The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.     

Amino acid sequence of a protease inhibitor isolated from Sarcophaga bullata determined by mass spectrometry.

The amino acid sequence of a protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae was determined by tandem mass spectrometry. Homology considerations with respect to other protease inhibitors with known primary structures assisted in the choice of the procedure followed in the sequence determination and in the alignment of the various peptides obtained from specific chemical cleavage at cysteines and enzyme digests of the S. bullata protease inhibitor. The resulting sequence of 57 residues is as follows: Val Asp Lys Ser Ala Cys Leu Gln Pro Lys Glu Val Gly Pro Cys Arg Lys Ser Asp Phe Val Phe Phe Tyr Asn Ala Asp Thr Lys Ala Cys Glu Glu Phe Leu Tyr Gly Gly Cys Arg Gly Asn Asp Asn Arg Phe Asn Thr Lys Glu Glu Cys Glu Lys Leu Cys Leu.     

Amino acid sequence of seminalplasmin, an antimicrobial protein from bull semen

Analytical ultracentrifugation of highly purified seminalplasmin revealed a molecular mass of 6300. Amino acid analysis of the protein preparation indicated the absence of sulfur-containing amino acids cysteine and methionine. The amino acid sequence of seminalplasmin was determined by manual Edman degradation of peptides obtained by proteolytic enzymes trypsin, chymotrypsin and thermolysin: NH₂-Ser Asp Glu Lys Ala Ser Pro Asp Lys His His Arg Phe Ser Leu Ser Arg Tyr Ala Lys Leu Ala Asn Arg Leu Ser Lys Trp Ile Gly Asn Arg Gly Asn Arg Leu Ala Asn Pro Lys Leu Leu Glu Thr Phe Lys Ser Val-COOH. The number of amino acids according to the sequence were 48, the molecular mass 6385. As predicted from the sequence, seminalplasmin very likely contains two α-helical domains in which residues 8-17 and 40-48 are involved. No evidence for the existence of β-sheet structures was obtained. Treatment of seminalplasmin with the above proteases as well as with amino peptidase M and carboxypeptidase Y completely eliminated biological activity.     

The primary structure of the β-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.     

Effects of orally administrated amino acids on myofibrillar proteolysis in chicks

We examined the effects of orally administrated amino acids on myfibrillar proteolysis in food-deprived chicks. Plasma N(tau)-methylhistidine concentration, as an index of myofibrillar proteolysis, was decreased by the administration of Glu, Gly, Ala, Leu, Ile, Ser, Thr, Met, Trp, Asn, Gln, Pro, Lys and Arg but not by Asp, Val, Phe, Tyr or His to chicks. Orally administrated Cys was fatal to chicks. These results indicate that oral Glu, Gly, Ala, Leu, Ile, Ser, Thr, Met, Trp, Asn, Gln, Pro, Lys and Arg administration suppressed myofibrillar proteolysis in chicks.     

Polymers produced by heating an amino acid mixture in sea water enriched with transition elements

A mixture of eighteen protein amino acids was heated in sea water medium enriched with transition metal ions. Small granules were obtained as precipitates. Both dialyzable polymers and undialyzable polymers were obtained from the supernatant. Dialyzable polymers yielded mainly Glu, Asp, Ser, and Thr on hydrolysis; undialyzable polymers (C, 29.45; H, 3.87; N, 4.87; and ash, 31.5 wt%) yielded Thr, Asp, Glu, Gly, Leu, Ser, Lys, Pro, His, Phe, and a few unidentified ninhydrin positive peaks after acid hydrolysis. Five wt% of the undialyzable polymers consist of acid-hydrolyzable protein amino acids.     

Substitution of aspartic acid with glutamic acid increases the unfolding transition temperature of a protein

Proteins from thermophiles are more stable than those from mesophiles. Several factors have been suggested as causes for this greater stability, but no general rule has been found. The amino acid composition of thermophile proteins indicates that the content of polar amino acids such as Asn, Gln, Ser, and Thr is lower, and that of charged amino acids such as Arg, Glu, and Lys is higher than in mesophile proteins. Among charged amino acids, however, the content of Asp is even lower in thermophile proteins than in mesophile proteins. To investigate the reasons for the lower occurrence of Asp compared to Glu in thermophile proteins, Glu was substituted with Asp in a hyperthermophile protein, MjTRX, and Asp was substituted with Glu in a mesophile protein, ETRX. Each substitution of Glu with Asp decreased the Tm of MjTRX by about 2 degrees C, while each substitution of Asp with Glu increased the Tm of ETRX by about 1.5 degrees C. The change of Tm destabilizes the MjTRX by 0.55 kcal/mol and stabilizes the ETRX by 0.45 kcal/mol in free energy.     

