Elastolytic activity of Pseudomonas aeruginosa elastase

Elastolysis of insoluble elastin by Pseudomonas aeruginosa elastase was found to be less specific (higher apparent Km value) but more active (higher activity) than with pancreatic elastase. Furthermore, pancreatic and P. aeruginosa elastases act synergistically during the initial stages of elastolysis. After extensive hydrolysis, the size distribution of digestion products was lower with P. aeruginosa than with pancreatic elastase. The higher extent of hydrolysis may be explained by the fact that, if pancreatic elastase needs at least six sub-sites for activity, P. aeruginosa elastase may hydrolyse tetrapeptides such as tetraalanine, or synthetic substrates such as furylacryloyltripeptides FA-X-Leu-Y, X and Y being Gly and/or Ala.     

Specific inhibition of human granulocyte elastase with peptide aldehydes

The kinetic features of human granulocyte elastase, chymotrypsin, porcine pancreatic elastase and elastomucoproteinase were compared. Amino acyl ester substrates were assayed and Km and kcat values were defined. Aldehyde analogues of the p-nitroanilide substrates designed for granulocyte elastase as optimal for Km appeared to be potent inhibitors. Suc-D-Phe-Pro-valinal (Ki = 40 microM) was found to inhibit granulocyte elastase competitively and specifically when measured with synthetic substrates, and the Ki was 3 microM with the natural protein substrate, elastin.     

Effect of sodium oleate on the hydrolysis of human plasma fibronectin by proteinases

Oleic acid binds in a saturable fashion to human plasma fibronectin (FN). Analysis of the binding indicated the presence of a high affinity binding site with nKa approximately equal to 10 uM-1. Furthermore, it was found that binding of sodium oleate to FN modulated its susceptibility to degradation by various proteinases. FN saturated with sodium oleate was hydrolysed at a higher rate by trypsin, cathepsin D, thermolysin and pancreatic elastase than native FN. In contrast, sodium oleate inhibits the activity of two human granulocyte proteinases, human leucocyte elastase (HLE) and cathepsin G on either FN or on their respective specific synthetic substrates (at concentrations ranging from 10(-6) mM to 10 mM). Cathepsin G inhibition was non-competitive and gave a Ki in the 10 uM range similar to the previously reported inhibitory constant of oleic acid toward HLE.     

Degradation of elastin by a cysteine proteinase from Staphylococcus aureus

Staphylococcus aureus is known to produce three very active extracellular proteinases. One of these enzymes, a cysteine proteinase, after purification to homogeneity was found to degrade insoluble bovine lung elastin at a rate comparable to human neutrophil elastase. This enzyme had no detectable activity against a range of synthetic substrates normally utilized by elastase, chymotrypsin, or trypsin-like proteinases. However, it did hydrolyze the synthetic substrate carbobenzoxy-phenylalanyl-leucyl-glutamyl-p-nitroanilide (Km = 0.5 mM, kcat = 0.16 s-1). The proteolytic activity of the cysteine proteinase was rapidly and efficiently inhibited by alpha 2-macroglobulin and also by the cysteine-specific inhibitor rat T-kininogen (Ki = 5.2 X 10(-7) M). Human kininogens, however, did not inhibit. Human plasma apparently contains other inhibitors of this enzyme, since plasma depleted of alpha 2-macroglobulin retained significant inhibitory capacity. The elastolytic activity of this S. aureus proteinase and its lack of control by human kininogens or cystatin C may explain some of the connective tissue destruction seen in bacterial infections due to this and related organisms such as may occur in septicemia, septic arthritis, and otitis.     

