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1.
Proc Natl Acad Sci U S A ; 98(4): 1410-5, 2001 Feb 13.
Article in English | MEDLINE | ID: mdl-11171964

ABSTRACT

An additivity-based sequence to reactivity algorithm for the interaction of members of the Kazal family of protein inhibitors with six selected serine proteinases is described. Ten consensus variable contact positions in the inhibitor were identified, and the 19 possible variants at each of these positions were expressed. The free energies of interaction of these variants and the wild type were measured. For an additive system, this data set allows for the calculation of all possible sequences, subject to some restrictions. The algorithm was extensively tested. It is exceptionally fast so that all possible sequences can be predicted. The strongest, the most specific possible, and the least specific inhibitors were designed, and an evolutionary problem was solved.


Subject(s)
Algorithms , Ovomucin/metabolism , Serine Endopeptidases/metabolism , Trypsin Inhibitors/metabolism , Amino Acid Sequence , Animals , Bacterial Proteins , Binding Sites , Cattle , Chymotrypsin/metabolism , Humans , Leukocyte Elastase/metabolism , Molecular Sequence Data , Pancreatic Elastase/metabolism , Subtilisins/metabolism
2.
Chembiochem ; 2(1): 45-51, 2001 Jan 08.
Article in English | MEDLINE | ID: mdl-11828426

ABSTRACT

LUTI (Linum usitatissimum trypsin inhibitor), a member of the potato inhibitor I family, has been isolated from seeds of flax by ethanol fractionation, ion exchange chromatography on CM-Sephadex C-25, affinity purification on immobilized methylchymotrypsin (alpha-chymotrypsin in which His57 has been converted to 3-methylhistidine) in the presence of 5M NaCl, and finally by reversed-phase HPLC. The 7655 Da inhibitor consists of a single polypeptide chain of 69 residues with one disulfide bridge. The molecule is acetylated at the N terminus. Its primary structure has been determined after limited proteolysis of the native molecule with trypsin at the reactive site, cleavage with cyanogen bromide or arginyl endopeptidase (Arg-gingipain), and alcoholytic deacetylation of the N-terminally blocked serine. The association constants (K(a)) of LUTI with bovine beta-trypsin and alpha-chymotrypsin are 3.58x10(10) M(-1) and 5.02x10(5) M(-1), respectively. High NaCl concentration (3M) increased the association constant of LUTI with alpha-chymotrypsin to 6.64x10(7) M(-1). To our knowledge, LUTI is the first serine-proteinase-type inhibitor isolated from a plant of the Linaceae family.


Subject(s)
Flax/chemistry , Plant Proteins/chemistry , Serine Proteinase Inhibitors/chemistry , Amino Acid Sequence , Chromatography, Affinity , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Chymotrypsin/antagonists & inhibitors , Chymotrypsin/chemistry , Electrophoresis, Polyacrylamide Gel , Mass Spectrometry , Molecular Sequence Data , Plant Proteins/isolation & purification , Seeds/chemistry , Serine Proteinase Inhibitors/isolation & purification , Trypsin Inhibitors/chemistry , Trypsin Inhibitors/isolation & purification
3.
J Chromatogr A ; 852(1): 207-16, 1999 Aug 06.
Article in English | MEDLINE | ID: mdl-10480245

ABSTRACT

Two serine proteinase inhibitors, designated clTI-1 and clTI-2 were purified from livers of chickens to apparent homogeneity by a combination of ethanol-acetone fractionation, gel filtration and ion-exchange chromatography on CM-cellulose and Mono S columns. The inhibitor clTI-1 is a single polypeptide chain, low-molecular-mass protein (Mr about 6200), very stable to heat and ethanol. It inhibits chicken, porcine and bovine trypsins as well as human plasmin. The second protein, clTI-2 of Mr 17,000 was shown to be a very effective inhibitor of both trypsins and human cathepsin G. Since both inhibitors are sensitive to arginine modification with phenylglyoxal it is assumed that this amino acid residue is present at the P1 position of the reactive site peptide bond. The N-terminal amino acid sequence of 28 residues of clTI-2 (SVDVSKYPSTVSKDGRTLVACPRILSPV) revealed a high homology of this protein to the third domain of the chicken ovoinhibitor, whereas, the clTI-1 (APPAAEKYYSLPPGAPRYYSPVV) has some sequence identity to a fragment of the human inter-alpha-trypsin inhibitor.


