A serine protease from the bovine duodenal mucosa, chymotrypsin-like duodenase.

Chymotrypsin-like duodenase (ChlD), a new protease from the bovine duodenum mucosa was isolated and purified. The enzyme molecule is a single chain (25 kDa); the native enzyme is a monomer with an isoelectric point > 10.0. ChlD displays the chymotrypsin-like activity and cleaves 4-nitroanilide substrates of chymotrypsin, chymases and cathepsin G. ChlD hydrolyzes its best substrate 2-N-succinylvalylprolylphenylalanine 4-nitroanilide with k(cat) of 2.8 s(-1) and catalytic efficiency k(cat)/Km of 2300 M(-1) s(-1). The enzyme is stable with a pH range of 3-10 and exhibits the maximum activity at pH 8-10. ChlD is irreversibly inhibited by diisopropylphosphofluoridate and phenylmethanesulfonyl fluoride, which is indicative of an active-site serine in this protease. Alpha-N-tosyl-L-phenylalanine chloromethane, a specific reagent for a catalytically active His, markedly inhibited ChlD. The enzyme activity was strongly inhibited by several natural inhibitors of serine proteases (from soybean, potato, Lima bean, kidney bean). The N-terminal sequence of the native ChlD (23 amino acids) shows high similarity, but not identity, to those of duodenase, granzymes, chymases and cathepsin G.

[Duodenase--a potential activator of cascade of digestive proteases]. / Duodenaza--potentsial'nyi aktivator kaskada pishchevaritel'nykh proteinaz.

The substrate specificity of duodenase from bovine duodenum mucosa to synthetic and natural polypeptides was studied. Amino acid residues preferential for duodenase in the P1 and P2 positions of the substrate were determined. It was shown that the enzyme is synthesized in epithelial secretory cells of duodenal (Brunner's) glands and enters, as part of the secreta, into the lumen of the duodenum. The possible role of duodenase as an activator of proenteropeptidase is discussed.

Subcellular localization, substrate specificity and crystallization of duodenase, a potential activator of enteropeptidase.

Duodenase, a serine protease from bovine duodenum mucosa, was located in endoplasmic reticulum, the Golgi secretory granules of epithelial cells and ducts of Brunner's glands by the A-gold immunocytochemical method. Duodenase exhibits trypsin-like and chymotrypsin-like specificities with a preference for substrates having lysine at the P1 and proline at the P2 positions. The kinetic constants for the hydrolysis of 21 potential duodenase substrates are reported. The best substrates were found to be alpha-N-tosylglycylprolyllysine 4-nitroanilide (k[cat]/Km of 35000 M[-1] s[-1]), alpha-N-succinylthreonylprolyllysine 4-nitroanilide (k[cat]/Km of 18000 M[-1] s[-1]) and alpha-N-serylprolyllysine 4-nitroanilide (k[cat]/Km of 2600 m[-1] s[-1]), all of which contain the P1-P3 sequence of the enteropeptidase zymogen/activation site. On the basis of its catalytic properties and sites of localization, duodenase has been postulated to be an activator of the enteropeptidase precursor. A tetradecapeptide (LVTQEVSPKIVGGS) having the P9-P5'sequence of the cleavage site of zymogen activation of bovine proenteropeptidase was synthesized, and kinetic parameters of its hydrolysis by duodenase were determined (Km of 87 microM; k[cat] of 1.4 s[-1]; k[cat]/Km of 16000 M[-1] s[-1]). Crystals of duodenase frozen in a stream of liquid nitrogen diffracted synchrotron X-rays to 0.2-nm resolution.

[The immunochemical detection of the site of duodenase synthesis and secretion: its functional role in the proteolytic carrier system]. / Immunokhimicheskoe vyiavlenie mesta sinteza i sekretsii duodenazy: ee funktsional'naia rol' v proteoliticheskom konveire.

A new protease (duodenase) was discovered, isolated from the bovine duodenum, and purified. The protease manifested two types of specific features: (1) dual tripsin- and chymotripsin-like specifics in respect to exoproteolysis of peptides and their derivatives, and (2) conformational specifics in respect to endoproteolysis of peptides. The duodenase seems to be synthetized and secreted in the duodenal (Brunner) glands.

Duodenase, a new serine protease of unusual specificity from bovine duodenal mucosa. Purification and properties.

