scholarly journals A catalytically active high-Mr form of human cathepsin B from sputum

1988 ◽  
Vol 254 (3) ◽  
pp. 693-699 ◽  
Author(s):  
D J Buttle ◽  
B C Bonner ◽  
D Burnett ◽  
A J Barrett
Keyword(s):  
Lysosomal Enzyme ◽  
Active Site ◽  
Cathepsin B ◽  
Human Liver ◽  
Cysteine Proteinase ◽  
Truncated Form ◽  
Physiological Importance ◽  
Catalytically Active ◽  
Human Cathepsin ◽  
Chicken Cystatin

A cysteine proteinase from purulent sputum was partially purified by a method involving affinity chromatography on Sepharose-aminohexanoylphenylalanylglycinaldehyde semicarbazone. It was immunologically related to lysosomal cathepsin B from human liver and was similar in many, but not all, other aspects. It was catalytically active, as demonstrated by active-site-directed radioiodination, and hydrolysed three cathepsin B substrates, two with Km values similar to those of lysosomal cathepsin B. In addition, the rates of inactivation of the sputum and lysosomal forms of the enzyme by L-3-carboxy-2,3-transepoxypropionyl-leucylamido(4-guanidino) butane (Compound E-64) were very similar. However, the sputum enzyme differed from lysosomal cathepsin B in the following respects. Inhibition by chicken cystatin was much weaker for sputum cathepsin B than for the lysosomal enzyme. Sputum cathepsin B had greater stability at pH 7.5 and a higher apparent Mr, even after deglycosylation, than lysosomal cathepsin B. We conclude that the form of cathepsin B found in sputum is probably a truncated form of human procathepsin B, with some differences in properties that could be of physiological importance.

scholarly journals The two cathepsin B-like proteases of Arabidopsis thaliana are closely related enzymes with discrete endopeptidase and carboxydipeptidase activities

2018 ◽  
Vol 399 (10) ◽  
pp. 1223-1235 ◽  
Author(s):  
Andreas Porodko ◽  
Ana Cirnski ◽  
Drazen Petrov ◽  
Teresa Raab ◽  
Melanie Paireder ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Active Site ◽  
Cathepsin B ◽  
Cysteine Proteinase ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Substrate Specificities ◽  
Active Site Cleft ◽  
Molecular Modeling Studies ◽  
Human Cathepsin

Abstract The genome of the model plant Arabidopsis thaliana encodes three paralogues of the papain-like cysteine proteinase cathepsin B (AtCathB1, AtCathB2 and AtCathB3), whose individual functions are still largely unknown. Here we show that a mutated splice site causes severe truncations of the AtCathB1 polypeptide, rendering it catalytically incompetent. By contrast, AtCathB2 and AtCathB3 are effective proteases which display comparable hydrolytic properties and share most of their substrate specificities. Site-directed mutagenesis experiments demonstrated that a single amino acid substitution (Gly336→Glu) is sufficient to confer AtCathB2 with the capacity to tolerate arginine in its specificity-determining S2 subsite, which is otherwise a hallmark of AtCathB3-mediated cleavages. A degradomics approach utilizing proteome-derived peptide libraries revealed that both enzymes are capable of acting as endopeptidases and exopeptidases, releasing dipeptides from the C-termini of substrates. Mutation of the carboxydipeptidase determinant His207 also affected the activity of AtCathB2 towards non-exopeptidase substrates, highlighting mechanistic differences between plant and human cathepsin B. This was also noted in molecular modeling studies which indicate that the occluding loop defining the dual enzymatic character of cathepsin B does not obstruct the active-site cleft of AtCathB2 to the same extent as in its mammalian orthologues.

scholarly journals The active-site residue Cys-29 is responsible for the neutral-pH inactivation and the refolding barrier of human cathepsin B

FEBS Letters ◽  
2000 ◽  
Vol 475 (3) ◽  
pp. 157-162 ◽  
Author(s):  
Jianxing Song ◽  
Ping Xu ◽  
Hui Xiang ◽  
Zhengding Su ◽  
Andrew C. Storer ◽  
...  
Keyword(s):  
Active Site ◽  
Cathepsin B ◽  
Active Site Residue ◽  
Neutral Ph ◽  
Ph Inactivation ◽  
Human Cathepsin

scholarly journals A cysteine proteinase secreted from human breast tumours is immunologically related to cathepsin B

1982 ◽  
Vol 207 (3) ◽  
pp. 633-636 ◽  
Author(s):  
A D Recklies ◽  
A R Poole ◽  
J S Mort
Keyword(s):  
Cathepsin B ◽  
Human Liver ◽  
Cysteine Proteinase ◽  
Human Breast ◽  
Breast Tumours ◽  
Monospecific Antiserum ◽  
Thiol Proteinase

The stable cathepsin B-like cysteine (thiol) proteinase secreted from human breast tumours in culture was shown to be destabilized by mercurial compounds. After such treatment the enzyme cross-reacts in a radioimmunoassay with a monospecific antiserum to human liver cathepsin B. It is suggested that the secreted enzyme may be a precursor form of lysosomal cathepsin B.

