[39 Tetanus and Botulism Neurotoxins Isolation and Assay

By Giampietro Schiavo and Cesare Montecucco Introduction The paralysis associated with tetanus and botulism is caused by neurotoxins produced by bacteria of the genus Clostridium. These are the most powerful toxins known, the LD50 in mice being in the range 0.1-1 ng kg. Tetanus neurotoxin (TeNT) is produced by toxigenic strains of Clostridium tetani in a single type, whereas seven different serotypes (A, B, C, D, E, F, and G) of botulism neurotoxin (BoNT) are made by Clostridium botuli-num and...

[21 Snake Venom Metalloendopeptidases Reprolysins

Throughout the course of recorded history of human civilization mankind has shown a unique fascination for venomous snakes, both admiring and detesting the awesome powers residing within their serpentine embrace. The destructive effects of snake venoms on living organisms have been obvious to all, but their potential healing powers have long been suspected by some. Thus ancient emblems of some medical professions frequently have a snake in their design drawn from the caduceus, a winged staff...

Hnc

AMINO , TOO , 200 , 30 ,400 , 50Q ACIDS PRE-DOMAIN g CATALYTIC -DOMAIN Fig. 1. Schematic comparison of the structures of prepro-HFC and prepro-HNC. The three histidine residues responsible for binding the catalytic zinc as well as the three cysteine residues present in each protein are indicated. The two cysteines in the hemopexin domain are disulfide bonded. The third, unpaired cysteine is part of the conserved cysteine switch region in the propeptide domain. The sites of proteolysis between...

Kkiremqkflglevtgkldsdtlevmrkprcgvpdvgh Frtfpgipkw

Steps involved in prostromelysin 1 activation by proteinases and APMA. Initial cleavage sites by chymotrypsin (CT), human neutrophil elastase (HNE), and plasma kallikrein (PKK) as well as the autolytic site of cleavage promoted by APMA in the propeptide are indicated. The final activation is mediated by the action of stromelysin 1 intermediates generated by proteinases or APMA, and a 45-kDa stromelysin 1 is produced. The 45-kDa form undergoes autolysis to the 28-kDa enzyme. Trypsin (T)...

Info

Inhibitors synthesized in pharmaceutical industries were also reported to have potent inhibitory potencies against NEP and ACE.46,46a'46b Analgesic Responses Induced by Enkephalin-Degrading Enzyme Inhibitors Antinociceptive Effects of Selective or Mixed Inhibitors Inhibitors of NEP or APN are able to potentiate the analgesic effects of exogenous enkephalins and, more interestingly, possess a weak intrinsic opioidergic action after intracerebroventricular (i.c.v.) injection. This has been...

Tnpypnsters Tnaqpdsaers

Conservation of sequences around the zinc ligands and catalytic residues of clan MB. Residues are numbered according to astacin. Asterisks (*) indicate the zinc ligands, + indicates the catalytic Glu residue, and indicates the Met turn. Residues identical to those in the astacin sequence are shown in white on black. Key to sequences 1, astacin 2, mouse meprin a subunit 3, rat meprin 6 subunit 4, human bone morphogenetic protein 1 5, Oryzias latipes hatching enzyme (HCE1) 6, O. latipes...

[46 Purification and Characterization of Mitochondrial Processing Peptidase of Neurospora crassa

By Michael Brunner and Walter Neupert Introduction Many nuclear encoded mitochondrial proteins are synthesized with a cleavable N-terminal presequence which targets the protein to the mitochondrial matrix.1,2 This presequence is proteolytically removed in the mitochondrial matrix during or shortly after import of the precursor.3 The processing reaction is carried out by the mitochondrial processing peptidase (MPP).4-8 The enzyme is a heterodimeric protein which is composed of the two related...

Fractions

Enzymatic detection of leishmanolysin in fractions after anion-exchange chromatography. showing a comparison of fibrinogen-SDS-PAGE analysis and the milk assay. Fractions collected during elution of the strong anion exchanger Mono Q are assayed by substrate-containing and normal SDS-polyacrylamide gels. Ten microliters from each fraction is mixed with 3 fd of 5 X concentrated sample buffer and loaded onto 12 polyacrylamide gels containing or not 240 fj.g ml-1 copolymerized fibrinogen....

