High Throughput Substrate Specificity Profiling of Serine and Cysteine Proteases Using Solution-Phase Fluorogenic Peptide Microarrays*
Total Page:16
File Type:pdf, Size:1020Kb
Load more
Recommended publications
-
Enzymes Handling/Processing
Enzymes Handling/Processing 1 Identification of Petitioned Substance 2 3 This Technical Report addresses enzymes used in used in food processing (handling), which are 4 traditionally derived from various biological sources that include microorganisms (i.e., fungi and 5 bacteria), plants, and animals. Approximately 19 enzyme types are used in organic food processing, from 6 at least 72 different sources (e.g., strains of bacteria) (ETA, 2004). In this Technical Report, information is 7 provided about animal, microbial, and plant-derived enzymes generally, and more detailed information 8 is presented for at least one model enzyme in each group. 9 10 Enzymes Derived from Animal Sources: 11 Commonly used animal-derived enzymes include animal lipase, bovine liver catalase, egg white 12 lysozyme, pancreatin, pepsin, rennet, and trypsin. The model enzyme is rennet. Additional details are 13 also provided for egg white lysozyme. 14 15 Chemical Name: Trade Name: 16 Rennet (animal-derived) Rennet 17 18 Other Names: CAS Number: 19 Bovine rennet 9001-98-3 20 Rennin 25 21 Chymosin 26 Other Codes: 22 Prorennin 27 Enzyme Commission number: 3.4.23.4 23 Rennase 28 24 29 30 31 Chemical Name: CAS Number: 32 Peptidoglycan N-acetylmuramoylhydrolase 9001-63-2 33 34 Other Name: Other Codes: 35 Muramidase Enzyme Commission number: 3.2.1.17 36 37 Trade Name: 38 Egg white lysozyme 39 40 Enzymes Derived from Plant Sources: 41 Commonly used plant-derived enzymes include bromelain, papain, chinitase, plant-derived phytases, and 42 ficin. The model enzyme is bromelain. -
Biochemistry and the Genomic Revolution 1.1
Dedication About the authors Preface Tools and Techniques Clinical Applications Molecular Evolution Supplements Supporting Biochemistry, Fifth Edition Acknowledgments I. The Molecular Design of Life 1. Prelude: Biochemistry and the Genomic Revolution 1.1. DNA Illustrates the Relation between Form and Function 1.2. Biochemical Unity Underlies Biological Diversity 1.3. Chemical Bonds in Biochemistry 1.4. Biochemistry and Human Biology Appendix: Depicting Molecular Structures 2. Biochemical Evolution 2.1. Key Organic Molecules Are Used by Living Systems 2.2. Evolution Requires Reproduction, Variation, and Selective Pressure 2.3. Energy Transformations Are Necessary to Sustain Living Systems 2.4. Cells Can Respond to Changes in Their Environments Summary Problems Selected Readings 3. Protein Structure and Function 3.1. Proteins Are Built from a Repertoire of 20 Amino Acids 3.2. Primary Structure: Amino Acids Are Linked by Peptide Bonds to Form Polypeptide Chains 3.3. Secondary Structure: Polypeptide Chains Can Fold Into Regular Structures Such as the Alpha Helix, the Beta Sheet, and Turns and Loops 3.4. Tertiary Structure: Water-Soluble Proteins Fold Into Compact Structures with Nonpolar Cores 3.5. Quaternary Structure: Polypeptide Chains Can Assemble Into Multisubunit Structures 3.6. The Amino Acid Sequence of a Protein Determines Its Three-Dimensional Structure Summary Appendix: Acid-Base Concepts Problems Selected Readings 4. Exploring Proteins 4.1. The Purification of Proteins Is an Essential First Step in Understanding Their Function 4.2. Amino Acid Sequences Can Be Determined by Automated Edman Degradation 4.3. Immunology Provides Important Techniques with Which to Investigate Proteins 4.4. Peptides Can Be Synthesized by Automated Solid-Phase Methods 4.5. -
Solutions to Detect Apoptosis and Pyroptosis
Powerful Tools for In vitro Fluorescent Imaging in Cancer Research Jackie Carville May 11, 2016 About Us Products and Services • Custom Assay Services – Immunoassay design and development – Antibody purification/conjugation • Consumable Reagents – ELISA Solutions – Cell-permeant Fluorescent Probes for detection of cell death • Products for research use only. Not for use in diagnostic procedures. ELISA Solutions • ELISA Wash Buffer • PBS • Substrates • Stop Solutions • Plates • Coating Buffers • Blocking Buffers • Conjugate Stabilizers • Assay Diluents • Sample Diluents Custom Services • Antibody Conjugation • Immunoassay Design • Immunoassay