scholarly journals Peptide glyoxals: a novel class of inhibitor for serine and cysteine proteinases

1993 ◽  
Vol 293 (2) ◽  
pp. 321-323 ◽  
Author(s):  
B Walker ◽  
N McCarthy ◽  
A Healy ◽  
T Ye ◽  
M A McKervey
Keyword(s):  
Inhibitory Activity ◽  
Cathepsin B ◽  
Cysteine Proteinase ◽  
Cysteine Proteinases ◽  
Reversible Inhibitors ◽  
Pancreatic Elastase ◽  
Porcine Pancreatic Elastase

A series of novel synthetic dipeptides, containing a C-terminal glyoxal grouping (-COCHO), have been tested as inhibitors against typical members of the serine- and cysteine-proteinase families. For example, the sequences benzyloxycarbonyl (Cbz)-Pro-Phe-CHO (I) and Cbz-Phe-Ala-CHO (II), which fulfil the known primary and secondary specificity requirements of chymotrypsin and cathepsin B respectively, have been found to be potent reversible inhibitors of their respective target proteinase. Thus I was found to inhibit chymotrypsin with a Ki of approximately 0.8 microM, whereas II exhibits a Ki of approximately 80 nm against cathepsin B. These Ki values are some 10-fold and 3-fold lower than those reported for the corresponding peptide-aldehyde inhibitors of chymotrypsin and cathepsin B upon which the peptidyl-glyoxals were fashioned. Unexpectedly, the sequence Cbz-Pro-Ala-CHO, which was designed to inhibit elastase-like proteinases, exhibited no inhibitory activity towards porcine pancreatic elastase, even when used at concentrations as high as 200 microM.

Rapid screening for deficiency of alpha 1-proteinase inhibitor.

1989 ◽  
Vol 35 (9) ◽  
pp. 1971-1975 ◽  
Author(s):  
C Lloyd ◽  
J Travis
Keyword(s):  
Human Plasma ◽  
Inhibitory Activity ◽  
Proteinase Inhibitor ◽  
Rapid Screening ◽  
Early Screening ◽  
Pancreatic Elastase ◽  
Low Concentrations ◽  
Normal Individuals ◽  
Porcine Pancreatic Elastase ◽  
Alpha 1 Antitrypsin

Abstract This rapid screening procedure for detection of low but functional elastase-inhibitory activity in human plasma is based on the fact that incubation of excess porcine pancreatic elastase (EC 3.4.21.36) with plasma results in formation of a complex with active alpha 1-proteinase inhibitor (alpha 1PI, also called alpha 1-antitrypsin). In normal individuals all of the elastase is complexed, leaving no free enzyme to hydrolyze the elastase substrate, and the reaction mixture remains clear. Because individuals homozygous for the Z allele have relatively low concentrations of alpha 1PI, their plasma cannot complex all of the elastase in the assay. The uncomplexed enzyme hydrolyzes the elastase-specific p-nitroanilide substrate, producing a yellow reaction mixture. Use of this simple assay for early screening of individuals for alpha 1PI deficiency may substantially decrease the number of untreated cases of familial emphysema, a disorder that develops as a result of a genetically derived proteinase-proteinase inhibitor imbalance.

Elastase inhibitory activity in serum of patients with thyroid dysfunction

1991 ◽  
Vol 37 (1) ◽  
pp. 108-110 ◽  
Author(s):  
Jacobo Wortsman ◽  
LolS V Matsuoka ◽  
Friedrich Kueppers
Keyword(s):  
Inhibitory Activity ◽  
Elastic Fiber ◽  
Fiber Density ◽  
Free T4 ◽  
Pancreatic Elastase ◽  
Porcine Pancreatic Elastase ◽  
Fiber Fragmentation ◽  
Alpha 1 Antitrypsin ◽  
Hyperthyroid Patients ◽  
Normal Controls