Subject(s)
Liver/chemistry , Serine Proteinase Inhibitors/isolation & purification , Amino Acid Sequence , Animals , Cattle , Chickens , Chromatography, Gel/methods , Chromatography, Ion Exchange/methods , Electrophoresis, Polyacrylamide Gel , Humans , Molecular Sequence Data , Molecular Weight , Serine Proteinase Inhibitors/chemistry
4.
J Chromatogr A ; 852(1): 227-35, 1999 Aug 06.
Article in English | MEDLINE | ID: mdl-10480247

ABSTRACT

It has been shown that specific trypsin inhibitors exhibit also antichymotrypsin activity in the presence of high NaCl concentrations. Taking advantage of this phenomenon a simple procedure of separation of the virgin forms of trypsin inhibitors from squash seeds and porcine pancreas (Kazal) was elaborated. In a typical experiment the inhibitor sample was loaded onto immobilized chymotrypsin equilibrated with 5 M NaCl at pH 8. After washing out unadsorbed material the virgin forms of inhibitors could be eluted either with water, buffer pH 8.0 or 0.02 M citrate buffer pH 2.6 containing no NaCl.


Subject(s)
Chymotrypsin/chemistry , Enzymes, Immobilized/chemistry , Sodium Chloride/chemistry , Trypsin Inhibitors/isolation & purification , Amino Acid Sequence , Animals , Chromatography, Affinity , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Molecular Sequence Data , Pancreas/enzymology , Sequence Homology, Amino Acid , Swine , Trypsin Inhibitors/chemistry
5.
Article in English | MEDLINE | ID: mdl-10631805

ABSTRACT

DPTI II and DPTI IV, two trypsin inhibitors from duck pancreas, have been isolated by affinity chromatography on immobilized anhydrotrypsin, anion exchange and RP-HPLC. The complete amino acid sequence of both inhibitors was determined after reductive carboxymethylation and digestion with Staphylococcus aureus V8 protease or trypsin. The inhibitors were each found to be a single polypeptide chain comprised of 69 amino acid residues and their molecular masses were estimated at 7687 Da for DPTI II and 7668 Da for DPTI IV. The only difference in amino acid sequence between the two inhibitors is the replacement of Arg for His residue in the C-terminal position of DPTI IV.


Subject(s)
Pancreas/chemistry , Peptides/chemistry , Peptides/isolation & purification , Trypsin Inhibitors/chemistry , Trypsin Inhibitors/isolation & purification , Amino Acid Sequence , Amino Acids/analysis , Animals , Chromatography, Affinity , Chromatography, High Pressure Liquid , Ducks , Electrophoresis, Polyacrylamide Gel , Molecular Sequence Data , Molecular Weight , Peptides/metabolism , Protein Isoforms , Sequence Analysis, Protein , Serine Endopeptidases/metabolism , Species Specificity , Trypsin Inhibitors/metabolism , Trypsin Inhibitors/pharmacology
6.
FEBS Lett ; 436(2): 174-8, 1998 Oct 02.
Article in English | MEDLINE | ID: mdl-9781673

ABSTRACT

Five 26-peptide analogues of the trypsin inhibitor [Pro18]CMTI-III containing Leu or Tyr in position 7 and Val or Tyr in position 27: 1 (Leu7, Tyr27), 2 (Tyr7, Val27), 3 (Tyr7, Tyr27), 4 (Leu7, Val27) and 5 (Leu7, Ala18, Tyr27) were synthesized by the solid-phase method. Analogues 1-4 displayed Ka with bovine beta-trypsin of the same order of magnitude as the wild CMTI-III inhibitor, whereas for analogue 5, this value was lower by about 3 orders of magnitude. This indicated that for the analogues with Pro (but not with Ala) in position 18, the side-chain interactions between positions 7 and 27 did not play a critical role for the stabilization of the active structure. In addition, these results also suggest that Tyr7 is involved in an additional aromatic interaction with position 41 of the enzyme.


