A comparison of the tryptic peptides of hemoglobin from two microtine genera: Clethrionomys and Ondatra

The composition of tryptic peptides was determined for Hb (major or fast electrophoretic component) from Clethrionomys rutilus and from Ondatra zibethicus. Analysis of the separated and β-Hb chains made possible the definition of theT9 and βT12 peptides and the resolution of uncertainties in earlier Microtus data for peptides T9,T12b,βT9,βT12 and βT13. The previous postulate of two deletions in peptide βT5 is supported by the present data, and the independent dipeptide βT6 appears in the Ondatra Hb. A Microtus stem sequence is inferred and used for comparison with the new data, and cladograms are given to accomodate the extended phylogenetic progression.     

A comparison of hemoglobins in five species of Microtus

The composition of tryptic peptides was determined for Hb (major or fast electrophoretic component) from four additional species of Microtus; M. p. pennsylvanicus, M. abbreviatus, M. miurus, and M. oeconomus, The amino acids from the four Hb were compared with Hb from M. p. tananaensis, and, on the basis of the combination of analyses for the several Hb. Ca 95% of the overall amino acid composition was considered. The compositions of most of the homologous peptides were identical, and two deletions in the sequence of 21 amino acids β 41–61 are believed to characterize the Hb of all four species. From differences in peptide composition the following substitutions were inferred. In the β chain, M. pennsylvanicus (2 ssp) differed from other species at two positions, 8: Ser vs Thr and 12: Thr vs Ash. In the β chain M. pennsylvanicus (2 ssp) differed from other species at two positions, β45: Phe vs Leu, and β50: Ser-Glx vs Thr. M. p. pennsylvanicus differed from M. p. tananaensis at position β88: Val-Leu vs Leu. All species showed ambiguity among the amino acids Ala-Ser-Phe-Leu centred presumably in positions β129 and β130. On the basis of an incomplete examination, the slow electrophoretic component of M. abbreviatus Hb could not be seen to differ from the fast component in its peptide map or in the general composition of its and β peptides.     

A comparison of the tryptic peptides of hemoglobin from two cricetine genera: Peromyscus and Calomys

The composition of tryptic peptides was determined for the major hemoglobin from two cricetine rodents. Peromyscus maniculatus bairdii and Calomys callosus. Amino acid sequences are proposed from homologous alignment with the respective sequences of white mouse and a microtine ‘stem’ developed from previous studies. These two hemoglobins differed at 11 - and 17 β-positions, showing only one common position beyond the cricetid stems (Hesperomyini, Cricetinae) and only three common positions with the microtinae beyond the muroid or myomorph stem (Cricetidae).     

Evidence that the Yb subunits of hepatic glutathione transferases represent two different but related families of polypeptides

Three soluble rat liver glutathione (GSH) transferases A, C and one referred to as 'D', all of which are dimers of Yb subunits [Bass et al. (1977) Biochim. Biophys. Acta, 492, 163-175], have been compared with respect to C-terminal amino acids and tryptic peptide maps. GSH transferases A and 'D' gave different tryptic peptide maps and different C-terminal amino acids, lysine and proline respectively. In each case the number of tryptic peptides is about half of that expected from their lysine and arginine content, and there are 2 mol C-terminal amino acid/mol enzyme. This indicates that GSH transferases A and 'D' represent two different Yb homodimers, which we refer to here as Y1bY1b and Y2bY2b respectively. GSH transferase C is the corresponding heterodimer Y1bY2b since it gives all the tryptic peptides which arise from GSH transferase A and GSH transferase 'D' and also contains both C-terminal lysine and proline. These results provide a structural basis to similar conclusions drawn by Mannervik and Jensson [(1980) J. Biol. Chem. 257, 9909-9912] based on enzymic and immunological comparisons. Tryptic peptide maps show that GSH transferases A and 'D' have considerable homology since there are 23 peptides common to both, 12 peptides unique to A and 8 peptides unique to 'D'. Even so GSH transferase A is selectively induced by a phenobarbitone regime. It is, therefore, concluded that Y1b and Y2b are derived from separate but related genes. A similar conclusion has been drawn concerning the Ya and Yc subunits [Beale et al. (1982) Eur. J. Biochem. 126, 459-463], and a comparison of amino acid compositions, presented here, further suggests a genetic relationship between both pairs of subunits.     

