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M Orlowski

Publications and source records attributed to M Orlowski.

At least 55 records · Page 3Linked to original sources
PubMed1990

Use of site-directed mutagenesis to identify valine-573 in the S'1 binding site of rat neutral endopeptidase 24.11 (enkephalinase).

On the basis of the identity of a segment of the amino acid sequence within the active site of the bacterial enzyme thermolysin and the mammalian enzyme neutral endopeptidase 24.11, the possible involvement of valine-573 of neutral endopeptidase 24.11 in substrate binding was investigated. Valine-573 was changed to leucine and to alanine by site-directed mutagenesis. The effect of these mutations on inhibitor binding and substrate catalysis was examined with a series of compounds containing variable P'1 residues. With a small P'1 residue such as alanine, both mutant enzymes exhibited kinetic properties essentially the same as the wild-type enzyme. However, with larger P'1 residues such as phenylalanine, tyrosine, and leucine, the Val573Leu mutant showed a 24-100-fold decrease in inhibitor affinity. Similarly substrates containing bulky P'1 residues showed a 10-40-fold decrease in Vmax with little change in Km. In contrast, the Val573Ala mutant showed only modest changes in terms of inhibitor binding or substrate turnover. These results support the proposed role of valine-573 as a part of the hydrophobic binding pocket, S'1 binding subsite, of neutral endopeptidase 24.11.

Amino Acid Sequence↗
PubMed1990

Primary and secondary structure of the 25S rRNA from the dimorphic fungus Mucor racemosus.

A 9.76 Kb ribosomal DNA repeat unit from the nuclear genome of the dimorphic fungus Mucor racemosus (Zygomycetes) was identified using a hybridization probe from the yeast Saccharomyces cerevisiae (Ascomycetes). This material was cloned in Escherichia coli plasmids as four overlapping pieces and mapped with respect to cleavage sites for 12 restriction endonucleases. The nucleotide sequence of the complete 25S rRNA gene and flanking regions was determined. The 5' and 3' ends of the structural gene were identified by comparison with the published sequence for the S. cerevisiae gene. The Mucor gene was found to possess 3469 bp and have a GC content of 42.8%. It was compared with the homologous gene from several other eukaryotes and found to be most similar to that from Saccharomyces. A potential secondary structure of the putative RNA transcript consistent with the structures proposed for the E. coli and Saccharomyces molecules was constructed by computer modelling.

Base Sequence↗
PubMed1990

Immunohistochemical localization of endopeptidase 24.15 in rat trachea, lung tissue, and alveolar macrophages.

Endopeptidase 24.15 (EP 24.15; EC 3.4.24.15), a zinc-metalloendopeptidase purified from rat brain and testes and also present in many other tissues, including the lung, degrades substance P, neurotensin, bradykinin, luteinizing hormone-releasing hormone, and some other bioactive peptides. The enzyme, present both as soluble cytoplasmic and membrane-bound forms, also rapidly converts dynorphin, alpha- and beta-neoendorphin, and some other opioid peptides into their respective enkephalins. In this study, a rabbit antibody to EP 24.15 purified from rat testes was used to study distribution of the enzyme in rat trachea, lung tissue, and alveolar macrophages (AMs) by immunohistochemical techniques. We found intense immunoreactivity to EP 24.15 within the cytoplasm of ciliated epithelial cells of tracheobronchial mucosa extending from trachea to terminal bronchioles. In addition, large myelinated paratracheal and peribronchial nerve fibers showed immunoreactivity. Blood vessels and alveolar lining cells were negative. AMs also showed intense diffuse cytoplasmic immunoreactivity. The findings of EP 24.15 immunoreactivity in airway epithelium, AMs, and paratracheal and peribroncheal nerve fibers suggest that the enzyme may modulate the activities of bioactive peptides within the lung.

Amino Acid Sequence↗
PubMed1990

Inhibition of endopeptidase 24.15 greatly increases the release of luteinizing hormone and follicle stimulating hormone in response to luteinizing hormone/releasing hormone.

