Asp-N Digestion Research Articles

The Two Subunits of a Phospholipase A2 Inhibitor from the Plasma of Thailand Cobra Having Structural Similarity to Urokinase-Type Plasminogen Activator Receptor and Ly-6 Related Proteins

Amino acid sequences of the two subunits (31-kDa and 25-kDa subunits) of a phospholipase A2 (PLA2) inhibitory protein, purified from the blood plasma of Thailand cobra Naja naja kaouthia, were determined by alignment of peptides obtained by lysyl endopeptidase, staphylococcal V8 protease and endoproteinase Asp-N digestions. The respective subunits were composed of 188 and 185 amino acid residues, and the former contained one asparagine-linked sugar chain at the position 157. There was 29% identity between 31-kDa and 25-kDa subunits. The analysis of internal homology in each sequence of the two subunits revealed the existence of two repeats of approximately 90 amino acid residues. These sequence units were found to be significantly homologous to those of urokinase-type plasminogen activator receptor and of Ly-6 related proteins, such as Ly-6A/E, Ly-6C, ThB, and CD59 antigens.

Identification of the facile gas-phase cleavage of the Asp-Pro and Asp-Xxx peptide bonds in matrix-assisted laser desorption time-of-flight mass spectrometry

Abundant ions corresponding to the gas-phase cleavage of the Asp-Pro and Asp-Xxx bonds of peptides in the process of matrix-assisted laser desorption were observed using a time-of-flight mass spectrometer equipped with both linear and reflector mass analyzers. Peptides containing the N-terminal sequence, Asp-Pro ... from an endoproteinase Asp-N digest yielded one peak in the molecular ion region in the linear mode and two equally abundant peaks in the reflector mode TOF mass spectra. The lower molecular masses in the reflector mode mass spectra could be eliminated by removing the Asp residue or derivatizing its side-chain carboxyl group. The observed mass differences did not correspond to any amino acid; however, by lowering the potential of the reflector to correct for the energy loss the mass difference was determined to be 115 Da, i.e., Asp. The extent and rate of this decomposition was compared with that obtained using a four-sector tandem mass spectrometer in the MS/MS mode of operation without and with a collision gas at collision cell potentials of 3.0 and 9.86 kV. These data suggest the Asp-Pro peptide bond is more labile than other peptide bonds in the gas phase. Abundant metastable decomposition of internal Asp-Pro bonds was also observed in larger peptides and proteins. Based on these latter data, a mechanism for this gas-phase cleavage is proposed.(ABSTRACT TRUNCATED AT 250 WORDS)

Primary structure of Beijing duck apolipoprotein A-1

The primary structure of Beijing duck apolipoprotein A-1 was determined by sequencing peptide fragments derived from tryptic and endoproteinase Asp-N digestion of the protein, and alignment with homologous chicken apo A-1. All of the peptide fragments were isolated by high-pressure liquid chromatography (HPLC) with a Vydac C18 column using a trifluoroacetic acid (TFA) buffer system. The N-terminus of the protein was determined to be aspartic acid by directly sequencing 52 residues of the intact protein. The C-terminus was alanine. The protein contains 240 amino acid residues. By analysis of the whole protein and its tryptic peptides, a six amino acid (Arg-Tyr-Phe-Trp-Gln-His) prosegment was determined. No cross-reactivity between duck and human apo A-1 with a goat antiserum against human apo A-1 was found. Sequence analysis of apo A-1 of other species indicates that amino acid substitutions in rat are more extensive than in other mammals. Isoleucine residues in apo A-1 are inversely correlated to the homology of human to other species, except dog.

Affinity labeling of c-H-ras p21 consensus elements with periodate-oxidized GDP and GTP.

The amino acid sequence motifs of human c-H-ras p21 involved in the interaction with guanosine nucleotides were cross-linked to in situ periodate-oxidized [alpha-32P]GDP or [alpha-32P]GTP. Site-specific reaction was achieved by cross-linking conserved lysine residues close to the G-nucleotide binding site of p21 with the 2',3'-dialdehyde derivatives of GDP or GTP under kinetically controlled conditions. After endoproteinase Asp-N digestion, HPLC separation of 32P-labeled peptides and N-terminal microsequence analysis, two single lysine residues, namely, K117 and K147, which are parts of the N-K-X-D and S-A-K/L consensus elements of ras proteins, respectively, were identified. No significant divergences in the position and extent of covalent modification could be detected between p21.GDP and p21.GTP. This is in contrast to Thermus thermophilus EF-Tu.GDP and EF-Tu.GTP, which were investigated with the same technique [Peter, M. E., Wittmann-Liebold, B. & Sprinzl, M. (1988) Biochemistry 27, 9132-9139] and which exhibited considerable differences in cross-linking efficiency in the GTP form as compared to the GDP form of the protein. The described affinity labeling technique of cross-linking [alpha-32P]GTP with GTP-binding proteins can be used as a general analytical method for the detection and identification of consensus elements in GTPases from different organisms.