In this paper, data are presented on purification and properties of a new serine endopeptidase (duodenase) isolated from bovine duodenum mucosa. The enzyme has been purified to homogeneity by combinations of ammonium sulphate fractionation, carboxymethyl-cellulose 52 chromatography, and affinity chromatography on Sepharose 4B with Kunitz soybean trypsin inhibitor as a ligand. Some physicochemical properties of this protease have been investigated. The molecular mass of the purified duodenase was determined to be 29 +/- 0.5 kDa by SDS/PAGE and G-2000 SW column chromatography. The enzyme molecule is a single chain and the native enzyme is a monomeric protein. Its isoelectric point was estimated to be 10 +/- 0.2. Duodenase has two forms (I and II) which possess similar properties but differ in their amino acid composition. The new protease is a glycoprotein and contains approximately 3.5% sugars. The enzyme displays trypsin-like and chymotrypsin-like activities and hydrolyzes the amide bonds of substrates having Lys, Arg, Tyr, Phe and Leu residues at the P1 position. Duodenase is most active at pH 7.9-8.2. Duodenase was irreversibly inhibited by diisopropylphosphofluoridate and phenylmethanesulphonyl fluoride, indicative of an active-site serine in this protease. alpha-N-Tosyl-L-lysine chloromethane and alpha-N-tosyl-L-phenylalanine chloromethane, which react with an active His, caused marked inhibition of trypsin-like and chymotrypsin-like activities of duodenase. The enzyme activity was strongly suppressed by trypsin inhibitors from different sources (soybeans, bovine lungs and Lima beans). Chicken egg white ovomucoid had no effect on the duodenase activity. The N-terminal sequence of the native duodenase (24 amino acid residues) shows high similarity with those of human and murine cytotoxic T-lymphocyte granzymes, human leukocyte cathepsin G and rat mast cell chymases. The biological role of duodenase is discussed.

Duodenase, a new serine protease of unusual specificity from bovine duodenal mucosa. Primary structure of the enzyme.

The complete amino acid sequence of duodenase, a new serine endopeptidase from bovine duodenal mucosa, has been determined. The sequence was reconstructed by the automated sequence analysis of the peptides obtained after cleavage with trypsin, Staphylococcus aureus V8 protease, cyanogen bromide and duodenase. The enzyme is composed of 226 amino acid residues yielding a molecular mass of 29.06 kDa. The presence of six cysteine residues and one potential sugar-chain-binding site at Asn50 was revealed. A predicted catalytic triade characteristic of the serine proteases was traced in the duodenase primary structure at the corresponding positions (His44, Asp87 and Ser181 in the sequence). Comparison of the sequence of duodenase with the other known primary structures of mammalian serine proteinases reveales the duodenase identity to granzymes from human and mice, human cathepsin G and mast cell chymases from rat, and gives an overall sequence identity of 47-55% with the mentioned enzymes. Alignment of the known serine protease and duodenase primary structures showed unique amino acid residues within the duodenase substrate-binding pocket at positions 189 (Asn) and 226 (Asp) (the bovine chymotrypsinogen A numbering). These results are discussed with respect to the relation between the duodenase unique residues within the primary specificity pocket S1 and the unusual dual specificity of the enzyme.

[Proteinases of small intestine enterocytes of swine. Purification and properties of aspartyl proteinase similar to cathepsin D]. / Proteinazy énterotsitov tonkogo kishechnika svin'i. Ochistka i svoistva aspartil'noi proteinazy, skhodnoi s katepsinom D.

A new aspartic proteinase was isolated from porcine intestine mucosa by affinity chromatography on pepstatin-Sepharose 4B and gel filtration on Sephadex G-100. The enzyme was purified 1600-fold and appeared homogeneous upon polyacrylamide gel electrophoresis. The proteinase has a Mr 60 000 +/- 4000 Da. During sodium dodecyl sulfate polyacrylamide gel electrophoresis the enzyme produced a single protein band (Mr 30 000 +/- 3000 Da). Isoelectric focusing revealed that the enzyme has several multiple forms (pI 6.9, 7.5, 8,0). The enzyme is a glycoprotein containing 5.9% of carbohydrates; the mannose to galactose ratio is 1:3. The amino acid composition of the enzyme was studied. The proteinase splits an oxidized insulin B-chain and synthetic substrates. The pH optimum is 3.2. The enzyme is immunologically identical to porcine spleen cathepsin D.

[Purification and properties of cathepsin D from the mammary glands of lactating rabbits]. / Ochistka i svoistva katepsina D molochnykh zhelez laktiruiushchikh krol'chikh.