scholarly journals Human tumour cathepsin B. Comparison with normal liver cathepsin B

1992 ◽  
Vol 285 (2) ◽  
pp. 427-434 ◽  
Author(s):  
K Moin ◽  
N A Day ◽  
M Sameni ◽  
S Hasnain ◽  
T Hirama ◽  
...  
Keyword(s):  
Cathepsin B ◽  
Human Liver ◽  
Normal Liver ◽  
Polyacrylamide Gels ◽  
Single Chain ◽  
Human Tumour ◽  
Double Chain ◽  
Normal Human Liver ◽  
Human Cathepsin ◽  
Chain Form

Cathepsin B was purified from normal human liver and several human tumour tissues and partially characterized. Three forms of cathepsin B, with molecular masses of 25 kDa, 26 kDa (the two appearing as a doublet) and 30 kDa, were detected in SDS/polyacrylamide gels. The 25-26 kDa doublet was associated with the fractions from tumours and normal liver containing the highest cathepsin B activity. Cathepsin B from both sources showed similar pH optima. Both normal liver and tumour cathepsin B exhibited similar kinetics against selected synthetic substrates. At neutral pH and 24 degrees C, cathepsin B from both normal liver and tumour exhibited a lower Km and a higher kcat./Km than at pH 6.0. Their inhibitory profiles against synthetic inhibitors were also similar. Immunological studies with a monospecific antibody against the mature double-chain form of human liver cathepsin B and an antibody against a cathepsin B-derived synthetic peptide established the immunological similarity of liver and tumour enzymes. The N-terminal sequences of the 25 kDa and 26 kDa forms were identical with that of the heavy chain of the mature double-chain form of human cathepsin B, whereas the N-terminal sequence of the 30 kDa species was identical with that of the single-chain form of human cathepsin B. Treatment of the double-chain form of cathepsin B from normal liver and tumours with the endoglycosidase peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase converted the 26 kDa form into 25 kDa in SDS/polyacrylamide gels, suggesting that cathepsin B may exist as both glycosylated and unglycosylated forms. Our results, in contrast with those reported earlier for mouse cathepsin B, indicate that human liver and tumour cathepsin B are similar.

scholarly journals Human sputum cathepsin B degrades proteoglycan, is inhibited by α2-macroglobulin and is modulated by neutrophil elastase cleavage of cathepsin B precursor and cystatin C

1991 ◽  
Vol 276 (2) ◽  
pp. 325-331 ◽  
Author(s):  
D J Buttle ◽  
M Abrahamson ◽  
D Burnett ◽  
J S Mort ◽  
A J Barrett ◽  
...  
Keyword(s):  
Cystatin C ◽  
Neutrophil Elastase ◽  
Cathepsin B ◽  
Cysteine Proteinase ◽  
Fold Increase ◽  
Stable Form ◽  
Zymogen Activation ◽  
Truncated Form ◽  
Α2 Macroglobulin

The high-Mr alkali-stable form of cathepsin B was purified from purulent human sputum. It was shown to solubilize proteoglycan monomer entrapped in polyacrylamide at a rate comparable with that of human lysosomal cathepsin B. Like the enzyme from lysosomes, sputum cathepsin B was bound by human alpha 2-macroglobulin, which inhibited its action on proteoglycan. Cystatin C in purulent sputum was shown to be the N-terminally truncated form generated by neutrophil elastase cleavage, and sputum cathepsin B was only weakly inhibited by recombinant cystatin C that had been cleaved by neutrophil elastase in vitro. Addition of neutrophil elastase to mucoid sputum led to a 5-fold increase in cathepsin B activity concomitant with a lowering in Mr of the cysteine proteinase from 40,000 to 37,000, i.e. the size of the active enzyme purified from purulent sputum. It is concluded that the high-Mr form of cathepsin B present in purulent sputum is a functional proteinase, unlike similar forms of the enzyme secreted by mammary gland in organ culture. The activity of cathepsin B in sputum is modulated by neutrophil elastase, by a combination of inhibitor inactivation and zymogen activation.

scholarly journals Action of human liver cathepsin B on the oxidized insulin B chain

1983 ◽  
Vol 213 (2) ◽  
pp. 467-471 ◽  
Author(s):  
M J McKay ◽  
M K Offermann ◽  
A J Barrett ◽  
J S Bond
Keyword(s):  
Amino Acid ◽  
Cathepsin B ◽  
Human Liver ◽  
Degradation Products ◽  
Cysteine Proteinase ◽  
Cathepsin L ◽  
Amino Acid Content ◽  
Peptide Bond ◽  
Cleavage Sites ◽  
Amino Acid Residues

The lysosomal cysteine proteinase cathepsin B (from human liver) was tested for its peptide-bond specificity against the oxidized B-chain of insulin. Sixteen peptide degradation products were separated by high-pressure liquid chromatography and thin-layer chromatography and were analysed for their amino acid content and N-terminal amino acid residue. Five major and six minor cleavage sites were identified; the major cleavage sites were Gln(4)-His(5), Ser(9)-His(10), Glu(13)-Ala(14), Tyr(16)-Leu(17) and Gly(23)-Phe(24). The findings indicate that human cathepsin B has a broad specificity, with no clearly defined requirement for any particular amino acid residues in the vicinity of the cleavage sites. The enzyme did not display peptidyldipeptidase activity with this substrate, and showed a specificity different from those reported for two other cysteine proteinases, papain and rat cathepsin L.