Synthetic linear and cyclic glucagon antagonists.

The synthesis and biological activities of seven new glucagon analogues are reported. The design of compounds 2-5 is based on potent antagonists recently reported from this laboratory, where we have focused on modifications in the N-terminal region. In this report we have concentrated specifically on modifications to histidine-1. In addition we have prepared two cyclic compounds 7 and 8, related to a linear in vivo antagonist [Glu9]glucagon, reported by Merrifield (Unson et al. (1987) Proc. Natl. Acad. Sci. USA 84, 4083-4087). The N-terminal modifications involved substitution of His1 by the unnatural conformationally constrained residue (S)-5,6,7,8-tetrahydro-5-oxoimidazo(1,5-c)pyrimidine-7-carboxylic acid (Toc), desaminohistidine (dHis) and 3-(4-nitrobenzyl)histidine. The structures of the new compounds are as follows. [Toc1,D-Phe4,Tyr5,Arg12,Lys17,18,Glu21]glucagon (2); [Toc1,D-Phe4,Tyr5,Arg12,Lys17,18,Glu21]glucagon amide (3); [3-(4-nitrobenzyl)His1,D-Phe4,Tyr5,Arg12,Lys17,18,G lu21]glucagon (4); [dHis1,D-Phe4,Tyr5,Arg12,Lys17,18,Glu21]glucagon (5); [dHis1,Glu9]glucagon (6); (desHis1)[Glu9,Lys12]glucagon amide (7); (desHis1)-[Glu9,Lys12,Asp15]glucagon amide (8). The binding potencies of the linear analogues, as expressed a percentage of glucagon binding, are 2.6 (2), 0.13 (3), 0.8 (4), 0.8 (5), 2.2 (6). Both cyclic analogues 7 and 8 show biphasic binding curves. The IC50 values for 7 at the high and low affinity sites are 1.5 and 167 nM, respectively (IC50 of glucagon = 1.3 nM). The IC50 values for 8 at the high and low affinity sites are 4.7 and 3451 nM, respectively. The cyclic analogues are characterized by fast atom bombardment mass spectrometry of endoproteinase ASP-N digests. The specificity of the enzyme used in these studies enables differentiation of isomers of the cyclic glucagon analogues which differ only in the position of cyclic amide bond. Analogues 2, 3 and 5-8 are glucagon receptor antagonists with respect to the glucagon receptor coupled to the adenylate cyclase (AC) system. Analogue 4 is a partial agonist (5.7% compared to glucagon) of AC. Introduction of unusual amino acids which do not contain a primary alpha-amino group such as Toc at the N-terminus is expected to increase in vivo metabolic stability by protecting against degradation by aminopeptidases.

The Amino Acid Sequence of Pseudomonas putida Azurin

The low molecular weight "blue" copper protein, azurin, has been purified from Pseudomonas putida (NCIB 9869) to homogeneity using various chromatographic techniques including reverse-phase HPLC. The amino acid sequence of the N-terminus of the reduced and carboxymethylated protein yielded a single sequence corresponding to AECKV. The complete sequence, comprising 128 amino acid residues with a C-terminal sequence corresponding to TVTLK, was determined from the peptides obtained from a Staphylococcus aureus V8 digest of the protein and confirmed using peptides obtained following cyanogen bromide and endoprotease Asp-N digests. The amino acid sequence contained three cysteine residues at positions 3, 26, and 112, was devoid of tryptophan, and showed closest similarity (90% identical residues) to the previously determined sequence of azurin isolated from Pseudomonas fluorescens biotype B [Ambler, R. P. (1971) in Developpements Recents Dans L′Etude Chimique De La Structures Des proteins (Preverio, A., Pechere, J.-F., and Coletti-Preverio, M.-A., Eds.), pp. 289-305, INSERM, Paris]. Examination of the complete sequence indicated P. putida azurin contained unique Asp and Ala residues at positions 19 and 21, respectively, that have not been found in any other azurin sequence.

Characterization of linear and cyclic glucagon analogs by fast atom bombardment mass spectrometry

Fast atom bombardment mass spectral mapping of endoproteinase Asp-N digest mixtures is used for characterization of new synthetic linear and cyclic glucagon analogs. The results allow rapid identification of sequence modifications in linear glucagon analogs. For the cyclic compounds, the technique allows confirmation of the presence and position of the cyclic amide bond, as well as verification of the sequence of the modified glucagon analogs. The specificity of the Asp-N enables differentiation of isometric glucagon analogs which differ only in the position of the cyclic amide bond. Important information concerning the purity of the synthetic analogs is also available.