Investigation of the active center of rat pancreatic elastase

We have isolated rat pancreatic elastase I (EC 3.4.21.36) using a fast two-step procedure and we have investigated its active center with p-nitroanilide substrates and trifluoroacetylated inhibitors. These ligands were also used to probe porcine pancreatic elastase I whose amino acid sequence is 84% homologous to rat pancreatic elastase I as reported by MacDonald, et al. (Biochemistry 21, (1982) 1453-1463). Both proteinases exhibited non-Michaelian kinetics for substrates composed of three or four residues: substrate inhibition was observed for most enzyme substrate pairs, but with Ala3-p-nitroanilide, rat elastase showed substrate inhibition, whereas porcine elastase exhibited substrate activation. With most of the longer substrates, Michaelian kinetics were observed. The kcat/Km ratio was used to compare the catalytic efficiency of the two elastases on the different substrates. For both elastases, occupancy of subsite S4 was a prerequisite for efficient catalysis, occupancy of subsite S5 further increased the catalytic efficiency, P2 proline favored catalysis and P1 valine had an unfavorable effect. Rat elastase has probably one more subsite (S6) than its porcine counterpart. The rate-limiting step for the hydrolysis of N-succinyl-Ala3-p-nitroanilide by rat elastase was essentially acylation, whereas both acylation and deacylation rate constants participated in the turnover of this substrate by porcine elastase. For both enzymes, trifluoroacetylated peptides were much better inhibitors than acetylated peptides and trifluoroacetyldipeptide anilides were more potent than trifluoroacetyltripeptide anilides. A number of quantitative differences were found, however, and with one exception, trifluoroacetylated inhibitors were less efficient with rat elastase than with the porcine enzyme.     

Hydrolysis of ester substrates of trypsin and chymotrypsin by barley carboxypeptidase

Purified barley carboxypeptidase exhibits high activity against a number of N-substituted amino acid esters, which are commonly used as synthetic substrates for mammalian and microbial proteinases. The proteinases of barley, on the contrary, do not hydrolyse these compounds. Because many other plants contain carboxypeptidases closely resembling the barley enzyme, we conclude that synthetic ester substrates should not be used to detect proteinase activity in extracts of higher plants. Plant carboxypeptidases also liberate C-terminal tryptophan from α-casein. Therefore, casein also is an unreliable substrate for plant proteinases.     

Comparison of specificity of human and horse leucocyte proteinases with synthetic peptide substrates

Highly purified horse leucocyte proteinases 1, 2A and 2B hydrolyze synthetic substrates which are decomposed also by human leucocyte elastase but they are unable to hydrolyze typical substrates of cathepsin G. Thus in distinction to other mammalian species horse leucocytes are devoid of cathepsin G and contain only elastases.     

Proteinase inhibitory spectrum of mouse murinoglobulin and alpha-macroglobulin

The interactions of mouse murinoglobulin and alpha-macroglobulin with several proteinases were investigated by filtration and by assays of amidolytic activity towards synthetic substrates in the presence of proteinaceous enzyme inhibitors as well as assays of the inhibition of proteolytic activity. Mouse alpha-macroglobulin formed complexes with thrombin, clotting factor Xa, plasmin, pancreatic kallikrein, plasma kallikrein, submaxillary gland trypsin-like proteinase, neutrophil elastase, and pancreatic elastase. These complexes lost the proteolytic activities against high-molecular-weight substrates, but protected the active sites of the enzymes from inactivation by their proteinaceous inhibitors. Mouse murinoglobulin showed essentially the same properties except (i) that it did not form a complex with the clotting factor Xa, and (ii) that it did not protect plasma kallikrein, neutrophil elastase or submaxillary proteinase from inactivation by their proteinaceous inhibitors, although it formed complexes with these proteinases. No interaction was detected between Clostridium histolyticum collagenase and murinoglobulin or alpha-macroglobulin. These results indicate (i) that murinoglobulin has a proteinase-binding spectrum similar to that of alpha-macroglobulin, but is weaker in the ability to protect the bound proteinases from inactivation by the proteinaceous inhibitors than alpha-macroglobulin and (ii) that mouse alpha-macroglobulin has essentially the same inhibitory spectrum as the human homologue.     