Inhibitors of endopeptidase (EP) 24.15, an enzyme cleaving the Tyr5-Gly6 bond of LHRH, greatly increase the half-life of i.v. or i.c.v. administered luteinizing hormone-releasing hormone (LHRH) (Lasdun et al., J. Pharmacol. Exp. Ther. 251: 439-447, 1989). Concentrations of plasma luteinizing hormone (LH) and follicle stimulating hormone (FSH) were measured in rats after i.c.v. and i.v. administration of LHRH alone or in conjunction with inhibitors of EP 24.15. In animals treated with two potent EP 24.15 inhibitors, i.v. and i.c.v. LHRH injections induced a much greater and longer-lasting increase of plasma LH and FSH concentrations than in controls Two and 4 hr after administration of the inhibitors and LHRH, hormone concentrations were one order of magnitude greater than in controls. The magnitudes and durations of the increases were similar to those after administration of [D-Trp6]-LHRH or [D-Leu6, Des-Gly-NH2(10)]-LHRH ethylamide, two "superactive" analogs of LHRH, which are resistant to degradation by EP 24.15, due to the presence of a D-amino acid in position 6. It is concluded that LHRH degradation by EP 24.15 limits the magnitude and duration of the response of the pituitary to LHRH, and that increases in plasma LH and FSH similar to those obtained after administration of superactive analogs can be also obtained with the natural hormone, provided that its degradation is prevented by EP 24.15 inhibitors. Accordingly, the increased in vivo activity of the superactive LHRH analogs can be largely attributed to their resistance to degradation by EP 24.15.

Animals↗
PubMed1989

Pituitary multicatalytic proteinase complex. Specificity of components and aspects of proteolytic activity.

The 700-kDa multicatalytic proteinase complex from bovine pituitaries separates in polyacrylamide gel electrophoresis under dissociating and reducing conditions into 11 components with molecular masses ranging from 21 to 32 kDa. No higher molecular mass components were detected. A rabbit polyclonal antibody raised against the complex recognizes five immunoreactive components. As reported previously, the complex exhibits three distinct proteolytic activities designated as chymotrypsin-like, trypsin-like, and peptidylglutamyl-peptide hydrolyzing activities. All three activities are rather rapidly inactivated by 3,4-dichloroisocoumarin, a general serine protease inhibitor, however, the pseudo-first-order rate constants of inactivation of the three components differ within a wide range, with the chymotrypsin-like activity being most sensitive to inhibition. The peptidylglutamyl-peptide hydrolyzing activity is greatly activated by low concentrations of sodium dodecyl sulfate and fatty acids and seems to constitute the main component responsible for degradation of protein substrates. In addition to cleaving bonds on the carboxyl side of glutamyl residues, this activity also cleaves, albeit at a slower rate, bonds on the carboxyl side of hydrophobic residues; however, the secondary specificity of this component is clearly different from the chymotrypsin-like activity. Heparin selectively activates the chymotrypsin-like activity. The complex cleaves rapidly both native and dephosphorylated beta-casein in a reaction greatly accelerated by low concentrations of sodium dodecyl sulfate. The nature of proteolytic products, and also the rate of formation of acid-soluble, ninhydrin-reactive products, is different for the phosphorylated and dephosphorylated form of beta-casein, indicating that the degree of phosphorylation influences the rate and pattern of proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

Endopeptidase 24.15 from rat testes. Isolation of the enzyme and its specificity toward synthetic and natural peptides, including enkephalin-containing peptides.

Endopeptidase 24.15, a metalloendopeptidase (EC 3.4.24.15) with an Mr of about 70,000, was purified to homogeneity from rat testes. The enzyme cleaves preferentially bonds on the carboxyl side of hydrophobic amino acids. Secondary enzyme-substrate interactions at sites removed from the scissile bond are indicated by the finding that a hydrophobic or bulky residue in the P3' position greatly contributes to substrate binding and catalytic efficiency. The isolated enzyme is inhibited by metal chelators and by thiols. Loss of enzymic activity after dialysis against EDTA can be restored by low concentrations of Zn2+ and Co2+ ions. The rate of reaction of the Co2+ enzyme with a synthetic substrate was higher than that of the Zn2+ enzyme. These results are consistent with the classification of the enzyme as a metalloendopeptidase. N-Carboxymethyl peptides that fulfil the binding requirements of the substrate recognition site of the enzyme act as potent competitive inhibitors. Biologically active peptides such as luteinizing hormone-releasing hormone, bradykinin and neurotensin are cleaved at sites consistent with the specificity of the enzyme deduced from studies with synthetic peptides. Dynorphin A (1-8)-peptide, beta-neoendorphin, metorphamide, and Metenkephalin-Arg6-Gly7-Leu8 are rapidly converted to the corresponding enkephalins. The testis enzyme is catalytically and immunologically closely related to the previously identified brain enzyme.