Abstract Dermal elastic fiber fragmentation and decreased fiber density are characteristic cutaneous abnormalities in myxedema. We therefore evaluated elastase inhibitory activity in serum in thyroid dysfunctional states by measuring the protease inhibitor alpha 1-antitrypsin (A-1-AT), as well as by directly determining the inhibition of porcine pancreatic elastase activity. Overall there was a strong correlation between A-1-AT concentration and elastase inhibitory activity in serum (r = 0.95, P less than 0.001). Mean (+/- SE) A-1-AT concentrations were greatest in hyperthyroidism (39 +/- 3 mumol/L, n = 13), followed by normal controls (29 +/- 1, n = 11), subclinical hypothyroidism (27 +/- 2, n = 7), and hypothyroidism (25 +/- 1, n = 12). Concentrations of both A-1-AT and porcine pancreatic elastase inhibitory activity were significantly greater in subjects with hyperthyroidism than in the other groups (P less than 0.01). The correlations (r) between the overall free thyroxin (T4) index and A-1-AT and elastase inhibitory activity were 0.68 and 0.61, respectively (P less than 0.01), implying that free T4 variations account for 46% of the variance in A-1-AT concentrations and 37% of the variance in elastase inhibitory activity. We conclude that serum elastase inhibitory activity is increased in hyperthyroid patients.

scholarly journals Biotin-labelled peptidyl diazomethane inhibitors derived from the substrate-like sequence of cystatin: targeting of the active site of cruzipain, the major cysteine proteinase of Trypanosoma cruzi

1996 ◽  
Vol 318 (2) ◽  
pp. 395-399 ◽  
Author(s):  
Gilles LALMANACH ◽  
Roger MAYER ◽  
Carole SERVEAU ◽  
Julio SCHARFSTEIN ◽  
Francis GAUTHIER
Keyword(s):  
Trypanosoma Cruzi ◽  
Cathepsin B ◽  
Active Sites ◽  
Cysteine Proteinase ◽  
Cathepsin L ◽  
Cysteine Proteinases ◽  
Irreversible Inhibitors ◽  
Human Cystatin C ◽  
Spacer Arm ◽  
Biotin Labelling

Biotin-labelled peptidyl diazomethane inhibitors of cysteine proteinases, based on the N-terminal substrate-like segment of human cystatin C, a natural inhibitor of cysteine proteinases, were synthesized. These synthetic derivatives were tested as irreversible inhibitors of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, to compare the kinetics of the inhibition of the parasite proteinase with that of the mammalian cathepsins B and L. The accessibility of the active sites of these proteinases to these probes was also investigated. The inhibition of cruzipain by Biot-LVG-CHN2 (where Biot represents biotinyl and L,V and G are single-letter amino acid residue abbreviations) and Biot-Ahx-LVG-CHN2 (where Ahx represents 6-aminohexanoic acid) was similar to that of unlabelled inhibitor. Biotin labelling of the inhibitor slowed the inhibition of both cathepsin B and cathepsin L. Adding a spacer arm (Ahx) between the biotin and the peptide moiety of the derivative increased the inhibition of cathepsin B but not that of cathepsin L. The discrimination provided by this spacer is probably due to differences in the topologies of the binding sites of proteinases, a feature that can be exploited to improve targeting of individual cysteine proteinases. Analysis of the blotted proteinases revealed marked differences in the accessibility of extravidin–peroxidase conjugate to the proteinase-bound biotinylated inhibitor. Cruzipain molecules exposed to Biot-LVG-CHN2 or Biot-Ahx-LVG-CHN2 were readily identified, but the reaction was much stronger when the enzyme was treated with the spacer-containing inhibitor. In contrast with the parasite enzyme, rat cathepsin B and cathepsin L treated with either Biot-LVG-CHN2 or Biot-Ahx-LVG-CHN2 produced no detectable bands. Papain, the archetype of this family of proteinases, was poorly labelled with Biot-LVG-CHN2, but strong staining was obtained with Biot-Ahx-LVG-CHN2. These findings suggest that optimized biotinylated diazomethanes might considerably improve their selectivity for the T. cruzi target enzyme.

scholarly journals A general framework of cysteine-proteinase mechanism deduced from studies on enzymes with structurally different analogous catalytic-site residues Asp-158 and −161 (papain and actinidin), Gly-196 (cathepsin B) and Asn-165 (cathepsin H). Kinetic studies up to pH 8 of the hydrolysis of N-α-benzyloxycarbonyl-l-arginyl-l-arginine 2-naphthylamide catalysed by cathepsin B and of l-arginine 2-naphthylamide catalysed by cathepsin H.