Biochemical characterization of human enteropeptidase light chain

The synthetic gene encoding human enteropeptidase light chain (L-HEP) was cloned into plasmid pET-32a downstream from the gene of fusion partner thioredoxin immediately after the DNA sequence encoding the enteropeptidase recognition site. The fusion protein thioredoxin (Trx)/L-HEP was expressed in Escherichia coli BL21(DE3). Autocatalytic cleavage of the fusion protein and activation of recombinant L-HEP were achieved by solubilization of inclusion bodies and refolding of Trx/L-HEP fusion protein. The kinetic parameters of human and bovine enteropeptidases in the presence of different concentrations of Ca2+ and Na+ for cleavage of the specific substrate GD4K-na and nonspecific substrates such as small ester Z-Lys-SBzl and chromogenic substrates Z-Ala-X-Arg-pNA have been comparatively analyzed. It is demonstrated that positively charged ions increased the Michaelis constant (Km) for cleavage of specific substrate GD4K-na, while the catalytic constant (k(cat)) remained practically unchanged. L-HEP demonstrated secondary specificity to the chromogenic substrate Z-Ala-Phe-Arg-pNA with k(cat)/Km 260 mM(-1) x sec(-1). Enzymatic activity of L-HEP was suppressed by inhibitors of trypsin-like and cysteine (E-64), but not metallo-, amino-, or chymotrypsin-like proteinases. L-HEP was active over a broad range of pH (6-9) with optimum activity at pH 7.5, and it demonstrated high stability to different denaturing agents.     

N-glycohydrolysis of adenosine diphosphoribosyl arginine linkages by dinitrogenase reductase activating glycohydrolase (activating enzyme) from Rhodospirillum rubrum

The reaction catalyzed by the activating enzyme for dinitrogenase reductase from Rhodospirillum rubrum has been studied using an ADP-ribosyl hexapeptide, obtained from proteolysis of inactive dinitrogenase reductase, and synthetic analogs such as N alpha-dansyl-N omega-ADP-ribosylarginine methyl ester. The activating enzyme catalyzed N-glycohydrolysis of the ribosyl-guanidinium linkage releasing ADP-ribose and regenerating an unmodified arginyl guanidinium group. Optimal glycohydrolysis of the low molecular weight substrates occurred at pH 6.6 and required 1 mM MnCl2, but did not require ATP. The ADP-ribosyl hexapeptide (Km 11 microM), N alpha-dansyl-N omega-ADP-ribosylarginine methyl ester (Km 12 microM), N alpha-dansyl-N omega-ADP-ribosylarginine (Km 12 microM), N alpha-dansyl-N omega-1,N6-etheno-ADP-ribosylarginine methyl ester (Km 11 microM), and N alpha-dansyl-N omega-GDP-ribosylarginine methyl ester (Km 11 microM) were comparable substrates. N omega-ADP-ribosylarginine (Km 2 mM) was a poor substrate, and the activating enzyme did not catalyze N-glycohydrolysis of N alpha-dansyl-N omega-5'-phosphoribosylarginine methyl ester or N alpha-dansyl-N omega-ribosylarginine methyl ester. 13C NMR of N alpha-tosyl-N omega-ADP-ribosylarginine methyl ester established that the activating enzyme specifically hydrolyzed the alpha-ribosyl-guanidinium linkage. The beta-linked anomer was hydrolyzed only after anomerization to the alpha configuration. We recommend [arginine(N omega-ADP-alpha-ribose)]dinitrogenase reductase N-glycohydrolase (dinitrogenase reductase activating) and dinitrogenase reductase activating glycohydrolase as the systematic and working names for the activating enzyme.     

Elastinolytic activity of horse leukocyte proteinases. Comparison with elastases from human leukocytes and porcine pancreas

Three proteinases from the azurophilic granules of horse leucocytes are typical elastases degrading elastin at neutral pH. Both proteinases: 1 and 2A exhibit similar elastinolytic activity, comparable with human leucocyte elastase (HLE). In relation to human enzyme, elastase 2B shows several-fold higher activity, which is comparable to the porcine pancreatic elastase activity (PPE). Similarly to HLE elastinolytic activity of the horse proteinases increases at higher ionic strength: twofold in case of 1 or 2A and fivefold for 2B. Significant activity observed during degradation of homologous lung elastin, implies the possible role of these enzymes during pathological injury of connective tissue in the lower respiratory tract and suggests similar pathogenesis of horse and human pulmonary emphysema.     