Identification of surface-exposed segments of apolipoprotein B-100 in the LDL particle

The isolation and amino acid sequence of eleven peptides liberated by tryptic treatment from surface-exposed regions of apolipoprotein B-100 in the native low-density lipoprotein particle are described. These peptides represent eight segments in the sequence of the B-100 protein, one of which was localised to the amino-terminal thrombolytic fragment T4 (1297 amino acids), four to the T3 fragment (2052 residues) and three to the carboxylterminal fragment T2 (1287 residues). An exposed segment was identified on each side of the T2/T3 cleavage site, in close proximity to two segments enriched in basic amino acids (residues 3147-3157 and 3359-3367 respectively). The surface exposure of this region is consistent with its contribution to the putative apo-B,E receptor binding domain. Four of the eight tryptic segments contribute to regions of proline-rich clusters. Homology between the sequence of the tryptic peptides and those predicted by cDNA cloning was complete.     

Amino acid sequence of human plasma apolipoprotein C-III from normolipidemic subjects

The complete amino acid sequence of human plasma apolipoprotein C-III (apoC-III) isolated from normal subjects is described. ApoC-III is a linear polypeptide chain of 79 amino acids. Tryptic digestion of intact apoC-III produced 5 major peptides, while tryptic digestion of the citraconylated protein yielded two peptides. The complete amino acid sequence of apoC-III was determined by the automated Edman degradation of the intact protein as well as the various tryptic peptides. Phenylthiohydantoin amino acids were identified by high-performance liquid chromatography and chemical ionization mass spectrometry. The amino acid sequence of apoC-III isolated from normolipidemic subjects is identical to the apoC-III sequence derived from the cDNA sequence and differs at 4 positions from the previously reported sequence of apoC-III derived from a patient with type V hyperlipoproteinemia.     

TRYPTIC PEPTIDES FROM BOVINE WHITE MATTER PROTEOLIPIDS

Abstract— The amino acid composition of the fractions obtained after tryptic digestion of performic acid oxidized and non-oxidized white matter proteolipids was studied. The acid-soluble fraction from the tryptic digest represented between 25 and 30% of the starting material and was relatively enriched in hydrophilic amino acids and deficient in hydrophobic amino acids. The acid-soluble peptides were separated by high voltage paper electrophoresis, and the amino acid compositions of 16 peptides were determined; three additional peptides were obtained from the acid-soluble digest of the oxidized proteolipid. The sequence of 7 peptides including the N- and C-terminal peptides is reported. The results suggest that the protein is segregated into hydrophilic and hydrophobic regions and that small hydrophilic regions are separated by large hydrophobic areas.     

Amino acid sequence studies on lactate dehydrogenase C4 isozymes from mouse and rat testes

Carboxymethylated sperm-specific lactate dehydrogenase isozyme C4 (LDH-C4) proteins from mouse and rat testes were cleaved with cyanogen bromide and trypsin. Proteins were also citraconylated and digested with trypsin. In the case of mouse LDH-C4 isozyme, all 7 CNBr and 11 limited tryptic (arginine) peptides were isolated and sequenced. Some of the CNBr peptides were further fragmented with trypsin and chymotrypsin and their compositions and/or sequences characterized. Also, 34 of the 36 expected tryptic peptides were purified, and their compositions and sequences determined. Amino acid sequences of these peptides purified from mouse LDH-C4 were overlapped into a complete covalent structure of the 330 residues. For rat LDH-C4, 5 of 6 expected CNBr peptides, 5 of 8 expected arginine peptides, and 28 of the 34 expected tryptic peptides were isolated, and their compositions and sequences were determined. Some of the CNBr and arginine peptides were further fragmented with chymotrypsin, thermolysin, or V8 protease, and their compositions and/or sequences characterized. The amino acid sequence of 85% of the 330 residues from rat LDH-C subunit has been unambiguously determined, and the sequences of the remaining regions were tentatively aligned on the basis of peptide compositions and sequence homologies with the other known lactate dehydrogenase sequences, including mouse LDH-C. A comparison of the proposed rat LDH-C sequence with the complete covalent structure of mouse LDH-C indicates that 27 differences are located in the established rat LDH-C sequence of 280 residues and that 5 additional differences are in the tentative sequence of the remaining 50 amino acids.     

Rule of antibody structure. Primary structure of a human monoclonal IgAl-immunoglobulin (myeloma protein Tro). VI. Amino acid sequence of the L-chain, lambda-type, subgroup II]

The primary structure of the L-chain of an IgA1-immunoglobulin (Myeloma protein Tro) has been determined by means of cleavage with trypsin and, if necessary, with alpha-chymotrypsin. The tryptic peptides of the variable part were characterized by amino acid analysis, Dansyl-Edman degradation and cleavage with carboxypeptidase; the peptides of the constant part were identified by amino acid analyses and determination of its N- and C-terminal residues. The sequence of the remaining amino acids and the arrangement of the peptides were established in homology to known structures. The protein comprises 216 amino acids. The homology of the variable part clearly characterizes it as belonging to subgroup II of lambda-chains. In positions 27a, b and c, there are the subgroup-specific additional residues and in position 96 is the characteristic deletion. The constant part of the chain is Kern- and Oz- which indicates that it has serine in position 154 and arginine in position 191.     

