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PubMed1992

Isotype-restricted hyperimmunity in a murine model of the toxic oil syndrome.

The toxic oil syndrome is characterized by IgE elevation and eosinophilia, as well as scleroderma-like skin manifestations and other symptoms of autoimmune disease. Fatty acid anilides, found in large amounts in adulterated cooking oil, were suspected to be the etiologic agent in this disease. The capacity of oleic acid anilide to induce features of autoimmunity in vivo was investigated. B10.S mice were continuously treated i.p. with oleic acid anilide for 6 wk by using osmotic pumps. A significant increase in IgE and IgM serum levels was observed after 1 to 3 wk; subsequently five of six mice developed IgG1 levels 3.5- to 10-fold higher than the controls. Anilide-treated mice developed splenomegaly with a 2.1- and a 3.5-fold increase in IgM- and IgG-bearing splenocytes, respectively, and a 5.6- and 29-fold elevation in functional IgM- and IgG-secreting cells, respectively. Increased serum levels of predominantly IgM antibodies to histone, denatured DNA, and DNP as well as rheumatoid factor were detected. In vivo expression in the spleen of 10 cytokine genes was also examined, and mRNA encoding IL-1 beta and IL-6 were significantly elevated in splenocytes of anilide-treated mice. The enhanced Ig production suggests that anilide induced a cytokine-mediated polyclonal activation of B cells. Elicitation of IgM antibodies to denatured forms of autoantigens indicates that anilide treatment partially broke autoimmune tolerance in these mice. Anilide-treated mice may be a useful animal model for further exploring the mechanism and pathogenesis of systemic autoimmunity in the toxic oil syndrome.

Anilides↗
PubMed1993

Rates of utilization of intravenous oleylanilide administered chronically to the rat.

The constant injection of [14C]oleylanilide into the vena cava of Wistar rats for 7 days with osmotic minipumps was used to estimate tissue distribution of chronically administered anilides. The largest concentrations of the anilide were found in the brown adipose tissue; all other tissues showed much lower proportions. When anilide distribution was expressed as nmol/g lipid, the high concentration of the compound in the brown adipose tissue contrasted with its very low presence in the white adipose tissue; all other tissues showed intermediate concentrations. Anilide-treated rats consumed more food and lost more weight than did controls; their energy balance showed higher energy inefficiency. The results suggest a possible effect of anilides on the thermogenic pathway of the rat brown adipose tissue. In addition, there was a large individual variability in the proportions of anilide present in all tissues, from as little as 0.3% of the total anilide injected in some animals to 6% in others.

Adipose Tissue↗
PubMed1985

The use of [3H]aniline to identify the essential carboxyl group in the bovine mitochondrial F1-ATPase that reacts with 1-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline.

The inactivation of the bovine heart mitochondrial F1-ATPase with 1-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) in the presence of [3H]aniline at pH 7.0 led to the covalent incorporation of 3H into the enzyme. When the ATPase was inactivated by 94% with 0.9 mM EEDQ in the presence of 3.6 mM [3H]aniline in a large-scale experiment in which the protein concentration was 21 mg/ml, 4.2 mol [3H]anilide were formed per mol enzyme, of which 0.35 mol was incorporated per mol of the alpha subunit and 1.0 mol was incorporated per mol of the beta subunit. Examination of a tryptic digest of the isolated alpha subunit revealed that the majority of the 3H was contained in a single tryptic peptide, which, when purified, was shown to contain the [3H]anilide of a glutamic acid residue which corresponds to alpha-Glu-402 of the Escherichia coli F1-ATPase. This residue was labeled to the extent of about 1.0 mol/mol enzyme. Analysis of tryptic peptides purified from the isolated beta subunit showed that 0.8 and 1.5 mol, respectively, of the [3H]anilides of beta-Glu-341 and beta-Glu-199 were formed per mol MF1 during the inactivation of the enzyme at 21 mg/ml. When the ATPase was inactivated by 90% at a protein concentration of 1.7 mg/ml by 0.9 mM EEDQ in the presence of 1.7 mM [3H]aniline, 3.1 mol [3H]anilide were formed per mol enzyme. From the analysis of the radioactive peptides purified from a tryptic digest of the labeled ATPase from this experiment it was estimated that 0.7 mol of the [3H]anilide of alpha-Glu-402, 0.3 mol of the [3H]anilide of beta-Glu-341, and 1.5 mol of the [3H]anilide of beta-Glu-199 were formed per mol F1-ATPase. Since beta-Glu-199 is labeled to the same extent in the two experiments while alpha-Glu-402 and beta-Glu-341 were not, suggests that the modification of beta-Glu-199 is responsible for inactivation of the enzyme by EEDQ.