Elastases from human and canine granulocytes, I. Some proteolytic and esterolytic properties.

The lysosome-like granules of human and canine granulocytes contain an enzyme with elastinolytic activity. The enzymatic behaviour of these elastases was further characterized using the protein substrates elastin-orcein and azocasein and the synthetic substrates tert.-butyloxycarbonyl-alanine p-nitrophenylester (Boc-Ala-ONp) and 3-carboxypropionyl-L-alanyl-L-alanyl-L-alanine p-nitroanilide (Suc-Ala3-NHNp) in photometric assays. The affinities of the granulocyte elastases and of porcine pancreatic elastase to these substrates are very similar, e.g. human granulocyte elastase: KM (Boc-Ala-ONp) = 0.35mM, KM (Suc-Ala3-NHNp) = 1.25mM, porcine pancreatic elastase: KM (Boc-Ala-ONp) = 0.3mM, KM (Suc-Ala3-NHNp) - 1.15mM. The most convenient substrate for the assay of human and dog granulocyte elastases and for kinetic measurements with these enzymes is Suc-Ala3-NHNp. Using this substrate, the dissociation constant of the complex of human granulocyte elastase with human alpha1-antitrypsin could be determined (Ki = 3.5 x 10(-10)M).     

Substrate specificity of human pancreatic elastase 2

The substrate specificity of human pancreatic elastase 2 was investigated by using a series of peptide p-nitroanilides. The kinetic constants, kcat and Km, for the hydrolysis of these peptides revealed that this serine protease preferentially hydrolyzes peptides containing P1 amino acids which have medium to large hydrophobic side chains, except for those which are disubstituted on the first carbon of the side chain. Thus, human pancreatic elastase 2 appears to be similar in peptide bond specificity to the recently described porcine pancreatic elastase 2 [Gertler, A., Weiss, Y., & Burstein, Y. (1977) Biochemistry 16, 2709] but differs significantly in specificity from porcine elastase 1. The best substrates for human pancreatic elastase 2 were glutaryl-Ala-Ala-Pro-Leu-p nitroanilide and succinyl-Ala-Ala-Pro-Met-p-nitroanilide. However, there was little difference among substrates with leucine, methionine, phenylalanine, tyrosine, norvaline, or norleucine in the P1 position. Changes in the hydrolysis rate of peptides with differing P5 residues indicate that this enzyme has an extended binding site which interacts with at least five residues of peptide substrates. The overall catalytic efficiency of human pancreatic elastase 2 is significantly lower than that of porcine elastase 1 or bovine chymotrypsin with the compounds studied.     

Isolation and Some Physical and Chemical Properties of Elastase and Cathepsin G from Dog Neutrophils

A new method for isolation of leukocyte serine proteinases has been developed. Elastase (EC 3.4.21.37) and cathepsin G (EC 3.4.21.20) have been isolated from dog neutrophils and purified to homogeneous state. The results of inhibitor analysis indicate that the enzymes belong to the group of serine proteinases. Some physical and chemical characteristics of the purified enzymes have been determined. The molecular weights of the enzymes are 24.5-26 kD for the elastase and 23.5-25.5 kD for the cathepsin G. The cathepsin G is a glycoprotein, while the elastase molecule lacks carbohydrate components. The cathepsin G exhibits a broad pH optimum of catalytic activity in the range of 7.0-9.0; the pH optimum for the elastase is 8.0-8.5. The Michaelis constant of the elastase for N-t-Boc-L-alanine p-nitrophenyl ester is 0.10 mM; the Michaelis constant of the cathepsin G for N-benzoyl-L-tyrosine ethyl ester is 0.42 mM.     

