The basic trypsin inhibitor of bovine pancreas IV. The linear sequence of the 58 amino acids

Kassell, Beatrice; Radicevic, Milla; Ansfield, Michael J.; Laskowski, M.

doi:10.1016/0006-291x(65)90749-7

Cited by 121 publications

(47 citation statements)

References 9 publications

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“…It is isolated from bovine lung and has a molecular weight of 6512 g/mol consisting of 58 amino acids. 61 Aprotinin was used in heart surgery with cardiopulmonary bypass to reduce bleeding by slowing down fibrinolysis through plasmin inhibition. 62 It was withdrawn from the market due to side effects in 2008.…”

Section: Known Wnv Ns2b-ns3 Protease Inhibitorsmentioning

confidence: 99%

Development and Characterization of New Peptidomimetic Inhibitors of the West Nile Virus NS2B–NS3 Protease

et al. 2013

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A series of new substrate analogue inhibitors of the WNV NS2B–NS3 protease containing decarboxylated arginine mimetics at the P1 position was developed. Among the various analogues, trans‐(4‐guanidino)cyclohexylmethylamide (GCMA) was identified as the most suitable P1 residue. In combination with dichloro‐substituted phenylacetyl groups at the P4 position, three inhibitors with inhibition constants of <0.2 μM were obtained. These GCMA inhibitors have a better selectivity profile than the previously described agmatine analogues, and possess negligible affinity for the trypsin‐like serine proteases thrombin, factor Xa, and matriptase. A crystal structure in complex with the WNV protease was determined for one of the most potent inhibitors, 3,4‐dichlorophenylacetyl‐Lys‐Lys‐GCMA (Ki=0.13 μM). The inhibitor adopts a horseshoe‐like conformation, most likely due to a hydrophobic contact between the P4 phenyl ring and the P1 cyclohexyl group, which is further stabilized by an intramolecular hydrogen bond between the P1 guanidino group and the P4 carbonyl oxygen atom. These inhibitors are stable, readily accessible, and have a noncovalent binding mode. Therefore, they may serve as suitable lead structures for further development.

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Section: Known Wnv Ns2b-ns3 Protease Inhibitorsmentioning

confidence: 99%

Development and Characterization of New Peptidomimetic Inhibitors of the West Nile Virus NS2B–NS3 Protease

et al. 2013

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“…Residue Lys-15 is the reactive-site residue . Acylation of the Eamino group of this residue inactivates the inhibitor [33,34]. However, removal of the positive charge by deamination keeps the inhibitor active [32].…”

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confidence: 99%

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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“…Fig. 5 shows the alignment of the structure analyzed with those of the bovine pancreatic inhibitor [15] and a previously reported inhibitor of another cobra venom [5]. Residue identities are 42% and To%, respectively, revealing a considerable variation of the inhibitor molecule, even between cobra species.…”

Section: Resultsmentioning

confidence: 98%

Primary structure and functional properties of cobra (Naja naja naja) venom Kunitz‐type trypsin inhibitor

Shafqat

Beg

Yin

et al. 1990

European Journal of Biochemistry

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A trypsin inhibitor from the venom of the cobra Nuja nuju nuja has been isolated by a single step of reversephase high-performance liquid chromatography. The protein strongly inhibits trypsin (Ki = 3.5 pM). The primary structure was determined by peptide analysis of the [ ''C]carboxymethylated inhibitor. The 57-residue polypeptide chain belongs to the family of Kunitz-type inhibitors, and exhibits 42% residue identity with bovine pancreatic trypsin inhibitor. The structure shows only 70% identity with the corresponding peptide from the Capa cobra (Naju neviu), establishing that the inhibitor molecule exhibits extensive variations. Functionally, a basic residue at position P3' correlates with strong inhibition.Basic protease inhibitors of the serine proteases are abundant in nature and appear to be present in all forms of life [l]. Different types have been isolated and extensively studied. Based on different properties, they have been classified into at least seven families [2].Studies of snake venoms reveal the presence of several protease inhibitors, particularly from Elapidae and Viperidae snakes. These inhibitors belong to the protein family of pancreatic trypsin inhibitors of the Kunitz type [3]. The amino acid sequences of the venom inhibitors from Viperu russelli (Russell's viper) [4], Nuju neviu (Cape cobra) [5], Dendroaspis polylepis (black mamba) [6], Vipera ummodytes (long-nosed viper) [7] and Hemachatus haemuchutus (Ringhalls cobra) [5] have been characterized. These analyses, correlated with the known tertiary structure of the pancreatic inhibitor [S], have facilitated understanding of the structure/function relationships for the inhibitors. Comparisons show conservation of residues at both the site of the main contacts with serine proteases [9] and the region of weak contacts [S]. The snake venom inhibitors can inhibit the activities of serine proteases, including bovine trypsin, kallikrein and plasmin [7].During investigation of the venom of the nominate race of cobra, Naja naja naja (from Pakistan), we have isolated a highly potent trypsin inhibitor of this venom. In this work, we report a single-step purification of the inhibitor on reversephase HPLC, enzymatic parameters for the protease inhibition, and the primary structure of the inhibitor, explaining its potency and antitrypsin activity.

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The basic trypsin inhibitor of bovine pancreas IV. The linear sequence of the 58 amino acids

Cited by 121 publications

References 9 publications

Development and Characterization of New Peptidomimetic Inhibitors of the West Nile Virus NS2B–NS3 Protease

Development and Characterization of New Peptidomimetic Inhibitors of the West Nile Virus NS2B–NS3 Protease

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

Primary structure and functional properties of cobra (Naja naja naja) venom Kunitz‐type trypsin inhibitor

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