scholarly journals Elastinolytic activity of human cathepsin L

1986 ◽  
Vol 233 (3) ◽  
pp. 925-927 ◽  
Author(s):  
R W Mason ◽  
D A Johnson ◽  
A J Barrett ◽  
H A Chapman
Keyword(s):  
Cathepsin B ◽  
Specific Activity ◽  
Cathepsin L ◽  
Cysteine Proteinases ◽  
Pancreatic Elastase ◽  
Optimum Ph ◽  
Human Cathepsin ◽  
Hydrolysis Of

The hydrolysis of a tritiated elastin substrate by the human cysteine proteinases cathepsins B and L has been studied. Cathepsin L was found to be at least 100-fold more active on this substrate than cathepsin B. The specific activity of cathepsin L at pH 5.5 for hydrolysis of elastin was about the same as that of pig pancreatic elastase at its optimum pH of 8.8.

scholarly journals A comparison of four cathepsins (B, L, N and S) with collagenolytic activity from rabbit spleen

1988 ◽  
Vol 256 (2) ◽  
pp. 433-440 ◽  
Author(s):  
R A Maciewicz ◽  
D J Etherington
Keyword(s):  
Cathepsin B ◽  
Specific Activity ◽  
Cathepsin L ◽  
Cross Reactivity ◽  
Enzymic Activity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Collagenolytic Activity ◽  
Cysteine Proteinases ◽  
Isoelectric Points ◽  
Multiple Charge

We have separated four cathepsins (B, L, N and S) from rabbit spleen. They are all collagen-degrading cysteine proteinases, with Mr values of 25,250, 23,500, 34,000 and 30,000 for cathepsin B, L, N and S respectively. Cathepsins B, N and S have isoelectric points of 5.4, 6.2 and 6.8 respectively, whereas cathepsin L exhibited multiple charge forms in the range 5.0-5.7. A comparison of their specific activity against a variety of protein and synthetic substrates shows many differences. These differences can be visually illustrated through isoelectric focusing and detection of enzymic activity with protein and synthetic-substrate overlays. By using an enzyme-linked immunosorbent assay based on the binding to chicken cystatin and detection with polyclonal and monoclonal antibodies to native cathepsins B and L, no cross-reactivity of the four native enzymes was observed. Studies on the co-operative or synergistic effect in degrading collagen indicated that, of the different combinations tested, only the combination of cathepsin B and N exhibited enhanced collagenolysis.

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.

scholarly journals The use of benzyloxycarbonyl[125I]iodotyrosylalanyldiazomethane as a probe for active cysteine proteinases in human tissues

1989 ◽  
Vol 263 (3) ◽  
pp. 945-949 ◽  
Author(s):  
R W Mason ◽  
L T Bartholomew ◽  
B S Hardwick
Keyword(s):  
Tissue Distribution ◽  
Cathepsin B ◽  
Cathepsin L ◽  
Molecular Size ◽  
Cathepsin S ◽  
Human Tissues ◽  
Cysteine Proteinases ◽  
Additional Protein ◽  
Human Cathepsin ◽  
Kidney Liver

The ability of benzyloxycarbonyl-(125I)Tyr-Ala-CHN2 to label cysteine proteinases in a variety of human tissues was investigated. The inhibitor bound only to cathepsin B in tissues homogenized at pH 5.0. When liver was autolysed at pH 4.0 for up to 4 h, the inhibitor also bound to a protein of Mr 25,000. This was identified immunologically and chromatographically as cathepsin L. Both cathepsins B and L were found primarily in kidney, liver and spleen. In spleen, an additional protein of Mr 25,000 was also labelled. This protein could not be precipitated by antibodies to any of cathepsins B, H and L. This protein has tentatively been identified as human cathepsin S by its tissue distribution, chromatographic properties and molecular size. This work clearly shows that peptidyldiazomethanes are specific probes for cysteine proteinases, and that benzyloxycarbonyl-(125I)Tyr-Ala-CHN2 binds to three such enzymes in human tissues.

The cysteine proteinases of Schistosoma mansoni cercariae

Parasitology ◽  
1997 ◽  
Vol 114 (2) ◽  
pp. 105-112 ◽  
Author(s):  
J. P. DALTON ◽  
K. A. CLOUGH ◽  
M. K. JONES ◽  
P. J. BRINDLEY
Keyword(s):  
Schistosoma Mansoni ◽  
Cathepsin B ◽  
Cathepsin L ◽  
Serine Proteinase ◽  
Skin Penetration ◽  
Scanning Electron Micrographs ◽  
Cysteine Proteinases ◽  
Similar Function ◽  
Substrate Preferences ◽  
Serine Proteinase Activity