Amino Acid Sequence↗
PubMed2005

On the generation and outcome of 3-(N-phenylamino)propane-1,2-diol derivatives in deodorized model oils related to toxic oil syndrome.

Toxic Oil Syndrome (TOS) was a massive food-born intoxication that occurred in Spain in 1981 and affected more than 20,000 people. TOS was attributed to the ingestion of rapeseed oil that had been adulterated with aniline, illegally refined, and delivered for human consumption. Two chemical species derived from aniline have been identified in oil batches: fatty acid anilides, qualified as biomarkers of the adulterated oil, and fatty acid esters of 3-(N-phenylamino)propane-1,2-diol (PAP), considered toxic oil biomarkers. These esters were generated by chemical processes during oil refining, specifically in the deodorization step, which involves treatment of the oil at high temperatures under vacuum to remove volatile contaminants. Since PAP derivatives are strongly associated with TOS, their formation and putative interconversion in a toxic oil model has been studied. The main results obtained are (i) only triglycerides and aniline are required to produce PAP esters, thus eliminating the possibility that unknown activators present in the deodorization tank were required for toxification of the oil; (ii) PAP and PAP mono- and diesters are chemically interrelated, as are anilides and PAP esters to an even higher degree. In addition to the reaction of aniline with triglycerides, anilides can be also formed via attack of PAP esters by aniline. However, the most important source of anilides during deodorization seems to be the thermal decomposition of PAP esters. Overall, these results suggest that the generation and outcome of PAP derivatives during deodorization is a complex scenario whereby PAP esters are not only generated from different reactions but decompose to produce anilides, among other compounds. In addition to providing a rapeseed oil model that reproduces the composition of case oils with respect to anilides and PAP derivatives, the results presented herein further support the hypothesis imputing PAP diesters or their metabolites for the intoxication episode.

Anilides↗
PubMed1983

Hydrolysis of oleylanilide in the rat.

The fate of oleylanilide, administered as a single intragastric dose in rape oil, has been studied in Porton Wistar rats. Following a dose of oleyl[U-14C]anilide approximately 60% of the label was recovered in the faeces over a 3 day period, and identified principally as unchanged anilide. Most of the remainder of the dose was recovered in urine as hydrophilic metabolites. p-hydroxyacetanilide, which is a major metabolite of aniline in the rat was identified in the hydrolysed samples of urine from anilide treated rats. Following administration of [9,10(n)-3H]oleyl[U-14C]-anilide to rats which had been fitted with a cannula in the thoracic lymph duct the [3H] label which was absorbed was recovered principally in triglycerides showing that extensive hydrolysis of the anilide occurs prior to or during uptake from the gut. Oleylanilide was identified in lymph by gas chromatography but amounted to less than 6% of the dose.

Acetanilides↗
PubMed1996

Toxic oil syndrome: traceback of the toxic oil and evidence for a point source epidemic.

Rapeseed oil denatured with aniline was the vehicle of the causal agent of the toxic oil syndrome (TOS) epidemic that occurred in Spain in 1981. Although the precise aetiologic agent remains unknown, researchers established that increasing concentrations of oleyl anilide and other fatty acid anilides were associated with an increased risk for disease. To examine the hypothesis that 5-litre plastic containers of rapeseed oil associated with TOS, and which contained oleyl anilide had a characteristic shape, we measured fatty acid, sterol and fatty acid anilide levels in oil from containers of different shapes. We identified 1673 bottles of oil that had been collected during the Spanish Government's oil exchange programme and linked these bottles to people with TOS as reported in the official government census of patients with TOS. Although rapeseed oil (identified by the presence of brassicasterol) was found in 798 (47.7%) of the 1673 bottles examined, contamination with fatty acid anilide occurred in only 329 (19.6%) of the 1673 bottles and 319 (97%) of the 329 were oil containers of the shape sold by RAELCA, an oil company in Madrid. The first aniline-denatured oil that RAELCA had purchased to be refined specifically for distribution was refined at the ITH refinery of Seville, and this oil has been most directly associated with the epidemic. Previous work has shown that the only toxic oil linked to a specific refinery was that associated with rapeseed oil from the ITH refinery in Seville, and the epidemic began shortly after this oil was delivered to RAELCA for retail sale. On the basis of these findings, we conclude that oil refined by ITH and distributed by RAELCA was the principal, and probably the only, oil responsible for the TOS epidemic. Information about the history and treatment of this oil may yield important clues towards identifying the aetiologic agent of TOS.