1985 ◽  
Vol 227 (2) ◽  
pp. 521-528 ◽  
Author(s):  
F Willenbrock ◽  
K Brocklehurst
Keyword(s):  
Catalytic Activity ◽  
Cathepsin B ◽  
Cysteine Proteinase ◽  
Ion Pairs ◽  
Preceding Paper ◽  
Structural Features ◽  
Catalytic Site ◽  
Cysteine Proteinases ◽  
Cathepsin H ◽  
Ph Range

The pH-dependences of kcat, Km and kcat./Km for the hydrolysis at 25 degrees C at I 0.1 of L-arginine 2-naphthylamide catalysed by cathepsin H from bovine spleen were determined in the pH range approx. 4-8. The pH-dependences of these kinetic parameters were determined also for the hydrolysis at 25 degrees C at I 0.1 of N-alpha-benzyloxycarbonyl-L-arginyl-L-arginine 2-naphthylamide catalysed by cathepsin B (EC 3.4.22.1) from bovine spleen in the pH range 7-8, which extends the studies in acidic media reported by Willenbrock & Brocklehurst [(1984) Biochem. J. 222, 805-814]. These results are discussed and related to those from the reactivity-probe kinetics reported in the preceding paper [Willenbrock & Brocklehurst (1985) Biochem. J. 227, 511-519] and to known structural features present in rat liver cathepsins B and H and in papain (EC 3.4.22.2) and actinidin (EC 3.4.22.14). Consideration of the kinetic data leads to the suggestion that in the cysteine proteinases rearrangement of intimate S-/ImH+ ion-pairs in catalytic sites is brought about by a combination of field effects in the immediate vicinity of the ion-pair and consequences of protonic dissociation of a group with pKa 5-6 remote from the catalytic site. The contributions of the two types of effect seem to differ from enzyme to enzyme. Of the four cysteine proteinases considered, only cathepsin B exerts an absolute requirement for the proton-deficient form of a group with pKa 5-6 for catalytic activity. Protonic dissociation with pKa 5-6 enhances catalytic activity in cathepsin H and in actinidin and appears to have little or no effect in papain. Only cathepsin B lacks a polar or negatively charged side chain in the residue analogous to Asp-158 in papain, and this is suggested to account for its total dependence on a protonic dissociation remote from the catalytic site.

Proteinases participating in the processing and activation of prolegumain in primary cultured rat macrophages

2004 ◽  
Vol 385 (6) ◽  
pp. 511-516 ◽  
Author(s):  
F. Lecaille ◽  
D. Muno ◽  
E. Kominami ◽  
K. Ishidoh
Keyword(s):  
Cathepsin B ◽  
Proteinase Inhibitors ◽  
Cysteine Proteinase ◽  
Vacuolar Atpase ◽  
Cysteine Proteinases ◽  
Bafilomycin A1 ◽  
Atpase Inhibitor ◽  
Peptide Bonds ◽  
Mature Form

Abstract The mammalian legumain is a recently identified lysosomal cysteine proteinase belonging to the clan CD and homologous to plant legumain. This enzyme has the characteristic of specifically hydrolyzing peptide bonds after asparagine residues. As in the case of papain-type cysteine proteinases, legumain is synthesized as an inactive zymogen, and processed into a mature form localized in lysosomes. However, the mechanism of its activation remains unclear. In this study, we analyze which types of proteinases may participate in the processing of legumain in rat primary cultured macrophages using various proteinase inhibitors after 24 h treatment with Bafilomycin A1, a vacuolar ATPase inhibitor. The processing of legumain in macrophages was accomplished by papain-type cysteine proteinases other than cathepsin B.

Effect of Cysteine Proteinase Inhibitors on Murine B16 Melanoma Cell Invasion in vitro

2002 ◽  
Vol 383 (5) ◽  
pp. 839-842 ◽  
Author(s):  
Natasa Sever ◽  
Metka Filipic ◽  
Joze Brzin ◽  
Tamara T. Lah
Keyword(s):  
Cell Invasion ◽  
Cathepsin B ◽  
Proteinase Inhibitor ◽  
Proteinase Inhibitors ◽  
Cysteine Proteinase ◽  
Cathepsin L ◽  
Cysteine Proteinase Inhibitor ◽  
Cysteine Proteinases ◽  
Cysteine Proteinase Inhibitors