Animals↗
PubMed1989

Substrate specificity and inhibitors of a capillary injury-related protease from sheep lung lymph.

A serine protease (Mr 70,000 to 75,000) appearing in sheep lung lymph after capillary damage induced by Escherichia coli endotoxin, oleic acid, or air emboli, was studied for its specificity toward a series of synthetic peptide and thioester substrates containing an Arg residue in the P1 position. High specificity constants (kcat/Km) were generally obtained with substrates having two or more basic amino acid residues, and with those having a Gln residues in the P2 position. Secondary enzyme-substrate interactions at sites more removed from the scissile bond are of importance, since a few peptides with two basic residues were hydrolyzed slowly, and the site of cleavage of natural peptides was influenced by the amino acid sequence beyond the immediate vicinity of the hydrolyzed bond. The properties of the enzyme and its pattern of specificity distinguish it from enzymes of the clotting cascade, from components of the complement system, and from lung and skin tryptase. The enzyme was inactivated by p-amidinophenylmethanesulfonyl fluoride and by a series of mechanism-based isocoumarin derivatives, the most potent inhibitor being 4-chloro-7-guanidino-3-(2-phenylethoxy)isocoumarin. Enzyme solutions inactivated by reaction with isocoumarin inhibitors could be completely reactivated after 30 h by treatment with hydroxylamine at neutral pH. Formation of a stable sheep lymph acyl enzyme--in contrast to thrombin and other trypsin-like enzymes--is not followed by alkylation of an active site nucleophile that leads to irreversible enzyme inactivation. The high activity toward substrates with two basic residues suggests that the enzyme may potentially function in processing of precursors of bioactive peptides.

Animals↗
PubMed1989

Lung lymph capillary injury-related protease.

Capillary damage induced in sheep by intravenous infusion of Escherichia coli endotoxin, oleic acid, or air emboli causes the appearance in lung lymph of a serine protease with trypsin-like activity. The time course of the appearance of the enzyme and the extent of its activity increase indicate a close association with capillary injury. The enzyme was isolated from active lymph after a 9,000-fold purification by affinity chromatography on Reactive Blue-agarose, aprotinin-agarose, and p-amino-benzamidine-agarose columns. The protein, molecular mass of 70-75 kDa, is composed of two polypeptide chains of 31 and 43 kDa linked by disulfide bonds. Studies with synthetic peptide and thioester substrates showed preferential cleavage of substrates having two or more basic amino acids and the importance for activity of secondary enzyme-substrate interactions at sites removed from the scissile bond. The specificity of the enzyme and its pattern of sensitivity to inhibition by a series of isocoumarin derivatives distinguish it from enzymes of the clotting and complement systems and also from tissue plasminogen activator and lung and skin tryptase. The origin of the enzyme, its role in capillary damage, and its physiological function remain to be established.

Amino Acid Sequence↗
PubMed1989

Inhibition of endopeptidase 24.15 slows the in vivo degradation of luteinizing hormone-releasing hormone.

Endopeptidase (EP) 24.15 cleaves the Tyr5-Gly6 bond of luteinizing hormone-releasing hormone (LHRH) (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2), and is the primary LHRH degrading enzyme in pituitary and hypothalamic membrane preparations. Potent and specific inhibitors were used to identify the enzymes involved in the in vivo degradation of LHRH. After i.c.v. administration of LHRH, only about 1% of the peptide was recovered from brain after 1 hr. Concurrent administration of LHRH and N-[1-(RS)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate (cFP-AAF-pAB), a specific inhibitor of EP 24.15, led to a more than 10-fold increase in LHRH recovery. Administration of N-[1-(RS)-carboxy-3-phenylpropyl]-Phe-pAB (cFP-F-pAB) or captopril, inhibitors of "enkephalinase" (EP 24.11) and angiotensin converting enzyme, respectively, did not significantly increase LHRH recovery. Intravenous administration of LHRH and either cFP-F-pAB or cFP-AAF-pAB but not captopril, led to an increase in the half-life of LHRH from 10 min to 15 and 20 min, respectively. Concurrent administration of both inhibitors resulted in a dramatic 8-fold increase in the half-life of LHRH, similar to values reported for "superactive" analogs of LHRH which are rendered resistant to enzymatic degradation by introduction of a D-amino acid in position 6. Concentrations of plasma LHRH 65 to 80 min after administration of inhibitors were 100- to 200-fold higher than those in controls. The potentiating effect of cFP-F-pAB resulted from inhibition of the in vivo degradation of cFP-AAF-pAB by EP 24.11.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Substrate-related potent inhibitors of brain metalloendopeptidase.