Anilides↗
PubMed1999

Epidemiologic evidence for a new class of compounds associated with toxic oil syndrome.

Toxic oil syndrome appeared in epidemic form in Spain in 1981. Epidemiologic studies have demonstrated that illness was caused by consumption of rapeseed oil that had been denatured with aniline. Chemical analyses of oil specimens conducted in conjunction with epidemiologic studies have established that consumption of specific oils containing fatty acid anilide contaminants was associated with increased risk for disease. New chemical analytic methods identified a family of compounds, the di-fatty acid esters of phenylamino propane-diol, and one of these compounds, the 1,2-di-oleyl ester of 3-(N-phenylamino)-1,2-propanediol (DPAP), has been found to be more strongly associated with disease status than the fatty acid anilides. We found the odds ratio for exposure to DPAP (OR = 26.4, 95% CI = 6.4-76.3) is much higher than the odds ratio for exposure to oleyl anilide (OR = 4.1, 95% CI = 2.2-7.8), implying that exposure to DPAP was a more relevant risk factor for development of toxic oil syndrome than exposure to oleyl anilide. In this paper, we review and present analyses of data from multiple studies of the possible etiologic role of DPAP in toxic oil syndrome. The presence of DPAP in oil collected from affected and unaffected households was a more specific correlate of case relatedness than was the presence of fatty acid anilides, and it was equally sensitive. Moreover, DPAP was found in oil from the only refinery whose oil was clearly associated with illness.

Anilides↗
PubMed2001

Determination of aniline derivatives in oils related to the toxic oil syndrome by atmospheric pressure ionization-tandem mass spectrometry.

In 1981, an unknown disease appeared in Spain, the Spanish Toxic Oil Syndrome. Nowadays and despite all efforts, the etiological agent is still unknown. Early studies showed a link between this illness and the consumption of denatured rapeseed oil fraudulently processed and marketed as edible oil. Two families of aniline derivatives present in these oils (fatty acid anilides and acylated phenyl amino propanediol derivatives or PAPs) were found to be good chemical markers of toxic oils. In this work, a new method has been developed to analyze these aniline derivatives in oil samples by HPLC-MS and HPLC-MS/MS with an API source. For their quantification, three different internal standards were used, one for anilides and two for PAPs. Quantification limits were 8 ppm for anilides and 0.2 ppm for PAPs. Anilides and PAPs were found in marker-positive samples at levels up to 50,000 and 330 ppm, respectively. The relative abundance of the different fatty acid anilides and PAPs correlates with the fatty acid composition of the oils. More than 2,600 different samples were analyzed by this method in the most exhaustive screening of suspected toxic oils carried out to date.

Aniline Compounds↗
PubMed1994

Studies on the kinetic and chemical mechanism of inhibition of stromelysin by an N-(carboxyalkyl)dipeptide.

We have investigated the inhibition of the human matrix metalloproteinase stromelysin (SLN) by the N-(carboxyalkyl)dipeptide Ala[N]hPhe-Leu-anilide and find that it is a competitive, slow-binding inhibitor of this enzyme with Ki = 3 x 10(-8) M (pH 6.0, 25 degrees C). The dependence of k(obs), the observed first-order rate constant for the approach to steady state, on Ala[N]hPhe-Leu-anilide concentrations less than 10(-5) M is linear and suggests a simple, one-step mechanism with kon = 3.4 x 10(4) M-1 s-1 and k(off) = 1.2 x 10(-3) s-1 (pH 6.0, 25 degrees C). Using rapid kinetic techniques, we extended the concentration range of Ala[N]hPhe-Leu-anilide to 2 x 10(-3) M and found that the [Ala[N]hPhe-Leu-anilide] dependence of K(obs) suggests saturation kinetics with a Ki' near 5 x 10(-4) M. Detailed analysis of these data reveal that the dependence of k(obs) on [Ala[N]hPhe-Leu-anilide] is, in fact, sigmoidal. To probe the chemical mechanism of inhibition, we determined pH and temperature dependencies and solvent deuterium isotope effects. For k(on), delta H not equal to = 12.4 kcal/mol and -T delta S not equal to = 6.2 kcal/mol (T = 298 K; [I]steady-state = 10(-6) M), while for k(off), delta H not equal to = 12.5 kcal/mol and -T delta S not equal to = 8.9 kcal/mol (T = 298 K). pH dependencies of the kinetic parameters for inhibition are complex but reflect greater potency at lower pH and suggest a mechanism involving the same active-site groups that are involved in catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Factors associated with pathogenicity of oils related to the toxic oil syndrome epidemic in Spain.