A repressor element in the 5'-untranslated region of human Pax5 exon 1A

Five members of the RecQ helicase family, RECQL, WRN, BLM, RTS and RECQL5, have been found in human and three of them (WRN, BLM and RTS) were disclosed to be the genes responsible for Werner, Bloom and Rothmund–Thomson syndromes, respectively. RECQL5 (RecQ helicase protein-like 5) was isolated as the fifth member of the family in humans through a search of homologous expressed sequence tags. The gene is expressed with at least three alternative splicing products, , β and γ. Here, we isolated mouse RECQL5β and determined the DNA sequence of full-length cDNA as well as the genome organization and chromosome locus. The mouse RECQL5β gene consists of 2949 bp coding 982 amino acid residues. Comparison of amino acid sequence among human (Homo sapiens), mouse (Mus musculus), Drosophila melanogaster and Caenorhabditis elegans RECQL5β homologs revealed three portions of highly conserved regions in addition to the helicase domain. Nineteen exons are dispersed over 40 kbp in the genome and all of the acceptor and donor sites for the splicing of each exon conform to the GT/AG rule. The gene is localized to the mouse chromosome 11E2, which has a syntenic relation to human 17q25.2-q25.3 where human RECQL5β exists. Our genetic characterizations of the mouse RECQL5β gene will contribute to functional studies on the RECQL5β products.     

A structural comparison of the A and B subunits of Griffonia simplicifolia I isolectins

A structural comparison between the A and B subunits of the five tetrameric Griffonia simplicifolia I isolectins (A4, A3B, A2B2, AB3, B4) was undertaken to determine the extent of homology between the subunits. The first 25 N-terminal amino acids of both A and B subunits were determined following the enzymatic removal of N-terminal pyroglutamate blocking groups with pyroglutamate aminopeptidase. Although 21 amino acids were common to both subunits, there were four unique amino acids in the N-terminal sequence of A and B. Residues 8, 9, 17, and 19 were asparagine, leucine, lysine, and asparagine in subunit A and threonine, phenylalanine, glutamic acid, and serine in subunit B. The last six C-terminal amino acids, released by digestion with carboxypeptidase Y, were the same for both subunits: Arg-(Phe, Val)-Leu-Thr-Ser-COOH. Subunit B, which contains one methionyl residue, was cleaved by cyanogen bromide into two fragments, a large (Mr = 31,000) and a small (Mr = 2700) polypeptide. Failure of the small fragment to undergo manual Edman degradation indicated an N-terminal blocking group, presumably pyroglutamate. Both subunits were digested with trypsin and the tryptic peptides were analyzed using reverse-phase HPLC. Tryptic glycopeptides were identified by labeling the carbohydrate moiety of the A and B subunit using sodium [3H] borohydride. Cysteine-containing tryptic peptides were similarly identified by using [1-14C]iodoacetamide. Approximately 30% of the tryptic peptides were common to both subunits. Thus, although the N- and C-terminal regions of A and B are similar, the subunits each possess unique sequences.     

The primary structure of the cytotoxin alpha-sarcin

The primary structure of the cytotoxin alpha-sarcin was determined. Eighteen of the 19 tryptic peptides were purified; the other peptide has arginine only. The complete sequence of 17 of the peptides was determined; the sequence of the remaining peptide was determined in part. The sequence of the 39 NH2-terminal residues was obtained by automated Edman degradation. The carboxyl-terminal amino acids were identified after carboxypeptidase treatment. The assignment of the amino acids in the tryptic peptides was confirmed and their alignment established from the sequence of the secondary tryptic peptides obtained after cleavage of citraconylated alpha-sarcin, from the sequence of a 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine peptide, from the sequence of a chymotryptic peptide, and from the sequence of a peptide obtained with Staphylococcus aureus V8 protease. alpha-Sarcin contains 150 amino acid residues; the molecular weight is 16,987. There are disulfide bridges between cysteine residues at positions 6 and 148 and between residues 76 and 132.     