Polymers produced by heating an amino acid mixture in sea water enriched with transition elements.

A mixture of eighteen protein amino acids was heated in sea water medium enriched with transition metal ions. Small granules were obtained as precipitates. Both dialyzable polymers and undialyzable polymers were obtained from the supernatant. Dialyzable polymers yielded mainly Glu, Asp, Ser, and Thr on hydrolysis; undialyzable polymers (C, 29.45; H, 3.87; N, 4.87; and ash, 31.5 wt%) yielded Thr, Asp, Glu, Gly, Leu, Ser, Lys, Pro, His, Phe, and a few unidentified ninhydrin positive peaks after acid hydrolysis. Five wt% of the undialyzable polymers con-ist of acid-hydrolyzable protein amino acids.     

Mechanistic investigations of the dehydration reaction of lacticin 481 synthetase using site-directed mutagenesis

Lantibiotic synthetases catalyze the dehydration of Ser and Thr residues in their peptide substrates to dehydroalanine (Dha) and dehydrobutyrine (Dhb), respectively, followed by the conjugate addition of Cys residues to the Dha and Dhb residues to generate the thioether cross-links lanthionine and methyllanthionine, respectively. In this study ten conserved residues were mutated in the dehydratase domain of the best characterized family member, lacticin 481 synthetase (LctM). Mutation of His244 and Tyr408 did not affect dehydration activity with the LctA substrate whereas mutation of Asn247, Glu261, and Glu446 considerably slowed down dehydration and resulted in incomplete conversion. Mutation of Lys159 slowed down both steps of the net dehydration: phosphorylation of Ser/Thr residues and the subsequent phosphate elimination step to form the dehydro amino acids. Mutation of Arg399 to Met or Leu resulted in mutants that had phosphorylation activity but displayed greatly decreased phosphate elimination activity. The Arg399Lys mutant retained both activities, however. Similarly, the Thr405Ala mutant phosphorylated the LctA substrate but had compromised elimination activity. Finally, mutation of Asp242 or Asp259 to Asn led to mutant enzymes that lacked detectable dehydration activity. Whereas the Asp242Asn mutant retained phosphate elimination activity, the Asp259Asn mutant was not able to eliminate phosphate from a phosphorylated substrate peptide. A model is presented that accounts for the observed phenotypes of these mutant enzymes.     

Metabolism of some amino acids in relation to the photorespiratory nitrogen cycle of pea leaves

¹⁵N-labelled (amino group) asparagine (Asn), glutamate (Glu), alanine (Ala), aspartate (Asp) and serine (Ser) were used to study the metabolic role and the participation of each compound in the photorespiratory N cycle ofPisum sativum L. leaves. Asparagine was utilised as a nitrogen source by either deamidation or transamination, Glu was converted to Gln through NH₃ assimilation and was a major amino donor for transamination, and Ala was utilised by transamination to a range of amino acids. Transamination also provided a pathway for Asp utilisation, although Asp was also used as a substrate for Asn synthesis. In the photorespiratory synthesis of glycine (Gly), Ser, Ala, Glu and Asn acted as sources of amino-N, contributing, in the order given, 38, 28, 23, and 7% of the N for glycine synthesis; Asp provided less than 4% of the amino-N in glycine. Calculations based on the incorporation of¹⁵N into Gly indicated that about 60% (Ser), 20% (Ala), 12% (Glu) and 11% (Asn) of the N metabolised from each amino acid was utilised in the photorespiratory nitrogen cycle.Abbreviations Ala alamine - Asn asparagine - Asp aspartate - Glu glutamate - MOA methoxylamine - Ser serine     

Subcellular distribution of aminoacyl-transfer RNA synthetases in Chinese hamster ovary cell culture

Chinese hamster ovary cells grown in cell culture were broken and fractionated by differential centrifugation. Four principal fractions: nuclear and membrane, microsomal, postribosomal, and supernatant were obtained. The distribution of aminoacyl-tRNA synthetases in these four fractions was determined for all twenty amino acids.It was shown that there is a differential distribution of synthetases. Activities specific for eight amino acids: Ala, Ser, Gly, Cys, His, Arg, Thr and Pro were found mainly in the supernatant fraction. Activities specific for eleven amino acids: Asp, Asn, Glu, Gln, Ile, Leu, Lys, Met, Phe, Tyr and Val were found mainly in the postribosomal fraction. Four activities were found at significant levels in the microsomal fraction: Asp, Phe, Lys and Pro. The nuclear and membrane fraction contained activity for Lys, His, Asp and Thr.Changes in aminoacyl-tRNA synthetase activities in various fractions from preparations made by breaking cells with a membrane-dissociating detergent showed that some of the aminoacyl-tRNA synthetase activities may be membrane bound.     