Hydrolysis of artificial substrates by enterokinase and trypsin and the development of a sensitive specific assay for enterokinase in serum.

The activities of highly purified human enterokinase (enteropeptidase, EC 3.4.21.9) and bovine trypsin were tested against three synthetic substrates alpha-N-Benzoyl-L-arginine ethyl ester HCl, alpha-N-Benzoyl-DL-arginine-p-nitroanilide HCl and alpha-N-Benzoyl-DL-arginine-2-naphthylamide HCl. There was no detectable hydrolysis of these substrates by enterokinase whereas the kinetic parameters obtained for trypsin were in close agreement with those previously described by other workers. The values for Km and kcat were dependent on the Ca2+ concentration. Hydrolysis of glycine-tetra-L-aspartyl-L-lysyl-2-naphthylamide (Gly(Asp)4-Lys-Nap) by these protease was also studied. Enterokinase-catalysed hydrolysis obeyed simple steady-state kinetics and values for Km of 0.525 mM and 0.28 mM and for kcat of 21.5 s-1 and 28.3 s-1 were obtained in 0.1 mM and 10 mM Ca2+, respectively. Trypsin-catalysed hydrolysis was complex and the response to Ca2+ was sigmoidal partly due to the lability of trypsin at low Ca2+ concentrations. A sensitive specific assay for enterokinase was developed and applied to the measurement of the enzyme in serum; interference by nonspecific arylamidases was eliminated by the addition of Zn2+.     

Synthesis and analytical use of 3-carboxypropionyl-alanyl-alanyl-valine-4-nitroanilide: a specific substrate for human leukocyte elastase

A simple synthesis is described for 3-carboxypropionyl-Ala-Ala-Val-4-nitroanilide, a convenient and very specific substrate for human leukocyte elastase (Km = 1.0mM, kcat = 8.7 s-1). The substrate does not undergo appreciable spontaneous hydrolysis. It is not cleaved by trypsin or chymotrypsin and only rather slowly by porcine pancreatic elastase (Km = 9.1mM, kcat = 1.4 s-1).     

Active site mapping of the serine proteases human leukocyte elastase, cathepsin G, porcine pancreatic elastase, rat mast cell proteases I and II. Bovine chymotrypsin A alpha, and Staphylococcus aureus protease V-8 using tripeptide thiobenzyl ester substrates

The primary subsite specificities of human leukocyte elastase, cathepsin G, porcine pancreatic elastase, rat mast cell proteases I and II, bovine chymotrypsin A alpha, and the protease from strain V-8 of Staphylococcus aureus have been mapped with a series of tripeptide thiobenzyl ester substrates of the general formula Boc-Ala-Ala-AA-SBzl, where AA represents one of 13 amino acids. In addition, the effects of a P2 Pro and P4 methoxysuccinyl and succinyl groups were investigated. In an attempt to introduce specificity and/or reactivity into the substrate Boc-Ala-Ala-Leu-SBzl(X), the 4-chloro-, 4-nitro-, and 4-methoxythiobenzyl ester derivatives were studied. Enzymatic hydrolyses of the substrates were measured in the presence of 4,4'-dithiobis(pyridine) or 5,5'-dithiobis(2-nitrobenzoic acid), which provided a highly sensitive assay method for free thiol. The thio esters were excellent substrates for the enzymes tested, and in many cases, the best substrates reported here have kcat/KM values higher than those reported previously. The best substrate for human leukocyte elastase was Boc-Ala-Pro-Nva-SBzl(Cl), which has a kcat/KM of 130 X 10(6) M-1 s-1. A very reactive rat mast cell protease substrate, Boc-Ala-Ala-Leu-SBzl(NO2), was also found. The S. aureus V-8 protease was the most specific enzyme tested since it hydrolyzed only Boc-Ala-Ala-Glu-SBzl. Substituents on the thiobenzyl ester moiety of Boc-Ala-Ala-Leu-SBzl resulted in decreased KM values with human leukocyte elastase and rat mast cell protease I when compared to the unsubstituted derivative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hypodermin B, a trypsin-related enzyme from the insect Hypoderma lineatum. Comparison with hypodermin A and Hypoderma collagenase, two serine proteinases from the same source