Covalent structure of collagen: amino acid sequence of three cyanogen bromide-derived peptides from human alpha 1(V) collagen chain

Type V collagen was prepared from human amnionic/chorionic membranes and separated into alpha 1(V) and alpha 2(V) polypeptide chains. The alpha 1(V) chain was digested with cyanogen bromide and nine peptides were obtained and purified. Three of the peptides, alpha 1(V)CB1, CB4, and CB7 having molecular weights of 5000, 8000, and 6000, respectively, were further analyzed by amino acid sequence analysis and thermolytic or tryptic digestions. CB1 contained 54 amino acids and identification of its complete sequence was aided by thermolysin digestion and isolation of two peptides, Th1 and Th2. CB4 contained 81 amino acids and sequence analysis of intact CB4 and five tryptic peptides provided us with its complete amino acid sequence. The peptide CB7 contained 67 amino acids and was cleaved into four tryptic peptides that were used for complete sequence analysis. The above results represent the first available covalent structure information on the alpha 1(V) collagen chain. These data enabled us to establish the location of these peptides within the helical structure of other collagen chains. CB4 was homologous to residues 66-145 in the collagen chain while CB1 represented residues 146-200 and CB7 was homologous with residues 201-269. This alignment was facilitated by identification of a helical collagen crossing site consisting of Hyl-Gly-His-Arg located at positions 87-90 in all collagen chains of this size thus far identified. Seventy-one percent homology (excluding Gly residues) was found between amino acids in this region of the alpha 1(XI) and of alpha 1(V) collagen chains while only 21 and 19% identity was calculated for the same region of alpha 2(V) and alpha 1(I) collagen chains, respectively.     

The primary structure of rat liver ribosomal protein L37. Homology with yeast and bacterial ribosomal proteins

The covalent structure of the rat liver 60 S ribosomal subunit protein L37 was determined. Twenty-four tryptic peptides were purified and the sequence of each was established; they accounted for all 111 residues of L37. The sequence of the first 30 residues of L37, obtained previously by automated Edman degradation of the intact protein, provided the alignment of the first 9 tryptic peptides. Three peptides (CN1, CN2, and CN3) were produced by cleavage of protein L37 with cyanogen bromide. The sequence of CN1 (65 residues) was established from the sequence of secondary peptides resulting from cleavage with trypsin and chymotrypsin. The sequence of CN1 in turn served to order tryptic peptides 1 through 14. The sequence of CN2 (15 residues) was determined entirely by a micromanual procedure and allowed the alignment of tryptic peptides 14 through 18. The sequence of the NH2-terminal 28 amino acids of CN3 (31 residues) was determined; in addition the complete sequences of the secondary tryptic and chymotryptic peptides were done. The sequence of CN3 provided the order of tryptic peptides 18 through 24. Thus the sequence of the three cyanogen bromide peptides also accounted for the 111 residues of protein L37. The carboxyl-terminal amino acids were identified after carboxypeptidase A treatment. There is a disulfide bridge between half-cystinyl residues at positions 40 and 69. Rat liver ribosomal protein L37 is homologous with yeast YP55 and with Escherichia coli L34. Moreover, there is a segment of 17 residues in rat L37 that occurs, albeit with modifications, in yeast YP55 and in E. coli S4, L20, and L34.     

Comparative studies on partial microsequence-analysis of Bence-Jones proteins

1. Sequence analyses of Bence-Jones proteins up to 15 amino acids from the N-terminus provide decision of subgroups. 2. Investigation of primary structure of 3 Bence-Jones proteins, monomer and dimer of a kappa type, is limited at position 9-10 using DABITC reagent, whereas DABITC/PITC double coupling method allows sequencing up to 20-21 amino acids. 3. Sequencing of tryptic peptides allows only determination of 6-9 amino acids. 4. Microsequencing of tryptic peptide T13a shows the allotypic variant, inv b+, of Bence-Jones proteins TRA and GAN.     

Biochemical studies on the H-2K mutant B6.C-H-2 bm10

The H-2K glycoprotein from the MHC mutant bm10 was analyzed biochemically to determine where primary structural differences distinguished it from the parental standard molecule, K^b. Comparative peptide maps showed differences in two peptides known to be part of the parental CNBr fragment spanning amino acids 139 to 228. Partial sequence analyses of CNBr fragments and tryptic peptides identified two tightly clustered amino acid substitutions at amino acids 165 (Val to Met) and 173 (Lys to unknown). The substitutions in bm10 represent the most carboxy-terminal substitutions characterized in the K^b molecules of the spontaneous, histogenically active H-2 mutants.     

