scholarly journals Cathepsin S from bovine spleen. Purification, distribution, intracellular localization and action on proteins

1989 ◽  
Vol 264 (2) ◽  
pp. 467-473 ◽  
Author(s):  
H Kirschke ◽  
B Wiederanders ◽  
D Brömme ◽  
A Rinne
Keyword(s):  
Cathepsin L ◽  
Intracellular Localization ◽  
Gel Filtration ◽  
Similar Rate ◽  
Cathepsin S ◽  
Purification Method ◽  
Cytoplasmic Granules ◽  
Bovine Kidney ◽  
Ph Values ◽  
C 50

Cathepsin S was detected in bovine kidney, spleen, lymph nodes and lung by immunochemical methods. The immunostaining of cathepsin S in kidney was concentrated to the cells of the proximal tubule, where the enzyme was present in cytoplasmic granules. The purification method for cathepsin S from bovine spleen involved (NH4)2SO4 fractionation, chromatography on CM-Sephadex C-50, gel filtration on Sephacryl S-200 and chromatofocusing (pH 8.0-6.0). The enzyme was partially destroyed by autolysis of the homogenate at pH 4.2. The isoelectric point of cathepsin S was 7.0. Cathepsin S was found to hydrolyse proteins at a similar rate to cathepsin L below pH 7.0. At pH values of 7.0-7.5 cathepsin S retained most of its activity, whereas cathepsin L was completely inactive.

Physico-chemical and cytotoxic analysis of a novel large molecular weight bacteriocin produced by Lactobacillus casei TA0021

2019 ◽  
Author(s):  
Elham Noroozi ◽  
Naheed Mojgani ◽  
Elahe Motevaseli ◽  
Mohammad Hossein Modarressi ◽  
Majid Tebianian
Keyword(s):  
Molecular Weight ◽  
Lactobacillus Casei ◽  
Pathogenic Bacteria ◽  
Proteolytic Enzymes ◽  
Shigella Flexneri ◽  
Gel Filtration ◽  
Antimicrobial Protein ◽  
Purification Method ◽  
Ph Values ◽  
Subsequent Decrease

Background and Objectives: Antimicrobial peptides produced by lactic acid bacteria have gained enormous attention owing to their health benefits. This study aimed to isolate, purify and characterize the antibacterial protein produced by au- tochthonous Lactobacillus casei TA0021 strain. Materials and Methods: The antagonistic activity of L. casei TA0021 against a number of pathogenic bacteria was tested by agar well diffusion assay. The antimicrobial agent in the neutralized supernatant fluids was subjected to the action of proteolytic enzymes, catalase, lipase and lysozyme, and their tolerance to variable pH and temperature was estimated. The proteinaceous antagonistic compound was precipitated by 60% w/v ammonium sulphate, desalted and subjected to cation exchange and gel filtration chromatography. Approximate molecular weight of Lactocin was determined by SDS-PAGE and non-denaturing gel electrophoresis. Hemoglobin release assay and cytotoxicity effect of Lactocin TA0021 was determined. The results were statistically analyzed. Results: The antagonistic agent active against Salmonella Typhimurium and Shigella flexneri appeared resistant to catalase and lipase treatments, while sensitive to the tested proteolytic enzymes. Lactocin TA0021 resisted acidic pH values of 3.0, while alkaline pH values of ˃9 completely destroyed the activity. The antibacterial peptide was approximately 68 KDa and heat labile as lost its activity at 100°C after 5 minutes. The bacteriocin was non-toxic to MRC-5 cell lines and non-hemolytic. Purification method lead to increase in antibacterial activity while, subsequent decrease in recovery and yield was observed with increasing purification fold. Conclusion: The purified antimicrobial protein from L. casei TA0021 might be used for application in medicinal and food products.

scholarly journals Cathepsin S The cysteine proteinase from bovine lymphoid tissue is distinct from cathepsin L (EC 3.4.22.15)