Dtvleemnlp

DTIIKENDLQ EVVLKNIHWD TSVLTTAHYN iKTI0TgHDMg iRT IyRYHDNg .QHSVLTRPDGP ITIISEEDWP LTVIgQTLVP KTCIAGRDWP ISVISDKYWP IATVISYTHWP Fig. 1. Conservation of sequences around the catalytic residues in aspartic endopeptidases. Sequences are numbered according to that of pig pepsin A P. Sepulveda, J. Marciniszyn, D. Liu, and J. Tang, J. Biol. Chem. 250, 5082 (1975) . One cDNA sequence for the gene of a variant of pig pepsin A (PEPA-PIG) has an extra residue (Ile-229A) not seen in pepsins A from other...

[31 Quantification of Matrix Metalloproteinases in Tissue Samples

A number of papers have reported on the assay of matrix metalloproteinases (MMPs) in tissue extracts or homogenates. Many reports contain one or more serious errors the extraction of enzyme is incomplete, latent enzymes are not activated, and inhibitors of MMPs are ignored. If a comparison is made between enzyme levels in two different situations, for example, normal and disease state, then the errors may become serious. If the extraction is incomplete, changes in the extracellular matrix may...

[47 OSialoglycoprotease from Pasteurella haemolytica

A novel endopeptidase, which hydrolyzes peptide bonds within glycoprotein substrates, has been described in culture supernatants of the bacterium Pasteurella haemolytica The P. haemolytica enzyme is designated as an O-sialoglycoprotein endopeptidase (E.C. 3.4.24.57) or glycoprotease because of its specificity for the proteolytic cleavage of proteins which have a significant number of O-sialoglycans attached to serine and threonine residues.2 The amino acid sequence predicted from the nucleotide...

[36 Neurolysin Purification and Assays

Dauch, V. Dive, B. Vincent, and J. P. Vincent Neurolysin (EC 3.4.24.16, also known as neurotensin-degrading neutral metalloendopeptidase) inactivates neurotensin by cleaving the peptide at the Pro10-Tyru bond, leading to the biologically inert catabolites neurotensin 1-10 and neurotensin 11-13.1 Previous studies indicated that substitution of the Tyr-11 residue by an aromatic D-amino acid led to highly potent neurotensin analogs in vivo,2'4 although they behaved as...

Wat Is D Enzyme Aggressin

THERM AD( ADNQFFASYD ) APAVDAHYYAGVTYDYYKNVHNR( LSYDGNNA)AIRSSVHYSQGYNNAFWNGSEH 120 PSEUD 120 THERM VSVVG) IGRDKLGKIFYRALTOYLTPTSNFSOLRAAAVOSATDLf YGSTSQE) VASVKQAFDAVGVK 316 a > < 1 H> 0 , > Fig. 2. Sequence alignment of thermolysin (THERM) and pseudolysin (PSEUD) implied by superposition of the structures, fi strands are denoted by arrows, helices by cylinders identical residues are highlighted with bars between the sequences. For segments of sequence included in parentheses, the...

Hflek Lxngli

id. 16 , si. 28 id. < 10 524 a Fig. 3. Schematic comparison of NRD convertase with related proteins. rIDE, Rat insulin-degrading enzyme13 hIDE, human insulin-degrading enzyme14 dIDE, Drosophila insulin-degrading enzyme15 protease III, E. coli protease III16 pqqF, open reading frame F of the pyrroloquinoline quinone operon of Klebsiella pneumoniae 1 B.s.protease, Bacillus subtilis processing proteinase18 ra-MPP, rat mitochondrial matrix processing peptidase a subunit.19 Putative signal...

Analgesia

Hypothetical model of interactions between CCK, via CCKA and CCKb receptors, and the opioid system via -opioid and -opioid receptors. This overlapping distribution has focused investigations on the role of CCK in nociception. The existence of regulatory mechanisms between CCK and enkephalin systems in the control of pain have been proposed. Schematically, activation of CCKB receptors could negatively modulate the opioid system, either directly (via binding of opioid agonists or...

