Cucumisin like protease from the sarcocarp of Benincasa hispida var. Ryukyu

A protease has been purified from the sarcocarp of Benincasa hispida (Thunb.) Cogn. var. Ryukyu by two steps of chromatography. Its M(r) was estimated by SDS-PAGE to be about 67,000. The enzyme was strongly inhibited by diisopropyl fluorophosphate, but not by EDTA and cysteine protease inhibitors. The substrate having alanine at the position of P1 was the best among the Ala-Ala-Pro-X-pNAs (X = Ala, Lys, Phe, Glu, and diaminopropionic acid (Dap)). The N-terminal sequence of the first 33 residues was determined and 25 of the residues agreed with that of cucumisin [EC 3.4.21.25], a protease from the sarcocarp of melon fruit (Cucumis melo L. var. Prince). The results indicated that the B. hispida protease is a cucumisin like serine protease.     

Purification and partial characterization of a neutral protease from a virulent strain of Bacillus cereus

The factors involved in the pathogenesis of Bacillus cereus (B. cereus) in non-gastrointestinal diseases are poorly investigated. Some researchers suggest that B. cereus proteases may be involved in these illnesses. The aim of this work was to purify and characterize a protease isolated from a virulent strain of B. cereus to explain its assumptive damaging effect. The enzyme was purified in a four-step procedure involving ammonium sulfate fractionation, acetone precipitation, Bio-Gel filtration and column chromatography on DEAE-cellulose (DE-52 cellulose). The enzyme appeared homogenous using disc electrophoresis. The specific activity of the protease was 72 U/mg of protein. The enzyme was shown to have a relative molecular mass of 29 kDa. The protease was most active at pH 7.0 and 40 degrees C with haemoglobin as the substrate. The enzyme was made completely inactive by ethylenediaminetetraacetic acid (EDTA), beta-mercaptoethanol, dithiothreitol (DTT) and benzamidine (at a concentration of 1 mM) and by diisopropylfluorophosphate (DIPF), L-cysteine, L-histidine, 1,10-phenanthroline (at a concentration of 10 mM). Divalent cations, especially Ca2+ increased enzyme activity. The enzyme hydrolysed haemoglobin, albumin and casein as the substrates. With haemoglobin and albumin as the substrates Michaelis-Menten kinetics was observed. The obtained Km values were 86 +/- 40 microM (SD, n = 3) and 340 +/- 100 microM (SD, n = 3) for haemoglobin and albumin, respectively. The corresponding Vmax values were 1.26 +/- 0.1 (SD, n = 3) and 0.38 +/- 0.07 (SD, n = 3) mumol of tyrosine liberated per min, per ml, and per mg, while those for casein were not determined. It is concluded that this enzyme is a metal-chelator-sensitive, neutral protease damaging haemoglobin and albumin.     

Antibody selection for immunohistochemical survey of equine tissue

A membrane-bound protease induced by sulfur mustard in cultured normal human epidermal keratinocytes (NHEK) was purified and partially characterized. Maximum enzyme stimulation occurred at 16 hr after normal human epidermal keratinocytes were exposed to 300 microM sulfur mustard. Purification to homogeneity of the protease was accomplished by Triton X-100 solubilization, ultracentrifugation, and dialysis, followed by ion-exchange chromatography through DEAE-cellulose and finally hydrophobic column chromatography through phenyl Sepharose. Analysis of the purified enzyme by SDS-PAGE revealed a single polypeptide at the 80 kDa region. Further investigation of biochemical properties showed that a synthetic serine-specific Chromozym TRY peptide and the physiological protein laminin were good substrates for this enzyme. Moreover, this enzyme was inhibited mostly by the serine-protease inhibitors leupeptin and di-isopropyl fluorophosphate and not by the cysteine protease inhibitor E-64 or the metalloprotease inhibitor 1,10-phenanthroline (Component H, CH), indicating the serine protease nature of this enzyme. This enzyme had a pH optimum in the range of 7.0 to 8.0. Amino acid sequencing of the purified enzyme revealed that this enzyme belongs to the endopeptidase family (serine protease), and is homologous with a mammalian-type bacterial serine endopeptidase that can preferentially cleave K-X, including K-P. These results suggest that serine-protease stimulation may be one of the mechanisms of mustard-induced skin blister formation, and that some specific serine-protease inhibitors may be useful for the treatment of this sulfur mustard toxicity.     

