibromo-p-xylenesulfonic acid (DBX) at five different pH values. I was alkylated by DBX at room temperature (28°C) with decrease in enzyme activity. The rate of inactivation depended upon the pH at which alkylation was carried out. The highest rate was seen at alkaline pH values; the lowest at more acidic pH values. Amino acid analyses showed that two lysines and two tryptophan residues had been modified at pH 9; two lysines, one tryptophan and one methionine had reacted at pH 8. A histidine residue was bound at pH 6.5 together with a tryptophan residue. At the lower pH values (2.7, 4.5, 6.5), alkylation occurred with a single tryptophan residue each. Fluorescence and CD data both ruled out the participation of tryptophans 62 or 108. Labeling experiments showed that two residues of DBX-³⁵S were bound per molecule of I at both pH 9 and pH 8; one residue of DBX was bound per molecule of I at the other pH values. Sedimentation coefficients were characteristic of native lysozyme. The stoichiometry of binding and residue modification indicated that intra-molecular cross links were established. The pH dependence of the cross-linking provides means to measure several allowed intramolecular distances. The results presented here are consistent with the existence of side chain motion in lysozyme.     

Synthesis of [3α‐3H] 17α‐Hydroxy pregnenolone and [3α‐3H] Pregnenolone

For the first time, [3α‐³H] 17α‐hydroxy pregnenolone (1) was synthesized through a multiple step sequence. The presence of [3β‐³H] isomer in RP‐HPLC purified product was identified by tritium NMR. The [3β‐³H] isomer was then separated from [3α‐³H] 17α‐hydroxy pregnenolone with chiralPAK AD‐H column. [3α‐³H] pregnenolone (2) was synthesized from commercial available 5‐pregnen‐3,20‐dione in one step with an improved procedure.     

Isolation and identification of thymosin α-1 from calf spleen using high performance liquid chromatography

A peptide isolated from calf spleen has been identified as thymosin α₁. The isolation involved defatting of the desiccated glands with acetone, extraction of the acetone power with pyridine acetate pH 5.5, heat denaturation, reverse phase chromatography on an RP-8 column, anion exchange chromatography on a Partisil SAX column and, finally, reverse phase purification on a μBondapak C₁₈ column. The identification was based on: 1) amino acid analysis; 2) thermolysin digest; and 3) retention time in two different HPLC systems. The amount isolated from the spleen was 10–20% of that isolated from calf thymus glands.     

PREPARATION OF TRITIATED α-MELANOTROPIN WITH HIGH SPECIFIC RADIOACTIVITY

α-Metanotropin (α-MSH) was iodinated and 3,5-diiodotyr² -α-MSH was isolated by reverse phase high performance liquid chromatography (HPLC). Catalytic dehalogenation of the diiodo derivative in the presence of tritium resulted in the formation of 3,5-ditritiotyr² -α-MSH. The tritiated peptide was purified by ion exchange and partition chromatography. The radioactive peptide was found to be homogeneous and identical to α-MSH by paper electrophoresis and HPLC. The tritiated α-MSH stimulated lipolysis in rabbit adipocytes nearly as well as α-MSH. The specific radioactivity of tritiated α-MSH was 42 Ci/mmol or 73% of the theoretical value.     

CONFORMATION CHANGES IN α-HELICAL MUSCLE PROTEINS AFTER REACTION WITH MALEIC ANHYDRIDE

The conformation changes on reaction of maleic anhydride with two tropomyosins (vertebrate and invertebrate) and paramyosin are reported. Maleylation of tropomyosin leads to more than 90% loss of the α-helical conformation and dissociation into subunits. Only one third of the helix is lost on maleylation of paramyosin and it is not dissociated into subunits. The ORD and CD spectra of maleyl tropomyosin are reported and compared with the spectra of other unordered polypeptide chains. The α-helix can be restored after maleylation of tropomyosin by increase of the ionic strength, variation of pH, or addition of helix-forming solvents such as ethanol, 2-chloroethanol, and trifluoroethanol.     

Expedient synthesis of [18F]‐labeled α‐trifluoromethyl ketones

Several [¹⁸F]‐labeled α‐trifluoromethyl ketones have been synthesized. Reactions of 2,2‐difluoro‐1‐aryl‐1‐trimethylsiloxyethenes ( 1a–d ) with [¹⁸F]‐F₂ at low temperature produced [¹⁸F]‐labeled α‐trifluoromethyl ketones ( 2a–d ). Radio‐labeled products were isolated by purification with column chromatography in 22–28% yields, decay corrected (d.c.) in three runs per compound. Radiochemical purity was >99% with specific activities 15–20 GBq/mmol at the end of synthesis (EOS). The synthesis time was 35–40 min from the end of bombardment (EOB). This one‐step simple method is highly useful for the radiochemical synthesis of potential biologically active [¹⁸F]‐labeled α‐trifluoromethyl ketones for PET imaging. Copyright © 2003 John Wiley & Sons, Ltd.