scholarly journals Isolation and amino-acid sequence of two inhibitors of serine proteinases, members of the squash inhibitor family, from Echinocystis lobata seeds.

1996 ◽  
Vol 43 (3) ◽  
pp. 507-513 ◽  
Author(s):  
D Stachowiak ◽  
A Polanowski ◽  
G Bieniarz ◽  
T Wilusz
Keyword(s):  
Amino Acid ◽  
Proteinase Inhibitors ◽  
Sequence Similarity ◽  
Serine Proteinase ◽  
Exchange Chromatography ◽  
Association Constants ◽  
Cathepsin G ◽  
Amino Acid Residues ◽  
Mature Seeds ◽  
Echinocystis Lobata

Two serine proteinase inhibitors (ELTI I and ELTI II) have been isolated from mature seeds of Echinocystis lobata by ammonium sulfate fractionation, methanol precipitation, ion exchange chromatography, affinity chromatography on immobilized anhydrotrypsin and HPLC. ELTI I and ELTI II consist of 33 and 29 amino-acid residues, respectively. The primary structures of these inhibitors are as follows: ELTI I KEEQRVCPRILMRCKRDSDCLAQCTCQQSGFCG ELTI II RVCPRILMRCKRDSDCLAQCTCQQSGFCG The inhibitors show sequence similarity with the squash inhibitor family. ELTI I differs from ELTI II only by the presence of the NH2-terminal tetrapeptide Lys-Glu-Glu-Gln. The association constants (Ka) of ELTI I and ELTI II with bovine-trypsin were determined to be 6.6 x 10(10) M-1, and 3.1 x 10(11) M-1, whereas the association constants of these inhibitors with cathepsin G were 1.2 x 10(7) M-1, and 1.1 x 10(7) M-1, respectively.

scholarly journals Sheep mast-cell proteinases-1 and -3: cDNA cloning, primary structure and molecular modelling of the enzymes and further studies on substrate specificity

1998 ◽  
Vol 333 (3) ◽  
pp. 801-809 ◽  
Author(s):  
Sybil M. McALEESE ◽  
Alan D. PEMBERTON ◽  
Mary E. McGRATH ◽  
John F. HUNTLEY ◽  
Hugh R. P. MILLER
Keyword(s):  
Mast Cell ◽  
Amino Acid ◽  
Salt Concentration ◽  
Catalytic Domain ◽  
Serine Proteinase ◽  
Binding Pocket ◽  
Cathepsin G ◽  
Amino Acid Residues ◽  
Human Thrombin ◽  
Amino Acid Signal Peptide

Sheep mast-cell proteinase-1 (sMCP-1) is a serine proteinase expressed predominantly by mucosal mast cells, with specificity for cleavage C-terminal to basic and hydrophobic amino acid residues. A cDNA encoding sMCP-1 has been cloned using reverse transcriptase (RT)-PCR. It appears to be translated as a pre-proenzyme with a 17-amino-acid signal peptide, a basic 2-amino-acid propeptide and a 226-amino-acid catalytic domain. A second cDNA, encoding a serine proteinase 90% identical with sMCP-1, was also cloned and named sMCP-3. Molecular models were constructed for both enzymes using coordinates for the refined X-ray structures of human cathepsin G, chymase and rat mast-cell proteinase-2. The model for sMCP-1 suggests that the acidic Asp-226 side chain extends into the substrate-binding pocket, hydrogen-bonding with Ser-190 on the opposite side and bisecting the pocket. The location of an acidic moiety in this position would favour interaction with basic substrate residues and binding of aromatic residues is rationalized by interaction of the positively charged equatorial plane with Asp-226. The balance between chymotryptic and tryptic activities of sMCP-1 was found to be sensitive to salt concentration, with increasing univalent cation concentration favouring chymotryptic activity relative to the tryptic. Using a peptide substrate representing residues 36–59 of the human thrombin receptor, increasing salt concentration favoured cleavage at Phe-43 rather than at Arg-41.

scholarly journals Cloning, post-translational modifications, heterologous expression and ligand-binding of boar salivary lipocalin