Rat brain metalloendopeptidase (EC 3.4.24.15) generates Leu- and Met-enkephalin from several larger opioid peptides and is capable of degrading a number of neuropeptides. Substrate-related N-(1-carboxy-3-phenylpropyl) peptide derivatives were synthesized and tested for enzyme inhibition. The best of these derivatives, N-[1(RS)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate, inhibited the enzyme in a competitive manner with a Ki of 16 nM. The data indicate that the carboxyl group of the N-(1-carboxy-3-phenylpropyl) moiety coordinates with the active site zinc atom and that the remaining part of the inhibitor is necessary for interaction with the substrate recognition site of the enzyme. Replacement of the 1-carboxy-3-phenylpropyl group by a carboxymethyl group decreased the inhibitory potency by more than 3 orders of magnitude, emphasizing the importance of the hydrophobic phenyl group for inhibitor binding to a hydrophobic pocket at the S1 subsite. Replacement of the Tyr residue by an Ala residue decreased the inhibitory potency by more than 20-fold. Changes in the structure of the residue interacting with the S1' subsite could cause a more than 60-fold change in inhibition. The inhibitors were either ineffective or only weakly inhibitory against membrane-bound metalloendopeptidase ("enkephalinase", EC 3.4.24.11), an enzyme highly active in rabbit kidney but also present in brain. The data indicate the presence of an extended binding site in the enzyme with residues interacting with S1, S1', and S3' subsites largely determining inhibitor binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Endopeptidase-24.15 is the primary enzyme that degrades luteinizing hormone releasing hormone both in vitro and in vivo.

The concentration of luteinizing hormone releasing hormone (LHRH) (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2), which reaches the anterior pituitary via the hypothalamo-hypophyseal portal system, appears to be controlled in part by the rate of LHRH degradation within the hypothalamus and/or pituitary. Specific, active site-directed endopeptidase inhibitors synthesized in our laboratory were used to identify the enzyme(s) involved in LHRH degradation by hypothalamic and pituitary membrane preparations, and by an intact anterior pituitary tumor cell line (AtT20). Incubation of LHRH with pituitary and hypothalamic membrane preparations led to the formation of pGlu-His-Trp (LHRH1-3) as the main reaction product. Under the same conditions, addition to the incubation mixtures of captopril, an inhibitor of the angiotensin converting enzyme, led to accumulation of pGlu-His-Trp-Ser-Tyr (LHRH1-5) and, to a lesser extent, pGlu-His-Trp-Ser-Tyr (LHRH1-6). The degradation of LHRH and the formation of the N-terminal tri- and pentapeptides was blocked by N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate (cFP-AAF-pAB), a specific, active site directed inhibitor of endopeptidase-24.15. Some inhibition of LHRH degradation and formation of the N-terminal hexapeptide was also obtained in the presence of N-[1-carboxy-2-phenylethyl]-Phe-p-aminobenzoate (cFE-F-pAB), an inhibitor of endopeptidase-24.11. Similar results were obtained with AtT20 cell membranes and with intact AtT20 cells in monolayer culture. Following cleavage by endopeptidases the C-terminal part of LHRH was rapidly degraded by aminopeptidases. Superactive analogs of LHRH in which Gly6 was replaced by a D-amino acid are resistant to degradation by both endopeptidase-24.11 and -24.15. In vivo, when LHRH was injected directly into the third ventricle of rats, the presence of cFP-AAF-pAB inhibited LHRH degradation. It is concluded that LHRH degradation is primarily initiated by the membrane-bound form of endopeptidase-24.15 to yield pGlu-His-Trp-Ser-Tyr and to a lesser extent by endopeptidase-24.11 to yield pGlu-His-Trp-Ser-Tyr-Gly.

Animals↗
PubMed1988

High cathepsin B activity in alveolar macrophages occurs with elastase-induced emphysema but not with bleomycin-induced pulmonary fibrosis in hamsters.