1986 ◽  
Vol 240 (2) ◽  
pp. 455-459 ◽  
Author(s):  
H Kirschke ◽  
I Schmidt ◽  
B Wiederanders
Keyword(s):  
Rat Liver ◽  
Polyclonal Antibody ◽  
Lymphoid Tissue ◽  
Cysteine Proteinase ◽  
Rat Kidney ◽  
Cathepsin L ◽  
Bovine Liver ◽  
Cathepsin S ◽  
Irreversible Inhibitor ◽  
C 50

Cathepsin S was purified from bovine spleen by acid autolysis, (NH4)2SO4 fractionation and chromatography on CM-Sephadex C-50, CM-cellulose and activated-thiol-Sepharose. Cathepsin L was isolated from lysosomal fractions of rat liver, rat kidney and bovine liver. Generally, cathepsin L was bound tightly to CM-Sephadex C-50. Preparations of cathepsin L from rat liver, rat kidney and bovine liver were shown to have kinetic constants for the substrate benzyloxycarbonyl-Phe-Arg-7-(4-methyl)coumarylamide in the same range (Km 2-3 microM). Benzyloxycarbonyl-Phe-Phe-diazomethane proved to be a sensitive irreversible inhibitor of cathepsin L from different species. Cathepsin S differed in all these characteristics from cathepsin L. A polyclonal antibody to cathepsin L from rat reacted with bovine cathepsin L but not with bovine cathepsin S.

scholarly journals The purification and properties of cathepsin L from rabbit liver

1984 ◽  
Vol 217 (1) ◽  
pp. 209-217 ◽  
Author(s):  
R W Mason ◽  
M A J Taylor ◽  
D J Etherington
Keyword(s):  
Cathepsin B ◽  
Cathepsin L ◽  
Gel Filtration ◽  
Subcellular Fractionation ◽  
Rabbit Liver ◽  
Purification And Properties ◽  
Chemical Inhibitors ◽  
Michaelis Constants ◽  
Ammonium Sulphate Fractionation ◽  
C 50

Cathepsin L was purified from rabbit liver by a method involving whole-tissue homogenization, pH precipitation, ammonium sulphate fractionation and chromatography on CM-Sephadex C-50, phenyl-Sepharose and Sephadex G-75. Pure enzyme was obtained without the necessity of laborious subcellular fractionation techniques. The Mr of the enzyme was determined to be 29 000 by gel filtration, and affinity for concanavalin A-Sepharose indicated that it was a glycoprotein. A novel technique for detection of enzyme activity in agarose isoelectrofocusing gels showed that the enzyme existed in multiple isoenzymic forms with pI values ranging from 5.0 to 5.9. The enzyme catalysed the hydrolysis of azocasein, collagen and Z-Phe-Arg-NMec (where Z and NMec indicate benzyloxycarbonyl and N-methylcoumarin derivative respectively) optimally at pH 5.2, 3.3 and 6.0 respectively. In addition, cathepsin L was found to degrade benzoyl-Phe-Val-Arg-NMec and 3-carboxypropionyl-Ala-Phe-Lys-NMec. However, cathepsin B also cleaved all of these substrates. One major difference between these two enzymes was in their Michaelis constants for Z-Phe-Arg-NMec; cathepsin B had Km 75 microM whereas that of cathepsin L was 0.7 microM. Cathepsin L was inhibited by all of the usual chemical inhibitors of thiol proteinases as well as the more specific inhibitors Z-Phe-Phe-CHN2, Z-Phe-Ala-CHN2, compound E-64 and compound Ep-475. Active-site titration with compound E-64 showed that the purified sample contained 80% active protein, which had kcat. 20s-1 for the substrate Z-Phe-Arg-NMec. Antibodies were raised to active cathepsin L, and these did not cross-react with cathepsin B, thus demonstrating that these two enzymes are immunologically distinct.