Based on substrate preferences, cercariae of Schistosoma mansoni were seen to express both cathepsin L and cathepsin B cysteine proteinases, although the former activity was many -fold greater. Two cathepsin L activities identified in cercarial extracts by zymography co-migrated with activities in extracts of 3 h and 24 h schisotosomula and in extracts of adult worms. Since these enzymes have been implicated in haemoglob in digestion by adult worms, they may perform a similar function in schistosomula. Immunolocalization using scanning electron micrographs showed that cathepsin L and cathepsin B proteinases were present in the cercarial post-acetabular glands. In addition, cercarial serine proteinase activities considered to facilitate skin penetration efficiently cleaved the substrates Z-Gly-Pro-Arg-NHMec and Z-Gly-Pro-Lys-NHMec. Cercariae release most of this serine proteinase activity when induced to secrete the contents of their acetabular glands. In contrast, newly transformed 3 h and 24 h schistosomula did not express this activity.

scholarly journals Purification of cathepsin B by a new form of affinity chromatography

1986 ◽  
Vol 235 (3) ◽  
pp. 731-734 ◽  
Author(s):  
D H Rich ◽  
M A Brown ◽  
A J Barrett
Keyword(s):  
Affinity Chromatography ◽  
Cathepsin B ◽  
Single Step ◽  
Starting Material ◽  
High Yield ◽  
Cysteine Proteinases ◽  
New Form ◽  
Human Cathepsin ◽  
Sepharose 4B

Human cathepsin B was purified by affinity chromatography on the semicarbazone of Gly-Phe-glycinal linked to Sepharose 4B, with elution by 2,2′-dipyridyl disulphide at pH 4.0. The product obtained in high yield by the single step from crude starting material was 80-100% active cathepsin B. The possibility that this new form of affinity chromatography may be of general usefulness in the purification of cysteine proteinases is discussed.

scholarly journals The identification of active forms of cysteine proteinases in Kirsten-virus-transformed mouse fibroblasts by use of a specific radiolabelled inhibitor

1989 ◽  
Vol 257 (1) ◽  
pp. 125-129 ◽  
Author(s):  
R W Mason ◽  
D Wilcox ◽  
P Wikstrom ◽  
E N Shaw
Keyword(s):  
Cathepsin B ◽  
Cathepsin L ◽  
Active Form ◽  
Culture Conditions ◽  
Secreted Protein ◽  
Cysteine Proteinases ◽  
Tumour Invasion ◽  
Specific Antibodies ◽  
Mouse Fibroblasts

The major active forms of cathepsins B and L were identified in Kirsten-virus-transformed mouse fibroblasts by the use of a specific radiolabelled inhibitor, benzyloxycarbonyl-Tyr(-125I)-Ala-CHN2. No other proteins were labelled, demonstrating the specificity of this inhibitor for cysteine proteinases. Cathepsins B and L were distinguished by the use of specific antibodies. One active form of cathepsin B, Mr 33,000-35,000, and two active forms of cathepsin L, Mr 30,000 and 23,000, were identified. The intracellular precursors of these proteins had higher Mr values of 39,000 and 36,000 for cathepsins B and L respectively, as shown by pulse-chase experiments with [35S]methionine-labelled proteins. These did not react with the inhibitor under our culture conditions. The precursor of cathepsin L was secreted whereas the precursor of cathepsin B was not, demonstrating that secretions of the two enzymes are regulated differently. In contrast with results found previously for the purified protein [Mason, Gal & Gottesman (1987) Biochem. J. 248, 449-454], the secreted precursor form of cathepsin L did not react with the inhibitor either, indicating that it is not active and therefore, as such, cannot be directly involved in tumour invasion. The secreted protein did react with the inhibitor when incubated at pH 3.0, showing that the protein can be activated, although this did not occur under our culture conditions.

scholarly journals Quantification of cathepsins B and L in cells

1998 ◽  
Vol 332 (2) ◽  
pp. 499-505 ◽  
Author(s):  
Ruye XING ◽  
Adele K. ADDINGTON ◽  
Robert W. MASON
Keyword(s):  
Active Site ◽  
Total Protein ◽  
Cathepsin B ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Cathepsin L ◽  
Breast Tumour ◽  
Cathepsin S ◽  
Cysteine Proteinases ◽  
Breast Tumour Cells