Subject(s)
Peptide Fragments/chemistry , Peptides/chemistry , Plant Proteins/chemistry , Protein Conformation , Trypsin Inhibitors/chemistry , Trypsin/chemistry , Amino Acid Sequence , Amino Acid Substitution , Animals , Catalytic Domain , Cattle , Disulfides , Kinetics , Models, Molecular , Molecular Sequence Data , Peptides/chemical synthesis , Plant Proteins/metabolism , Thermodynamics , Trypsin/metabolism , Trypsin Inhibitors/metabolism
7.
Acta Biochim Pol ; 43(3): 445-53, 1996.
Article in English | MEDLINE | ID: mdl-8922026

ABSTRACT

Insect hemolymph, like vertebrate serum, contains several different types of polypeptides that are able to inhibit the catalytic function of proteolytic enzymes, however studies on proteins possessing this capability have been limited to a relatively few species. A comparative examination of the inhibition of trypsin, chymotrypsin, neutrophil elastase and cathepsin G and pancreatic elastase by the hemolymph of 14 insect species belonging to six orders showed great diversity in terms of both total proteinase inhibitory capacity and specificity. Most of the inhibitors examined fall into two groups: low molecular mass proteins (below 10 kDa) related to Kunitz type inhibitors, and proteins of about 45 kDa which belong to the serpin superfamily of serine proteinase inhibitors. This minireview describes the properties, characteristics and possible biological significance of selected inhibitors.


Subject(s)
Hemolymph/chemistry , Insecta/chemistry , Serine Proteinase Inhibitors/blood , Animals , Substrate Specificity
8.
Acta Biochim Pol ; 43(3): 503-6, 1996.
Article in English | MEDLINE | ID: mdl-8922033

ABSTRACT

Elastase, cathepsin G and azurocidin from human neutrophils are key components of body inflammatory defense. Perturbations in regulation of their activities lead to many serious pathological states. The paper describes a simple, fast and efficient method of joint purification of these proteins with the use of sequential affinity chromatography on squash trypsin inhibitor (CMTI I) and bovine pancreatic trypsin inhibitor (BPTI).


Subject(s)
Blood Proteins/isolation & purification , Carrier Proteins , Cathepsins/isolation & purification , Leukocyte Elastase/isolation & purification , Neutrophils/enzymology , Antimicrobial Cationic Peptides , Cathepsin G , Chromatography, Affinity/methods , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Humans , Serine Endopeptidases
9.
Acta Biochim Pol ; 43(3): 507-13, 1996.
Article in English | MEDLINE | ID: mdl-8922034

ABSTRACT

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.


Subject(s)
Serine Proteinase Inhibitors/isolation & purification , Vegetables/chemistry , Amino Acid Sequence , Chromatography, Affinity , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Molecular Sequence Data , Serine Proteinase Inhibitors/chemistry
10.
Acta Biochim Pol ; 42(1): 109-14, 1995.
Article in English | MEDLINE | ID: mdl-7653151

ABSTRACT

A chemically synthesized gene coding for the serine proteinase inhibitor CPTI II was cloned in E. coli and its expression was investigated in cytoplasmic and secretion systems. Under all conditions investigated the biologically active form of the inhibitor was found only in the latter system, although the yield was rather low.


Subject(s)
Escherichia coli/metabolism , Plant Proteins/biosynthesis , Serine Proteinase Inhibitors/biosynthesis , Trypsin Inhibitors/biosynthesis , Vegetables/genetics , Amino Acid Sequence , Bacterial Outer Membrane Proteins/genetics , Base Sequence , Cloning, Molecular , Escherichia coli/genetics , Genes, Synthetic , Genetic Vectors , Isopropyl Thiogalactoside/pharmacology , Molecular Sequence Data , Plant Proteins/genetics , Protein Sorting Signals , Restriction Mapping , Serine Proteinase Inhibitors/genetics , Temperature , Time Factors , Trypsin/metabolism , Trypsin Inhibitors/genetics
11.
Biol Chem Hoppe Seyler ; 375(4): 289-91, 1994 Apr.
Article in English | MEDLINE | ID: mdl-8060538

ABSTRACT

Three new CMTI-III analogues containing the Val residue in the reactive site (position 5) were synthesized by the solid-phase method. The analogues displayed an elastase inhibitory activity. It is shown that the removal of the N-terminal Arg residue and the introduction of the Gly-Pro-Gln tripeptide in the region 23-25 decreases the antielastase activity by two orders of magnitude. The removal of the disulfide bridge in positions 16-28 and the substitution of Ala for Cys16 and Gly for Cys28 decreases the activity (measured as Ka with HLE) by five orders of magnitude as compared with [Val5]CMTI-III.


