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Mal-, Suc- and Glt-(Ala)3-NAp were prepared as new substances for determining pancreatic elastase. The kinetic constants show them to be more satisfactory than the previously described Ac-(Ala)3-NAp. The mean elastase output values after pancreozymin and secretin stimulation of the exocrine pancreas were significantly higher in the control subjects than in patients in whom other secretion values were altered. In agar electrophoresis, hog pancreatic elastase (Merck) formed a single cathodal fraction. One cathodal and one anodal fraction were found in human duodenal contents. Serum, plasma, alpha1-antitrypsin and alpha2-macroglobulin inhibit pancreatic elastase. Elastase-alpha1-antitrypsin complexes are enzymatically inactive, whereas the enzymatic activity of elastic-alpha2-macroglobulin complexes is partly preserved. Both types of complexes are stable and are not dissociated to a major extent in the presence of an excess amount of the other inhibitor.
Hamsters were intratracheally instilled with saline solutions containing a high dose (145 to 220 micrograms) or a low dose (1.3 to 1.5 micrograms) of 3H-methylated pancreatic elastase or N-acetyl-(L-alanyl)3-L-alanine chloromethyl ketone-inactivated 3H-methylated pancreatic elastase. Only the lysyl residues of the elastase molecule were methylated and radiolabeled in a nonlabile manner. The 3H-methylated elastase preparation exhibited esterolytic and elastolytic activity, spectral properties, and emphysema-inducing properties indistinguishable from those of unmodified pancreatic elastase. There was no detectable hemorrhagic or emphysematous reaction with the inactivated 3H-methylated elastase, and this material was cleared from the lungs 11 times faster than the corresponding enzymatically active high dose of 3H-methylated elastase and 18 times faster than the corresponding enzymatically active low dose of 3H-methylated elastase. There were correspondingly higher amounts of radioactivity in the urine of hamsters treated with the inactivated elastase. All of the 3H radioactivity recovered from the urine was associated with epsilon-N-methyllysyl and epsilon-N,N-dimethyllysyl residues. Significant levels of radioactivity were found in the cells, primarily alveolar macrophages, lavaged from the lungs. The low dose of enzymatically active elastase caused neither detectable hemorrhage nor emphysema, both of which were associated with the high dose. At 144 days significant radioactivity (1,200 cpm) remained in the lungs of animals treated with high or low doses of enzymatically active elastase, whereas virtually no radioactivity (100 cpm) was found in the lungs of those treated with high or low doses of inactivated elastase. The data presented support the hypothesis that the formation of elastase complexes with alpha 1-protease inhibitor and alpha 2-macroglobulin is associated with the slower clearance and the retention of significant amounts of radioactivity in the lungs. Some of the residual radioactivity found in the hamster lungs might represent enzymatically active elastase complexed with alpha 2-macroglobulin and might offer an explanation for the progressive nature of emphysema induced by a single dose of elastase.
Beagle dogs exposed to cigarette smoke for 600 d experience a significant change in pancreatic elastase levels, as measured in tissue homogenates, compared with their sham-exposed controls. Greater elastase activity was found in the high-nicotine cigarette smokers than in the low-nicotine cigarette smokers. Levels of serum alpha 1-antitrypsin, an antiprotease capable of complexing the excess elastase, were also investigated. Animals smoking high-nicotine cigarettes had significantly lower serum alpha 1-antitrypsin activities than controls. Low-nicotine smokers showed alpha 1-antitrypsin activities that were not significantly different from those of controls. The importance of these observations is reinforced by a number of studies suggesting that proteases, their inhibitors, and an imbalance thereof may be related to the onset of neoplastic lesions. Studies have indicated that antiprotease levels follow the patterns of Mendelian inheritance. Severe deficiency states predispose human subjects to emphysema. A similar relationship may exist between antiprotease levels and susceptibility or resistance to neoplasms of the pancreas, a concept that deserves investigation in light of the findings reported here.
The partition of labelled rat pancreatic elastase (EC 3.4.21.11) between the different protease inhibitors of rat plasma was studied at different levels of saturation of the inhibitors of rat plasma was studied at different levels of saturation of the inhibitor capacity of plasma with the enzyme. The reaction mixtures were analysed by immunoelectrophoretic methods utilizing specific antisera against the different inhibitors and by gel filtration on Sephadex G-200. Rat serum was shown to contain four elastase binding proteins. alpha 1-antitrypsin, alpha 1-macroglobulin and alpha 2-acute phase protein and alpha 1-inhibitor 3 which exhibits immunologic cross-reaction with human inter-alpha-trypsin inhibitor and is of similar molecular weight. With minute amounts of labelled elastase the partition among the binding protein was alpha 1-macroglobulin 60%, alpha 1-antitrypsin 24% and alpha 1-I3 16%. The 60% value of alpha 1-M bound radioactivity in normal serum corresponds to the sum of alpha 1-M and alpha 2-AP labelling in inflammatory serum.
Porcine elastase II (EC 3.4.21.-), a pancreatic proteinase with elastolytic activity, hydrolyses the oxidized beta-chain of insulin with major cleavages occurring at Leu17-Val18, Phe24-Phe25, Phe25-Tyr26 and Tyr26-Thr27. Canine leucocytic elastase splits the same substrate with major sites at Val12-Glu13 and Val18-Cys19 O3H. This indicates similarity of elastase II to chymotrypsins (EC 3.4.21.1 or 3.4.21.2) and of dog leucocyte enzyme to human granulocyte elastase and porcine pancreatic elastase I (EC 3.4.21.11).
