Simple preparation of pacific cod trypsin for enzymatic Peptide synthesis.

Trypsin from the pyloric caeca of Pacific cod (Gadus macrocephalus) was easily prepared by affinity chromatography on Benzamidine Sepharose 6B and gel filtration on Superdex 75. Pacific cod trypsin was composed of three isozymes, and their molecular masses were estimated 23,756.34 Da, 23,939.

Variation of glycyrrhizin and liquiritin contents within a population of 5-year-old licorice (Glycyrrhiza uralensis) plants cultivated under the same conditions.

Cultivated licorice plants (Glycyrrhiza uralensis FISCH.) contain smaller amounts of the triterpene saponin glycyrrhizin than wild licorice plants. To resolve this problem and to breed strains with high-glycyrrhizin content we determined the glycyrrhizin content of 100 samples of G.

Identification of a novel carbohydrate-mimicking octapeptide from chemical peptide library and characterization as selectin inhibitor.

We found a novel octapeptide (H-YRNWFGRW-NH₂) mimicking sialyl Lewis X (sLe(X)) carbohydrate from a chemical peptide library with anti-sLe(X) monoclonal antibody (MAb) 2H5. The peptide libraries were constructed by Fmoc-based solid-phase methodology using the mix-split method. The octapeptide sequence was determined by the iterative deconvolution method using anti-sLe(X) MAb 2H5.

Phenolics in the seed coat of wild soybean (Glycine soja) and their significance for seed hardness and seed germination.

Hardseededness in annual wild soybean (Glycine soja Sieb. Et Zucc.) is a valuable trait that affects the germination, viability, and quality of stored seeds.

Atlantic cod trypsin-catalyzed peptide synthesis with inverse substrates as acyl donor components.

Atlantic cod trypsin-catalyzed peptide synthesis has been studied by using p-amidino- and p-guanidinophenyl esters of N-(tert-butyloxycarbonyl)amino acid as acyl donor components. The reaction temperature was optimized at 0 degrees C. The method was shown to be successful as effectively for synthesizing the peptide and useful for preparing dipeptide between D-amino acid with D-amino acid and beta-amino acid with beta-amino acid, respectively.

A structural comparison of three isoforms of anionic trypsin from chum salmon (Oncorhynchus keta).

Three anionic salmon trypsin isoforms (CST-1, CST-2 and CST-3) were isolated from the pyloric caeca of chum salmon (Oncorhynchus keta). The order of catalytic efficiency (K(m)/k(cat)) of the isoforms during BAPA hydrolysis was CST-2 > CST-1 > CST-3. In order to find a structural rationalization for the observed difference in catalytic efficiency, the X-ray crystallographic structures of the three isoforms were compared in detail.

Identification of Armillaria nabsnona in gastrodia tubers.

The symbiosis between Armillaria species and an achlorophylous orchid Gastrodia elata BLUME has been reported. The main species described as a symbiont is Armillaria mellea (VAHL: FR.) KUMMER, known widely as a primary root rot pathogen.

Trypsin-catalyzed synthesis of dipeptide containing alpha-aminoisobutyric acid using p- and m-(amidinomethyl)phenyl esters as acyl donor.

Two series of inverse substrates, p- and m-(amidinomethyl)phenyl esters derived from N-(tert-butyloxycarbonyl)amino acid, were prepared as acyl donor components for enzymatic peptide synthesis. They were found to be readily coupled with an acyl acceptor such as L-alanine p-nitroanilide to produce dipeptide. An alpha-aminoisobutyric acid containing dipeptide was especially obtained in satisfactory yield.

A facile synthesis of p- and m-(amidinomethyl)phenyl esters derived from amino acid and tryptic hydrolysis of these synthetic inverse substrates.

A facile synthetic method for p- and m-(amidinomethyl)phenyl esters derived from a variety of amino acids is presented. We analyzed the kinetic behavior of trypsin towards these synthetic esters, which are inverse substrates. The substituent (meta- and para-isomers) and isosteric effects of (amidinomethyl)phenyl esters are discussed.

Kinetic properties of three isoforms of trypsin isolated from the pyloric caeca of chum salmon (Oncorhynchus keta).

Three isoforms of anionic chum salmon trypsin (ST-1, ST-2, and ST-3) were purified from the pyloric caeca of chum salmon (Oncorhynchus keta). The molecular weights of the three isoforms were about 24 kDa as determined by SDS-PAGE. The isoelectric points of ST-1, ST-2, and ST-3 were 5.