Serum ferritin, hemoglobin, and Helicobacter pylori infection: a seroepidemiologic survey comprising 2794 Danish adults

Subtilisin-like serine protease, which is associated with the dormant spores of Bacillus cereus, was solubilized by washing the spores with 2 M KCl and purified to homogeneity by carbobenzoxy-D-phenylalanine-liganded affinity column chromatography and hydrophobic interaction column chromatography. Enzyme activity was completely inhibited by reagents for sulfhydryl groups such as HgCl2 as well as by conventional subtilisin inhibitors, suggesting the enzyme to be cysteine-dependent. The enzyme retained activity in 5 M urea at 4 degrees C for at least 2 months, and the specific activity was 50 times that of subtilisin BPN when measured for a common chromogenic substrate, carbobenzoxy-glycyl-glycyl-L-leucine p-nitroanilide. The gene encoding this protease was cloned in Escherichia coli, and its nucleotide sequence was analyzed. The deduced amino acid sequence suggested that the protease is produced as a precursor comprising three portions; a signal sequence (28 amino acid residues), a prosequence (80 amino acid residues) and a mature enzyme (289 amino acid residues). The mature region of the enzyme had high similarity with a thermitase from Thermoactinomyces vulgaris (72% identity) and a thermostable alkaline protease from Thermoactinomyces sp. E79 (66% identity), which have the N-terminal sequence showing scarcely noticeable similarity with corresponding stretches of subtilisins and mercuric ion-sensitive free cysteine in the equivalent position of the primary structure.     

Dimerization and activation of the herpes simplex virus type 1 protease

The quaternary state of the herpes simplex virus type 1 (HSV-1) protease has been analyzed in relation to its catalytic activity. The dependence of specific activity upon enzyme concentration indicated that association of the 27-kDa subunits strongly increased activity. Size-exclusion chromatography identified the association as a monomer-dimer equilibrium. Isolation of monomeric and dimeric species from a size-exclusion column followed by immediate assay identified the dimer as the active form of the enzyme. Activation of the protease by antichaotropic cosolvents correlated with changes in the monomer-dimer equilibrium. Thus, dimerization of the enzyme was enhanced in solvents containing glycerol or the anions citrate or phosphate. These are substances previously identified as activators of HSV-1 protease (Hall, D. L., and Darke, P. L. (1995) J. Biol. Chem. 270, 22697-22700). The relative potencies of these cosolvents as enzyme activators correlated with their efficiency in promoting dimerization. Under all solvent conditions examined, the dependence of specific activity upon enzyme concentration was consistent with a kinetic model in which only the dimer is active. Dissociation constants for the HSV-1 protease dimer determined with this model at 15 degrees C, pH 7.5, were 964 and 225 nM in 20% glycerol with 0.2 and 0.5 M citrate present, respectively. The activation of the HSV-1 protease by antichaotropic cosolvents was hereby shown to be similar in nature to the activation of the other well characterized herpesvirus protease, that from human cytomegalovirus.     

Catalytic contribution of flap-substrate hydrogen bonds in "HIV-1 protease" explored by chemical synthesis

An analogue of "HIV-1 protease" was designed in which the ability to donate important water-mediated hydrogen bonds to substrate was precisely and directly deleted. Chemical ligation of unprotected peptide segments was used to synthesize this "backbone-engineered" enzyme. The functionally relevant amide -CONH- linkage between residues Gly49-Ile50 in each flap of the enzyme was replaced by an isosteric thioester -COS- bond. The backbone-engineered enzyme had normal substrate specificity and affinity (Km). However, the catalytic activity (kcat) was reduced approximately 3000-fold compared to the native amide bond-containing enzyme. Inhibition by the reduced peptide bond substrate analogue MVT-101 was unaffected compared with native enzyme. By contrast, the normally tight-binding hydroxyethylamine inhibitor JG-365 bound to the backbone-engineered enzyme with an approximately 2500-fold reduction in affinity. The reduced catalytic activity of the -Gly49-psi(COS)-Ile50-backbone-engineered enzyme analogue provides direct experimental evidence to support the suggestion that backbone hydrogen bonds from the enzyme flaps to the substrate are important for the catalytic function of the HIV-1 protease.     