Plasminogen-Activator in Human Early Milk: Its Partial Purification and Characterization

1981 ◽  
Vol 45 (02) ◽  
pp. 121-126 ◽  
Author(s):  
Utako Okamoto ◽  
Noboru Horie ◽  
Yoko Nagamatsu ◽  
Jun-Ichiro Yamamoto
Keyword(s):  
Plasminogen Activator ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Partial Purification ◽  
Order Kinetic ◽  
Diisopropyl Fluorophosphate ◽  
Step Procedure ◽  
Activity Band ◽  
C 50 ◽  
Gel Isoelectric Focusing

SummaryMilk plasminogen-activator was partially purified from human transitional milk collected at about 10 days after delivery, by a five-step procedure involving chloroform treatment, ammonium sulfate precipitation, and column chromatography on Sephadex G-150, CM Sephadex C-50 and DEAE Sephadex A-50. This gave milk-activator with a maximum purification factor of about 2,400-fold with respect to the skimmed milk. The CM Sephadex-step preparation showed, on polyacrylamide gel electrophoresis, a single plasminogen-activator activity band located between the bands of albumin and prealbumin of human serum. This preparation exhibited no kinin forming activity. The activator hydrolyzed acetyl-glycyl-L-lysine methyl ester with similar order kinetic constants to urokinase, and was inhibited strongly by diisopropyl-fluorophosphate. The molecular weight of the activator as estimated by gel filtration was approximately 86,000, the isoelectric points as estimated by gel isoelectric focusing were pH 7.2, 6.9 and 6.6, and the activator activity was not quenched by antiurokinase globulin, indicating that the milk-activator is a different entity from urokinase.

scholarly journals Differential Regulation of Cathepsin S and Cathepsin L in Interferon γ–treated Macrophages

2003 ◽  
Vol 197 (2) ◽  
pp. 169-179 ◽  
Author(s):  
Courtney Beers ◽  
Karen Honey ◽  
Susan Fink ◽  
Katherine Forbush ◽  
Alexander Rudensky
Keyword(s):  
Cathepsin L ◽  
Specific Inhibitor ◽  
Cell Types ◽  
Interferon Γ ◽  
Cathepsin S ◽  
Antigen Presenting Cell ◽  
Relative Contribution ◽  
Helper Cell Type ◽  
Ifn Γ ◽  
Antigen Presenting

Cathepsin S (catS) and cathepsin L (catL) mediate late stages of invariant chain (Ii) degradation in discrete antigen-presenting cell types. Macrophages (Mϕs) are unique in that they express both proteases and here we sought to determine the relative contribution of each enzyme. We observe that catL plays no significant role in Ii cleavage in interferon (IFN)-γ–stimulated Mϕs. In addition, our studies show that the level of catL activity is significantly decreased in Mϕs cultured in the presence of IFN-γ whereas catS activity increases. The decrease in catL activity upon cytokine treatment occurs despite the persistence of high levels of mature catL protein, suggesting that a specific inhibitor of the enzyme is up-regulated in IFN-γ–stimulated peritoneal Mϕs. Similar inhibition of activity is observed in dendritic cells engineered to overexpress catL. Such enzymatic inhibition in Mϕs exhibits only partial dependence upon Ii and therefore, other mechanisms of catL inhibition are regulated by IFN-γ. Thus, during a T helper cell type 1 immune response catL inhibition in Mϕs results in preferential usage of catS, such that major histocompatibility complex class II presentation by all bone marrow–derived antigen-presenting cell is regulated by catS.

Cleavage of immunoglobulin G by excretory–secretory cathepsin S-like protease of Spirometra mansoni plerocercoid

Parasitology ◽  
1994 ◽  
Vol 109 (5) ◽  
pp. 611-621 ◽  
Author(s):  
Y. Kong ◽  
Y.-B. Chung ◽  
S.-Y. Cho ◽  
S.-Y. Kang
Keyword(s):  
Immunoglobulin G ◽  
Cathepsin L ◽  
Immunoblot Analysis ◽  
Cathepsin S ◽  
Terminal Amino Acid ◽  
Optimal Activity ◽  
Spirometra Mansoni ◽  
Terminal Amino ◽  
Secretory Products ◽  
Ph Range