Development • Lyophilization • Plate Coating Overview of ICT’s Fluorescent Probes FOR DETECTION OF: • Active caspases • Caspases/Cathepsins in real time • Mitochondrial Health • Cytotoxicity • Serine Proteases Today’s Agenda • FLICA • FLISP • Magic Red • Cell Viability • Mito PT • Oxidative Stress • Cytotoxicity FLICA • FLICA – Fluorescent Labeled Inhibitor of Caspases • In vitro, whole cell detection of caspase activity in apoptotic or caspase-positive cells • Available in green, red, and far red assays FLICA Cancer Research Applications • FLICA is the most accurate and sensitive method of apoptosis detection • Can identify four stages of cell death in one sample • Detect poly caspase activity or individual caspase enzymes How FLICA A. INACTIVE CASPASE (ZYMOGEN) works… prodomain B. CASPASE PROCESSING FMK Fluorescent Reporter YVAD tag C. ACTIVATED CASPASE (HETERO-TETRAMER) D. BINDING OF FLICA Sample -
The Osteoclast-Associated Protease Cathepsin K Is Expressed in Human Breast Carcinoma1
CANCER RESEARCH57. 5386-5390. December I, 19971 The Osteoclast-associated Protease Cathepsin K Is Expressed in Human Breast Carcinoma1 Amanda J. Littlewood-Evans,' Graeme Bilbe, Wayne B. Bowler, David Farley, Brenda Wlodarski, Toshio Kokubo, Tetsuya Inaoka, John Sloane, Dean B. Evans, and James A. Gallagher Novartis Pharina AG, CH-4002 Base!. Switzerland (A. I. L-E., G. B., D. F., D. B. El; Human Bone Cell Research Group, Department of Human Anatomy and Cell Biology 1W. B. B., B. W., J. A. G.J, and Department of Pathology If. S.), University of Liverpool, Liverpool 5.69 3BX, United Kingdom [W. B. B., B. W., J. S., J. A. G.]; and Takarazuka Research Institute, Novartis Pharma lid., Takarazuka 665, Japan IT. K., T. LI ABSTRACT (ZR75—l,Hs578Tduct, BT474, ZR75—30,BT549, and T-47D), adenocarci noma (MDA-MB-231, SK-BR3, MDA-MB-468, and BT2O), and breast car Human cathepsin K is a novel cysteine protease previously reported cinoma[MVLNandMTLN,transfectedMCF-7-derivedclonesobtainedfrom to be restricted in its expression to osteoclasts. Immunolocalization of Dr. J. C. Nicolas (Unit de Recherches sur Ia Biochemie des Steroides Insern cathepsin K in breast tumor bone metastases revealed that the invading U58, Montpelier, France), and MCF-7]. The MCF-7 cells were obtained from breast cancer cells expressed this protease, albeit at a lower intensity two different sources, ATCC and Dr. Roland Schuele (Tumor Klinik, Freiburg, than in osteoclasts. In situ hybridization and immunolocalization stud Germany). These are indicated in the figure legend as clone 1 (ATCC) and ies were subsequently conducted to demonstrate cathepsin K mRNA clone 2 (Dr. -
Application of Plant Proteolytic Enzymes for Tenderization of Rabbit Meat
Biotechnology in Animal Husbandry 34 (2), p 229-238 , 2018 ISSN 1450-9156 Publisher: Institute for Animal Husbandry, Belgrade-Zemun UDC 637.5.039'637.55'712 https://doi.org/10.2298/BAH1802229D APPLICATION OF PLANT PROTEOLYTIC ENZYMES FOR TENDERIZATION OF RABBIT MEAT Maria Doneva, Iliana Nacheva, Svetla Dyankova, Petya Metodieva, Daniela Miteva Institute of Cryobiology and Food Technology, Cherni Vrah 53, 1407, Sofia, Bulgaria Corresponding author: Maria Doneva, e-mail: [email protected] Original scientific paper Abstract: The purpose of this study is to assess the tenderizing effect of plant proteolytic enzymes upon raw rabbit meat. Tests are performed on rabbit meat samples treated with papain and two vegetal sources of natural proteases (extracts of kiwifruit and ginger root). Two variants of marinade solutions are prepared from each vegetable raw materials– 50% (w/w) and 100 % (w/w), with a duration of processing 2h, 24h, and 48h. Changes in the following physico- chemical characteristics of meat have been observed: pH, water-holding capacity, cooking losses and quantity of free amino acids. Differences in values of these characteristics have been observed, both between control and test samples, as well as depending of treatment duration. For meat samples marinated with papain and ginger extracts, the water-holding capacity reached to 6.74 ± 0.04 % (papain), 5.58 ± 0.09 % (variant 1) and 6.80 ± 0.11 % (variant 2) after 48 hours treatment. In rabbit meat marinated with kiwifruit extracts, a significant increase in WHC was observed at 48 hours, 3.37 ± 0.07 (variant 3) and 6.84 ± 0.11 (variant 4). -