A systematical analysis of tryptic peptide identification with reverse phase liquid chromatography and electrospray ion trap mass spectrometry

In this study we systematically analyzed the elution condition of tryptic peptides and the characteristics of identified peptides in reverse phase liquid chromatography and electrospray tandem mass spectrometry (RPLC-MS/MS) analysis. Following protein digestion with trypsin, the peptide mixture was analyzed by on-line RPLC-MS/MS. Bovine serum albumin (BSA) was used to optimize acetonitrile (ACN) elution gradient for tryptic peptides, and Cytochrome C was used to retest the gradient and the sensitivity of LC-MS/MS. The characteristics of identified peptides were also analyzed. In our experiments, the suitable ACN gradient is 5% to 30% for tryptic peptide elution and the sensitivity of LC-MS/MS is 50 fmol.Analysis of the tryptic peptides demonstrated that longer (more than 10 amino acids) and multi-charge state ( 2, 3) peptides are likely to be identified, and the hydropathicity of the peptides might not be related to whether it is more likely to be identified or not. The number of identified peptides for a protein might be used to estimate its loading amount under the same sample background. Moreover, in this study the identified peptides present three types of redundancy, namely identification, charge, and sequence redundancy, which may repress low abundance protein identification.     

Sequence diversity of O-superfamily conopetides from Conus marmoreus native to Hainan

The full-length cDNAs of six new O-superfamily conotoxins (CTX) were cloned and sequenced from Conus marmoreus native to Hainan in China South Sea using RT-PCR and 3′-RACE. Six novel conotoxin precursors encoded by these cDNAs consist of three typical regions of signal, pro-peptide and mature peptide. All the six toxin regions share a common O-superfamily cysteine pattern (C-C-CC-C-C, with three disulfide bridges). The predicted precursors are composed of 73–88 amino acids, and the predicted mature peptides consist of 26–34 amino acids. Phylogenetic analysis of new conotoxins from C. marmoreus from the present study and published homologue T-superfamily sequences from other Conus species was performed systematically. Patterns of sequence divergence for three regions of signal, pro-region and mature peptides, as well as Cys codon usage define the major O-superfamily branches and suggest how these separate branches arose. Percent identities of the amino acid sequences of the signal region exhibited high conservation, whereas the sequences of the mature peptides ranged from almost identical to highly divergent between inter- and intra-species. Notably, the diversity of the pro-region was also high with intermediate divergence between that observed in signal and toxin regions. Amino acid sequences and their mode of action (target) of previously identified conotoxins from molluscivorous C. marmoreus for the known conotoxins classes are discussed in detail. The data presented are new and should pave the way for chemical synthesis of these unique conotoxins for to allow determination of the molecular targets of these peptides, and also to provide clues for a better understanding of the phylogeny of these peptides.     

Amino acid sequence of the light chain derived from a rabbit anti-p-azobenzoate antibody of restricted heterogeneity

The amino acid sequence of the light chain from a specifically purified rabbit (No. 2717) anti-p-azobenzoate antibody preparation (b4 allotype) of restricted heterogeneity has been determined. This light chain is composed of 216 residues, including seven half-cystine residues located at positions 23, 80, 88, 134, 171, 194 and 216. Three intrachain disulfide bonds appear to be present in contrast to only two disulfide bonds as has been so far described for Bence Jones protein and light chains of human and mouse. This light chain was sequenced by isolating the tryptic peptides, sequencing the peptides and establishing their order within the molecule. Unambiguous identification of the overlaps was achieved by taking into account the partially characterized tryptic peptides from citraconic anhydride-treated light chains and chymotryptic and peptic peptides from digests of both untreated and citraconylated light chains. Comparison of this amino acid sequence with the amino acid sequence of the car?ylterminal half of the b4 light chains from unimmunized rabbits reveals differences at positions 165, 166, 169 and 176 indicating the existence of more than one sequence in the b4 “constant” region. There is substantial sequence homology between the variable half of 2717 light chain and human Bence Jones protein. Indeed, 46 positions in the V region (42%) are occupied by the same residues in this light chain and in human subgroup V_κIII.     

The primary structure of the hemoglobin alpha-chain of the arctic ground squirrel

The amino acid sequence of the alpha-chain from the arctic ground squirrel Citellus parryii) is reported. The tryptic peptides prepared from the hemoglobin were isolated by reverse phase HPLC and sequenced. Data from the tryptic peptides were supported by that from cyanogen bromide peptides and acid cleavage peptides which were partially sequenced. Comparison with other rodent alpha-chains shows 15 differences with mouse, 20 with rat, 25 with muskrat, 16 with mole rat, 33 with the guinea-pig and 23 with the hamster. Comparison of arctic ground squirrel hemoglobin alpha-chain with the amino-terminal 25 residues of the marmot shows one amino acid difference at position 13.