[5 Theoretical and Practical Aspects of Proteinase Inhibition Kinetics

Many physiological and pathological processes are controlled by proteinases whose activity is itself regulated by protein proteinase inhibitors. The endogenous inhibitors usually exhibit a good proteinase class specificity, for example, serine proteinase inhibitors do not inhibit cysteine proteinases and vice versa. However, when tested with a rough in vitro assay, a given proteinase inhibitor will usually inhibit many proteinases belonging to the same class. The only way to define the...

[1 Peptide Thioester Substrates for Serine Peptidases and Metalloendopeptidases

Synthetic peptide substrates are widely used in biochemical and physiological studies of proteolytic enzymes. Synthetic peptide substrates can be used to detect enzyme during isolation, to assay enzyme activity, to determine enzyme concentrations, to investigate enzyme specificity, and to determine inhibitor potency. The three most commonly used synthetic substrates are peptide 4-nitroanilides, peptide thioesters, and peptide derivatives of 7-amino-4-methylcoumarin. Amino acid and peptide...

[ 14 Removal and Replacement of Metal Ions in Metallopeptidases

Metal chelators contain anionic or neutral oxygen, nitrogen, and sulfur atoms spatially arranged to give bi-, tri-, or tetradentate ligation to a metal atom. Chelators (C) can inhibit metalloenzyme (EM) catalysis either by Copyright 1995 by Academic Press, Inc. METHODS IN BNZYMOLOGY, VOL. 248 All rights of reproduction in any form reserved. removal of the metal (M) from the enzyme or by directly binding to it. The former mechanism may be written where Kx MC ( M C ), K2 MC2 ( MC C ), K3 MC3 (...

[2 Fluorimetric Assays of Proteolytic Enzymes

Fluorogenic substrates of proteolytic enzymes may be divided into three groups. The first group employs aromatic amines, such as 7-amino-4-methyl-coumarin, which change fluorescence characteristics on acylation by an Copyright 1995 by Academic Press, Inc. All rights of reproduction in any form reserved. amino acid. Cleavage of the amide bond by a peptidase is accompanied by an increase in fluorescence, when monitored at the appropriate wavelengths. In most substrates of this type, the...

[12 Bacterial Prolipoprotein Signal Peptidase

Wu Since the discovery and structural elucidation of the major outer membrane protein of Escherichia coli (also known as Braun's or murein lipoprotein),'-2 more than 130 lipoproteins have been identified in bacteria.3 The common feature shared by this structurally and functionally diverse group of membrane proteins in bacteria is the presence of an extensively lipid-modified N-terminal amino acid, namely, cysteine substituted on sulfur with the CH2(OOCR)...

[13 Evolutionary Families of Metallopeptidases

Barrett Metallopeptidases form the most diverse of the catalytic types of peptidases, about 30 families being recognized by our criteria (see 1 , Volume 244, this series, for definitions of the terms family and clan). About half of the families comprise enzymes containing the His-Glu-Xaa-Xaa-His (or HEXXH) motif that has been shown by X-ray crystallography to form part of the site for binding of the metal (normally zinc) atom in some families, and almost...

D E

VPSILMEYFS LPSHMEHFL VPSCJ4LENWV APSQtfLENWV APSQYILEFWT LPSQFMHSWC LPSQFMHMC FPSQUN1EHWA. FPSQINEHWA PS E VPSILMEYFS LPSHMEHFL VPSCJ4LENWV APSQtfLENWV APSQYILEFWT LPSQFMHSWC LPSQFMHMC FPSQUN1EHWA. FPSQINEHWA PS E ndykwsqfa nsrqvlslfh wdtdslrrls wekeplmrms wnknelinls wepealafis wepealgfis shpkvferya thpqvfak a khyqtcqplp adsessssqp khykcgspit ( yrtogeap shykigekip qkeigeplp (byeigeplp rhvdsgekmp khyqsgaamp Fig. 2. Amino acid sequence comparison of the region surrounding the putative Zn2 binding...