scholarly journals Probing the specificity of cysteine proteinases at subsites remote from the active site: analysis of P4, P3, P2′ and P3′ variations in extended substrates

2000 ◽  
Vol 347 (1) ◽  
pp. 123-129 ◽  
Author(s):  
Fernada C. Vieira PORTARO ◽  
Ana Beatriz F. SANTOS ◽  
Maria Helena S. CEZARI ◽  
Maria Aparecida JULIANO ◽  
Luiz JULIANO ◽  
...  
Keyword(s):  
Amino Acids ◽  
Significant Influence ◽  
Active Site ◽  
Cathepsin B ◽  
Cathepsin L ◽  
Cysteine Proteinases ◽  
Aminobenzoic Acid ◽  
Site Analysis ◽  
Hydrophobic Amino Acids ◽  
Human Cathepsin

We have determined the kinetic parameters for the hydrolysis by papain, cathepsin B and cathepsin L of internally quenched fluorescent peptides derived from the lead peptides Abz-AAFRSAQ-EDDnp [in which Abz and EDDnp stand for o-aminobenzoic acid and N-(2,4-dinitrophenyl)ethylenediamine respectively], to map the specificity of S4 and S3 subsites, and Abz-AFRSAAQ-EDDnp, to identify the specificity of S2ʹ and S3ʹ. Abz and EDDnp were the fluorescent quencher pair. These two series of peptides were cleaved at the Arg-Ser bond and systematic modifications at P4, P3, P2ʹ and P3ʹ were made. The S4 to S2ʹ subsites had a significant influence on the hydrolytic efficiencies of the three enzymes. Only papain activity was observed to be dependent on S3ʹ, indicating that its binding site is larger than those of cathepsins B and L. Hydrophobic amino acids were accepted at S4, S3, S2ʹ and S3ʹ of the three enzymes. The best substrates for cathepsins L and B had Trp and Asn at P2ʹ respectively; variations at this position were less accepted by these enzymes. The best substrates for papain were peptides containing Trp, Tyr or Asn at P3ʹ. Basic residues at P3 and P4 were well accepted by cathepsin L and papain. We also explored the susceptibility of substrates Abz-AFRSXAQ-EDDnp, modified at P2ʹ (X), to human cathepsin B mutants from which one or two occluding loop contacts had been removed. The modifications at His111 (H111A) and His110 (H110A) of cathepsin B led to an increase in kcat values of one or two orders of magnitude. The hydrolytic efficiencies of these cathepsin B mutants became closer to those of papain or cathepsin L.

Human Cathepsin B Is a Metastable Enzyme Stabilized by Specific Ionic Interactions Associated with the Active Site

Biochemistry ◽  
1994 ◽  
Vol 33 (49) ◽  
pp. 14800-14806 ◽  
Author(s):  
Boris Turk ◽  
Iztok Dolenc ◽  
Eva Zerovnik ◽  
Dusan Turk ◽  
Franc Gubensek ◽  
...  
Keyword(s):  
Active Site ◽  
Cathepsin B ◽  
Ionic Interactions ◽  
Human Cathepsin

scholarly journals Activity of a Carboxyl-Terminal Truncated Form of Catechol 2,3-Dioxygenase fromPlanococcussp. S5

2014 ◽  
Vol 2014 ◽  
pp. 1-9
Author(s):  
Katarzyna Hupert-Kocurek ◽  
Danuta Wojcieszyńska ◽  
Urszula Guzik
Keyword(s):  
Active Site ◽  
Nonsense Mutation ◽  
Alkaline Ph ◽  
Optimal Temperature ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Truncated Form ◽  
Catalytically Active ◽  
Monoaromatic Hydrocarbons ◽  
Carboxyl Terminal

Catechol 2,3-dioxygenases (C23Os, E.C.1.13.12.2) are two domain enzymes that catalyze degradation of monoaromatic hydrocarbons. The catalytically active C-domain of all known C23Os comprises ferrous ion ligands as well as residues forming active site pocket. The aim of this work was to examine and discuss the effect of nonsense mutation at position 289 on the activity of catechol 2,3-dioxygenase fromPlanococcusstrain. Although the mutant C23O showed the same optimal temperature for activity as the wild-type protein (35°C), it exhibited activity slightly more tolerant to alkaline pH. Mutant enzyme exhibited also higher affinity to catechol as a substrate. ItsKm(66.17 µM) was approximately 30% lower than that of wild-type enzyme. Interestingly, removal of the C-terminal residues resulted in 1.5- to 1.8-fold(P<0.05)increase in the activity of C23OB61 against 4-methylcatechol and 4-chlorocatechol, respectively, while towards catechol the activity of the protein dropped to about 80% of that of the wild-type enzyme. The results obtained may facilitate the engineering of the C23O for application in the bioremediation of polluted areas.

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