Morris Zimmerman

Sensitive substrates for human leukocyte and porcine pancreatic elastase: a study of the merits of various chromophoric and fluorogenic leaving groups in assays for serine proteases.

Abstract Five sensitive substrates of human leukocyte and porcine pancreatic elastase having the sequence MeO-Suc-Ala-Ala-Pro-Val-X where -X is -NA (4-nitroanilide), -SBzl (thiobenzyl ester), -OEt, -AMC (4-methyl-7-coumarylamide), or -NNapOMe (1-methoxy-3-naphthylamide), were synthesized. The kinetic constants for the enzymatic hydrolysis as well as the sensitivity of each substrate are reported. Hydrolysis of the peptide -AMC and -NNapOMe derivatives were followed by monitoring spectrofluorometrically the release of H-AMC and H-NNapOMe, respectively. Cleavage of the thiobenzyl ester yields benzyl mercaptan as the hydrolysis product. Its release was monitored at 412 nm by reaction with Ellmans reagent [5,5′-dithiobis(2-nitrobenzoic acid)] in the assay mixture to produce the 3-carboxy-4-nitrothiophenoxide anion or at 324 nm by reaction with 4,4′-dithiodipyridine to produce 4-thiopyridone. Hydrolysis of the ethyl ester was measured using a coupled assay with NAD+ and liver alcohol dehydrogenase. The NADH+ formed upon oxidation of the ethanol released from cleavage of the ester was followed at 340 nm. MeO-Suc-Ala-Ala-Pro-Val-SBzl, the best substrate of the series, was capable of detecting as little as 2.4 p m (0.072 ng/ml) of active-site titrated human leukocyte elastase and 5.8 p m (0.15 ng/ml) of active-site titrated porcine pancreatic elastase using 4,4′-dithiodipyridine. The corresponding values with Ellmans reagent were 5.0 p m (0.15 ng/ml) and 7.4 p m (0.19 ng/ml), respectively. Advantages of this substrate are its high k cat K m values and ease of synthesis. One disadvantage is the interference of high concentration of thiols with the assay. The peptidyl-AMC is almost as sensitive as the thiobenzyl ester and can detect 11 p m of HL elastase and 18 p m of PP elastase. An advantage of this substrate is the fact that cleavage involves a peptide bond. Disadvantages are relatively low k cat K m values and greater difficulty in synthesis. The peptidyl-NNapOMe has possible utility in histochemical studies due to the low intrinsic fluorescence of the substrate relative to the peptidyl-AMC. For a rate assay it has a lower sensitivity than either the thiobenzyl ester or peptidyl-AMC. The coupled liver alcohol dehydrogenase-ethyl ester assay offers no advantages except in cases where the ester substrate is commercially available. The 4-nitroanilide assay enjoys moderate sensitivity and is extremely convenient for routine use. Except for the peptidyl ethyl ester assay, all of the human leukocyte elastase assays reported in this paper are vastly more sensitive than any other existing assay for this protease.

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.

A new fluorogenic substrate for chymotrypsin

A new sensitive assay for amidase activity of chymotrypsin has been developed using 7-glutarylphenylalaninamido-4-methylcoumarin as substrate. Release of 7-amino-4-methylcoumarin was determined fluorometrically. As little as 0.5 μg/ml of chymotrypsin could be detected; at pH 8.0, K m = 0.7 m m . The substrate was not hydrolyzed by either trypsin or elastase and was capable of measuring chymotrypsin-like activity in tissue extracts. Hydrolysis of the substrate by chymotrypsin was blocked by specific inhibitors of the enzyme.

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.

Protease activities present in wheat germ and rabbit reticulocyte lysates

Abstract Rabbit reticulocyte lysates and wheat germ lysates were found to contain significant neutral protease activity when assayed against the highly sensitive 7-amino-4-methylcoumarin (AMC) peptide substrates Phe-AMC, succinyl-Ala-Ala-Phe-AMC and t-boc-Ala-Ala-Pro-Ala-AMC (substrates for aminopeptidase, chymotrypsin and elastase-like enzymes, respectively). Additionally, wheat germ lysates contain a trypsin-like activity when assayed against CBZ-Gly-Gly-Arg-AMC and a post-proline cleaving activity which hydrolyzed the Pro-Ala bond of t-boc-Ala-Ala-Pro-Ala-AMC.

Inhibition of procine kidney “enkephalinase” by substituted-N-carboxymethyl dipeptides

Abstract A design effective for generating inhibitors of angiotensin converting enzyme has been successfully extended to inhibitors of another Zn ++ peptidase. A series of potent inhibitors for the zinc metalloendopeptidase associated with porcine kidney microsomes has been developed. Most contain the (S)-N-(1-carboxy-3-phenylpropyl)-L-Phe-X backbone. The most effective inhibitors with ID 50 values in the 10 −7 M range at pH 7.5 and 22°C were those where X = glycine, β-alanine, γ-amino butyric acid. A simple nomenclature designating substituted N-carboxy-methyl amino acid derivatives as -[N]- sharing amino acid derivatives is described.

Cytochalasin B-dependent release of azurophil granule enzymes from human polymorphonuclear leukocytes.

The dose-response effects of phorbol myristate acetate and cytochalasin B on secretion of azurophil and specific granule enzymes from viable human polymorphonuclear leukocytes have been examined. Secretion of the azurophil granule enzymes elastase andβ-glucuronidase from cells exposed to 50 ng/ml of phorbol myristate acetate is dependent on prior exposure of the cells to greater than 0.5 mg/ml of cytochalasin B. In contrast, the secretion of the specific granule enzyme lysozyme is not dependent on pretreatment with cytochalasin B. The concentration of phorbol myristate acetate needed to elicit maximal secretion of specific versus azurophil granule enzymes differs, being 5.0 ng/ml and 50 ng/ml, respectively. The results suggest that cytochalasin B-sensitive cellular components, possibly microfilaments, may selectively modulate some step in the exocytosis of azurophil granule enzymes from human polymorphonuclear leukocytes exposed to phorbol myristate acetate.

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.