Subject(s)
Pancreatic Elastase/antagonists & inhibitors , Plant Proteins/pharmacology , Plants, Medicinal/chemistry , Serine Proteinase Inhibitors/pharmacology , Amino Acid Sequence , Animals , Humans , Molecular Sequence Data , Plant Proteins/chemistry , Plant Proteins/isolation & purification , Serine Proteinase Inhibitors/chemistry , Serine Proteinase Inhibitors/isolation & purification , Swine
12.
Biol Chem Hoppe Seyler ; 375(1): 21-3, 1994 Jan.
Article in English | MEDLINE | ID: mdl-8003251

ABSTRACT

Four new analogues of trypsin inhibitor CMTI-III(3-28) = [desArg1,desVal2,desGly29]CMTI-III which was recently shown to be fully active, were synthesized by the solid-phase method. The introduction of glycine in position 9 (peptide 1) and Gly-Pro-Gly (peptide 2) and Gly-Pro-Asn (peptide 3) in the regions 17-19 and 23-25, respectively, did not change the antitrypsin activity of all modified peptides. All of these substitutions are presumed to be outside the trypsin-binding loop as judged from the X-ray structure of the complex between beta-trypsin and the related inhibitor CMTI-I. Also the fourth analogue which was substituted in all the positions mentioned, exhibited the full activity.


Subject(s)
Plant Proteins/chemistry , Trypsin Inhibitors/chemistry , Amino Acid Sequence , Amino Acids/analysis , Animals , Cattle , Crystallography, X-Ray , Molecular Sequence Data , Plant Proteins/chemical synthesis , Plant Proteins/metabolism , Trypsin/metabolism , Trypsin Inhibitors/chemical synthesis , Trypsin Inhibitors/metabolism
15.
Biochemistry ; 33(1): 208-13, 1994 Jan 11.
Article in English | MEDLINE | ID: mdl-8286342

ABSTRACT

Porcine pepsin hydrolyzes the Leu7-Met8 (P2'-P3') peptide bond in Cucurbita maxima trypsin inhibitor I (CMTI I) in the pH range 2.0-4.8. The reaction proceeds to equilibrium between intact CMTI I and its cleaved form. The pH-independent value of the equilibrium constant (Khyd0 = 0.78) indicates that both forms of the inhibitor have similar Gibbs energies. The pH dependence of this constant shows that the peptide bond hydrolysis does not perturb ionization constants of any preexistent groups. The same equilibrium values can also be reached from the cleaved inhibitor side through pepsin-catalyzed resynthesis of the Leu7-Met8 peptide bond. Catalytic rate constants for the forward (hydrolysis) and reverse (resynthesis) reactions are similar. Both catalytic rate constants are strongly pH dependent, approaching the highest values at pH 2.0. Michaelis constant values for hydrolysis and resynthesis reactions depend much less on pH and are within values typical for oligopeptide substrates of pepsin. The influence of the binding loop rigidity on slow proteolysis by pepsin and other proteinases is discussed.


Subject(s)
Pepsin A/metabolism , Plant Proteins/metabolism , Trypsin Inhibitors/metabolism , Amino Acid Sequence , Hydrogen-Ion Concentration , Hydrolysis , Kinetics , Plant Proteins/isolation & purification , Serine Proteinase Inhibitors/metabolism , Substrate Specificity
16.
Comp Biochem Physiol B ; 102(4): 757-60, 1992 Aug.
Article in English | MEDLINE | ID: mdl-1395509

ABSTRACT

1. The inhibition of trypsin, chymotrypsin, neutrophil elastase and cathepsin G, and pancreatic elastase by the hemolymph of 14 insect species in six orders has been investigated. 2. All samples showed great diversity in terms of both total proteinase inhibitory capacity and specificity. 3. The highest total inhibitory capacity was found in the larval hemolymph of species in the beetle family Tenebrionidae and the lowest in that of an adult coreid bug, Acanthocephala femorata.


Subject(s)
Hemolymph/enzymology , Insecta/enzymology , Serine Proteinase Inhibitors/physiology , Amino Acid Sequence , Animals , Cathepsin G , Cathepsins/antagonists & inhibitors , Chymotrypsin/antagonists & inhibitors , Electrophoresis, Polyacrylamide Gel , Endopeptidases/metabolism , Molecular Sequence Data , Pancreatic Elastase/antagonists & inhibitors , Serine Endopeptidases , Serine Proteinase Inhibitors/metabolism , Trypsin Inhibitors/physiology
17.
Biol Chem Hoppe Seyler ; 372(1): 63-8, 1991 Jan.
Article in English | MEDLINE | ID: mdl-2039608

ABSTRACT

Five new analogues of squash trypsin inhibitor CMTI III were obtained by the solid-phase method, exhibiting antitrypsin, antichymotrypsin or antielastase activity. Modification in the reactive site region changes dramatically the specificity, whereas substitution in non-contact positions facilitates the refolding of the reduced form of the peptides and does not have a significant influence on the association equilibrium constants of the inhibitor analogues with cognate enzymes.