1

is the most potent NEP inhibitor described so far (K 0.03 nM), a property that has been used to visualize NEP directly in crude membrane fractions after gel electrophoresis.33 Another interesting series of inhibitors are the phosphorus-containing dipeptides,34,34a among which is the natural competitive inhibitor of NEP, phosphoramidon. All the inhibitors generally have a limited ability to penetrate the gastrointestinal and blood-brain barriers. Protection of the thiol and or carboxyl groups...

O O

Hydroxyapatite chromatography of post-DEAE D1 and D2 pooled proteins. Post-DEAE pooled proteins (Dl, top profile D2, bottom profile) were concentrated, dialyzed, and loaded on the hydroxyapatite column. All fractions were tested for neurotensin degradation and analyzed by HPLC as described in the text (system 2). Insets correspond to HPLC analysis of neurotensin degradation by HI or H2 pooled proteins. steps give a purification factor of about 550-fold with an overall recovery of 15...

[6 Active Site Titration of Peptidases

Measurement of the active-site molarity of a peptidase is a valuable step toward defining the molecular properties of the enzyme. In the absence of such a method, catalytic activity (micromoles of substrate cleaved per second) can only be defined in terms of protein concentration. Protein concentration may be measured directly or derived from an established assay for which a specific activity was calculated on the basis that the purified protein was fully active. This assumption is likely to be...

[44 Insulysin and Pitrilysin Insulin Degrading Enzymes of Mammals and Bacteria

Roth Introduction Insulin regulates a wide variety of metabolic and cellular processes in target tissues. Most, if not all, of the varied effects of insulin appear to be mediated through a specific cell-surface receptor.1,2 After insulin binds to its receptor, the receptor-hormone complex is internalized in endocytic vesicles.35 When the vesicles are acidified, the complex dissociates, with most of the insulin receptors being recycled to the cell surface and...

L 2500

Phylogenetic tree for the interstitial collagenase family (M10). The alignment of peptidase domain sequences and construction of the phylogenetic tree were as for Fig. 4. The tree has been calibrated for evolutionary distance in millions of years ago by assuming that the divergence between soybean leaf metalloendopeptidase and the other matrixins represents 1000 million years ago. Key 1, soybean leaf metalloendopeptidase 2, Paracentrotus lividus envelysin 3, Xenopus stromelysin 3 4,...

Easson Stedman Equation

1 2 3 A 5 6 INHIBITOR ENZYME (mol mol) Fig. 2. Experimental illustration of the influence of E 0 on the shape of the inhibition curve. The effect of increasing concentrations of recombinant eglin c on the activity of constant concentrations of porcine pancreatic elastase was studied using two widely different concentrations of enzyme. The inhibition curve was linear with 5 iM elastase (curve 1) and concave with 30 nAi elastase (curve 2). Other conditions were as follows for curve 1,E + I...

[ 15 Pseudolysin and Other Pathogen Endopeptidases of Thermolysin Family

Pseudomonas aeruginosa is an opportunistic pathogen which can cause fatal infection in vulnerable hosts.1 Several products of the organism that are related to virulence have been identified and characterized.2 Among them, elastase has long been thought to be of major significance.3 The elastase is now called pseudolysin (EC 3.4.24.26) by recommendation of the IUBMB. Pseudolysin is a Zn-metalloendopeptidase belonging to the thermolysin family (or family MH, see 13 in this volume).3 Similar...

Cd O

Simulated tight-binding titrations of 20 nM cathepsin D by pepstatin analogs, calculated using Eq. (11), at different values of EJo Kj. When the titration is analyzed by curve-fitting, an EJo ivj ratio of 2 is probably sufficient to give accurate values of E 0 and K . Much higher values of the ratio are necessary for a manual extrapolation to be accurate. obtained was the same as that determined with A'- ra .v-cinnamoy imidazole,33 showing that the inhibitor was 100 active. An...

[17 Inhibitors of Neprilysin Design Pharmacological and Clinical Applications

Roques, Florence Noble, Philippe Crine, and Marie-Claude Fournie-Zaluski There has been considerable interest in ectoenzymes (membrane-bound peptidases) since the discovery that inhibition of angiotensin-converting enzyme (ACE), the enzyme implicated in the formation of angiotensin II from angiotensin I, led to antihypertensive effects,1 and that the inhibition of another membrane-bound Zn-metallopeptidase, involved in the inactiva-tion of the opioid enkephalins in the brain,...

