Bonnie M. Ashe

Sensitive assays for trypsin, elastase, and chymotrypsin using new fluorogenic substrates.

Abstract Sensitive fluorogenic substrates for trypsin, chymotrypsin, and elastase were prepared. These substrates are amides of an acyl amino acid or peptide with 7-amino-4-methylcoumarin (AMC). The substrates with their respective K cat K m ratios given in parentheses are: for chymotrypsin, glutaryl-Phe-AMC (78) and Ala-Ala-Phe-AMC · TFA (1660); for trypsin, benzoyl- dl -Arg-AMC (800) and Carbobenzoxy (Cbz)- l -Arg-AMC (5300); for elastase, N-acetyl-Ala-Ala-Pro-Ala-AMC (15,000). The detection limits obtained by using the best substrates and short incubation times are: chymotrypsin, 25 ng; trypsin, 5 ng; and elastase, 2 ng.

Substrate specificity of the elastase and the chymotrypsin-like enzyme of the human granulocyte

Human granulocyte elastase (EC 3.4.21.11) differs from hog pancreatic elastase in its specificity for synthetic substrates. Although hydrolyzing peptide bonds adjacent to the carboxyl group of alanine, the granulocyte enzyme prefers valine at the cleaved bond, in contrast to the pancreatic enzyme which prefers alanine. Peptide bonds involving the carboxyl group of isoleucine can be hydrolyzed by the granulocyte enzyme but are not hydrolyzed to any significant extent extent by pancreatic elastase. This difference in specificty could explain the lower sensitivity of the granulocyte enzyme to inhibitors containing alanine analogs, such as the peptide chloromethyl ketones and elastatinal. The human granulocyte chymotrypsin-like enzyme differs from pancreatic chymotrypsin by being able to cleave substrates containing leucine in addition to those containing the aromatic amino acids.

Specific inhibition of human granulocyte elastase by cis-unsaturated fatty acids and activation by the corresponding alcohols

Abstract Human granulocyte elastase is markedly inhibited by cis-unsaturated fatty acids, whereas trypsin, chymotrypsin, pancreatic elastase, and the granulocyte chymotrypsin-like enzyme are totally unaffected. The most potent of the acids tested is oleic acid (K i = 9×10 −6 M ). The inhibition is noncompetitive, affecting K cat but not K m with a tetrapeptide nitroanilide substrate. On the other hand, the corresponding alcohols and nitriles markedly stimulate the activity of granulocyte elastase on synthetic substrates but not on elastin, in contrast to the inhibitors which affect activity on both types of substrate. These data further define the differences between the granulocyte and pancreatic elastases and suggest that part of the difference may reside in the presence of an unusual hydrophobic binding site on the enzyme affecting its activity.

PMN elastases: A comparison of the specificity of human isozymes and the enzyme from other species toward substrates and inhibitors

The human elastases isolated from polymorphonuclear neutrophils (PMN) and purulent sputum displayed identical kinetic constants toward substrates and inhibitors. The elastases from the two sources yield identical N-terminal sequences and were recognized by antiserum prepared against human sputum elastase (HSE) isozyme-4 (I-4). The data support the proposal put forth by Twumasi and Liener (1977, J. Biol. Chem. 252, 1917-1926) that the human elastase from sputum is of PMN origin. PMN elastases from other species displayed kinetic constants toward both substrates and inhibitors significantly different from the human enzyme. Therefore, extrapolation of inhibitor profiles from these elastases to the human source should be avoided. Four groups of isozymes were resolved from HSE by FPLC. Only the most basic isozyme (I-4) was obtained as a single species. The isozymes displayed identical macroscopic kinetic constants toward several substrates and two classes of inhibitors. The similar partition ratios observed with a cephalosporin-derived inhibitor suggest that the microscopic rate constants are also identical. The data support the proposal suggested by Baugh and Travis (1976, Biochemistry 15, 836-841) that HLE isozymes differ only in carbohydrate content. Whatever the source of human PMN elastase heterogeneity, it does not result in heterogeneous catalytic properties. In addition, a new protein was identified in elastase preparations derived from human sputum. This protein displayed homology to serine proteases and properties suggesting that it is identical to azurocidin.

Inhibition of human leukocyte elastase by C-2 substituted cephalosporin sulfones

Abstract Cephalosporin sulfones with a number of substituents at the C-2 position were prepared and tested as inhibitors of human leukocyte elastase (HLE), an enzyme implicated in the tissue destruction associated with pulmonary emphysema. Nearly all substituents gave a substantial increase in activity against the enzyme over the unsubstituted parent. The enzyme can accommodate a number of functional groups at this position, but is not very discriminating. Both α- and β-methyl compounds have comparable activity, as do α-phenylthiomethyl and α-methoxy. Substitution at this position has led to the preparation of several compounds with exceptional potency against HLE.

Inhibition of human leukocyte elastase. 6. Inhibition by 6-substituted penicillin esters.

Abstract Penicillin amides substituted at C-6 with either an α- or β-trifluoroacetamido or an α-alkoxy functionality are reported as human leukocyte elastase (HLE) inhibitors. The structure activity relations for these derivatives are discussed and compared to the corresponding known cephalosporin structures in terms of chemical stability, HLE inhibition, and efficacy in an intratracheal (IT) lung hemorrhage assay.

The effect of N-acyl substituents on the stability of monocyclic β-lactam inhibitors of human leukocyte elastase

Abstract Substituted monocyclic β-lactam have recently been reported as inhibitors of human leukocyte elastase (HLE). Simple N-acetyl-2-azetidinone lead structures were found to undergo N-deacylation as well as β-lactam ring opening. The development of the N-carbamoyl-2-azetidinone nucleus was crucial to the stability of these compounds for effective oral bioavailability.

Monocyclic β-lactam inhibitors of human leukocyte elastase. Stereospecific synthesis and activity of 3,4-disubstituted-2-azetidinones.

Abstract The synthesis of the four stereoisomers of 3-ethyl-4-[(4-carboxyphenyl)oxy]-1-[[(phenylmethyl)amino]carbonyl]-2-azetidinone ( 1 ) starting from either D or L-aspartic acid is reported. The trans (3R,4R) isomer 7a , prepared from L-aspartic acid had the most inhibitory activity against human leukocyte elastase (HLE). This monocyclic β-lactam was very resistant to hydrolysis and was found to be orally bioavailable in marmosets.

Proteinase activities of the golden hamster eggs and cells of the cumulus oophorus.

Abstract Proteinase activities of eggs and cells of the cumulus oophorous of the golden hamster were investigated with highly sensitive fluorogenic amide substrates. Eggs contain a neutral endopeptidase which hydrolyzed Suc-Ala-Ala-Phe-7-amino-4-methylcoumarin amide between the Ala and the Phe residues. Endopeptidase action on this substrate resulted in the accumulation of Phe-7-amino-4-methylcoumarin amide which was monitored by tlc identification. Hamster eggs also contained aminopeptidase and elastase-like activities but no detectable trypsin-like activity. Aminopeptidase, endopeptidase, trypsin-like, and elastase-like activities were detected in cumulus cells.