Photoinactivation of the bovine heart mitochondrial F1-ATPase by [14C]dequalinium cross-links phenylalanine-403 or phenylalanine-406 of an alpha subunit to a site or sites contained within residues 440-459 of a beta subunit.

Synthesis of [14C]dequalinium, 1,1'-(1,10-[1,10-14C]decanediyl)bis[4-amino-2-methylquinolinium ], is described, which photoinactivates the bovine heart mitochondrial F1-ATPase (MF1). Maximal photoinactivation occurs on incorporation of about 1.5 mol of [14C]dequalinium/mol of MF1. Three radioactive species were resolved when photoinactivated enzyme was submitted to polyacrylamide gel electrophoresis at pH 4.0 in the presence of tetradecyltrimethylammonium bromide, which correspond to the alpha and beta subunits and a cross-linked species with an M(r) of 116,000. Fractionation of a tryptic digest of photoinactivated enzyme by high-performance liquid chromatography led to isolation of a radioactive peptide which contains residues 399-420 of a alpha subunit. Two fragments containing equal amounts of radioactivity were obtained on fractionation of an endoproteinase Asp-N digest of the isolated radioactive tryptic peptide by high-performance liquid chromatography. Amino acid sequence analysis showed that both fragments contained residues 399-408 of the alpha subunit, but one was missing Phe-alpha 403 and the other was lacking Phe-alpha 406. Fractionation of a cyanogen bromide digest of photoinactivated enzyme followed by trypsin digestion of partially purified cyanogen bromide fragments and fractionation of the resulting radioactive tryptic fragments yielded several radioactive species comprised of residues 399-420 of the alpha subunit cross-linked to residues 440-459 of the beta subunit and a radioactive fragment containing residues 399-420 of the alpha subunit. Partial sequence analyses of the cross-linked fragments suggest that Phe-alpha 403 and Phe-alpha 406 participate in cross-links, whereas no information was obtained on the site or sites of cross-linking in the beta subunit fragment.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning, sequencing, and high expression of the proline iminopeptidase gene from Bacillus coagulans.

The gene coding for proline iminopeptidase in Bacillus coagulans was cloned and expressed in Escherichia coli. Nucleotide sequencing revealed an 861-bp open reading frame with an unusual TTG initiation codon, encoding a 287-amino-acid protein. The calculated molecular weight of the product was 32,415. The amino acid sequences of the amino-terminal region and those of some peptide fragments obtained by endoproteinase Asp-N digestion of the purified enzyme completely coincided with those deduced from the nucleotide sequence. The rare TTG initiation codon that normally codes for leucine was translated as a formal initiation codon; a methionine residue was found at the amino terminus of the enzyme. By using a vector bearing the strong tac promoter, an expression level as high as 200-fold that of the first clone was achieved. The replacement of the TTG initiation codon with ATG and a simultaneous reduction of the distance to the tac promoter resulted in a further increase of 2.5-fold. The expressed enzyme was easily purified to homogeneity by hydrophobic chromatography on a Toyopearl HW-65C column and crystallization, with a recovery of activity of 36%. The molecular weight was found to be 33,000 by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration on a Hi-Load 16/60 Superdex 200 fast protein liquid chromatography column. The expressed enzyme showed the same catalytic and physicochemical properties as those of the wild type, specifically cleaving the N-terminal proline from small substrates.

Correlation between carbohydrate-binding specificity and amino acid sequence of carbohydrate-binding regions of Cytisus-type anti-H(O) lectins

A carbohydrate-binding peptide of the di- N-acetylchitoblose-binding Cytisus sessilifolius anti-H(O) lectin I (CSA-I) was isolated from the endoproteinase Asp-N digest of CSA-I by affinity chromatography on a column of N-acetyl- d-glucosamine oligomer-Sepharose (GIcNAc oligomer-Sepharose). The amino acid sequence of the carbohydrate-binding peptide of CSA-I was determined to be DTYFGKTYNPW using a gas-phase protein sequencer. This sequence corresponds to the sequence from Asp-129 to Trp-139 based on the primary structure of CSA-I, and shows a high degree of homology to those of the putative carbohydrate-binding peptide of the Laburnum alpinum lectin I (LAA-I) (DTYFGKAYNPW) and of the Ulex europaeus lectin II (UEA-II) (DSYFGKTYNPW). The binding of these three anti-H(O) lectins is known to be inhibited by di- N-acetylchitobiose but not by l-fucose. These results strongly suggest that there is a good correlation between the carbohydrate-binding specificity and the amino acid sequence of the carbohydrate-binding regions of di- N-acetylchitobiose-binding lectins.