When immunoglobulin G (IgG) was incubated with Spirometra mansoni plerocercoid (sparganum), it was cleaved into Fab and Fc fragments. Fab/c fragments were also hydrolysed. The digestion was accelerated by dithiothreitol (DTT), indicating that cleavage of IgG heavy chain was due to a cysteine protease secreted into the medium. The responsible enzyme, of Mr 27 (± 0·8) kDa, was purified by a series of thiopropyl affinity, Sephacryl S-300 HR and DEAE-anion exchange chromatographies, either from worm extracts or from excretory–secretory products (ESP). The purified, thiol-dependent protease showed an optimal activity at pH 5·7 with 0·1 M sodium acetate but was active over the pH range 4·5–8·0. Its activity was inhibited completely by 10−5 M L-trans-epoxysuccinylleucylamido(4-guanidino) butane (E-64) and 1 mM iodoacetamide (IAA), but by only 53% using the specific cathepsin L inhibitor, Z-Phe-Phe-CHN2 (5 × 10−5 M). Partial NH2-terminal amino acid sequence was Leu-Pro-Asp-Ser-Val-Asn-Trp-Arg-Glu-Gly-Ala-Val-Thr-Ala-Val which showed 80% homology to human cathepsin S. Immunoblot analysis showed that sera from infected patients exhibited IgE antibody reaction. It is proposed that cleavage of immunoglobulin by an excreted–secreted, cathepsin S-like, allergenic protease is a mechanism of immune evasion used by the sparganum.

scholarly journals Conserved Motifs within Ebola and Marburg Virus VP40 Proteins Are Important for Stability, Localization, and Subsequent Budding of Virus-Like Particles

2009 ◽  
Vol 84 (5) ◽  
pp. 2294-2303 ◽  
Author(s):  
Yuliang Liu ◽  
Luis Cocka ◽  
Atsushi Okumura ◽  
Yong-An Zhang ◽  
J. Oriol Sunyer ◽  
...  
Keyword(s):  
Self Assembly ◽  
Mammalian Cells ◽  
Ebola Virus ◽  
Intracellular Localization ◽  
Gel Filtration ◽  
Point Mutations ◽  
Marburg Virus ◽  
Structure Stability ◽  
Wild Type ◽  
Virus Like Particles

ABSTRACT The filovirus VP40 protein is capable of budding from mammalian cells in the form of virus-like particles (VLPs) that are morphologically indistinguishable from infectious virions. Ebola virus VP40 (eVP40) contains well-characterized overlapping L domains, which play a key role in mediating efficient virus egress. L domains represent only one component required for efficient budding and, therefore, there is a need to identify and characterize additional domains important for VP40 function. We demonstrate here that the 96LPLGVA101 sequence of eVP40 and the corresponding 84LPLGIM89 sequence of Marburg virus VP40 (mVP40) are critical for efficient release of VP40 VLPs. Indeed, deletion of these motifs essentially abolished the ability of eVP40 and mVP40 to bud as VLPs. To address the mechanism by which the 96LPLGVA101 motif of eVP40 contributes to egress, a series of point mutations were introduced into this motif. These mutants were then compared to the eVP40 wild type in a VLP budding assay to assess budding competency. Confocal microscopy and gel filtration analyses were performed to assess their pattern of intracellular localization and ability to oligomerize, respectively. Our results show that mutations disrupting the 96LPLGVA101 motif resulted in both altered patterns of intracellular localization and self-assembly compared to wild-type controls. Interestingly, coexpression of either Ebola virus GP-WT or mVP40-WT with eVP40-ΔLPLGVA failed to rescue the budding defective eVP40-ΔLPLGVA mutant into VLPs; however, coexpression of eVP40-WT with mVP40-ΔLPLGIM successfully rescued budding of mVP40-ΔLPLGIM into VLPs at mVP40-WT levels. In sum, our findings implicate the LPLGVA and LPLGIM motifs of eVP40 and mVP40, respectively, as being important for VP40 structure/stability and budding.

scholarly journals Characterization of Melanoidins and Color Development in Dulce de Leche, a Confectionary Dairy Product With High Sucrose Content: Evaluation of pH Effect, an Essential Manufacturing Process Parameter

2021 ◽  
Vol 8 ◽  
Author(s):  
Analía Rodríguez ◽  
Patricia Lema ◽  
María Inés Bessio ◽  
Guillermo Moyna ◽  
Cristina Olivaro ◽  
...  
Keyword(s):  
Enzymatic Hydrolysis ◽  
Ph Effect ◽  
Dairy Product ◽  
Gel Filtration ◽  
Significant Degree ◽  
Product Analysis ◽  
Molecular Weights ◽  
Ph Values ◽  
Initial Ph