Cathepsin D was purified from the lactating rabbit mammary gland by a rapid procedure, which included fractionation with (NH4)2SO4, acid precipitation, double affinity chromatography on pepstatin-Sepharose 4B and gel filtration on Sephadex G-100, resulting in approximately 360-fold purification of the enzyme over the homogenate and approximately 16% recovery. After isoelectric focusing, the enzyme dissociated into four (pI 5.8, 6.3, 6.5 and 7.2) multiple forms, but appeared homogeneous on polyacrylamide gel electrophoresis. Cathepsin D has a Mr of 45 kDa as determined by Sephadex G-100 column chromatography. On sodium dodecylsulfate/polyacrylamide gel electrophoresis the enzyme gave a single protein band, corresponding to Mr of 45 kDa. The amino acid composition of the enzyme is similar to that of cathepsins D from other tissues. A single N-terminal amino acid was glycine. Cathepsin D contains 6.4% carbohydrates consisting of mannose, galactose, fucose and glucosamine at a ratio of 3:9:2:2. Cathepsin D is inhibited by pepstatin with Ki of 2.5 X 10(-9) M and irreversibly by N-diazoacetyl-N'-2.4-dinitrophenyl-ethylene diamine. The enzyme hydrolyzes bovine hemoglobin with the maximal activity at pH 3.0 with Km = 10(-5) M and HLeu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe with Km = 4 X 10(-5) M and Rcat = 0.95 s-1. The major cleavage sites were Leu15-Tyr16, Phe24-Phe25 and Phe25-Tyr26 during hydrolysis of the oxidized insulin B-chain by cathepsin D.

[Proteases from the enterocytes of the porcine small intestine. Role of aminopeptidase N in the transport of dipeptides]. / Proteazy énterotsitov tonkogo kishechnika svin'i. Rol' aminopeptidazy N v transporte dipeptidov.

The kinetics of radioactive label uptake from [U-14C]Gly, L-[4.5-3H]Leu and dipeptide [U-14C]Gly-L-[4.5-3H]Leu by the brush border membrane vesicles of porcine small intestine have been studied. The effect of aminopeptidase N inhibitors and of leucine-binding protein on the accumulation rates has been investigated. A comparison of the kinetic parameters for the uptake and hydrolysis of Gly-L-Leu, has demonstrated that the dipeptide transfer includes two conjugated steps, i. e., hydrolysis catalyzed by aminopeptidase N and transport of the resultant free amino acids by a specific carrier.

[Proteases of swine small intestine enterocytes. Kinetics of substrate hydrolysis and inhibition of aminopeptidase N from brush border vesicles]. / Proteazy énterotsitov tonkogo kishechnika svin'i. Kinetika gidroliza substratov i ingibirovanie aminopeptidazy N vezikul shchetochnoi kaimy.

The kinetics of hydrolysis of L-leucine p-nitroanilide and some p-nitrophenylalanine dipeptides by vesicular aminopeptidase N from the porcine small intestine brush border membrane was studied. It was shown that the catalytic properties of the vesicular enzyme are very similar to those known for its solubilized counterpart. Both enzymes are inhibited by o-phenanthroline, ZnCl2 and puromycin with Ki = 10(-5)-10(-6) M. The data obtained offer new possibilities for investigating the role of aminopeptidase N in the amino acid and peptide transport across the enterocyte membrane.

Role of intestinal brush border membrane aminopeptidase N in dipeptide transport.

The kinetics of uptake of radioactive label from [U-14C]Gly, L-[4,5-3H]Leu and the dipeptide [14C]Gly-L-[4,5-3H]Leu by the brush border membrane vesicles of porcine small intestine have been studied. The effect of aminopeptidase N inhibitors and leucine-binding protein on accumulation rates has also been tested. Comparison of the kinetic parameters for uptake and hydrolysis of Gly-L-Leu makes it possible to conclude that the dipeptide transfer includes two conjugated steps, viz., hydrolysis catalysed by aminopeptidase N and transport of the resultant free amino acids by a specific carrier.

[Determination of leucine-binding protein content of Escherichia coli cells]. / Metody testirovaniia leitsinsviazyvaiushchego belka i opredelenie ego soderzhania v kletkakh E. coli.

A radioimmunochemical method of determination of leucine-binding protein and a method, based on the selective absorption of the protein and its complex with leucine on DEAE-cellulose, has been developed. The protein content in the E. coli cells at different stage of growth has been determined by the radioimmunochemical and equilibrium dialysis methods. It was shown that the protein content in the cells is practically independent of the growth-phase.