2000 ◽  
Vol 350 (2) ◽  
pp. 369-379 ◽  
Author(s):  
Dietrich LOEBEL ◽  
Andrea SCALONI ◽  
Sara PAOLINI ◽  
Carlo FINI ◽  
Lino FERRARA ◽  
...  
Keyword(s):  
Amino Acid ◽  
Sequence Similarity ◽  
Three Dimensional ◽  
Protein Isoforms ◽  
Cysteine Residues ◽  
Protein Chemistry ◽  
Single Individual ◽  
Amino Acid Residues ◽  
Three Dimensional Model ◽  
Submaxillary Glands

Boar submaxillary glands produce the sex-specific salivary lipocalin (SAL), which binds steroidal sex pheromones as endogenous ligands. The cDNA encoding SAL was cloned and sequenced. From a single individual, two protein isoforms, differing in three amino acid residues, were purified and structurally characterized by a combined Edman degradation/MS approach. These experiments ascertained that the mature polypeptide is composed of 168 amino acid residues, that one of the three putative glycosylation sites is post-translationally modified and the structure of the bound glycosidic moieties. Two of the cysteine residues are paired together in a disulphide bridge, whereas the remaining two occur as free thiols. SAL bears sequence similarity to other lipocalins; on this basis, a three-dimensional model of the protein has been built. A SAL isoform was expressed in Escherichiacoli in good yields. Protein chemistry and CD experiments verified that the recombinant product shows the same redox state at the cysteine residues and that the same conformation is observed as in the natural protein, thus suggesting similar folding. Binding experiments on natural and recombinant SAL were performed with the fluorescent probe 1-aminoanthracene, which was efficiently displaced by the steroidal sex pheromone, as well as by several odorants.

Purification of large peptides using Chemoselective tags

1999 ◽  
Author(s):  
Paolo Mascagni
Keyword(s):  
Amino Acid ◽  
Synthetic Peptides ◽  
Solid Phase ◽  
Solid Phase Peptide Synthesis ◽  
Exchange Chromatography ◽  
Target Sequence ◽  
Coupling Reactions ◽  
Amino Acid Residues ◽  
Quaternary Structures ◽  
Specific Stationary Phase

In solid phase peptide synthesis (SPPS), deletion sequences are generated at each addition of amino acid due to non-quantitative coupling reactions. Their concentration increases exponentially with the length of the peptide chain, and after many cycles not only do they represent a large proportion of the crude preparation, but they can also exhibit physicochemical characteristics similar to the target sequence. Thus, these deletion-sequence contaminants present major problems for removal, or even detection. In general, purification of synthetic peptides by conventional chromatography is based on hydrophobicity differences (using RP-HPLC) and charge differences (using ion-exchange chromatography). For short sequences, the use of one or both techniques is in general sufficient to obtain a product with high purity. However, on increasing the number of amino acid residues, the peptide secondary and progressively tertiary and quaternary structures begin to play an important role and the conformation of the largest peptides can decisively affect their retention behaviour. Furthermore, very closely related impurities such as deletion sequences lacking one or few residues can be chromatographically indistinguishable from the target sequence. Therefore, purification of large synthetic peptides is a complex and time-consuming task that requires the use of several separation techniques with the inevitable dramatic reduction in yields of the final material. Permanent termination (capping) of unreacted chains using a large excess of an acylating agent after each coupling step prevents the formation of deletion sequences and generates N-truncated peptides. However, even under these more favourable conditions, separation of the target sequence from chromatographically similar N-capped polypeptides requires extensive purification. If the target sequence could be specifically and transiently labelled so that the resulting product were selectively recognized by a specific stationary phase, then separation from impurities should be facilitated. This chapter deals with such an approach and in particular with the purification of large polypeptides, assembled by solid phase strategy, using lipophilic and biotin-based 9-fluorenylmethoxycarbonyl (Fmoc) chromatographic probes. Assuming that the formation of deletion sequences is prevented by capping unreacted chains, a reciprocal strategy can be applied that involves functional protection of all polymer-supported peptide chains that are still growing, with a specially chosen affinity reagent or chromatographic probe.

scholarly journals A high-molecular-mass neutral endopeptidase-24.5 from human lung

