Biochemistry of Natural Proteinase Inhibitors

Tschesche, Harald

doi:10.1002/anie.197400101

Cited by 89 publications

(11 citation statements)

References 173 publications

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“…This results has been generally proposed for trypsin inhibitors by Laskowski, Jr [1,2]. Only virgin inhibitor was obtained when modified inhibitor was incubated with trypsin for 1 h at pH 7.8 and trypsin and inhibitor were separated by gel filtration on Sephadex G-50 at pH 1.7.…”

Section: Characterization Of Modified Inhibitorsupporting

confidence: 82%

“…The bond Lys-15 -Ala-16 is resynthesized in modified inhibitor when the complex with trypsin is formed and subjected to kinetic control dissociation at acidic pH. This results has been generally proposed for trypsin inhibitors by Laskowski, Jr [1,2]. Only virgin inhibitor was obtained when modified inhibitor was incubated with trypsin for 1 h at pH 7.8 and trypsin and inhibitor were separated by gel filtration on Sephadex G-50 at pH 1.7.…”

Section: Des-ala16-inhibitor and De~-(ala'~arg''ile'~)-in-mentioning

confidence: 70%

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Preparation and Characterization of the Active Derivative of Bovine Trypsin‐Kallikrein Inhibitor (Kunitz) with the Reactive Site Lysine‐15 – Alanine‐16 Hydrolyzed

Jering¹,

Tschesche²

1976

European Journal of Biochemistry

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The derivative of the trypsin-kallikreinm inhibitor (Kunitz), TKI + , was prepared with the reactivesite peptide bond Lys-15 -Ala-16 hydrolyzed. This was achieved by selective borohydride reduction of the Cys-14-Cys-38 disulfide bond, followed by tryptic cleavage of the reactive-site peptide bond, air reoxidation of the half-cystine residues, and purification by ion-exchange chromatography. The derivative corresponds to the hypothetical 'modified' inhibitor TKI' , which so far could not be obtained from virgin inhibitor by a direct modification reaction (partial proteolysis).The derivative isolated was homogeneous as revealed by amino acid analysis, disc electrophoresis, inactivation by carboxypeptidase B, and inactivation by sodium borohydride reduction. The inhibitory activity of the sodium-borohydride-reduced inhibitor was fully recovered after air reoxidation. The site of cleavage in the inhibitor was confirmed by performic acid oxidation and subsequent isolation of the two corresponding peptides containing residues 1 -15 and 16-58 of the entire polypeptide chain. From several aminopeptidases tested only aminopeptidase K rapidly cleaved Ala-16 and Arg-17 from the modified inhibitor and at a reduced rate Ile-18. Des-(Ala16,Arg17)-inhibitor and des-Ala16-inhibitor are both lacking a strong inhibitory activity against bovine trypsin. This indicates a decrease in the association constant by factor of at least 10'-lo1'. The reactive-site-modified inhibitor is not subject to further enzymic breakdown and therefore is a permanent inhibitor of trypsin. However, the modified inhibitor forms the inactive complex much slower than virgin inhibitor.In the modified inhibitor the hydrolyzed peptide bond was resynthesized to yield virgin inhibitor by forming the complex with trypsin and subjecting the complex to kinetic control dissociation. This proves that the bond Lys-15-Ala-16 is at the reactive site of this inhibitor. Preparation of a modified and still active inhibitor (Kunitz) is in agreement with the general model proposed for the interaction of proteinase-inhibitor -proteinase interactions. This presents new evidence that this model is generally applicable. .

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Section: Characterization Of Modified Inhibitorsupporting

confidence: 82%

Section: Des-ala16-inhibitor and De~-(ala'~arg''ile'~)-in-mentioning

confidence: 70%

Preparation and Characterization of the Active Derivative of Bovine Trypsin‐Kallikrein Inhibitor (Kunitz) with the Reactive Site Lysine‐15 – Alanine‐16 Hydrolyzed

Jering¹,

Tschesche²

1976

European Journal of Biochemistry

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“…Its inhibitory properties and primary structure are well characterised (Tschesche, 1974a(Tschesche, , 1974bFritz and Wunderer, 1983) and its tertiary structure was determined by high-resolution X-ray crystal structural analysis (Huber et al. 1970;Deisenhofer and Steigemann, 1975).…”

