Proteasic polyseritis: effects of intraperitoneal injections of collagenase, alone or together with the administration of trypsin]

The authors showed previously that the effusions of the experimental polyseritis after intraperitoneal injections of trypsin and elastase, go from peritoneum to the pleural cavities and never from thorax to the peritoneum. The intraperitoneal injections of collagenase or of collagenase and trypsin can also cause polyseritis in the rat; but more often they provoke heavy hemorrhagic lesions of the wall of the abdomen and the diaphragm. Perforations of the diaphragm were observed in 8 rats of 32, with in 6, a intrathoracic hernia of the liver or the stomach. After the intrapleural injection of collagenase or of collagenase and trypsin, hemorrhagic lesions were seen in the thorax, but not in the abdomen. These facts are a new proof for the transdiaphragmatic propagation of the proteasic solutions injected in the peritoneum.     

Increased survival of rat hepatocytes in serum-free medium by inhibition of a trypsin-like protease associated with their plasma membranes

Bovine pancreatic trypsin-inhibitor (bPTI) is required for survival of adult rat hepatocytes for more than 2 days in primary cultures in serum-free medium. Of the various protease inhibitors tested, all trypsin inhibitors increased the survival of rat hepatocytes in serum-free medium, their potencies being in the order bPTI greater than alpha 2-plasmin inhibitor greater than leupeptin greater than soybean trypsin inhibitor greater than alpha 1-antitrypsin = alpha 2-macroglobulin. Elastatinal, a specific inhibitor of elastase, was also effective. bPTI did not inhibit the degradation of proteins with short or long lives, suggesting that it did not increase the survival of hepatocytes by inhibiting cellular protein degradation. alpha 2-Plasmin inhibitor immobilized on Sepharose 4B caused dose-dependent increase in survival. Plasma membranes purified from adult rat liver had significant protease activity, about 80% of which was sensitive to bPTI, alpha 2-plasmin inhibitor and leupeptin. From its specificity for substrates and sensitivity to inhibitors, the membrane-bound protease was characterized as a trypsin-like protease. The effects of various inhibitors on the membrane-bound protease correlated well with their abilities to increase survival of rat hepatocytes. Therefore, it seems that bPTI acts on the cell surface and increases hepatocyte survival in serum-free cultures by inhibiting a trypsin-like protease associated with the plasma membranes.     

Resistance of cathepsin L compared to elastase to proteolysis when complexed with the serpin endopin 2C, and recovery of cathepsin L activity

This study demonstrates unique differences in the conformational nature of cathepsin L compared to elastase when complexed with the serpin endopin 2C, assessed by susceptibilities of protease/endopin 2C complexes to proteolysis by trypsin. Complexed and uncomplexed cathepsin L were resistant to degradation by trypsin, which indicated that trypsin cleavage sites within cathepsin L remain inaccessible when this cysteine protease is complexed with the endopin 2C serpin. In contrast, elastase in complexes with endopin 2C was degraded by trypsin, but uncomplexed elastase was not degraded. These results demonstrate a change in the conformational properties of trypsin cleavage sites within elastase when it is complexed with endopin 2C, compared to uncomplexed elastase. Cathepsin L complexes with endopin 2C were short-lived, but elastase complexes were stable. Furthermore, cathepsin L dissociated from complexes demonstrated recovery of cathepsin L activity, and reducing conditions provided optimum recovery of cathepsin L activity. These findings suggest that cathepsin L, when complexed with endopin 2C, maintains its general conformation in a manner that allows recovery of cathepsin L activity upon dissociation from endopin 2C. These results demonstrate differences in the relative conformational properties of the cysteine protease cathepsin L, compared to the serine protease elastase, in complexes with the serpin endopin 2C.     

Apical trypsin increases ion transport and resistance by a phospholipase C-dependent rise of Ca2+

We investigated the mechanisms by which serine proteases alter lung fluid clearance in rat lungs and vectorial ion transport in airway and alveolar epithelial cells. Inhibition of endogenous protease activity by intratracheal instillation of soybean trypsin inhibitor (SBTI) or alpha(1)-antitrypsin decreased amiloride-sensitive lung fluid clearance across rat fluid-filled lungs; instillation of trypsin partially restored this effect. Gelatin zymography demonstrated SBTI-inhibitable trypsin-like activity in rat lung lavage fluid. Apical trypsin and human neutrophil elastase, but not agonists of protease activated receptors, increased Na(+) and Cl(-) short-circuit currents (I(sc)) and transepithelial resistance (R(TE)) across human bronchial and nasal epithelial cells and rat alveolar type II cells, mounted in Ussing chambers, for at least 2 h. The increase in I(sc) was fully reversed by amiloride and glibenclamide. The increase in R(TE) was not prevented by ouabain, suggesting that trypsin decreased paracellular conductance. Apical trypsin also induced a transient increase in intracellular Ca(2+) in human airway cells; treatment of these cells with BAPTA-AM mitigated the trypsin-induced increases of intracellular Ca(2+) and of I(sc) and R(TE). Increasing intracellular Ca(2+) in airway cells with either ionomycin or thapsigargin reproduced the increase in I(sc), whereas inhibitors of phospholipase C (PLC) prevented the increases in both Ca(2+) and I(sc). These data indicate trypsin-like proteases and elastase, either present in lung cells or released by inflammatory cells into the alveolar space, play an important role in the clearance of alveolar fluid by increasing ion transport and paracellular resistance via a PLC-initiated rise of intracellular Ca(2+).     