Correlation of x-ray deduced and experimental amino acid sequences of trimethylamine dehydrogenase.

The amino acid sequence of the iron-sulfur-flavoprotein, trimethylamine dehydrogenase, isolated from the bacterium W3A1 has been deduced from the x-ray diffraction pattern obtained at 2.4-A resolution. This sequence has been compared to portions of the primary sequence derived by gas-phase sequencing of isolated peptides obtained from cyanogen bromide and endoprotease Arg-C and Asp-N digestions of the purified enzyme. A consensus sequence has resulted and is comprised of 729 amino acids with Ala at both NH2- and COOH-terminal positions. The consensus sequence contains 13 cysteine residues. Approximately 80% of the sequence has been confirmed by direct sequencing with approximately 81% agreement with the x-ray deduced sequence. The calculated subunit molecular mass of the apoenzyme is 78,899 Da, in good agreement with published values of approximately 83,000. The anomalous scattering map from the native protein has also been shown to provide accurate information about the positions of most of the weak anomalous scattering centers such as sulfur or phosphorus atoms and to complement x-ray or chemical sequencing methods.

Determination of the carbohydrate-binding site of Bauhinia purpurea lectin by affinity chromatography

To determine the carbohydrate-binding site of Bauhinia purpurea lectin (BPA), a d-galactose- and lactose-binding lectin, a peptide which interacts with lactose was purified from endoproteinase Asp-N digests of BPA by chromatography on a lactose—Sepharose column. It consists of nine amino acids and its amino acid sequence is Asp-Thr-Trp-Pro-Asn-Thr-Glu-Trp-Ser. A tryptic fragment with the ability to interact with lactose was also purified and found to contain this sequence, consisting of nine amino acids. This nonapeptide was aligned in a part of the metal-binding region conserved in all legume lectins. The chemical synthesis of the nonapeptide was carried out by a solid-phase method and the synthetic peptide showed a lactose-specific binding activity in the presence of calcium. A chimeric lectin gene was constructed using a cDNA coding BPA in which the nonapeptide sequence was replaced by the corresponding region of the α- d-mannose binding Lens culinaris lectins. Although BPA is specific for β- d-galactose, the chimeric lectin expressed in Escherichia coli was found to bind α- d-mannosyl—bovine serum albumin and this binding was inhibited by d-mannose.

<b>GALANIN STIMULATES CORTICOSTERONE SECRETION BY ISOLATED RAT ADRENOCORTICAL CELLS </b>

Glucagon was biotinyl-e-aminocaproylated as one major derivative which was isolated by reverse phase high performance liquid chromatography and proved to be homogeneous upon capillary electrophoresis. The estimated yield of this glucagon-derivative was 50% and its molecular weight (3826) as determined by plasma desorption mass spectroscopy (PDMS) corresponded to a monobiotinyl-e-aminocaproylated glucagon. The position of biotinly-e-aminocaproylation at lysine 12 was revealed by PDMS of endoproteinase Asp-N digest of the derivative which indicated the presence of biotinyl-e-aminocaproylated fragment 9-14

The Primary Structure of a Protein Carboxyl Methyltransferase from Bovine Brain That Selectively Methylates L-Isoaspartyl Sites

Protein L-isoaspartyl methyltransferase (PIMT) transfers the methyl group of S-adenosyl-L-methionine to free alpha-carboxyl groups of atypical L-isoaspartyl residues in proteins. The complete primary structure of the type I isoform of bovine brain PIMT was determined by sequence analysis of peptides generated by endoprotease Lys-C, trypsin, cyanogen bromide, and endoprotease Asp-N digests. The correct composition of every peptide was verified by fast atom bombardment mass spectrometry. The efficiency of sequencing by tandem mass spectrometry was examined for several peptides by comparing its speed and accuracy with automated Edman degradation. Tandem mass spectrometry was used to determine the structure of the NH2-terminal blocked peptide derived from a hydroxylamine cleavage. PIMT is 226 residues with Mr = 24,500 and contains acetyl alanine as the amino-terminal residue. The partial sequence (141 residues from 8 tryptic peptides) of a homologous human red cell PIMT (Gilbert, J. M., Fowler, A., Bleibaum, J., and Clarke, S. (1988) Biochemistry 27, 5227-5233) shows a 97% identity with the corresponding peptides of the bovine brain enzyme. The complete brain enzyme sequence reported here bears no significant homology to any other known class of methyltransferase including those which methylate the side chain gamma-carboxyl group of receptor proteins involved in bacterial chemotaxis.