1987 ◽  
Vol 241 (1) ◽  
pp. 129-135 ◽  
Author(s):  
R Zolfaghari ◽  
C R Baker ◽  
P C Canizaro ◽  
A Amirgholami ◽  
F J Bĕhal
Keyword(s):  
Metal Ions ◽  
Human Lung ◽  
Proteinase Inhibitors ◽  
Gel Filtration ◽  
Serine Proteinase ◽  
Neutral Endopeptidase ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Ph Optimum ◽  
Apparent Homogeneity

A high-Mr neutral endopeptidase-24.5 (NE) that cleaved bradykinin at the Phe5-Ser6 bond was purified to apparent homogeneity from human lung by (NH4)2SO4 fractionation, ion-exchange chromatography and gel filtration. The final enzyme preparation produced a single enzymically active protein band after electrophoresis on a 5% polyacrylamide gel. Human lung NE had an Mr of 650,000 under non-denaturing conditions, but after denaturation and electrophoresis on an SDS/polyacrylamide gel NE dissociated into several lower-Mr components (Mr 21,000-32,000) and into two minor components (Mr approx. 66,000). The enzyme activity was routinely assayed with the artificial substrate Z-Gly-Gly-Leu-Nan (where Z- and -Nan represent benzyloxycarbonyl- and p-nitroanilide respectively). NE activity was enhanced slightly by reducing agents, greatly diminished by thiol-group inhibitors and unchanged by serine-proteinase inhibitors. Human lung NE was inhibited by the univalent cations Na+ and K+. No metal ions were essential for activity, but the heavy-metal ions Cu2+, Hg2+ and Zn2+ were potent inhibitors. With the substrate Z-Gly-Gly-Leu-Nan a broad pH optimum from pH 7.0 to pH 7.6 was observed, and a Michaelis constant value of 1.0 mM was obtained. When Z-Gly-Gly-Leu-Nap (where -Nap represents 2-naphthylamide) was substituted for the above substrate, no NE-catalysed hydrolysis occurred, but Z-Leu-Leu-Glu-Nap was readily hydrolysed by NE. In addition, NE hydrolysed Z-Gly-Gly-Arg-Nap rapidly, but at pH 9.8 rather than in the neutral range. Although human lung NE was stimulated by SDS, the extent of stimulation was not appreciable as compared with the extent of SDS stimulation of NE from other sources.

A NEW PEPTIDE THROMBIN INHIBITOR FROM STREPTOMYCES GRISEUS

1987 ◽  
Author(s):  
Hua-zhen Liu ◽  
Wei Chen ◽  
Qi-ying Liu ◽  
Xia Zhang ◽  
Li-xiu Wang ◽  
...  
Keyword(s):  
Amino Acid ◽  
High Performance ◽  
Antithrombin Iii ◽  
Streptomyces Griseus ◽  
Exchange Chromatography ◽  
Performic Acid ◽  
Thrombin Inhibitor ◽  
Amino Acid Residues ◽  
Macroporous Resin ◽  
Terminal Residue

A new peptide thrombin inhibitor was found in the Streptomyces griseus strain 254 isolated from a soil sample from Tongan, Fujian province, China, the inhibitor being a secondary metabolic product. The production of the inhibitor reached a maximum after 3 days culture of bacteria at 28°C in a rotary shaker. The inhibitor excreted in the culture filtrate was purified by absorption on macroporous resin, followed by ion exchange chromatography on DEAE-52, CM-32 cellulose, affinity chromatography on the immobilized thrombin and high performance liquid chromatography. The amino acid composition of the inhibitor was determined to be Val(2), Met(l), Ile(l), Leu(2) and Arg (1), similar to that of the amino acid residues around the reactive site of human antithrombin III, the critical plasma inhibitor of thrombin. The NH2-terminal residue of the inhibitor seems to be blocked by the alkyl group due to the negative reaction to ninhydrin, whereas the COO-terminal residue is most likely to be arginal because of that Arg was not found in the amino acid analysis, unless the peptide was oxidized by performic acid before acid hydrolysis. The chromogen substrates Bz-Phe-Val-Arg-PNA and Bz-Gly-Pro-Lys-PNA were used to determine the thrombin and plasmin activities, respectively. Besides thrombin, the purified inhibitor also exhibits a weak inhibitory activities on trypsin and much weak on plasmin, but not on chymotrypsin and other protein-ases.