Cathepsin B activity was quantitated in alveolar macrophages obtained from hamsters 10, 21, and 105 days after the intratracheal instillation of porcine pancreatic elastase, bleomycin, or normal saline. Alveolar macrophages lavaged from animals receiving elastase contained significantly higher enzyme levels at 21 and 105 days (16,200 and 17,000 U/mg protein/hr, respectively) as compared with saline-treated animals (12,300 units). In contrast, cells from animals receiving bleomycin showed a decrease in activity at 21 and 105 days (9700 and 9900 units, respectively). At 10 days enzyme levels did not differ significantly. The results suggest that cathepsin B levels in alveolar macrophages reflect differences in lung destruction and connective tissue repair in vivo. In addition, the finding of high cathepsin B activity in animals with emphysema suggests the possibility that cysteine proteases contribute to progressive lung destruction initiated by the intratracheal instillation of elastase.

Animals↗
PubMed1987

Intracerebroventricular infusion of inhibitors of endopeptidase-24.11 ('enkephalinase') increases the spontaneous firing frequency of an identifiable set of cells in the substantia nigra.

The effect of inhibitors of the membrane-bound metalloendopeptidase-24.11 ('enkephalinase') on the activity of electrophysiologically identifiable neurons in the substantia nigra is described. Dopaminergic and non-dopaminergic cells were examined. Cells were classified by their responses to striatal stimulation. Only those cells in which the stimulation evoked excitation (alone or mixed with inhibition) responded to the inhibitors. Those cells in which the evoked response was only inhibition did not respond to the drugs. Infusion of 1 mumol of N-[1-(R,S)-carboxy-2-phenyl-ethyl]Phe-pAB (CPAB), 1 or 2 mumol of N-[1-(R,S)-carboxy-3-phenylpropyl]Phe-pAB (CPPAB) into the lateral ventricle produced statistically significant increases (pre- to post-drug treatment) in the spontaneous activity of cells exhibiting excitatory evoked responses: average increases were 33.3%. The increase in spontaneous activity reached an apparent maximum 20 min after the end of the infusion. The increased firing frequency was shown to result from the inhibition of the enzyme, rather than a non-specific effect, as the infusion of 2 mumol of N-[1-(R,S)-carboxy-2-phenyl-ethyl]Leu-pAB, an inhibitor structurally related to CPAB and CPPAB yet two orders of magnitude less potent, was without effect on the activity of nigral neurons. The inhibition of the enzyme by 1 mumol CPAB was verified through in vitro assay. We hypothesize that inhibition of the enzyme enhances peptide-modulated (tachykinin and/or enkephalin) excitation in select neurons of the substantia nigra.

Animals↗
PubMed1987

Isolation and properties of a capillary injury-related protease from lung lymph.

Lung microvascular injury induced in sheep by intravenous infusion of Escherichia coli endotoxin, oleic acid, or air emboli caused the appearance in lung lymph of high levels of a protease with trypsin-like activity. The enzyme was isolated as an apparently homogeneous protein from pooled samples of active lung lymph, after an almost 9000-fold purification by affinity chromatography on columns of Reactive Blue 2-agarose, aprotinin-agarose, and p-aminobenzamidine-agarose, and chromatography on a column of Sephadex G-100. A molecular weight of about 70,000 to 75,000 was determined from mobility in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The pH optimum was between 7.3 and 7.6. The isolated enzyme was quite labile, rapidly losing activity at both 37 and 25 degrees C. Addition of albumin to enzyme solutions protected against inactivation. Inhibition by diisopropylfluorophosphate and phenylmethanesulfonyl fluoride indicated that the enzyme belongs to the class of serine proteases. The enzyme cleaved peptide bonds on the carboxyl side of arginine residues and showed a relatively high affinity toward peptides containing several basic amino acid residues. Bonds involving the carboxyl group of lysine were cleaved at a much slower rate. The enzyme showed no plasminogen activator activity and its substrate specificity was quite different from that of several proteases of the clotting cascade. Its appearance in lymph was not influenced by lymph clotting and the isolated enzyme was not capable of correcting the clotting defect of plasmas deficient in factors XII, XI, IX, VII, and X.

Animals↗
PubMed1987

Regulation of gene expression during aerobic germination of Mucor racemosus sporangiospores.