Evaluation of Anthelmintic Activity of Carica Papaya Latex Using Pheritima Posthuma
Research Article Vol 2/Issue 1/Jan-Mar 2012 EVALUATION OF ANTHELMINTIC ACTIVITY OF CARICA PAPAYA LATEX USING PHERITIMA POSTHUMA LAKSHMI KANTA KANTHAL1*, PRASENJIT MONDAL2, SOMNATH DE4, SOMA JANA3, S. ANEELA4 AND K. SATYAVATHI1 1Koringa college of Pharmacy, Korangi, Tallarevu (M), East Godavari Dist., A.P. 2Vaageswari College Of Pharmacy, Karimnagar, A.P. 3Vaageswari Institute Of Pharmaceutical sciences, Karimnagar, A.P 4Dr.Samuel George Institute Of Pharmaceutical Sciences, Markapur, A.P. ABSTRACT The aim of present study is to evaluate Anthelmintic potential of latex of Carica papaya using Pheretima posthuma as test worms. Various concentrations (100%, 50%, and 20%) of Carica papaya latex were tested in the assay, which involved determination of time of paralysis (P) and time of death (D) of the worms. It show shortest time of paralysis (P=24.5 min) and death (D=56min) in 100% concentration, while the time of paralysis and death will increase in 50% concentration (P=28min&D=64min) and in 20% concentration (P=34min&D=74min) respectively as compare to Piperazine citrate (10mg/ml) used as standard reference (P= 24 min& D= 54) and distilled water as control. The results of present study indicated that the latex of Carica papaya showed significantly demonstrated paralysis, and also caused death of worms especially at higher concentration as compared to standard reference Piperazine citrate and control.From the result it is conclude that the latex of Carica papaya showed significant Anthelmintic activity. Key words : Pheretima posthuma, Anthelmintic, Carica papaya latex, Piperazine citrate. 1. INTRODUCTION Helminthiasis is a disease in which a part of the .2005).The papaya is a short-lived, fast-growing, body is infested with worms such as pinworm, woody, large herb to 10 or 12 feet in height. -
Data Sheet Cathepsin V Inhibitor Screening Assay Kit Catalog #79589 Size: 96 Reactions
6405 Mira Mesa Blvd Ste. 100 San Diego, CA 92121 Tel: 1.858.202.1401 Fax: 1.858.481.8694 Email: [email protected] Data Sheet Cathepsin V Inhibitor Screening Assay Kit Catalog #79589 Size: 96 reactions BACKGROUND: Cathepsin V, also called Cathepsin L2, is a lysosomal cysteine endopeptidase with high sequence homology to cathepsin L and other members of the papain superfamily of cysteine proteinases. Its expression is regulated in a tissue-specific manner and is high in thymus, testis and cornea. Expression analysis of cathepsin V in human tumors revealed widespread expression in colorectal and breast carcinomas, suggesting a possible role in tumor processes. DESCRIPTION: The Cathepsin V Inhibitor Screening Assay Kit is designed to measure the protease activity of Cathepsin V for screening and profiling applications. The Cathepsin V assay kit comes in a convenient 96-well format, with purified Cathepsin V, its fluorogenic substrate, and Cathepsin buffer for 100 enzyme reactions. In addition, the kit includes the cathepsin inhibitor E- 64 for use as a control inhibitor. COMPONENTS: Catalog # Component Amount Storage 80009 Cathepsin V 10 µg -80 °C Fluorogenic Cathepsin Avoid 80349 Substrate 1 (5 mM) 10 µl -20 °C multiple *4X Cathepsin buffer 2 ml -20 °C freeze/thaw E-64 (1 mM) 10 µl -20 °C cycles! 96-well black microplate * Add 120 µl of 0.5 M DTT before use. MATERIALS OR INSTRUMENTS REQUIRED BUT NOT SUPPLIED: 0.5 M DTT in aqueous solution Adjustable micropipettor and sterile tips Fluorescent microplate reader APPLICATIONS: Great for studying enzyme kinetics and screening small molecular inhibitors for drug discovery and HTS applications. -
Serine Proteases with Altered Sensitivity to Activity-Modulating