Programming the Rous sarcoma virus protease to cleave new substrate sequences

The Rous sarcoma virus protease displays a high degree of specificity and catalyzes the cleavage of only a limited number of amino acid sequences. This specificity is governed by interactions between side chains of eight substrate amino acids and eight corresponding subsite pockets within the homodimeric enzyme. We have examined these complex interactions in order to learn how to introduce changes into the retroviral protease (PR) that direct it to cleave substrates. Mutant enzymes with altered substrate specificity and wild-type or greater catalytic rates have been constructed previously by substituting single key amino acids in each of the eight enzyme subsites with those residues found in structurally related positions of human immunodeficiency virus (HIV)-1 PR. These individual amino acid substitutions have now been combined into one enzyme, resulting in a highly active mutant Rous sarcoma virus (RSV) protease that displays many characteristics associated with the HIV-1 enzyme. The hybrid protease is capable of catalyzing the cleavage of a set of HIV-1 viral polyprotein substrates that are not recognized by the wild-type RSV enzyme. Additionally, the modified PR is inhibited completely by the HIV-1 PR-specific inhibitor KNI-272 at concentrations where wild-type RSV PR is unaffected. These results indicate that the major determinants that dictate RSV and HIV-1 PR substrate specificity have been identified. Since the viral protease is a homodimer, the rational design of enzymes with altered specificity also requires a thorough understanding of the importance of enzyme symmetry in substrate selection. We demonstrate here that the enzyme homodimer acts symmetrically in substrate selection with each enzyme subunit being capable of recognizing both halves of a peptide substrate equally.     

Protease activities during preparation and handling of nuclear particles containing hnRNA

Conditions were devised to avoid protease activity during the preparation and the subsequent handling of nuclear particles containing hnRNA. During all the steps of preparation of rat liver particles, the presence of phenylmethyl sulfonyl fluoride (PMSF) was required for the reproducibility of the results. It probably inhibited the cellular serine proteases before the separation of the particles from the other cellular structures. Protease activity was detected in the rat liver particles. The enzyme(s) preferentially hydrolyzed a few particle polypeptides. It was not inhibited by PMSF, nor by two trypsin and chymotrypsin-like protease inhibitors, nor by iodoacetamide, but was inhibited by sodium bisulfite and para-hydroxymercury benzoate (PHMB). PHMB was preferred above bisulfite because it could be used at lower concentration. It proved useful when particles were to be incubated at 37 degrees C. A protease hydrolysing the same polypeptides as the liver enzyme was also detected in rat brain particles. However, its activity was much lower in this tissue and the presence of protease inhibitors was not absolutely required under the standard conditions of preparation and handling of brain particles.     

Intracellular proteolytic cleavage of 9-cis-retinoic acid receptor alpha by cathepsin L-type protease is a potential mechanism for modulating thyroid hormone action

We previously reported that the responsiveness of hepatocytes to thyroid hormone is markedly attenuated when they were cultured as monolayers rather than spheroids. To elucidate the mechanisms underlying the altered responsiveness, thyroid hormone receptor auxiliary proteins in the hepatocytes were analyzed by electrophoretic mobility shift assay. The major thyroid hormone receptor auxiliary protein was identified as 9-cis-retinoic acid receptor alpha (RXRalpha) in the hepatocytes regardless of the culture conditions. The cytoplasmic fraction was shown to contain a protease(s) that cleaves RXRalpha at its amino terminus. The presence of the protease in the cytosol, but not in the nucleus, was ascertained by incubating full-length 35S-labeled RXRalpha with each fraction. Using various protease inhibitors, it was shown that cathepsin L-type protease could participate in the cleavage of the RXRalpha. The enzyme activity was much higher in the monolayers than the spheroids. Inhibition of this enzyme activity in the monolayer hepatocyte resulted in the increase of nuclear RXRalpha protein and the augmentation of T3-dependent induction of spot 14 mRNA. These results suggest that the changes in cathepsin L-type protease activity in the cytosol may alter the turnover of RXRalpha in the nucleus and modify the function of steroid receptor superfamilies that heterodimerize with RXRalpha.     

Salt-tolerant and thermostable alkaline protease from Bacillus subtilis NCIM no. 64

The proteolytic activity produced by a Bacillus subtilis isolated from a hot spring was investigated. Maximum protease production was obtained after 38 h of fermentation. Effects of various carbon and nitrogen sources indicate the requirement of starch and bacteriological peptone to be the best inducers for maximum protease production. Requirement for phosphorus was very evident, and the protease was secreted over a wide range of pH 5-11. The partially purified enzyme was stable at 60 degrees C for 30 min. Calcium ions were effective in stabilizing the enzyme, especially at higher temperature. The enzyme was extremely salt tolerant and retained 100% activity in 5M NaCl over 96 h. The molecular weight of the purified enzymes as determined by SDS-PAGE was 28,000. The enzyme was completely inactivated by PMSF, but little affected by urea, sodium dodecyl sulfate, and sodium tripoly phosphate.     