Preparation of soluble peptide libraries: application to studies of platelet adhesion sequences.

We describe chemical synthesis of peptide mixtures that equally express many sequence combinations. Using 65 couplings of single amino acids, five mixtures were prepared with the sequences Tyr-Gly-Arg-Gly-Yyy-Xxx-Xxx, where Yyy is Ser, Asp, Arg, Asn, or Glu, and Xxx is any amino acids. Compositional and sequence analyses supported full representation of all amino acids, except isoleucine was deficient in the sixth position. The data suggest formation of a repertoire of 1,900 sequence combinations (5 x 19 x 20). The mixture with Asp as the fifth residue inhibited platelet adhesion to fibronectin more effectively than the other mixtures. Peptide libraries offer a new tool for investigating bioactive peptides.     

Thermal denaturation of eukaryotic class 1 translation termination factor eRF1. Relationship between stability of the eRF1 molecule and alteration of functional activity of its mutants

Thermal denaturation of eukaryotic class-1 translation termination factor eRF1 and its mutants was examined using differential scanning microcalorimetry (DSK). Changes of free energy caused by mutants in the N domain of human eRF1 were calculated. Melting of eRF1 molecule composed of three individual domains is cooperative. Some amino acid substitutions did not affect protein thermostability and in some other cases even slightly stabilize the protein globule. These imply that these amino acid residues are not involved in maintenance of the 3D structure of human eRF1. Thus, in Glu55Asp, Tyr125Phe, Asn61Ser, Glu55Arg, Glu55A1a, Asn61Ser + Ser64Asp, Cys127Ala and Ser64Asp mutants selective inactivation of release activity is not caused by a destabilization of protein 3D structure and, most likely, is associated with local stereochemical changes introduced by substitutions of amino acid side chains in the functionally essential sites of N-domain molecule. Some residues (Asn129, Phe131) as shown by calorimetric measurements are essential for preservation of stable protein structure, but at the same time they affect selective stop codon recognition probably via their neighboring amino acids. Recognition of UAG and UAA stop codons in vitro is more sensitive to preservation of protein stability than the UGA recognition.     

Characterization of the active site and thermostability regions of endoxylanase from Thermoanaerobacterium saccharolyticum B6A-RI.

Deletion mutants were constructed from pZEP12, which contained the intact Thermoanaerobacterium saccharolyticum endoxylanase gene (xynA). Deletion of 1.75 kb from the N-terminal end of xynA resulted in a mutant enzyme that retained activity but lost thermostability. Deletion of 1.05 kb from the C terminus did not alter thermostability or activity. The deduced amino acid sequence of T. saccharolyticum B6A-RI endoxylanase XynA was aligned with five other family F beta-glycanases by using the PILEUP program of the Genetics Computer Group package. This multiple alignment of amino acid sequences revealed six highly conserved motifs which included the consensus sequence consisting of a hydrophobic amino acid, Ser or Thr, Glu, a hydrophobic amino acid, Asp, and a hydrophobic amino acid in the catalytic domain. Endoxylanase was inhibited by EDAC [1-(3-dimethylamino propenyl)-3-ethylcarbodiimide hydrochloride], suggesting that Asp and/or Glu was involved in catalysis. Three aspartic acids, two glutamic acids, and one histidine were conserved in all six enzymes aligned. Hydrophobic cluster analysis revealed that two Asp and one Glu occur in the same hydrophobic clusters in T. saccharolyticum B6A-RI endoxylanase and two other enzymes belonging to family F beta-glycanases and suggests their involvement in a catalytic triad. These two Asp and one Glu in XynA from T. saccharolyticum were targeted for analysis by site-specific mutagenesis. Substitution of Asp-537 and Asp-602 by Asn and Glu-600 by Gln completely destroyed endoxylanase activity. These results suggest that these three amino acids form a catalytic triad that functions in a general acid catalysis mechanism.     