A unique trypsin-like protease associated with plasma membranes of rat liver

One way in which serum promotes survival of primary cultured hepatocytes is by inhibiting plasma membrane protease (Nakamura, T., Asami, O., Tanaka, K., and Ichihara, A. (1984) Exp. Cell Res. 155, 81-91). One of these proteases was solubilized from the plasma membranes of rat liver with 4% octyl glucoside and purified to a homogeneous state by affinity chromatography on bovine pancreatic trypsin inhibitor linked to Sepharose 4B. The protease had an apparent Mr = 120,000 by Sephacryl S-200 gel filtration and the Mr of its subunits was 30,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. It appeared to be a glycoprotein. A high concentration of detergent was necessary to keep the protein soluble. The purified enzyme readily hydrolyzed synthetic tripeptide nitroanilides at sites adjacent to Arg or Lys residues, but did not degrade synthetic substrates of chymotrypsin, elastase, or aminopeptidase. It showed endopeptidase activity, hydrolyzing various proteins such as casein, hemoglobin, and denatured albumin. The enzyme was strongly inhibited by diisopropyl fluorophosphate, phenylmethanesulfonyl fluoride, bovine pancreatic trypsin inhibitor, leupeptin, antipain, and alpha 1-antitrypsin, but not by chymostatin, elastatinal, or inhibitors of carboxyl, thiol, or metallo proteases, suggesting that it is a seryl trypsin-like protease. This protease was found in plasma membranes of rat and mouse liver and in small amounts in those of kidney, but not in those of brain, red cells, Ehrlich ascites tumor, or two Morris hepatomas, suggesting that it may be involved in differentiated functions of normal hepatocytes.     

Evaluation of assays for detecting alpha-1-protease inhibitor during purification from rat serum

We purified the R1 alpha-1-protease inhibitor from rat serum and developed a convenient assay for its detection during purification procedures. Purification was accomplished by desalting, DEAE-Sephacel, zinc chelate, and reactive green-agarose columns. The resultant antiprotease had a molecular weight of 54,000 and inhibited elastase, chymotrypsin, and trypsin. By isoelectric focusing, five bands were produced with pI values from 4.3 to 4.7. Functional assays utilizing protease substrates imbedded in agarose plates were evaluated for the ability to distinguish the R1 alpha-1-protease inhibitor from the other serum antiproteases eluted in column chromatography fractions. This technique of screening for anti-protease activity was compared to conventional spectrophotometric methods and was found to correlate well when quantifying inhibition of elastase and chymotrypsin, but not trypsin. The presence of alpha-1-protease inhibitor was most reliably detected by testing for anti-elastase activity. Technician time and expense were saved by employing protease substrate plates to test chromatogrpahy fractions. This technique may facilitate purification of other protease inhibitors.     

Trypsin inhibitor paradoxically stabilizes trypsin activity in sodium dodecyl sulfate, facilitating proteolytic fingerprinting

Normally trypsin has negligible activity after being dissolved in sodium dodecyl sulfate (SDS), and so it has had little utility for proteolytic fingerprinting during gel electrophoresis. Here it is demonstrated that trypsin retained activity in SDS if it was first complexed to either of two soybean-derived protease inhibitors: trypsin inhibitor (Kunitz) or trypsin-chymotrypsin inhibitor (Bowman-Birk). The inhibitors alone did not cause proteolysis. Heating or acidification in SDS inactivated the inhibitor-dependent tryptic activity, as did prior treatment with tosyl lysine chloromethyl ketone, a covalent affinity reagent for trypsin. Quenching of samples with acid at intervals prior to gel electrophoresis revealed that proteolysis did not occur in sample buffer (pH 6.8), but only at higher pH and during gel electrophoresis. Exposure of trypsin to SDS prior to addition of trypsin inhibitor resulted in an irreversible loss of activity with a half-life of about 10 s. It is proposed that the trypsin inhibitors stabilize trypsin by retarding its denaturation in SDS. The substrate for these experiments was the alpha subunit of the Na,K-ATPase. The same pattern of Na,K-ATPase fragments was obtained with bovine and porcine trypsin and with rat and porcine Na,K-ATPases. Different fragments resulted when chymotrypsin or elastase were substituted for trypsin; these proteases were active in the absence of an inhibitor, and were not markedly stabilized by interaction with soybean trypsin-chymotrypsin inhibitor (Bowman-Birk).     