Expression of a murine leukemia virus Gag-Escherichia coli RNase HI fusion polyprotein significantly inhibits virus spread

The antiviral strategy of capsid-targeted viral inactivation (CTVI) was designed to disable newly produced virions by fusing a Gag or Gag-Pol polyprotein to a degradative enzyme (e.g., a nuclease or protease) that would cause the degradative enzyme to be inserted into virions during assembly. Several new experimental approaches have been developed that increase the antiviral effect of the CTVI strategy on retroviral replication in vitro. A Moloney murine leukemia virus (Mo-MLV) Gag-Escherichia coli RNase HI fusion has a strong antiviral effect when used prophylactically, inhibiting the spread of Mo-MLV and reducing virus titers 1,500- to 2,500-fold. A significant (approximately 100-fold) overall improvement of the CTVI prophylactic antiviral effect was produced by a modification in the culture conditions which presumably increases the efficiency of delivery and expression of the Mo-MLV Gag fusion polyproteins. The therapeutic effect of Mo-MLV Gag-RNase HI polyproteins is to reduce the production of infectious Mo-MLV up to 18-fold. An Mo-MLV Gag-degradative enzyme fusion junction was designed that can be cleaved by the Mo-MLV protease to release the degradative enzyme.     

Development of thymic carcinoma in transgenic mice expressing SV40 T antigen

The strongest fibrinolytic protease (F-III-2) in the six enzyme proteins purified from earthworm, Lumbricus rubellus [N. Nakajima et al., Biosci. Biotech. Biochem., 57, 1726-1730 (1993)] has been modified chemically with fragmented human serum albumin (mol. wt., 10,000-30,000). The modified enzyme lost the antigenicity of the native enzyme and reacted with the antisera against human serum albumin, the human serum albumin fragments, and the conjugate with the native enzyme to form precipitation lines, which fused with each other. The conjugate was significantly more resistant to inactivation by protease inhibitors in rat plasma. The enzyme was a non-hemorrhagic protein and did not induce platelet aggregation. The enzyme kept potent proteolytic activity for fibrin and fibrinogen than that of human plasmin. The enzyme easily solubilized actual fibrin clots (thrombi) of whole blood induced by thrombin in a rat's vena cava. The continuous fibrinolysis for fibrin suspension in an enzyme reactor system using the modified enzyme immobilized to oxirane-activated acrylic beads has been achieved without any inactivation of the activity at least for more than 1 month. The N-terminal amino acid sequence of the protein was also investigated and the sequence showed local similarity to those of the serine proteases such as plasmin and chymotrypsin.     

Trypsin-like neutral protease associated with soluble elastin

Isolation of tropoelastin is complicated by the presence of a neutral protease closely associated with tropoelastin that is capable of sequentially degrading tropoelastin to small peptides. Substrate and inhibitor specificities of this neutral protease associated with purified tropoelastin were examined. The enzyme displayed proteolytic activity against casein, and esterase activity was detected when assayed against N-tosyl-L-arginine methyl ester but not against tert-butyl-oxycarbonyl-L-alanine p-nitrophenyl ester. No appreciable elastinolytic activity was detectable against either insoluble sodium dodecyl sulfate treated elastin or maleylated tropoelastin. The enzyme was not inhibited by the chymotrypsin inhibitor toluenesulfonylphenylalanine chloromethyl ketone. The enzyme was inhibited by phenylmethanesulfonyl fluoride and, to various degrees, by metal chelators. Tosyllysyl chloromethyl ketone, epsilon-aminocaproic acid, and Aprotinin (pancreatic trypsin inhibitor--Kunitz type), all inhibitors of trypsin-like enzymes, were very effective inhibitors, as were soybean trypsin inhibitor and human alpha-1-antitrypsin. The data suggest that the tropoelastin-associated enzyme is a neutral serine protease with trypsin-like specificity.     