The toxic oil syndrome (TOS), which affected over 20,000 persons in Spain in 1981, has been linked to the consumption of aniline-denatured rapeseed oil, but the precise etiologic agent is still unknown. We attempted to validate the use of high concentrations of oleyl anilide as a marker for oils that contain (or contained) the causal agent. We compared the chemical compositions of oils obtained from ill (N = 59) and unaffected (N = 70) families in 1981. Case oils had higher concentrations of fatty acids and sterols in which rapeseed oil is particularly rich. In addition, case oils had more frequent and extensive contamination with oleyl anilide and other fatty acid anilides. We observed a dose-response effect; risk increased sharply with increasing concentrations of oleyl anilide, and no control oil had more than about 825 micrograms per liter of that compound. We conclude that high concentrations of oleyl anilide specifically mark oils that contain (or used to contain) the TOS etiologic agent.

Aniline Compounds↗
PubMed1984

Similarities of the substrate cleavage catalyzed by proline specific endopeptidase and dipeptidyl peptidase IV.

16 substrates of the types succinyl-alanyl-alanine-pX-anilide, and succinyl-alanyl-proline-pX anilide having different substituents in para-position of the aryl residue were synthesized and characterized. The influence of electronic as well as hydrophobic substituent constants, sigma and pi, on the hydrolysis of the substrates catalyzed by the enzyme Proline Specific Endopeptidase (PSE) has been investigated. In the Hansch approach, the catalytic constants lg kcat and lg (kcat/KM) of succinyl-alanyl-alanine-pX-anilides hydrolyzed by PSE correlate significantly with electronic substituent constants sigma, whereas no correlation in the case of succinyl-alanyl-proline-pX-anilides. The intercorrelation of the constants of the former substrates with corresponding data from Dipeptidyl Peptidase IV (DP IV) catalyzed hydrolysis of alanyl-alanine-pX-anilides suggest that both enzymes act by similar catalytic mechanism.

Chemical Phenomena↗
PubMed1989

Highly conformationally constrained halogenated 6-spiroepoxypenicillins as probes for the bioactive side-chain conformation of benzylpenicillin.

The halogenated 6-spiroepoxypenicillins are a series of novel semisynthetic beta-lactam compounds with highly conformationally restricted side chains incorporating an epoxide. Their biological activity profiles depend crucially on the configuration at position C-3 of that epoxide. In derivatives with aromatic-containing side chains, e.g., anilide, the 3R-compounds possess notable Gram-positive antibacterial activity and potent beta-lactamase inhibitory properties. The comparable 3S-compounds are antibacterially inactive, but retain beta-lactamase inhibitory activity. Using the molecular simulation programs COSMIC and ASTRAL, we attempted to map a putative, lipophilic accessory binding site on the PBPs that must interact with the side-chain aromatic residue. Comparative computer-assisted modelling of the 3R-, and 3S-anilides, along with benzylpenicillin, indicated that the available conformational space at room temperature for the side chains of the 3R- and the 3S-anilides was mutually exclusive. The conformational space for the more flexible benzylpenicillin could accommodate the side chains of both the constrained penicillin derivatives. By a combination of van der Waals surface calculations and a pharmacophoric distance approach, closely coincident conformers of the 3R-anilide and benzylpenicillin were identified. These conformers must be related to the antibacterial, 'bioactive' conformer for the classical beta-lactam antibiotics. From these proposed bioactive conformations, a model for the binding of benzylpenicillin to the PBPs relating the three-dimensional arrangement of a putative lipophilic S2-subsite, specific for the side-chain aromatic moiety, and the 3 alpha-carboxylate functionality is presented.

Epoxy Compounds↗
PubMed1990

Enantiomeric resolution of amino acid derivatives on molecularly imprinted polymers as monitored by potentiometric measurements.