Sequences of tryptic peptides containing the five cysteinyl residues of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Tryptic peptides which account for all five cysteinyl residues in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have been purified and sequenced. Collectively, these peptides contain 94 of the approximately 500 amino acid residues per molecule of subunit. Due to one incomplete cleavage at a site for trypsin and two incomplete chymotryptic-like cleavages, eight major radioactive peptides (rather than five as predicted) were recovered from tryptic digests of the enzyme that had been carboxymethylated with [³H]iodoacetate. The established sequences are: GlyTyrThrAlaPheValHisCys¹Lys TyrValAspLeuAlaLeuLysGluGluAspLeuIleAla GlyGlyGluHisValLeuCys¹AlaTyr AlaGlyTyrGlyTyrValAlaThrAlaAlaHisPheAla AlaGluSerSerThrGlyThrAspValGluValCys¹ ThrThrAsxAsxPheThrArg AlaCys¹ThrProIleIleSerGlyGlyMetAsnAla LeuArg ProPheAlaGluAlaCys¹HisAlaPheTrpLeuGly GlyAsnPheIleLys In these peptides, radioactive carboxymethylcysteinyl residues are denoted with asterisks and the sites of incomplete cleavage with vertical wavy lines. None of the peptides appear homologous with either of two cysteinyl-containing, active-site peptides previously isolated from spinach ribulosebisphosphate carboxylase/oxygenase.     

NH2-terminal sequences of DNA-, heparin-, and gelatin-binding tryptic fragments from human plasma fibronectin

NH₂-terminal sequence analysis was performed on subregions of human plasma fibronectin including 24,000-dalton (24K) DNA-binding, 29,000-dalton (29K) gelatin-binding, and 18,000-dalton (18K) heparin-binding tryptic fragments. These fragments were obtained from fibronectin after extensive trypsin digestion followed by sequential affinity purification on gelatin-Sepharose, heparin-agarose, and DNA-cellulose columns. The gelatin-binding fragment was further purified by gel filtration on Sephadex G-100, and the DNA-binding and heparin-binding fragments were further purified by high-performance liquid chromatography. The 29K fragment had the following NH₂-terminal sequence: AlaAlaValTyrGlnProGlnProHisProGlnProPro (Pro)TyrGlyHis HisValThrAsp(His)(Thr)ValValTyrGly(Ser) ?(Ser)?-Lys. The NH₂-terminal sequence of a 50K, gelatin-binding, subtilisin fragment by L. I. Gold, A. Garcia-Pardo, B. Prangione, E. C. Franklin, and E. Pearlstein (1979, Proc. Nat. Acad. Sci. USA76, 4803–4807) is identical to positions 3–19 (with the exception of some ambiguity at position 14) of the 29K fragment. These data strongly suggest that the 29K tryptic fragment is included in the 50K subtilisin fragment, and that subtilisin cleaves fibronectin between the Ala²Val³ residues of the 29K tryptic fragment. The 18K heparin-binding fragment had the following NH₂-terminal sequence: (Glu)AlaProGlnProHisCysIleSerLysTyrIle LeuTyrTrpAspProLysAsnSerValGly?(Pro) LysGluAla?(Val)(Pro). The 29K gelatin-binding and 18K heparin-binding fragments have proline-rich NH₂-terminal sequences suggesting that they may have arisen from protease-sensitive, random coil regions of fibronectin corresponding to interdomain regions preceding macromolecular-binding domains. Both of these fragments contain the identical sequence ProGlnProHis, a sequence which may be repeated in other interdomain regions of fibronectin. The 24K DNA-binding fragment has the following NH₂-terminal sequence: SerAspThrValProSerProCysAspLeuGlnPhe ValGluValThrAspVal LysValThrIleMetTrpThrProProGluSerAla ValThrGlyTyrArgVal AspValCysProValAsnLeuProGlyGluHisGly Gln(Cys)LeuProIleSer. The sequence of positions 9–22 are homologous to positions 15–28 of the α chain of DNA-dependent RNA polymerase from Escherichia coli. The homology observed suggests that this stretch of amino acids may be a DNA-binding site.     

Conformational rearrangement of beta-lactoglobulin upon interaction with an anionic membrane

The recent availability of the SHV-1 beta-lactamase crystal structure provides a framework for the understanding of the functional role of amino acid residues in this enzyme. To that end, we have constructed by site-directed mutagenesis 18 variants of the SHV beta-lactamase: an extended spectrum group: Gly238Ser, Gly238Ser-Glu240Lys, Asp104Lys-Gly238Ser, Asp104Lys-Thr235Ser-Gly238Ser, Asp179Asn, Arg164His, and Arg164Ser; an inhibitor resistant group: Arg244Ser, Met69Ile, Met69Leu, and Ser130Gly; mutants that are synergistic with those that confer resistance to oxyimino-cephalosporins: Asp104Glu, Asp104Lys, Glu240Lys, and Glu240Gln; and structurally conserved mutants: Thr235Ser, Thr235Ala and Glu166Ala. Among the extended spectrum group the combination of high-level ampicillin and cephalosporin resistance was demonstrated in the Escherichia coli DH10B strains possessing the Gly238Ser mutation: Gly238Ser, Gly238Ser-Glu240Lys, Asp104Lys-Gly238Ser, and Asp104Lys-Thr235Ser-Gly238Ser. Of the inhibitor resistant group, the Ser130Gly mutant was the most resistant to ampicillin/clavulanate. Using a polyclonal anti-SHV antibody, we assayed steady state protein expression levels of the SHV beta-lactamase variants. Mutants with the Gly238Ser substitution were among the most highly expressed. The Gly238Ser substitution resulted in an improved relative k(cat)/K(m) value for cephaloridine and oxyimino-cephalosporins compared to SHV-1 and Met69Ile. In our comparative survey, the Gly238Ser and extended spectrum beta-lactamase variants containing this substitution exhibited the greatest substrate versatility against penicillins and cephalosporins and greatest protein expression. This defines a unique role of Gly238Ser in broad-spectrum beta-lactam resistance in this family of class A beta-lactamases.     