Hypodermin B, a serine proteinase with a molecular weight of 23000, was purified to homogeneity from the larvae Hypoderma lineatum. It is stoichiometrically inhibited by diisopropylfluorophosphate and fully inactivated by N-tosyllysine chloromethyl ketone and soya bean and bovine pancreatic trypsin inhibitors. N-Tosylphenylalanine chloromethyl ketone and ovomucoid are without effect on its activity. Hypodermin B hydrolyses both amide and ester substrates of trypsin but does not display any chymotryptic activity on synthetic substrates. Its specificity on the B chain of insulin is slightly broader than that of bovine trypsin. Its amino acid composition and N-terminal sequence suggest structural homology with serine proteinases of the trypsin family and with two other serine proteinases, hypodermin A and Hypoderma collagenase, previously isolated from the same larvae. Hypodermins A and B are very similar with respect to their inhibition and specificity, they differ however strongly from Hypoderma collagenase.     

The purification and properties of yeast proteinase B from Candida albicans.

A serine proteinase (ycaB) from the yeast Candida albicans A.T.C.C. 10261 was purified to near homogeneity. The enzyme was almost indistinguishable from yeast proteinase B (EC 3.4.21.48), and an Mr of 30,000 for the proteinase was determined by SDS/polyacrylamide-gel electrophoresis. The initial site of hydrolysis of the oxidized B-chain of insulin, by the purified proteinase, was the Leu-Tyr peptide bond. The preferential degradation at this site, analysed further with N-blocked amino acid ester and amide substrates, demonstrated that the specificity of the proteinase is determined by an extended substrate-binding site, consisting of at least three subsites (S1, S2 and S'1). The best p-nitrophenyl ester substrates were benzyloxycarbonyl-Tyr p-nitrophenyl ester (kcat./Km 3,536,000 M-1 X S-1), benzyloxycarbonyl-Leu p-nitrophenyl ester (kcat./Km 2,250,000 M-1 X S-1) and benzyloxycarbonyl-Phe p-nitrophenyl ester (kcat./Km 1,000,000 M-1 X S-1) consistent with a preference for aliphatic or aromatic amino acids at subsite S1. The specificity for benzyloxycarbonyl-Tyr p-nitrophenyl ester probably reflects the binding of the p-nitrophenyl group in subsite S'1. The presence of S2 was demonstrated by comparison of the proteolytic coefficients (kcat./Km) for benzyloxycarbonyl-Ala p-nitrophenyl ester (825,000 M-1 X S-1) and t-butyloxycarbonyl-Ala p-nitrophenyl ester (333,000 M-1 X S-1). Cell-free extracts contain a heat-stable inhibitor of the proteinase.     

Hydrolysis of alanine oligomers and of elastin by P. aeruginosa proteinases and thermolysin

The hydrolysis of alanine oligomers by P. aeruginosa proteinases, thermolysin and porcine pancreatic elastase was studied. The concentrations of substrates and cleavage products were determined using reverse phase high pressure liquid chromatography. Tetraalanine was the shortest oligomer for which we could demonstrate hydrolysis by all the proteinases, except for porcine pancreatic elastase which only significantly hydrolyzed peptides longer than hexaalanine. Porcine pancreatic elastase hydrolyzes hexaalanine at a single site, whereas the other enzymes may split it either into two trialanine molecules, or into di- and tetraalanine, the latter being further cleavable to dialanine. A kinetic model based on first-order kinetic rate constants is proposed and the individual constants determined. Although P. aeruginosa elastase and thermolysin are closely similar in structure, they have shown a marked difference in their hydrolysis of either elastin or tetraalanine. Elastolytic activity of thermolysin was higher than that of elastase but tetraalanine was hydrolyzed more slowly by thermolysin.