Abstract Various types of proteinases are implicated in the malignant progression of human and animal tumors. Proteinase inhibitors may therefore be useful as therapeutic agents in antiinvasive and antimetastatic treatment. The aims of this study were (1) to estimate the relative importance of proteinases in B16 cell invasion in vitro using synthetic, classspecific proteinase inhibitors and (2) to assess the inhibitory effect of some naturally occurring cysteine proteinase inhibitors. Serine proteinase inhibitor reduced invasiveness by up to 24%, whereas inhibition of aspartic proteinases reduced invasion by 11%. Synthetic inhibitors of cysteine proteinases markedly impaired invasion: cathepsin B inhibitors, particularly Ca 074Me, inhibited invasion from 20 40%, whereas cathepsin L inhibitor Clik 148 reduced invasion by 11%. The potato cysteine proteinase inhibitor PCPI 8.7 inhibited invasion by 21%, whereas another potato inhibitor, PCPI 6.6, and the mushroom cysteine proteinase inhibitor clitocypin had no effects. As the inhibitors that inhibited cathepsin B were in general more efficient at impairing the invasiveness, we conclude that of the two cysteine proteinases, cathepsin B plays a more important role than cathepsin L in murine melanoma cell invasion.

scholarly journals The synthesis of peptidylfluoromethanes and their properties as inhibitors of serine proteinases and cysteine proteinases

1986 ◽  
Vol 239 (3) ◽  
pp. 633-640 ◽  
Author(s):  
P Rauber ◽  
H Angliker ◽  
B Walker ◽  
E Shaw
Keyword(s):  
Cathepsin B ◽  
Cysteine Proteinase ◽  
Bond Formation ◽  
Serine Proteinase ◽  
Covalent Bond ◽  
Serine Proteinases ◽  
Cysteine Proteinases ◽  
Active Centre ◽  
Covalent Bond Formation ◽  
The Difference

A synthesis of peptidylfluoromethanes is described that utilizes the conversion of phthaloyl amino acids into their fluoromethane derivatives. These can be deblocked and elongated. The inactivation of chymotrypsin by Cbz-Phe-CH2F (benzyloxycarbonylphenylalanylfluoromethane) was found to be considerably slower than that of the analogous chloromethane. The fluoromethane analogue inactivates chymotrypsin with an overall rate constant that is 2% of that observed for the inactivation of the enzyme with the chloromethane. However, the result is the same. The reagent complexes in a substrate-like manner, with Ki = 1.4 × 10(-4) M, and alkylates the active-centre histidine residue. Cbz-Phe-Phe-CH2F and Cbz-Phe-Ala-CH2F were investigated as inactivators of the cysteine proteinase cathepsin B. The difference in reactivity between fluoromethyl ketones and chloromethyl ketones is less pronounced in the case of the cysteine proteinase than for the serine proteinase. Covalent bond formation takes place in this case also, as demonstrated by the use of a radiolabelled reagent.

scholarly journals ‘pH-jump’ crystallographic analyses of γ-lactam–porcine pancreatic elastase complexes

2000 ◽  
Vol 351 (2) ◽  
pp. 335-340
Author(s):  
Penny A. WRIGHT ◽  
Rupert C. WILMOUTH ◽  
Ian J. CLIFTON ◽  
Christopher J. SCHOFIELD
Keyword(s):  
Crystallographic Analysis ◽  
Side Chain ◽  
Enzyme Complex ◽  
Peptide Hydrolysis ◽  
Reversible Inhibitors ◽  
Pancreatic Elastase ◽  
Ph Jump ◽  
Porcine Pancreatic Elastase ◽  
Enzyme Complexes ◽  
Similar Conformation

β-Lactams inhibit a range of enzymes via acylation of nucleophilic serine residues. Certain γ-lactam analogues of monocyclic β-lactams have also been shown to be reversible inhibitors of porcine pancreatic elastase (PPE), forming acyl-enzyme complexes that are stable with respect to hydrolysis. Crystallographic analysis at pH 5 of an acyl-enzyme complex formed with PPE and one of these inhibitors revealed the ester carbonyl located in the oxyanion hole in a similar conformation to that observed in the structure of a complex formed between a heptapeptide (β-casomorphin-7) and PPE. Only weak electron density was observed for the His-57 side chain in its ‘native’conformation. Instead, the His-57 side chain predominantly adopted a conformation rotated approx. 90° from its normal position. PPE–γ-lactam crystals were subjected to ‘pH-jumps’by placing the crystals in a buffer of increased pH prior to freezing for data collection. The results indicate that the conformation of the γ-lactam-derived acyl-enzyme species in the PPE active site is dependent on pH, a result having implications for the analysis of other serine protease–inhibitor structures at non-catalytic pH values. The results help to define the stereoelectronic relationship between the ester of the acyl-enzyme complex, the side chain of His-57 and the incoming nucleophile during the reversible (de)acylation steps, implying it is closely analogous to the hydrolytic deacylation step during catalytic peptide hydrolysis.

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