scholarly journals Towards a classification of glycosyltransferases based on amino acid sequence similarities: prokaryotic α-mannosyltransferases

1996 ◽  
Vol 318 (1) ◽  
pp. 133-138 ◽  
Author(s):  
Roberto A GEREMIA ◽  
E Alejandro PETRONI ◽  
Luis IELPI ◽  
Bernard HENRISSAT
Keyword(s):  
Amino Acid ◽  
Sequence Similarity ◽  
Amino Acid Residues ◽  
Glycosyl Hydrolases ◽  
Hydrophobic Cluster Analysis ◽  
Alignment Algorithms ◽  
Automatic Alignment ◽  
Genes Encoding ◽  
Sequence Similarities

A number of genes encoding bacterial glycosyltransferases have been sequenced during the last few years, but their low sequence similarity has prevented a straightforward grouping of these enzymes into families. The sequences of several bacterial α-mannosyltransferases have been compared using current alignment algorithms as well as hydrophobic cluster analysis (HCA). These sequences show a similarity which is significant but too low to be reliably aligned using automatic alignment methods. However, a region spanning approx. 270 residues in these proteins could be aligned by HCA, and several invariant amino acid residues were identified. These features were also found in several other glycosyltransferases, as well as in proteins of unknown function present in sequence databases. This similarity most probably reflects the existence of a family of proteins with conserved structural and mechanistic features. It is argued that the present IUBMB classification of glycosyltransferases could be complemented by a classification of these enzymes based on sequence similarities analogous to that which we proposed for glycosyl hydrolases [Henrissat, B. (1991) Biochem. J. 280, 309–316].

scholarly journals Isolation and a partial amino acid sequence of insulin from the islet tissue of cod (Gadus callarias)

1968 ◽  
Vol 106 (2) ◽  
pp. 531-541 ◽  
Author(s):  
P T Grant ◽  
K. B. M. Reid
Keyword(s):  
Amino Acid ◽  
Ion Exchange ◽  
Gel Filtration ◽  
Bovine Insulin ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Performic Acid ◽  
Amino Acid Residues ◽  
Islet Tissue ◽  
A Chain

1. Insulin has been isolated by gel filtration and ion-exchange chromatography from extracts of the discrete islet tissue of cod. The final preparation yielded a single band on electrophoresis at two pH values. The biological potency was 11·5 international units/mg. in mouse-convulsion and other assay procedures. 2. Glycine and methionine were shown to be the N-terminal amino acids of the A and B chains respectively. An estimate of the molecular weight together with amino acid analyses indicated that cod insulin, like the bovine hormone, consists of 51 amino acid residues. In contrast, the amino acid composition differs markedly from bovine insulin. 3. Oxidation of insulin with performic acid yielded the A and B peptide chains, which were separated by ion-exchange chromatography. Sequence studies on smaller peptides isolated from enzymic digests or from dilute acetic acid hydrolysates of the two chains have established the sequential order of 14 of the 21 amino acid residues of the A chain and 25 of the 30 amino acid residues of the B chain.

scholarly journals Cloning and sequencing of the cDNA species for mammalian dimeric dihydrodiol dehydrogenases

1999 ◽  
Vol 342 (3) ◽  
pp. 721-728 ◽  
Author(s):  
Eiji ARIMITSU ◽  
Shinya AOKI ◽  
Syuhei ISHIKURA ◽  
Kumiko NAKANISHI ◽  
Kazuya MATSUURA ◽  
...  
Keyword(s):  
Amino Acid ◽  
Reductase Activity ◽  
Sequence Similarity ◽  
Japanese Monkey ◽  
Amino Acid Identity ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Animal Tissues ◽  
Dihydrodiol Dehydrogenase ◽  
Low Degrees