The effect on color of the initial pH employed in dulce de leche (DL) production was evaluated through physicochemical and spectroscopical characterization of the melanoidins formed in the process. Melanoidins originated at pH values of 6.5, 7.0, and 7.5, and they were released by the enzymatic hydrolysis of the protein backbone and purified by gel filtration. They showed a significant degree of polydispersity, in general, with molecular weights (MWs) below 1,800 Da. DL produced at a higher pH released melanoidins with higher average MW after the enzymatic hydrolysis. They also presented darker colors (dE*ab, C*), more closely resembling those typical of the commercial product. Analysis of the fractions isolated by gel filtration using HPLC-DAD and multinuclear NMR showed an heterogeneous and complex composition. Even though structurally related, the 1H NMR spectra of melanoidins showed a higher degree of aromaticity at higher pH values. In conclusion, the pH employed in DL production affects the amount and structure of the colored products originated by MR reactions, and thus the color of the final product.

scholarly journals Metabolism of parathyroid hormone. Degradation of 125I-labelled hormone by kidney enzyme

1969 ◽  
Vol 111 (4) ◽  
pp. 509-514 ◽  
Author(s):  
T. J. Martin ◽  
R. A. Melick ◽  
M. de Luise
Keyword(s):  
Parathyroid Hormone ◽  
Trichloroacetic Acid ◽  
Rat Kidney ◽  
Gel Filtration ◽  
Void Volume ◽  
Control Medium ◽  
Ph Values ◽  
Kidney Enzyme ◽  
Rapid Destruction ◽  
Kidney Microsomes

A study was made of the enzymic degradation of 125I-labelled parathyroid hormone by rat kidney microsomes. Incubation with microsomes resulted in rapid destruction of the labelled hormone. The microsomal factor was not separable by dialysis, and the reaction was favoured by pH values in the physiological range. Velocity of the reaction varied directly as the substrate concentration, and additional crude parathyroid hormone (trichloroacetic acid-precipitated, 3·68mg./ml.) inhibited destruction of labelled hormone. There was much less inhibition with added trichloroacetic acid-precipitated calcitonin (3·92mg./ml.) and virtually none with added pig insulin (3·80mg./ml.). Gel filtration of control medium on P6 (Bio-Gel) yielded one radioactive peak at the void volume. After incubation with microsomes three further peaks were obtained on gel filtration. Only the void-volume peak contained intact 125I-labelled parathyroid hormone, indicating that the microsomal enzyme degraded labelled hormone to a number of smaller fragments.

Ultrastructural localization of cationic proteins in cytoplasmic granules of chicken and rabbit polymorphonuclear leukocytes

1975 ◽  
Vol 17 (1) ◽  
pp. 79-94
Author(s):  
E.K. Macrae ◽  
J.K. Spitznagel
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Intracellular Localization ◽  
Ultrastructural Localization ◽  
Cationic Protein ◽  
Cationic Proteins ◽  
Cytoplasmic Granules ◽  
Electron Dense Deposit ◽  
Dense Deposit ◽  
Neutrophilic Polymorphonuclear Leukocytes ◽  
Ammoniacal Silver

Cytoplasmic granules known to contain cationic arginine-rich proteins can be identified by the ammoniacal silver reaction (ASR) which provides a cytochemical marker detectable under the electron microscope. Only the large rod-shaped granules of the chicken polymorphonuclear leukocytes (heterophils) and the large spherical azurophilic granules of the rabbit neutrophilic polymorphonuclear leukocytes show the ASR product as a discrete particulate electron-dense deposit. The other smaller granules are devoid of reaction product, as are membranes and mitochondria. The intracellular localization of the ASR product, as are membranes and mitochondria. The intracellular localization of the ASR product on the large granules coincides with the ASR product localization on the same isolated granule populations, when the ammoniacal silver reaction is applied to these granules after their separation by sucrose-density gradients. The cationic proteins may have intraleukocytic bacteriolytic properties, since ASR product, presumably indicating cationic protein from discharged granules, appears to surround ingested bacteria within cytoplasmic phagosomes.

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