(19) & (11) EP 2 045 321 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 08.04.2009 Bulletin 2009/15 C12N 9/00 (2006.01) C12N 15/00 (2006.01) C12Q 1/37 (2006.01) (21) Application number: 09150549.5 (22) Date of filing: 26.05.2006 (84) Designated Contracting States: • Haupts, Ulrich AT BE BG CH CY CZ DE DK EE ES FI FR GB GR 51519 Odenthal (DE) HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI • Coco, Wayne SK TR 50737 Köln (DE) •Tebbe, Jan (30) Priority: 27.05.2005 EP 05104543 50733 Köln (DE) • Votsmeier, Christian (62) Document number(s) of the earlier application(s) in 50259 Pulheim (DE) accordance with Art. 76 EPC: • Scheidig, Andreas 06763303.2 / 1 883 696 50823 Köln (DE) (71) Applicant: Direvo Biotech AG (74) Representative: von Kreisler Selting Werner 50829 Köln (DE) Patentanwälte P.O. Box 10 22 41 (72) Inventors: 50462 Köln (DE) • Koltermann, André 82057 Icking (DE) Remarks: • Kettling, Ulrich This application was filed on 14-01-2009 as a 81477 München (DE) divisional application to the application mentioned under INID code 62. (54) Serine proteases with altered sensitivity to activity-modulating substances (57) The present invention provides variants of ser- screening of the library in the presence of one or several ine proteases of the S1 class with altered sensitivity to activity-modulating substances, selection of variants with one or more activity-modulating substances. A method altered sensitivity to one or several activity-modulating for the generation of such proteases is disclosed, com- substances and isolation of those polynucleotide se- prising the provision of a protease library encoding poly- quences that encode for the selected variants. -
Current IUBMB Recommendations on Enzyme Nomenclature and Kinetics$
Perspectives in Science (2014) 1,74–87 Available online at www.sciencedirect.com www.elsevier.com/locate/pisc REVIEW Current IUBMB recommendations on enzyme nomenclature and kinetics$ Athel Cornish-Bowden CNRS-BIP, 31 chemin Joseph-Aiguier, B.P. 71, 13402 Marseille Cedex 20, France Received 9 July 2013; accepted 6 November 2013; Available online 27 March 2014 KEYWORDS Abstract Enzyme kinetics; The International Union of Biochemistry (IUB, now IUBMB) prepared recommendations for Rate of reaction; describing the kinetic behaviour of enzymes in 1981. Despite the more than 30 years that have Enzyme passed since these have not subsequently been revised, though in various respects they do not nomenclature; adequately cover current needs. The IUBMB is also responsible for recommendations on the Enzyme classification naming and classification of enzymes. In contrast to the case of kinetics, these recommenda- tions are kept continuously up to date. & 2014 The Author. Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/). Contents Introduction...................................................................75 Kinetics introduction...........................................................75 Introduction to enzyme nomenclature ................................................76 Basic definitions ................................................................76 Rates of consumption and formation .................................................76 Rate of reaction .............................................................76 -
Specificity of Aza-Peptide Electrophile Activity-Based Probes of Caspases
Cell Death and Differentiation (2006), 1–6 & 2006 Nature Publishing Group All rights reserved 1350-9047/06 $30.00 www.nature.com/cdd Specificity of aza-peptide electrophile activity-based probes of caspases KB Sexton1, D Kato1, AB Berger3, M Fonovic1,4, SHL Verhelst1 and M Bogyo*,1,2,3 Activity-Based Probes (ABPs) are small molecules that form stable covalent bonds with active enzymes thereby allowing detection and quantification of their activities in complex proteomes. A number of ABPs that target proteolytic enzymes have been designed based on well-characterized mechanism-based inhibitors. We describe here the evaluation of a novel series of ABPs based on the aza-aspartate inhibitory scaffold. Previous in vitro kinetic studies showed that this scaffold has a high degree of selectivity for the caspases, clan CD cysteine proteases activated during apoptotic cell death. Aza-aspartate ABPs containing either an epoxide or Michael acceptor reactive group were potent labels of executioner caspases in apoptotic cell extracts. However they were also effective labels of the clan CD protease legumain and showed unexpected crossreactivity with the clan CA protease cathepsin B. Interestingly, related aza peptides containing an acyloxymethyl ketone reactive group were relatively weak but highly selective labels of caspases. Thus azapeptide electrophiles are valuable new ABPs for both detection of a broad range of cysteine protease activities and for selective targeting of caspases. This study also highlights the importance of confirming the specificity of covalent protease inhibitors in crude proteomes using reagents such as the ABPs described here. Cell Death and Differentiation advance online publication, 15 December 2006; doi:10.1038/sj.cdd.4402074 Most proteolytic enzymes are regulated by a series of tightly We recently developed a solid-phase synthesis methodo- controlled posttranslational modifications. -