Cynomolgus and Japanese monkey kidneys, dog and pig livers and rabbit lens contain dimeric dihydrodiol dehydrogenase (EC 1.3.1.20) associated with high carbonyl reductase activity. Here we have isolated cDNA species for the dimeric enzymes by reverse transcriptase-PCR from human intestine in addition to the above five animal tissues. The amino acid sequences deduced from the monkey, pig and dog cDNA species perfectly matched the partial sequences of peptides digested from the respective enzymes of these animal tissues, and active recombinant proteins were expressed in a bacterial system from the monkey and human cDNA species. Northern blot analysis revealed the existence of a single 1.3 kb mRNA species for the enzyme in these animal tissues. The human enzyme shared 94%, 85%, 84% and 82% amino acid identity with the enzymes of the two monkey strains (their sequences were identical), the dog, the pig and the rabbit respectively. The sequences of the primate enzymes consisted of 335 amino acid residues and lacked one amino acid compared with the other animal enzymes. In contrast with previous reports that other types of dihydrodiol dehydrogenase, carbonyl reductases and enzymes with either activity belong to the aldo-keto reductase family or the short-chain dehydrogenase/reductase family, dimeric dihydrodiol dehydrogenase showed no sequence similarity with the members of the two protein families. The dimeric enzyme aligned with low degrees of identity (14-25%) with several prokaryotic proteins, in which 47 residues are strictly or highly conserved. Thus dimeric dihydrodiol dehydrogenase has a primary structure distinct from the previously known mammalian enzymes and is suggested to constitute a novel protein family with the prokaryotic proteins.

Primary Structure and Anticandidal Activity of the Major Histatin from Parotid Secretion of the Subhuman Primate, Macaca fascicularis

1990 ◽  
Vol 69 (11) ◽  
pp. 1717-1723 ◽  
Author(s):  
T. Xu ◽  
E. Telser ◽  
R.F. Troxler ◽  
F.G. Oppenheim
Keyword(s):  
Amino Acid ◽  
High Performance ◽  
Macaca Fascicularis ◽  
Sequence Similarity ◽  
Gel Filtration ◽  
Alpha Helix ◽  
Reversed Phase ◽  
Amino Acid Residues ◽  
Beta Turns ◽  
Anticandidal Activity

A major macaque histatin (M-histatin 1) from the parotid secretion of the subhuman primate, Macaca fascicularis, was isolated by gel filtration on Bio-Gel P-2 and purified to homogeneity by reversed-phase high-performance liquid chromatography on a TSK-ODS C18 column. The complete amino acid sequence of M-histatin 1, determined by automated Edman degradation, is: 1 10 20 Asp-Pse-His-Glu-Glu-Arg-His-His-Gly-Arg-His-Gly-His-His-Lys-Tyr-Gly-Arg-Lys-Phe 21 30 38 His-Glu-Lys-His-His-Ser-His-Arg-Gly-Tyr-Arg-Ser-Asn-Tyr-Leu-Tyr-Asp-Asn M-histatin 1 contains 38 amino acid residues, a phosphoserine at residue 2, has a molecular weight of 4881.8, a calculated pI of 8.5, and histidine forms 26.3% of the mass. The hydropathicity plot of M-histatin 1 predicts that the molecule is entirely hydrophilic, and Chou-Fasman secondary prediction indicates that the polypeptide is devoid of alpha-helix and beta-sheet conformation in aqueous solutions but contains a series of beta turns. M-histatin 1 includes a six-amino-acid insert (residue 10-15) not present in human histatins and, with the introduction of gaps to maximize homology, it displays 89% and 91% sequence similarity with human histatins 1 and 3, respectively. M-histatin 1 exhibited fungicidal and fungistatic effects against the dimorphic pathogen, Candida albicans, in three separate bioassays. Its anticandidal effects were comparable with or greater than those of human histatins 1, 3, and 5. M-histatins 2, 3, and 4 were not sequenced directly because insufficient materials were available, but the amino acid composition of M-histatin 3 was nearly identical to that of the N-terminal 20 amino acid residues of M-histatin 1. There appears to be only one major histatin in macaque parotid secretion in contrast to the family of histatins in human parotid and submandibular secretions, and the significance of this in the context of evolution and mechanism of action in anticandidal assays is discussed.

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