Joseph G. Bieth

The synthesis and analytical use of a highly sensitive and convenient substrate of elastase

Abstract This paper describes the synthesis and analytical use of succinyl-( l -alanine) 3 - p -nitroanilide, a new elastase substrate. At pH 8.0 and 25°C, the kinetic constants Km and k cat are 1.15 m m and 16.6 sec −1 , respectively, in the absence of organic solvent and 2.4 m m and 21.2 sec −1 in the presence of 0.92% ( v v ) N -methylpyrrolidone. An expeditious and highly sensitive spectrophotometric method for measuring elastase activity is described: enzyme concentrations as low as 0.05 μg/ml may be assessed accurately within a few minutes. The procedure may be used to assay elastase in crude biological material since the substrate is virtually not attacked by trypsin or chymotrypsin and does not undergo appreciable spontaneous hydrolysis. The new substrate may also be used to detect elastase on polyacrylamide gels.

In vivo significance of kinetic constants of protein proteinase inhibitors

We describe the in vivo significance of the kinetic parameters which characterize the interaction between proteinases and protein proteinase inhibitors. Knowledge of the second-order association rate constant kass and in vivo inhibitor concentration allows the calculation of the delay time of inhibition, i.e., the time required for complete inhibition of a proteinase in vivo. The influence of biological substrates on the delay time is also analyzed. The extent of substrate breakdown during the delay time of inhibition may be computed from the various constants describing the proteinase/substrate/inhibitor interactions and the biological concentrations of proteinase and inhibitor. The in vivo partition of a proteinase between two inhibitors may be calculated if the kinetic parameters are known. We define a stability time for enzyme-inhibitor complexes as a minimal time during which the complexes may be considered as stable. This time is related to kdiss the dissociation rate constant of the reversible enzyme-inhibitor complex or to k, the breakdown rate constant of the complex formed with temporary inhibitors. The overall stability of the complex depends upon the ratio between the inhibitor concentration and Ki, the equilibrium dissociation constant of the complex. If this ratio is higher than 1000, a reversible inhibitor behaves like an irreversible one in vivo whatever the enzyme concentration.

Kinetics of the inhibition of leukocyte elastase by the bronchial inhibitor.

The rate constant for the association between human leukocyte elastase (EC 3.4.21.11) and human bronchial inhibitor has been determined by competition experiments with alpha 1-proteinase inhibitor. This constant (1.1.10(7) M(-1) . s(-1)) is 6-times lower than that for the association of leukocyte elastase and alpha 1-proteinase inhibitor. The latter inhibitor is able to dissociate the leukocyte elastase-bronchial inhibitor complex with a rate constant 1.3.10(-4) s-1. The equilibrium dissociation constant Ki of the complex is 1.2.10(-11) M. The physiopathological significance of these constants is discussed.

The functional activity of α1-proteinase inhibitor in bronchoalveolar lavage fluids from healthy human smokers and non-smokers

Cigarette smoking is an important risk factor for pulmonary emphysema. In vitro experiments document cigarette smoke-induced inactivation of alpha 1-proteinase inhibitor, a protein which is thought to protect the lung interstitium against the deleterious action of neutrophil elastase. To assess the relevance of this in in vitro findings, we have measured the functional activity of alpha 1-proteinase inhibitor collected by bronchoalveolar lavage from twenty healthy volunteers (10 heavy smokers and 10 non-smokers). The total inhibitor concentration was measured by radial immunodiffusion. The active inhibitor concentration was determined by virtue of its elastase inhibitory capacity. We used pure and active site-titrated porcine pancreatic elastase and a kinetic assay with succinyl-trialanine-p-nitroanilide in order to get reliable and accurate results. In smokers and in non-smokers the percentage of functionally active alpha 1-proteinase inhibitor in bronchoalveolar lavage fluid is 54 +/- 12 and 38 +/- 14%, respectively. This difference is not significant. Serum alpha 1-proteinase inhibitor is 100% active in both groups. Our data disagree with previous reports suggesting the presence of fully active alpha 1-proteinase inhibitor in bronchoalveolar lavage fluid of non-smokers and of partially active inhibitor in the lavage fluid of smokers.