1. A cationic protease has been purified from the granule fraction of blood-donor leukocytes by a preparative method including precipitation by acetone and chromatography on Bio-Gel A 1.5 m, CM-Sephadex C-50 and Sephadex G-G-75. 2. The pH optimum against denatured bovine hemoglobin is 7.4. Gel chromatography indicated a molecular weight close to 23 000. 3. This neutral protease (EC 3.4.-.-) is able to split the synthetic esters Z-Ala-NPh and AcAla3OMe, its activity on the former substrate being 2.2 times greater than that of pancreatic elastase, on the latter the same. It differs crucially from pancreatic elastase in having small elastinolytic activity. 4. In cationic disk electrophoresis, neutral protease resolves into three protein bands with lower mobility than lysozyme: all bands exhibit esterolytic activity against 2-acetoxy-3-naphthoic acid o-toluidide, strongly suggesting that they represent isoenzymes. 5. The enzyme is completely inhibited by iPr2P-F, partially so by soybean trypsin inhibitor and Trasylol. Cysteine, EDTA and TosLysCH2Cl have no effect. 6. During chromatography on CM-Sephadex C-50 a more positively charged enzyme(s) was identified. This had hemoglobinolytic activity at pH 7.4 but only a small esterolytic effect on Z-Ala-NPh; it showed only traces of activity against AcAla3OMe.
The effect of I- and C1- on the enzymatic degradation of elastin pancreatic elastase has been studied. At low halide concentrations both ions show a marked stimulation of elastolysis, I- being significantly more effective than Cl-. Kinetic plots of enzyme activity indicate that anions enhance the susceptibility of the substrate to enzymatic and non-enzymatic degradation. If the halide concentration is raised inhibition of elastolysis occurs starting at 30 mM NaCl and 300 mM NaI. This inhibitory effect is explained by a direct attack of the halides leading to increasing destabilization and inactivation of the enzyme.
Intratracheal injection of rabbit whole leukocyte homogenate induced emphysema-like lesions in rabbits. The lesions were produced only by preparations having elastinolytic activity. The pathological aspects appeared similar to those induced by the administration of porcine pancreatic elastase. The pulmonary changes, resembling several of the anatomic appearances of panacinar human emphysema, may be a suitable experimental model for studying histogenesis of panacinar human emphysema. Numerous abnormal fenestrations were present in air spaces walls and were a constant feature in opposition to that has been reported in elastase-treated hamsters. For the presence of a prominent dilatation of the periarterial lymphatic network, this experimental model might be used also for studying the ultrastructural features of pulmonary lymphatic vessels.
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19F NMR investigations of the interactions between elastase and the reversible inhibitors CF3CO-Ala3, CF3CO-Lys-Ala2 and CF3CO-Ala4 show that these peptides have a single mode of binding to the enzyme. Furthermore the results indicate that the CF3CO-group experiences the same environment in all of the reversible complexes formed with these inhibitors. This agrees with the higher affinity of these peptides for the enzyme as compared to the corresponding acetylated inhibitors and confirms our earlier hypothesis of the existence of a specific binding site for the CF3CO-group on the enzyme. The interactions between elastase and the irreversible inhibitors CF3CO-Alan chloromethyl ketone (n = 2, 3, 4, 5) and CF3CO-Lys-Ala4 chloromethyl ketone have been investigated by enzymatic measurements and 19F NMR spectroscopy. The kinetic constants k2 and KI describing the irreversible inhibition are significantly lower for all the CF3CO-peptide chloromethyl ketones with exception of CF3CO-Ala2 chloromethyl ketone, than for the corresponding acetylated ones. Moreover, 19F NMR spectroscopy enabled us to demonstrate, for the tri- and tetrapeptide derivatives, the parallel formation of reversible nonproductive enzyme.inhibitor complexes. The spectroscopic properties of these complexes are completely different from those of the irreversible complexes but are similar to those observed with the reversible complexes described above. In the case of the pentapeptide chloromethyl ketones, fast hydrolysis of the peptide or fast inactivation of the enzyme does not allow observations to be made but does not exclude the existence of reversible nonproductive complexes. In fact, their existence is strongly suggested by the enzyme reaction rate measurements. The similarity of the properties of all the reversible complexes, their striking differences with those of all the irreversible complexes, as well as their mutual exclusivity, permit the conclusion that the CF3CO-group does not bind at one of the classical S subsites of elastase.
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Porcine pancreatic elastase (EC 3.4.21.11) has been immobilized on polyacrylamide beads using glutaraldehyde ad bridging reagent without important loss of catalytic activity. A nitroxide spin label, 1-oxyl-2,2,5,5-tetramethyl-4-piperidinyl-ethylphosphonofluoridate, reacting covalently with the serine-195 residue of the active centre of free elastase was used as a conformational and dynamical electron spin resonance probe. This signal is quenched by (Cu2+) which bind specifically at the active site at a distance of 7 A from the nitroxide group. This distance is not significantly affected by the fixation on the solid support. The electron spin resonance lineshape analysis indicates some mobility of the spin label with respect to the native protein. This restricted motion, which is pH dependent, is not noticeably modified by the immobilization of the enzyme. This immobilization has therefore induced no large conformational change of the protein in the vicinity of the active centre. Thermal denaturation of elastase in homogeneous solution is irreversible. Immobilization on the polyacrylamide beads results in 70% reversibility, but the temperature of denaturation is not modified.