Application of several types of substrates to ficin-catalyzed peptide synthesis.

The capability of ficin, a cystine protease, to form peptide bonds was investigated using several types of N-Boc-amino acid phenyl and naphthyl esters as acyl donor components. Enzyme-catalyzed peptide synthesis was carried out under optimized reaction conditions of pH, acyl acceptor concentration and selection of the best yield organic solvent. It used a condensation of N-Boc-Ala-OpGu and Ala-pNA as a model reaction.

Amidino-containing Schiff base copper(II) and iron(III) chelates as a thrombin inhibitor.

Four series of Schiff base copper(II) and iron(III) chelates were synthesized from 4-formyl-3-hydroxybenzamidine or 3-formyl-4-hydroxybenzamidine and various L- or D-amino acids. Their inhibitory activities for bovine alpha-thrombin (abbreviated as thrombin) were determined. The most potent thrombin inhibitor in this series is copper(II) chelate (1g') derived from 4-formyl-3-hydroxybenzamidine and D-Trp.

Tracing of Xenopus tropicalis germ plasm and presumptive primordial germ cells with the Xenopus tropicalis DAZ-like gene.

A gamete is derived initially from a presumptive primordial germ cell (pPGC) and transmits genetic potential to the next generation. Xenopus tropicalis, which is a close relative of Xenopus laevis, has a diploid genome and advantages for genetic and genomic research; however, little is known about the developmental mechanism of its germinal lineage. Here, we identified the Xenopus tropicalis DAZ-like gene (Xtdazl), which encodes RNA-binding proteins homologous to Xdazl in Xenopus laevis and examined the expression patterns of Xtdazl transcripts during embryogenesis.

Xenopus tropicalis nodal-related gene 3 regulates BMP signaling: an essential role for the pro-region.

In vertebrates, nodal-related genes are crucial for specifying mesendodermal cell fates. Six nodal-related genes have been identified in Xenopus, but only one, nodal, has been identified in the mouse. The Xenopus nodal-related gene 3 (Xnr3), however, lacks the mesoderm-inducing activity of the other five nodal-related genes in Xenopus, and can directly induce neural tissue in animal caps by antagonizing BMP signals.

Photoregulation of deacylation rate of acyl trypsin derived from photoresponsive inverse substrate.

The acyl trypsin was prepared by use of an inverse substrate, which is comprise of a photoresponsive 4-phenylazobenzoyl moiety. The acyl group in acyl trypsin has been shown to isomerize from trans-form (4t-trypsin) to cis-form (4c-trypsin)/from cis-form to trans-form by irradiation of UV-vis light. The deacylation rate of the cis-form (4c-trypsin) has been shown to be 18.

Synthesis and evaluation of guanidine-containing Schiff base copper(II), zinc(II), and iron(III) chelates as trypsin inhibitors.

3-Formyl-4-hydroxyphenylguanidine hydrochloride and its Schiff base copper(II), zinc(II), and iron(III) chelates were synthesized and their inhibitory activity against bovine beta-trypsin were determined. Syntheses of Schiff base metal chelates were carried out from 3-formyl-4-hydroxyphenylguanidine, various L-amino acids, and divalent metal acetate. Their structures were established on the basis of spectroscopic evidence and elemental analysis.

Crystal structure and nucleotide sequence of an anionic trypsin from chum salmon (Oncorhynchus keta) in comparison with Atlantic salmon (Salmo salar) and bovine trypsin.

The nucleotide sequence and crystal structure of chum salmon trypsin (CST) are now reported. The cDNA isolated from the pyloric caeca of chum salmon encodes 222 amino acid residues, the same number of residues as the anionic Atlantic salmon trypsin (AST), but one residue less than bovine beta-trypsin (BT). The net charge on CST determined from the sum of all charged amino acid side-chains is -3.

Chromosome mapping of Xenopus tropicalis using the G- and Ag-bands: tandem duplication and polyploidization of larvae heads.

Developmental cytogenetic analyses of Xenopus tropicalis larvae from two origins were performed on stage 27-34 heads treated with colchicine. Standard G-band karyotyping using trypsin and chromosome mapping of 184 bands were examined. Although the main karyotype was 2n = 20, polyploidy (3n = 30 or 4n = 40) and aneuploidy were detected in each individual treated with colchicine, even those treated for only 1 h.