Potentiometric measurements have been applied to the detection of enantiomeric separations on molecularly imprinted polymers. A flow-through column electrode, based on the use of polymers imprinted against L-phenylalanine anilide, is described. The electrode consisted of a glass column in which the polymer was packed and where the end frits constituted the electrodes. The flow stream potential across the column can be continuously recorded as solvent is pumped through the system. The column resolved the enantiomers of phenylalanine anilide as detected by both UV absorption and potentiometric measurements and the recorded signals could be correlated with the concentration of phenylalanine anilide. The calibration graphs obtained for the UV absorption of phenylalanine anilide were linear over the concentration range investigated, whereas the potentiometric signal was shown to be exponentially linear with concentration. The application of molecular imprints to the preparation of supports suitable for chromatographic separations of enantiomers and for the preparation of specific electrodes is discussed.

Amino Acids↗
PubMed2001

T cells ignore aniline, a prohapten, but respond to its reactive metabolites generated by phagocytes: possible implications for the pathogenesis of toxic oil syndrome.

The most basic arylamine, aniline, belongs to a class of compounds notorious for inducing allergic and autoimmune reactions. In 1981 in Spain, many people succumbed to toxic oil syndrome (TOS), a disease caused by ingestion of cooking oil contaminated with aniline. Indirect evidence points toward an immune pathogenesis of TOS driven by T lymphocytes, but it is unclear to which antigens these cells could react. Here, using the popliteal lymph node (PLN) assay in mice, we analyzed the sensitizing potential of aniline, its metabolites, and some of the aniline-coupled lipids detected in the contaminated cooking oil. Whereas aniline itself and its non-protein-reactive metabolites nitrobenzene, p-aminophenol and N-acetyl-p-aminophenol, failed to elicit PLN responses, its reactive metabolites nitrosobenzene and N-hydroxylaniline did. The aniline-coupled lipids, namely, linoleic anilide and linolenic anilide, and a mixture of fatty acid esters of 3-(N-phenylamino)-1,2-propanediol, all implicated in TOS, induced significant PLN responses, whereas the respective aniline-free lipids, linoleic acid, linolenic acid, and triolein, did not. Hence, the aniline moiety plays a crucial role in the immunogenicity of the aniline-coupled lipids of TOS. PLN responses to the reactive aniline metabolites and the one aniline-coupled lipid that was tested, linolenic anilide, were T-cell-dependent. Secondary PLN responses to nitrosobenzene were detectable not only after priming with nitrosobenzene but, in some experiments, also after priming with linolenic anilide. This suggests that the aniline moiety was cleaved from the aniline-coupled lipid and metabolized to the intermediate nitrosobenzene that generated the prospective neoantigens. Consistent with this, in lymphocyte proliferation tests in vitro, T cells primed to nitrosobenzene reacted in anamnestic fashion to white bone marrow cells (WBMCs) pulsed with aniline. Hence, we propose that aniline is a prohapten that can be metabolized by WBMCs, which form neoantigens that are recognized by T cells. The possible significance of these findings for the pathogenesis of TOS is discussed.

Aniline Compounds↗
PubMed1993

A radiochemical assay for aminopeptidase N.

We developed an assay for aminopeptidase N (AmN) in which substrate, Arg-Phe-[3H]anilide (24.9 Ci/mmol), can be used at concentrations (1-200 nM) well below Km (12 microM) and at or below enzyme concentration ([E]). Such reaction conditions simulate those in vivo where peptide hormones in picomolar concentrations (<< Km) are degraded by nano- or micromolar concentrations of enzyme. The Arg-Phe-[3H]anilide:AmN reaction obeyed first-order enzyme kinetics when human serum, human seminal plasma, guinea pig serum, or homogeneous porcine kidney AmN was used as enzyme source and substrate was within the concentration range of 1-200 nM. For porcine AmN, kcat/Km was 1.47 x 10(9) M-1 min-1, kcat 17,640 min-1. Human serum AmN was in a concentration (about 4.6 nM) in great excess over those reported for substrates such as angiotensin III. Several advantages accrue under conditions of first-order enzyme kinetics: (1) Vmax/Km is measured directly. (2) When kcat/Km is known, [E] can be computed in mol/liter. (3) IC50 values for alternative substrates can be taken as Km values. (4) IC50 values for inhibitors are Ki values when Ki >> [E]. Arg-Phe-[3H]anilide can be used to measure AmN activity in the presence of chromophores and fluorophores that interfere with photometric and fluorometric assays. We have confirmed that alleged substrates such as angiotensin III and Met-Lys- and Lys-bradykinin are bound by AmN with high affinities (Km values, 5.7, 9.1, and 14.3 microM). Bovine pulmonary artery endothelial cell cultures were found to possess AmN-like activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1988

Determination of oxidation products of N-phenyllinoleamide: Spanish toxic oil syndrome studies.