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confidence: 99%

Recombinant aprotinin homologue with new inhibitory specificity for cathepsin G

Brinkmann

Schnierer

Tschesche

1991

European Journal of Biochemistry

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The substitution of amino acids in the reactive site of aprotinin, a bovine serine proteinase inhibitor with potent activity against trypsin, plasmin and tissue kallikrein, led to a change in specificity of the inhibitor.Twelve new aprotinin variants prepared by recombinant DNA technology and expressed in Escherichia coli clearly demonstrated that the neighbouring groups of the PI residue, in particular PL, contribute to the specificity of the inhibitor, while earlier investigations on semisynthetically prepared variants revealed the importance of the P I residue in dominating the inhibitory specificity.Recombinant aprotinin variants which act specifically against chymotrypsin-like proteinases, were obtained by substitution of the amino acids in position P, and P; by hydrophobic amino acids like phenylalanine, tyrosine and leucine. Some of these variants, particularly those with phenylalanine or leucine substitutions, were also found to exhibit inhibitory activity against cathepsin G with an equilibrium constant of dissociation Ki of lo-' M. Inhibitory specificity against cathepsin G was not found in any semisynthetic variant prepared earlier.The Kunitz-type inhibitor aprotinin is one of the most extensively studied serine proteinase inhibitors. Its inhibitory properties and primary structure are well characterised (Tschesche, 1974a(Tschesche, , 1974b Fritz and Wunderer, 1983) and its tertiary structure was determined by high-resolution X-ray crystal structural analysis (Huber et al.. 1970;Deisenhofer and Steigemann, 1975).During the last decade, more than 20 analogues of aprotinin with vastly changed specific inhibitory activity (Jering and Tschesche, 1976a, b ;Wenzel and Tschesche, 1981 ;Wenzel et al., 1982;Tschesche and Wenzel, 1982) have been prepared by semisynthetic substitution of the amino acid in position P, (Schechter/Berger notation, 1967) of the reactive site.The analogues were directed against chymotrypsin and different elastases, depending on the kind of substituted amino acids. However, the semisynthetic procedure allowed only substitution of a few positions in the reactive site. The use of an expression system for the synthesis of recombinant aprotinin in prokaryotes now permits modification of the inhibitor by amino acid substitution in any desired position of the molecule. The new variants could be designed to interact specifically with the chosen target enzyme, e.g. particularly with cathepsin G; thus, a new inhibitory specificity, so far not observed with any of the earlier prepared variants, could be evoked. This serine proteinase from human granulocytes is one of the deleterious enzymes responsible for tissue degradation in lung emphysema. MATERIALS A N D METHODS MaterialsThe expression system pNH04.1.1 was kindly donated by Bayer AG (Wuppertal, FRG) and the restriction enzymes ApaI, SruI, EcoRI, Hind111 and NciI were purchased from Bethesda Research Lab., USA, which also supplied the T4 ligase, polynucleotide kinase and exonuclease 111. The alkaline phosphatase and Klenow fragment were pu...

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“…The spectrophotometric method was preferred since the substrate could be used in concentrations below the Km value, thus only slightly affecting the dissociation of the enzyme:inhibitor complex. These values are relatively higher than those obtained for other enzyme:inhibitor complexes (17,19,20). Indeed, significantly higher values for residual elastase I1 activity in the different enzyme:inhibitor complexes were obtained by the titrimetric method though the Km values for both substrates are similar.…”

Section: Inhibition O F Elastase Zz By Ovoinhibitor By Modified Ovoinmentioning

confidence: 52%

Inhibition of Porcine Elastase Ii by Chicken Ovoinhibitor

Vered

Gertler

Burstein

1981

International Journal of Peptide and Protein Research

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Interaction o f porcine elastase II with native and modified chicken egg-white ovoinhibitor was studied by determining the residual activity of the partially inhibited enzyme and by direct measurement of the stoichiometry of interaction using affinity chromatography, electrophoresis and gel filtration. It was found that the chymotrypsin binding site that is not modified by mild oxidation with N-chlorosuccinimide (Shechter et al., Biochemistry, (1977) 16,[992][993][994][995][996][997] is capable o f binding elastase II as well. The binding o f chymotrypsin and elastase II is mutually exclusive and the affinity for chymotrypsin is stronger. Binding of 2 mol trypsin as 1 rnol elastase I by ovoinhibitor does not interfere with the binding of elastase II. There is also an indication that the second binding site for chymotrypsin is capable o f forming a complex with an additional molecule o f elastase I . but the binding is so weak that it could be detected only by electrophoresis.Key words: chicken ovoinhibitor; inhibition; porcine elastase 11; protein-protein inter- action.Chicken ovoinhibitor, an egg-white glyco-exhibits chymotrypsin-like specificity toward protein with a molecular weight of 48000, hydrolysis of synthetic substrates and its is a multiheaded inhibitor of several mammalian amino terminal sequence strongly resembles serine pancreatic proteinases (1-3). It has been the B chain of bovine chymotrypsin B. Howdemonstrated by kinetic studies (1, 2, 4, 5 ) ever, unlike chymotrypsin, it hydrolyzes and by direct stoichiometric measurements elastin and is not inhibited by turkey ovo-(6) that it can simultaneously inhibit two mucoid (8). molecules of trypsin, two molecules of c h y m e ,We have reported that ovoinhibitor is trypsin and one molecule of elastase I. capable of inhibiting elastase I1 (9). The present Porcine elastase I1 is a recently described study was devoted t o further characterization pancreatic proteinase (7, 8). This enzyme of the interaction between these proteins and identification of the ovoinhibitor binding site(s) Abbrevhti ons: Ac, N-acetyl; Glut, glutaryl; Tos, that reacts with elastase 11. This was achieved N-p-toluenesulfonyl; OMe, methyl ester; OEt, ethyl by using both kinetic studies and measuring ester. the stoichiometry of interaction by affinity 169 0367-8377/81/070169-11 $02.00/0 o 1981 Munkspmd, Copenhagen

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Biochemistry of Natural Proteinase Inhibitors

Cited by 89 publications

References 173 publications

Preparation and Characterization of the Active Derivative of Bovine Trypsin‐Kallikrein Inhibitor (Kunitz) with the Reactive Site Lysine‐15 – Alanine‐16 Hydrolyzed

Preparation and Characterization of the Active Derivative of Bovine Trypsin‐Kallikrein Inhibitor (Kunitz) with the Reactive Site Lysine‐15 – Alanine‐16 Hydrolyzed

Recombinant aprotinin homologue with new inhibitory specificity for cathepsin G

Inhibition of Porcine Elastase Ii by Chicken Ovoinhibitor

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