SERINE PROTEASE INHIBITION BY INSECT PEPTIDES CONTAINING A CYSTEINE KNOT AND A TRIPLE-STRANDED BETA -SHEET

Three insect peptides showing high sequence similarity and belonging to the same structural family incorporating a cysteine knot and a short three-stranded antiparallel β-sheet were studied. Their inhibitory effect on two serine proteases (bovine α-chymotrypsin and human leukocyte elastase) is reported. One of them, PMP-C, is a strong α-chymotrypsin inhibitor (Ki = 0.2 nM) and interacts with leukocyte elastase with a Ki of 0.12 μM. The other two peptides, PMP-D2 and HI, interact only weakly with α-chymotrypsin and do not inhibit leukocyte elastase. Synthetic variants of these peptides were prepared by solid-phase synthesis, and their action toward serine proteases was evaluated. This enabled us to locate the P1 residues within the reactive sites (Leu-30 for PMP-C and Arg-29 for PMP-D2 and HI), and, interestingly, variants of PMP-D2 and HI were converted into powerful inhibitors of both α-chymotrypsin and leukocyte elastase, the most potent elastase inhibitor obtained in this study having a Ki of 3 nM.

The action of elastase on p-nitroanilide substrates

Abstract The action of elastase has been studied on four p-nitroanilides: BOC-(Ala)2-NA, (Ala)3-NA, Ac-(Ala)3-NA and BOC-(Ala)3-NA. The second order rate constant kcat/Km increases considerably with the chain length of these substrates. With (Ala)3-NA, activation by excess substrate was observed. DMF and DMSO inhibit strongly the elastase catalyzed hydrolysis of Ac- and BOC-(Ala)3-NA. The later substrate may be used to assess rapidly elastase activity: concentrations as low as 0.2 μg/ml may be determined accurately.

Photometric or fluorometric assay of cathepsin B, L and H and papain using substrates with an aminotrifluoromethylcoumarin leaving group

N-trifluoromethylcoumarinylamide derivatives of benzyloxycarbonyl-Arg-Arg, benzyloxycarbonyl-Phe-Arg and Arg are convenient chromogenic and fluorogenic substrates of cathepsin B, L and H, respectively. Benzyloxycarbonyl-Phe-Arg-N-trifluoromethylcoumarinylamide is also a highly sensitive substrate for papain.

Mucus proteinase inhibitor: a fast-acting inhibitor of leucocyte elastase

Human mucus proteinase inhibitor is a fast-acting inhibitor of human leucocyte elastase (EC 3.4.21.37) and forms a stable, complex with this enzyme. At physiological ionic strength and temperature and in the presence of 10 mg/ml albumin, the kinetic constants characterizing the interaction between elastase and the non-degraded inhibitor are: kass = 6.4.10(6) M-1.s-1, kdiss = 2.3.10(-3) s-1, Ki = 3.10(-10) M. The partially degraded inhibitor isolated by chymotrypsin-Sepharose chromatography inhibits elastase with similar efficiency, suggesting that if partial proteolysis of the inhibitor occurs in vivo, the latter may still act as a potent antielastase. Mucus proteinase inhibitor therefore plays a physiological antielastase function in upper respiratory tract secretions, since it inhibits elastase with a delay time of 150 ms and behaves like an irreversible inhibitor.

The influence of α2-macroglobulin on the elastolytic and esterolytic activity of elastase

It has recently been shown that the elastolytic activity of elastase (pancreatopeptidase E, EC 3.4.4.7.) is inhibited by human cw2-macroglobulin [ 1,2] . Elastase thus belongs to a class of hydrolytic enzymes (trypsin, chymotrypsin, thrombin, plasmin) which form complexes with cr2-macroglobulin (cy2 M). These complexes are active against synthetic substrates but inactive against proteins; also, the qM-bound enzymes are not inhibited by high molecular weight inhibitors (see [3] and references therein). It was thus of interest to see whether the above properties can be extended to elastase. The results presented in this letter concern the effect of human and porcine 02M on porcine elastase. The 02M-elastase complexes are inactive against Remazol-brilliant-blue elastin but retain approximately 80% of the activity of free elastase on t-BOC-alaninep-nitrophenyl ester. One molecule of 02M binds two or three molecules of enzyme. The complex between human 02M and elastase is only partially protected against the inhibition by soybean trypsin inhibitor or o1 -antitrypsin.

A kinetic study of the inhibition of human and bovine trypsins and chymotrypsins by the inter-α-inhibitor from human plasma

Human plasma inter-alpha-inhibitor forms 1:1 inactive complexes with human and bovine trypsins (EC 3.4.21.4) and chymotrypsins (EC 3.4.21.1). The association and dissociation rate constants as well as the equilibrium dissociation constants (Ki) of the complexes formed of inter-alpha-inhibitor and the four proteases have been measured. The most stable complexes are those formed with the bovine enzymes. For instance, Ki = 2.1-10-11 M for bovine trypsin whereas Ki = 1.2 - 10-8 M for human trypsin. Whatever the species, the complexes formed with the chymotrypsins are less stable than those formed with the trypsins.