Trypsin-catalysed synthesis of oligopeptide amides: comparison of catalytic efficiency among trypsins of different origin (bovine, Streptomyces griseus and chum salmon).

A procedure has been developed for the synthesis of oligopeptide amide using inverse substrates as acyl donors with amino acid amide instead of p-nitroanilide as acyl acceptor and trypsins of different origin (bovine, Streptomyces griseus and chum salmon trypsins) as the catalyst. The effectiveness of this procedure was demonstrated by the synthesis of a pentapeptide, Boc-[Leu5]-enkephalin amide, as a model compound. The method was the first enzymatic method shown to be successful at each successive coupling step for the synthesis of the oligopeptide.

Chum salmon trypsin-catalyzed preferential formation of peptides containing D-amino acid.

Chum salmon trypsin-catalyzed peptide synthesis has been studied by using nine series of "inverse substrates," i.e., p-amidinophenyl, p- and m-guanidinophenyl, p- and m-(guanidinomethyl)phenyl, and four position isomers of guanidinonaphthyl esters derived from Nalpha-(tertbutyloxycarbonyl)amino acid as acyl donor components.

X-ray crystallographic analyses of complexes between bovine beta-trypsin and Schiff base copper(II) or iron(III) chelates.

To establish the structural basis underlying the activity of a novel series of metal-chelate trypsin inhibitors, the structures of p-amidinosalicylidene-l-alaninato(aqua)copper(II) (1a), m-amidinosalicylidene-l-alaninato(aqua)copper(II) (1b), bis(p-amidinosalicylidene-l-alaninato)iron(III) (2a), and bis(m-amidinosalicylidene-l-alaninato)iron(III) (2b) bound to bovine beta-trypsin were studied by X-ray crystallography. The amidinium group of the inhibitor donates hydrogen bonds to Asp189, Gly219 and Ser190, as seen before in trypsin-benzamidine complexes. The copper(II) ion of 1a is situated away from trypsin's catalytic triad residues, and is octahedrally coordinated by a Schiff base and three water molecules.

Anionic trypsin from chum salmon: activity with p-amidinophenyl ester and comparison with bovine and Streptomyces griseus trypsins.

An anionic trypsin from pyloric caeca of chum salmon (Oncorhynchus keta) was purified by ammonium sulfate and acetone fractionation followed by affinity chromatography, gel-filtration, and DEAE-anion exchange chromatography. The apparent molecular mass was about 24 kDa as determined by SDS-PAGE. The anionic chum salmon trypsin was moderately active toward esterase substrates such as tosyl-L-arginine methyl ester and tosyl-L-lysine methyl ester.

Trypsin-catalyzed peptide synthesis with m-guanidinophenyl and m-(guanidinomethyl)phenyl esters as acyl donor component.

Two series of inverse substrates, m-guanidinophenyl and m-(guanidinomethyl)phenyl esters derived from N-(tert-butyloxycarbonyl)-amino acid, were prepared as an acyl donor component for trypsin-catalyzed peptide synthesis. The kinetic behavior of these esters toward tryptic hydrolysis was analyzed. They were found to couple with an acyl acceptor such as L-alanine p-nitroanilide to produce dipeptide in the presence of trypsin.

Application of Schiff base copper(II) and iron(III) chelates to site-specific cleavage of a trypsin.

Amidine-containing Schiff base iron(III) and copper(II) chelates were prepared from alpha-amino acid, metal ion, and salicylaldehyde. These chelates behaved as specific inhibitors of trypsin, with Ki values in the range 10(-5)-10(-6) M. Selective cleavage of the trypsin backbone resulting from specific binding of the chelate to the trypsin active site was investigated.

Enzymatic peptide synthesis with p-guanidinophenyl and p-(guanidinomethyl)phenyl esters as acyl donors.

Two series of "inverse substrates", N-Boc-amino acid p-guanidinophenyl and p-(guanidinomethyl)phenyl esters, were prepared as acyl donor components for enzymatic peptide synthesis. The kinetic behavior of these esters toward bovine and Streptomyces griseus (SG) trypsin was analyzed. The spatial requirement of the active site of these enzymes for catalytic efficiency is discussed based on the steric characteristics of the substrates.