Subject(s)
Chymotrypsin/antagonists & inhibitors , Pancreatic Elastase/antagonists & inhibitors , Seeds/analysis , Trypsin Inhibitors/chemical synthesis , Amino Acid Sequence , Humans , Leukocytes/enzymology , Molecular Sequence Data , Oxidation-Reduction , Substrate Specificity , Trypsin Inhibitors/pharmacology , X-Ray Diffraction
18.
Planta ; 185(3): 344-9, 1991 Oct.
Article in English | MEDLINE | ID: mdl-24186416

ABSTRACT

Metallo-proteinase from 8-d-old seedlings of kale was isolated. The enzyme was extracted with 1% NaCl, concentrated by ammonium sulfate and finally purified by high-performance liquid chromatography. The isolated enzyme had a molecular weight of 22.4 kDa and showed a maximum activity at pH 9.0 using casein as a substrate. Proteolytic activity of proteinase was inhibited by chelators. The inhibition by ethylenediaminetetraacetate (EDTA) was abolished by some divalent metals ions, especially by Zn(2+). The enzyme showed activity against the synthetic peptides Suc-Ala-Ala-Pro-Leu-pNA and Suc-Ala-Ala-Pro-Phe-pNA, and hydrolized the following peptide bonds in the oxidized insulin B-chain: Leu6-Cya7, Leu15-Tyr16, Leu17-Val18 and Phe25-Tyr26.

19.
Biol Chem Hoppe Seyler ; 371(9): 889-95, 1990 Sep.
Article in English | MEDLINE | ID: mdl-2291813

ABSTRACT

A proteinase was purified from resting seeds of Cucurbita ficifolia by ammonium sulfate fractionation and successive chromatography on CM-cellulose, Sephacryl S-300 and TSK DEAE-2SW (HPLC) columns. Inhibition by DFP and PMSF suggests that the enzyme is a serine proteinase. The apparent molecular mass of this enzyme is ca. 77 kDa. The optimum activity for hydrolysis of casein and Suc-Ala-Ala-Pro-Phe-pNA is around pH 10.5. The following peptide bonds in the oxidized insulin B-chain were hydrolysed by the proteinase: Phe1-Val2, Asn3-Gln4, Gln4-His5, Cya7-Gly8, Glu13-Ala14, Ala14-Leu15, Cya19-Gly20, Pro28-Lys29 and Lys29-Ala30. The proteinase is more selective towards the native squash seed trypsin inhibitor (CMTI I) and primarily cuts off only its N-terminal arginine. The inhibitor devoided of the N-terminal arginine residue is still active against trypsin.


Subject(s)
Plant Proteins/metabolism , Seeds/enzymology , Serine Endopeptidases/metabolism , Trypsin Inhibitors/metabolism , Amino Acid Sequence , Animals , Cattle , Insulin/metabolism , Kinetics , Molecular Sequence Data , Peptides/metabolism , Serine Endopeptidases/isolation & purification , Substrate Specificity
20.
Biol Chem Hoppe Seyler ; 371(7): 589-94, 1990 Jul.
Article in English | MEDLINE | ID: mdl-2145863

ABSTRACT

The squash inhibitors of serine proteinases have been discovered as proteins, which inhibit the catalytic activity of bovine trypsin. In this report we show, that three human enzymes of trypsin-like specificity - i.e. plasmin, plasma kallikrein and thrombin - are also inhibited by squash inhibitors. Moreover, rather strong inhibition was demonstrated for human cathepsin G. Lower association constants were found for Streptomyces griseus proteinase B (SGPB) and subtilisin BPN'. No association was detected for bovine chymotrypsin, even at millimolar concentrations of the inhibitors. Porcine pancreatic elastase showed extremely weak inhibition by squash inhibitors. Most of the enzymes examined did not exhibit a clear discrimination between P1 Arg and P1 Lys inhibitors. However, human plasma kallikrein and human thrombin formed much stronger complexes with CMTI I (P1-Arg) than with CPTI II (P1-Lys).


Subject(s)
Fibrinolysin/antagonists & inhibitors , Kallikreins/antagonists & inhibitors , Serine Proteinase Inhibitors/pharmacology , Thrombin/antagonists & inhibitors , Amino Acid Sequence , Animals , Cattle , Humans , Kallikreins/blood , Kinetics , Molecular Sequence Data , Vegetables
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