GC constituents and relative codon expressed amino acid composition in cyanobacterial phycobiliproteins

The genomic as well as structural relationship of phycobiliproteins (PBPs) in different cyanobacterial species are determined by nucleotides as well as amino acid composition. The genomic GC constituents influence the amino acid variability and codon usage of particular subunit of PBPs. We have analyzed 11 cyanobacterial species to explore the variation of amino acids and causal relationship between GC constituents and codon usage. The study at the first, second and third levels of GC content showed relatively more amino acid variability on the levels of G3 + C3 position in comparison to the first and second positions. The amino acid encoded GC rich level including G rich and C rich or both correlate the codon variability and amino acid availability. The fluctuation in amino acids such as Arg, Ala, His, Asp, Gly, Leu and Glu in α and β subunits was observed at G1C1 position; however, fluctuation in other amino acids such as Ser, Thr, Cys and Trp was observed at G2C2 position. The coding selection pressure of amino acids such as Ala, Thr, Tyr, Asp, Gly, Ile, Leu, Asn, and Ser in α and β subunits of PBPs was more elaborated at G3C3 position. In this study, we observed that each subunit of PBPs is codon specific for particular amino acid. These results suggest that genomic constraint linked with GC constituents selects the codon for particular amino acids and furthermore, the codon level study may be a novel approach to explore many problems associated with genomics and proteomics of cyanobacteria.     

Ammonia cleaves polypeptides at asparagine proline bonds.

Polypeptides that contain the sequence Asn-Pro undergo complete cleavage at this amide bond with ammonia. One cleavage product possesses Pro as the new amino terminus and the other Asn or isoAsn as the new C-terminus, the formation of the latter probably arising by way of a cyclic succinimide intermediate. Other Asn-X bonds where X = Tyr, Gln, Ile, Glu, Ala, Gly, Asn or Phe did not exhibit any peptide bond cleavage, whereas when X = Leu, Thr and Ser partial cleavage was observed. Asn residues not involved in chain-cleavage underwent deamidation to Asp as shown by MALDI-ToF mass spectrometry (MS) analysis. The partial conversion of in-chain Asp residues to isoAsp under the reaction conditions was inferred from RP-HPLC and MS analysis of reaction mixtures.     

Chromatography and Atomic Absorption Spectrometry for the Assessment of Heavy Metal Distribution among Amino Acids Used in Parenteral Nutrition Formulations—Studies with Cadmium and Lead

The distribution of Cd (II) and Pb (II) among amino acids in parenteral nutrition formulations was investigated by coupling ion-chromatography (HPLC/IC) and electrothermal atomic absorption spectrometry. The methodology was based on ion-exchange separation and fluorimetric amino acid detection after post-column derivatization. Cd (II) and Pb (II) were assayed in 500-µL fractions of the column effluent. The distribution of Cd (II) and Pb (II) in alanine (Ala), aspartic acid (Asp), glutamic acid (Glu), glycine (Gly), histidine (His), methionine (Met), phenylalanine (Phe), serine (Ser), and threonine (Thr) were analyzed by monitoring changes in the concentration of free amino acids by HPLC/IC. The results indicated that Cd (II) and Pb (II) were distributed according to the following trend: Gly–Cd?>?Gly–Pb?>?Ala–Cd?>?Ala–Pb?>?His–Cd?～?His–Pb?>?Thr–Cd?>?Thr–Pb?>?Phe–Cd?～?Phe–Pb?～?Asp–Cd?～?Asp–Pb?～?Met–Cd?～?Met–Pb?～?Glu–Cd?～?Glu–Pb?>?Ser–Cd?～?Ser–Pb. The effects of amino acid concentration and stability constants of amino acid–metal complexes are discussed.