[16 Neprilysin Assay Methods Purification and Characterization

Neprilysin (neutral endopeptidase 24.11, EC 3.4.24.11) is a plasma membrane-bound zinc-containing enzyme which degrades and inactivates a number of bioactive peptides. Neprilysin is one of the many zinc metallopepti-dases (see 13 in this volume). The enzyme was first isolated from porcine kidney brush border as an endopeptidase hydrolyzing the B chain of insulin.1'2 It is an ectoenzyme composed of a 23-amino acid N-terminal cytoplasmic domain, a 28-amino acid membrane-spanning domain, and an...

4-methylumbelliferyl 4-guanidinobenzoate Mangel Wf

Tuhy, C.-M. Kam, and J. C. Powers, J. Biol. Chem. 259, 4279 1984 . h J. C. Powers, R. Boone, D. L. Carroll, B. F. Gupton, C.-M. Kam, N. Nishino, M. Sakamoto, and P. Tuhy, J. Biol. Chem. 259, 4288 1984 . ' R. R. Cook and J. C. Powers, Biochem. J. 215, 287 1983 . '' G. R. Schonbaum, B. Zerner, and M. L. Bender, J. Biol. Chem. 236, 2930 1961 . p M. L. Bender, M. L. Begue-Canton, R. L. Blakely, L. J. Brubacher, J. Feder, C. R. Gunter, F. J. Kezdy, J. V....

Xiii

Peptides I-VII are fluorogenic and VIII-XIII are chromogenic. Meprin A data are from R. L. Wolz, R. B. Harris, and J. S. Bond, Biochemistry 30, 8488 1991 , and astacin data are from R. L. Wolz, Arch. Biochem. Biophys. 310, 144 1994 . 6 Abbreviations 2ABz, 2-aminobenzoyl Hyp, hydroxyproline Npa, 4-nitrophenylalanine. H, Meprin A cleavage T - astacin cleavage. Numbers preceding the arrows indicate proportion of cleavage. ''Two cleavage sites kCM d S dt. don an inhibitor of thermolysin and...

Contributors to Volume 248

Article numbers are in parentheses following the names of contributors. Affiliations listed are current. Angela Anastasi 43 , Department of Pathology, The Medical School, St. Luke's Hospital, M sida, Malta David S. Auld 14 , Center for Biochemical and Biophysical Sciences and Medicine, and Department of Pathology, Harvard Medical School and Brigham and Women's Hospital, Boston, Massachusetts 02115 H l n Barelli 36 , Institut de Pharmacologie Mol culaire et Cellulaire, CNRS, Universit de...

[40 Human Carboxypeptidase N Lysine Carboxypeptidase

Human plasma carboxypeptidase N lysine carboxypeptidase EC 3.4.17.3 was discovered by Erdos and Sloane in the early 1960s1 as an enzyme that inactivates bradykinin by cleaving its C-terminal arginine. It was rediscovered and renamed many times as it was found to cleave C-terminal Arg or Lys from a variety of peptide and protein substrates.2,3 Carboxypeptidase N is an important plasma regulator of peptide hormone activity and protects against the deleterious actions of peptides released into the...

Sbq With Nasogastric Tube

0 20 40 60 80 100 Stromelysin 1 ng tube Fig. 1. Transferrin radioassay. Various concentrations of stromelysin 1 10 xl were mixed with 3H Cm-Tf 10 xl in TNC buffer and incubated for 2 hr and 4 hr A at 37 . The total radioactivity of 10 A of 3H Cm-Tf digested by 10 A of trypsin 100 xg ml was 23,000 cpm. 0 20 40 60 80 100 Stromelysin 1 ng tube Fig. 1. Transferrin radioassay. Various concentrations of stromelysin 1 10 xl were mixed with 3H Cm-Tf 10 xl in TNC buffer and incubated for 2 hr and 4 hr A...