Resistance to HIV protease inhibitors: a comparison of enzyme inhibition and antiviral potency

Resistance of HIV-1 to protease inhibitors has been associated with changes at residues Val82 and Ile84 of HIV-1 protease (HIV PR). Using both an enzyme assay with a peptide substrate and a cell-based infectivity assay, we examined the correlation between the inhibition constants for enzyme activity (Ki values) and viral replication (IC90 values) for 5 active site mutants and 19 protease inhibitors. Four of the five mutations studied (V82F, V82A, I84V, and V82F/I84V) had been identified as conferring resistance during in vitro selection using a protease inhibitor. The mutant protease genes were expressed in Escherichia coli for preparation of enzyme, and inserted into the HXB2 strain of HIV for test of antiviral activity. The inhibitors included saquinavir, indinavir, nelfinavir, 141W94, ritonavir (all in clinical use), and 14 cyclic ureas with a constant core structure and varying P2, P2' and P3, P3' groups. The single mutations V82F and I84V caused changes with various inhibitors ranging from 0.3- to 86-fold in Ki and from 0.1- to 11-fold in IC90. Much larger changes compared to wild type were observed for the double mutation V82F/I84V both for Ki (10-2000-fold) and for IC90 (0.7-377-fold). However, there were low correlations (r2 = 0.017-0.53) between the mutant/wild-type ratio of Ki values (enzyme resistance) and the mutant/wild-type ratio of viral IC90 values (antiviral resistance) for each of the HIV proteases and the viruses containing the identical enzyme. Assessing enzyme resistance by "vitality values", which adjust the Ki values with the catalytic efficiencies (kcat/Km), caused no significant improvement in the correlation with antiviral resistance. Therefore, our data suggest that measurements of enzyme inhibition with mutant proteases may be poorly predictive of the antiviral effect in resistant viruses even when mutations are restricted to the protease gene.     

Purification and molecular cloning of carp ovarian cystatin

Endothelin converting enzyme (ECE) from intact cells of a permanent human endothelial cell line, EA.hy926, was studied by examining the effects of phosphoramidon, an endothelin converting enzyme inhibitor, on the levels of secreted endothelin-1 and big endothelin-1. The specific ECE activity was demonstrated by a phosphoramidon dose-dependent decrease in ET-1 level with a concomitant increase in big ET-1 level. By using a specific neutral endopeptidase 24.11 (NEP 24.11) inhibitor, thiorphan, it was also shown that the phosphoramidon-sensitive ET-1 degrading activity in this cell line is due to the NEP 24.11 activity. Other serine, acid, and cysteine protease inhibitors had no effect on the endogenous synthesis of ET-1 and big ET-1 supporting the evidence that ECE is insensitive to these protease inhibitors as has been demonstrated with the isolated enzyme.     

Comparative analysis of changes in respiration and systemic hemodynamics during activation of GABA receptors in cats and rats]

Rat liver mitochondrial alanine aminotransferase (mALT) is known to be a very unstable enzyme, a property that has hindered efforts to purify it. In this report we examine the possibility of stabilizing mALT with ethanol, trehalose, and protease inhibitors. The presence of ethanol was shown to slow down the inactivation of mALT, increasing its half-life from 1 to 4 h. Trehalose was found to greatly enhance the stability of mALT in a concentration-dependent manner. In the presence of 36.5% trehalose, the half-life of mALT was 85 h. Of the protease inhibitors tested only antipain and chymostatin slowed down the inactivation of mALT but only within the first 24 h following preparation of the crude enzyme. It is concluded that the inclusion of ethanol and trehalose in purification protocols could aid the purification of the enzyme. It is also concluded that the inclusion of protease inhibitors in purification protocols of mALT may not be necessary as its inactivation does not seem to be due to protease activity.     

Molecular basis of proteolytic activation of Sendai virus infection and the defensive compounds for infection

It has been proposed that the pathogenicity of Sendai virus is primarily determined by a host cellular protease(s) that activates viral infectivity by proteolytic cleavage of envelope fusion glycoproteins. We isolated a trypsin-like serine protease, tryptase Clara, localized in and secreted from Clara cells of the bronchial epithelium of rats. The enzyme specifically cleaved the precursor of fusion glycoprotein F0 of Sendai virus at residue Arg116 in the consensus cleavage motif, Gln(Glu)-X-Arg, resulting in the presentation of the membrane fusion domain in the amino-terminus of the F1 subunit. Administration of an antibody against tryptase Clara in the airway significantly inhibited the activation of progeny virus and multiple cycles of viral replication, thus reducing the mortality rate. These findings indicate that tryptase Clara in the airway is a primary determinant of Sendai virus infection and that proteolytic activation occurs extracellularly. We identified two cellular inhibitory compounds against tryptase Clara in bronchial lavage. One was a mucus protease inhibitor, a major serine protease inhibitor of granulocyte elastase in the lining fluids of the human respiratory tract, and the other was a pulmonary surfactant which may adsorb the enzyme, resulting in its inactivation. These compounds inhibited virus activation by tryptase Clara in vitro and in vivo, but did not themselves affect the hemagglutination and the infectivity of the virus. The functional domain of the mucus protease inhibitor against the enzyme, which is organized in two homologous N- and C-terminal domains, is located in the C-terminal. Administration of these compounds in the airway may be useful for preventing infection with Sendai virus.     