The early identification of fatty acid anilides in suspect oils directed attention to their possible role in the Spanish toxic oil syndrome. These anilides or their oxidized derivatives could have been spontaneously formed during the handling and/or storage of the oil. The exact cause of the intoxication is still unknown but free radical and peroxidative mechanisms have been implicated in its etiology. Epoxy-hydroxylated derivatives from linoleic acid anilide were obtained using a model of accelerated oxidation. Their mass spectral patterns agree with the trimethylsilyl ethers of N-phenyl-9,10-epoxy-11-hydroxy-12-octadecenamide and N-phenyl-12,13-epoxy-11-hydroxy-9-octadecenamide, respectively. These compounds were also identified is rapeseed oil samples supplemented with N-phenyl-linoleamide and submitted to the reported accelerated oxidation method.

Anilides↗
PubMed1988

Chemical correlates of pathogenicity of oils related to the toxic oil syndrome epidemic in Spain.

The identity of the etiologic agent that caused the 1981 epidemic of toxic oil syndrome in Spain has not been established, and toxicologic study of oil specimens from the outbreak has been hampered by uncertainty about which oils were actually capable of causing illness. To identify chemical characteristics associated with pathogenicity, the authors compared specimens collected during the Spanish government's oil recall program in June and July 1981 from affected and unaffected households in the two contiguous towns of Alcorcón and Leganés (Madrid Province). Oils were blind-coded for laboratory analysis, and personnel with no knowledge of the laboratory results determined whether illness was present in a family. Contamination with free aniline and oleyl, linoleyl, and palmityl anilides was strikingly more frequent and extensive in oils collected from the case (affected) families. There was a clear-cut dose-response effect, with increasing concentrations of aniline and anilides associated with increasing risk of illness. Differences in fatty acid and sterol compositions among oils indicated more rapeseed oil admixture in the case group, but these indicators of rapeseed oil admixture did not contribute significantly to risk after the degree of aniline/anilide contamination had been taken into account. The authors conclude that the presence of relatively high levels of aniline and fatty acid anilides in oil specimens collected during the epidemic in the two towns studied indicates a high probability of the current or prior presence of the etiologic agent of toxic oil syndrome. Although these data do not necessarily indicate that any of the compounds measured actually caused the illness, further toxicologic work should concentrate on oils with substantial concentrations of the marker compounds.

Anilides↗
PubMed1983

Mapping of the S' subsites of porcine pancreatic and human leucocyte elastases.

Trifluoroacetyl dipeptide anilides have been synthesized and used to map the S' subsites of porcine pancreatic elastase and human leucocyte elastase. A confident mapping of these subsites, at least for the porcine enzyme, was possible since the x-ray crystallographic study of its complex with CF3CO-Lys-Ala-NH-Ph-p-CF3 at a resolution of 2.5 A (Hughes, D. L., Sieker, L. C., Bieth, J., and Dimicoli, J. L. (1982) J. Mol. Biol. 162, 645-658) shows the CF3CO group at the S1 subsite and the dipeptide anilide bound at sites close to the S'1-S'3 subsites. Furthermore the effect of substitution was easy to investigate since these ligands are reversible competitive inhibitors of elastases, whose mode of binding to the porcine enzyme has been shown by nuclear magnetic resonance spectroscopy (Dimicoli, J. L., Renaud, A., and Bieth, J. (1980) Eur. J. Biochem. 107, 423-432) to be essentially unique and common to all CF3CO peptide anilides. The total number of S' subsites was found to be three for both enzymes. The individual subsites have the following specificities: subsite S'1, in porcine pancreatic elastase this subsite prefers Lys to Ala or Glu. In human leucocyte elastase this subsite is less specific; subsite S'2, in porcine pancreatic elastase, this subsite has a marked specificity for Ala. It accommodates bulkier residues with some difficulty. In human leucocyte elastase there is a remarkable specificity for Leu at this subsite; subsite S'3, in porcine pancreatic elastase, this subsite has a high aromatic specificity. In human leucocyte elastase there is no such affinity in S'3 but favorable local interaction exists. These specificities are examined on the basis of the coordinates of the CF3CO-Lys-Ala-NH-Ph-p-CF3 . porcine pancreatic elastase complex. Furthermore the different specificities of the S'2 subsite found in our own work and proposed by Atlas (Atlas, D. (1975) J. Mol. Biol. 93, 39-53) are briefly discussed.

Anilides↗