Crystal Structure of Cathepsin X: a Flip–Flop of the Ring of His23
st8308.qxd 03/22/2000 11:36 Page 305 Research Article 305 Crystal structure of cathepsin X: a flip–flop of the ring of His23 allows carboxy-monopeptidase and carboxy-dipeptidase activity of the protease Gregor Guncar1, Ivica Klemencic1, Boris Turk1, Vito Turk1, Adriana Karaoglanovic-Carmona2, Luiz Juliano2 and Dušan Turk1* Background: Cathepsin X is a widespread, abundantly expressed papain-like Addresses: 1Department of Biochemistry and v mammalian lysosomal cysteine protease. It exhibits carboxy-monopeptidase as Molecular Biology, Jozef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia and 2Departamento de well as carboxy-dipeptidase activity and shares a similar activity profile with Biofisica, Escola Paulista de Medicina, Rua Tres de cathepsin B. The latter has been implicated in normal physiological events as Maio 100, 04044-020 Sao Paulo, Brazil. well as in various pathological states such as rheumatoid arthritis, Alzheimer’s disease and cancer progression. Thus the question is raised as to which of the *Corresponding author. E-mail: [email protected] two enzyme activities has actually been monitored. Key words: Alzheimer’s disease, carboxypeptidase, Results: The crystal structure of human cathepsin X has been determined at cathepsin B, cathepsin X, papain-like cysteine 2.67 Å resolution. The structure shares the common features of a papain-like protease enzyme fold, but with a unique active site. The most pronounced feature of the Received: 1 November 1999 cathepsin X structure is the mini-loop that includes a short three-residue Revisions requested: 8 December 1999 insertion protruding into the active site of the protease. The residue Tyr27 on Revisions received: 6 January 2000 one side of the loop forms the surface of the S1 substrate-binding site, and Accepted: 7 January 2000 His23 on the other side modulates both carboxy-monopeptidase as well as Published: 29 February 2000 carboxy-dipeptidase activity of the enzyme by binding the C-terminal carboxyl group of a substrate in two different sidechain conformations. -
Food Proteins Are a Potential Resource for Mining
Hypothesis Article: Food Proteins are a Potential Resource for Mining Cathepsin L Inhibitory Drugs to Combat SARS-CoV-2 Ashkan Madadlou1 1Wageningen Universiteit en Research July 20, 2020 Abstract The entry of SARS-CoV-2 into host cells proceeds by a two-step proteolysis process, which involves the lysosomal peptidase cathepsin L. Inhibition of cathepsin L is therefore considered an effective method to prevent the virus internalization. Analysis from the perspective of structure-functionality elucidates that cathepsin L inhibitory proteins/peptides found in food share specific features: multiple disulfide crosslinks (buried in protein core), lack or low contents of a-helix structures (small helices), and high surface hydrophobicity. Lactoferrin can inhibit cathepsin L, but not cathepsins B and H. This selective inhibition might be useful in fine targeting of cathepsin L. Molecular docking indicated that only the carboxyl-terminal lobe of lactoferrin interacts with cathepsin L and that the active site cleft of cathepsin L is heavily superposed by lactoferrin. Food protein-derived peptides might also show cathepsin L inhibitory activity. Abstract The entry of SARS-CoV-2 into host cells proceeds by a two-step proteolysis process, which involves the lyso- somal peptidase cathepsin L. Inhibition of cathepsin L is therefore considered an effective method to prevent the virus internalization. Analysis from the perspective of structure-functionality elucidates that cathepsin L inhibitory proteins/peptides found in food share specific features: multiple disulfide crosslinks (buried in protein core), lack or low contents of a-helix structures (small helices), and high surface hydrophobicity. Lactoferrin can inhibit cathepsin L, but not cathepsins B and H.