Estimates of the chromium(VI) reducing capacity in human body compartments as a mechanism for attenuating its potential toxicity and carcinogenicity

Cathepsin L was purified from carp hepatopancreas by a method involving ammonium sulfate precipitation and a series of column chromatographies, in which the enzyme had an affinity toward Concanavalin A and Cibacron Blue F3GA. Its homogeneity was established by Native-PAGE, but two protein bands corresponding to molecular masses of 30,000 (single chain) and 24,000 (heavy chain) migrated on SDS-PAGE. The enzyme exhibited a maximum activity for carbobenzoxy-L-phenylalanyl-L-arginyl-4-methylcoumaryl-7-amide (Z-Phe-Arg-MCA) at pH 5.5-6.0 and 50 degrees C and the remarkable stability at pH 5.0-6.5 and below 40 degrees C. All tested cysteine protease inhibitors and TLCK and chymostatin markedly inhibited its activity, whereas the other serine protease inhibitors and a metalloprotease inhibitor negligibly affected it. In addition, several metal compounds reduced either its activity or stability to differing extents. Although EDTA alone caused an only marginal activation of the enzyme, its maximum activation required both 2 mM cysteine and 1 mM EDTA. The enzyme had an ability to hydrolyze three peptidyl-MCA substrates including Z-Phe-Arg-MCA, but all kinetic constants indicate that Z-Phe-Arg-MCA is the optical substrate to the enzyme.     

Activation of caspase-3-like protease by digitonin-treated lysosomes

Apoptosis, a naturally occurring programmed cell death or cell 'suicide', has been paid much attention as one of the critical mechanisms for morphogenesis and tissue remodeling. Activation of cysteine aspartases (caspases) is one of the critical steps leading to apoptosis. Although a mitochondria-mediated pathway has been postulated to be one of the activation mechanism of caspase-3, another subcellular compartment might be involved in the activation of the enzyme. The present study shows that the supernatant fraction of digitonin-treated lysosomes strongly activates Ac-DEVD-CHO inhibitable caspase-3-like protease. Activation of caspase-3-like protease by digitonin-treated lysosomal fractions was specifically suppressed by leupeptin and E-64, inhibitors of cysteine protease. These results indicate that leakage of lysosomal cysteine protease(s) into the cytosolic compartment might be involved in the activation of caspase-3-like protease.     

Elastase and the LasA protease of Pseudomonas aeruginosa are secreted with their propeptides

Pseudomonas aeruginosa elastase and the LasA protease are synthesized as preproenzymes with long amino-terminal propeptides. The elastase propeptide is cleaved autocatalytically in the periplasm to form a transient, inactive elastase-propeptide complex. In contrast, the processing of proLasA does not involve autoproteolysis. In this study, we analyzed short-term P. aeruginosa cultures under conditions that minimize proteolysis and found that an elastase-propeptide complex is secreted, and then the propeptide is degraded extracellularly, apparently by elastase itself. LasA protease, on the other hand, was found to be secreted in its unprocessed 42-kDa proenzyme form. The processing of proLasA occurred extracellularly, and it involved the transient appearance of a 28-kDa intermediate and the respective 14-kDa LasA propeptide fragment. The processing of proLasA in P. aeruginosa strain FRD740, which does not express elastase, also proceeded via the 28-kDa intermediate, but the rate of processing was greatly reduced. This low rate of proLasA processing was further reduced when the activity of a secreted lysine-specific protease was blocked. Purified secreted proteases of P. aeruginosa (i.e. elastase, the lysine-specific protease, and alkaline proteinase) converted proLasA to the active enzyme. Processing by elastase and the lysine-specific enzyme, but not by alkaline proteinase, proceeded via the 28-kDa intermediate, and both were far more effective than alkaline proteinase in converting proLasA to the mature enzyme. We conclude that LasA protease and elastase are secreted with their propeptides, which are then degraded by secreted proteases of P. aeruginosa. In addition to their other functions, the propeptides may play a role in targeting their respective enzymes across the outer membrane.