A method for quantifying active cysteine proteinases in mammalian cells has been developed using an active-site-directed inhibitor. Fluoren-9-ylmethoxycarbonyl(di-iodotyrosylalanyl)-diazomethane (Fmoc-[I2]Tyr-Ala-CHN2) was prepared and shown to react irreversibly with cathepsins B and L, but not with cathepsin S. The non- and mono-iodo forms of the inhibitor reacted with all three enzymes. These results demonstrate that, unlike cathepsins B and L, cathepsin S has a restricted S2-binding site that cannot accommodate the bulky di-iodotyrosine. Fmoc-[I2]Tyr-Ala-CHN2 was able to penetrate cells and react with active enzymes within the cells. A radiolabelled form of the inhibitor was synthesized and the concentration of functional inhibitor was established by titration with papain. This inhibitor was used to quantify active cysteine proteinases in cultured cells. Active cathepsin B was found to be expressed by all of the cells studied, consistently with a housekeeping role for this enzyme. Active forms of cathepsin L were also expressed by all of the cells, but in different quantities. Two additional proteins were labelled in some of the cells, and these may represent other non-characterized proteinases. Higher levels of active cathepsins B and L, and an unidentified protein of Mr 39000, were found in breast tumour cells that are invasive, compared with those that are not invasive. From the data obtained, it can be calculated that the concentrations of both active cathepsins B and L in lysosomes can be as high as 1 mM, each constituting up to 20% of total protein in the organelle. This new technique provides a more direct procedure for determining the proteolytic potential of cellular lysosomes.

scholarly journals Human kidney cathepsins B and L. Characterization and potential role in degradation of glomerular basement membrane

1988 ◽  
Vol 252 (1) ◽  
pp. 301-304 ◽  
Author(s):  
W H Baricos ◽  
Y Zhou ◽  
R W Mason ◽  
A J Barrett
Keyword(s):  
Basement Membrane ◽  
Glomerular Basement Membrane ◽  
Cathepsin B ◽  
Cathepsin L ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Kidney ◽  
Ph Optimum ◽  
Dose Dependent ◽  
Hydrolysis Of

Cathepsins B and L were purified from human kidney. SDS/polyacrylamide-gel electrophoresis demonstrated that cathepsins B and L, Mr 27000-30000, consist of disulphide-linked dimers, subunit Mr values 22000-25000 and 5000-7000. The pH optimum for the hydrolysis of methylcoumarylamide (-NHMec) substrates (see below) is approx. 6.0 for each enzyme. Km and kcat. are 252 microM and 364s-1 and 2.2 microM and 25.8 s-1 for the hydrolysis of Z-Phe-Arg-NHMec (where Z- represents benzyloxycarbonyl-) by cathepsins B and L respectively, and 184 microM and 158 s-1 for the hydrolysis of Z-Arg-Arg-NHMec by cathepsin B. A 10 min preincubation of cathepsin B (40 degrees C) or cathepsin L (30 degrees C) with E-64 (2.5 microM) results in complete inhibition. Under identical conditions Z-Phe-Phe-CHN2 (0.56 microM) completely inhibits cathepsin L but has little effect on cathepsin B. Incubation of glomerular basement membrane (GBM) with purified human kidney cathepsin L resulted in dose-dependent (10-40 nM) GBM degradation. In contrast, little degradation of GBM (less than 4.0%) was observed with cathepsin B. The pH optimum for GBM degradation by cathepsin L was 3.5. Cathepsin L was significantly more active in degrading GBM than was pancreatic elastase, trypsin or bacterial collagenase. These data suggest that cathepsin L may participate in the lysosomal degradation of GBM associated with normal GBM turnover in vivo.

scholarly journals Insight into the Effects of Sous Vide on Cathepsin B and L Activities, Protein Degradation and the Ultrastructure of Beef

Foods ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 1441
Author(s):  
Yantao Yin ◽  
Jailson Pereira ◽  
Lei Zhou ◽  
Jose M. Lorenzo ◽  
Xiaona Tian ◽  
...  
Keyword(s):  
Cathepsin B ◽  
Sarcomere Length ◽  
Cathepsin L ◽  
Myofibrillar Protein ◽  
Potential Mechanism ◽  
Beef Tenderness ◽  
Fragmentation Index ◽  
Sous Vide ◽  
Hydrolysis Of ◽  
Insight Into

This study aimed to evaluate the effects of sous vide cooking (SV) on beef tenderness and its underlying potential mechanism. Beef semimembranosus (SM) were subjected to SV treatments at 45 °C, 55 °C and 65 °C for 4 h. Compared with control samples (CK, cooked at 75 °C until a core temperature of 72 °C was attained), SV treatment significantly promoted the release of cathepsin B and cathepsin L from lysosomes and decreased the shear force of beef SM (p < 0.05). In comparison with CK, samples treated with SV had more hydrolysis of myosin heavy chain and obtained higher myofibrillar fragmentation index, collagen solubility as well as longer sarcomere length (p < 0.05). The current study showed that the proteolysis of myofibrillar protein and collagen induced by cathepsin B and cathepsin L, and the limited longitudinal shrinkage together contributed to the improvement of beef tenderness upon SV.

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