The pool of mRNA stored in dormant sporangiospores of Mucor racemosus and expressed during early germination in air has been investigated. Total RNA was extracted from dormant and germinating spores and translated in a cell-free rabbit reticulocyte system containing L-[35S]methionine. Isotopically labelled in vitro translation products were analysed by PAGE and autoradiography and were compared with labelled proteins synthesized in vivo at the same stages of development. This comparison revealed several significant findings about the fates of individual mRNA populations as templates in translation: (i) a pool of mRNA, presumably represented entirely or in part by a recoverable polyadenylated RNA fraction, can be extracted from dormant spores in a translatable form; (ii) most of the differential gene expression displayed at the level of protein synthesis during germination results from concomitant changes in functional mRNA levels; (iii) some of the stored mRNA species may be activated and others inactivated by post-transcriptional processing mechanisms; and (iv) a small population of gene products may be regulated at the level of selective translation of pre-existing messages.

Autoradiography↗
PubMed1987

Synaptosomal membrane-bound form of endopeptidase-24.15 generates Leu-enkephalin from dynorphin1-8, alpha- and beta-neoendorphin, and Met-enkephalin from Met-enkephalin-Arg6-Gly7-Leu8.

Brain contains a membrane-bound form of endopeptidase-24.15, a metalloendopeptidase predominantly associated with the soluble protein fraction of brain homogenates. Subcellular fractionation of the enzyme in rat brain showed that 20-25% of the total activity is associated with membrane fractions including synaptosomes. Solubilization of the enzyme from synaptosomal membranes required the use of detergents or treatment with trypsin. The specific activity of the enzyme in synaptosomal membranes measured with tertiary-butoxycarbonyl-Phe-Ala-Ala-Phe-p-aminobenzoate as substrate was higher than that of endopeptidase-24.11 ("enkephalinase"), a membrane-bound zinc-metalloendopeptidase believed to function in brain neuropeptide metabolism. Purified synaptosomal membranes converted efficiently dynorphin1-8, alpha- and beta-neoendorphin into leucine enkephalin and methionine-enkephalin-Arg6-Gly7-Leu8 into methionine enkephalin in the presence of captopril, bestatin, and N-[1-(R,S)-carboxy-2-phenylethyl]-Phe-p-aminobenzoate, inhibitors of angiotensin converting enzyme (EC 3.4.15.1), aminopeptidase (EC 3.4.11.2), and membrane-bound metalloendopeptidase (EC 3.4.24.11), respectively. The conversion of enkephalin-containing peptides into enkephalins was virtually completely inhibited by N-[1-(R,S)-carboxy-2-phenylethyl]-Ala-Ala-Phe-p-aminobenzoate, a specific active-site-directed inhibitor of endopeptidase-24.15, indicating that this enzyme was responsible for the observed interconversions. The data indicate that synaptosomal membranes contain enzymes that can potentially generate and degrade both leucine- and methionine-enkephalin.

Animals↗
PubMed1987

Proteolytic activity in sheep lung lymph as marker of lung capillary injury.

Intravenous infusions of Escherichia coli endotoxin into sheep caused the appearance in lung lymph of high levels of an enzyme with trypsinlike activity. The time course of appearance of the enzyme and the extent of its increase corresponded to the known events of endotoxin-induced capillary injury. Accordingly, activity was low in the first phase of endotoxin-induced increased lung lymph flow caused by increased pressure filtration but was high in the second phase of increased lung lymph flow caused by increased permeability filtration. Recovery was associated with a decrease of activity to preinfusion levels. Capillary damage and increased permeability filtration induced by air emboli or oleic acid led to a similar increase in lung lymph proteolytic activity. By contrast lung lymph proteolytic activity remained virtually unchanged during increased pressure filtration induced by inflation of a balloon in the left atrium. Activity also remained unchanged in thoracic duct lymph, indicating that the increased activity in lung lymph is not an expression of a generalized response to endotoxin. The enzyme, a serine protease with a molecular weight of about 70,000 to 75,000 and a pH optimum between 7.3 and 7.6, was not related to lymph clotting and was not capable of correcting the clotting defects of plasmas deficient in enzymes of the clotting cascade. These results together with specificity studies indicate that the enzyme represents a new, hitherto unidentified, protease. Measurements of its activity in lung lymph represent a sensitive marker of lung capillary injury.

Animals↗