Structure and function of an isozyme of earthworm proteases as a new biocatalyst

The amino acid sequence of the earthworm-serine protease, isozyme C, which shows not only elastase-like activity but also trypsin-like activity, was determined. The catalytic triad of the trypsin family, His, Asp, Ser, was conserved in isozyme C, but the primary substrate determinant of trypsin, Asp, was missing in isozyme C, the same as in elastase. One of the two Gly at the entrance of the substrate-binding pocket of trypsin was replaced by Val as in elastase, however, the other was replaced by Ser whereas Thr is present in elastase. Furthermore, isozyme C also showed esterase-like activity, which was applicable for the synthesis of useful substances.     

Serine protease inhibitors of North American leeches

Serine protease inhibitors in extracts from three North American leeches, Nephelopsis obscura, Erpobdella punctata and Hemopis marmorata have been separated by anion exchange chromatography and the activity pattern against human granulocyte elastase and porcine chymotrypsin and trypsin determined. All three leech species contained a major peak with anti-trypsin activity, but Hemopis was unique in that the trypsin inhibitor was equally active against chymotrypsin. Nephelopsis was rich in anti-elastase activity of two types, one which was also active against chymotrypsin, and one which was a specific elastase inhibitor. Erpobdella contained inhibitors against elastase and chymotrypsin but with major activity against the latter.     

Development of a protease activity assay using heat-sensitive Tus-GFP fusion protein substrates

Proteases are implicated in various diseases and several have been identified as potential drug targets or biomarkers. As a result, protease activity assays that can be performed in high throughput are essential for the screening of inhibitors in drug discovery programs. Here we describe the development of a simple, general method for the characterization of protease activity and its use for inhibitor screening. GFP was genetically fused to a comparatively unstable Tus protein through an interdomain linker containing a specially designed protease site, which can be proteolyzed. When this Tus–GFP fusion protein substrate is proteolyzed it releases GFP, which remains in solution after a short heat denaturation and centrifugation step used to eliminate uncleaved Tus–GFP. Thus, the increase in GFP fluorescence is directly proportional to protease activity. We validated the protease activity assay with three different proteases, i.e., trypsin, caspase 3, and neutrophil elastase, and demonstrated that it can be used to determine protease activity and the effect of inhibitors with small sample volumes in just a few simple steps using a fluorescence plate reader.     

Characterisation of the α1-protease inhibitor system in Thoroughbred horse plasma by horizontal two-dimensional (ISO-DALT) electrophoresis. 2. Protease inhibition

The protease inhibitory spectra of the eight homozygous Thoroughbred Pi types against trypsin, elastase and chymotrypsin have been determined. The α1-protease inhibitor proteins exhibit three classes of inhibitory specificity towards these enzymes. The Pi types F, I, N and U exhibit class I (trypsin, elastase and chymotrypsin) and class II (trypsin and elastase) types of inhibition and fit Juneja et al.s (1979) classification of two separate genetic systems Pi 1 and Pi 2 based on differences in the inhibitory spectra against trypsin and chymotrypsin. The remaining four Pi types are exceptions to Juneja et al.s (1979) classification. Types G, L, S₁ and S₂ possess class I but not class II proteins. A third class of proteins (class III) which exclusively inhibit chymotrypsin was detected in all eight protease inhibitor types. Type G is well represented by class III proteins because two of the three major proteins of the ISO-DALT pattern inhibit only chymotrypsin and is thus an exception to Juneja et al.s (1979) classification.     

Tissue kallikrein activation of the epithelial Na channel

Epithelial Na Channels (ENaC) are responsible for the apical entry of Na(+) in a number of different epithelia including the renal connecting tubule and cortical collecting duct. Proteolytic cleavage of γ-ENaC by serine proteases, including trypsin, furin, elastase, and prostasin, has been shown to increase channel activity. Here, we investigate the ability of another serine protease, tissue kallikrein, to regulate ENaC. We show that excretion of tissue kallikrein, which is secreted into the lumen of the connecting tubule, is stimulated following 5 days of a high-K(+) or low-Na(+) diet in rats. Urinary proteins reconstituted in a low-Na buffer activated amiloride-sensitive currents (I(Na)) in ENaC-expressing oocytes, suggesting an endogenous urinary protease can activate ENaC. We next tested whether tissue kallikrein can directly cleave and activate ENaC. When rat ENaC-expressing oocytes were exposed to purified tissue kallikrein from rat urine (RTK), ENaC currents increased threefold in both the presence and absence of a soybean trypsin inhibitor (SBTI). RTK and trypsin both decreased the apparent molecular mass of cleaved cell-surface γ-ENaC, while immunodepleted RTK produced no shift in apparent molecular mass, demonstrating the specificity of the tissue kallikrein. A decreased effect of RTK on Xenopus ENaC, which has variations in the putative prostasin cleavage sites in γ-ENaC, suggests these sites are important in RTK activation of ENaC. Mutating the prostasin site in mouse γ-ENaC (γRKRK186QQQQ) abolished ENaC activation and cleavage by RTK while wild-type mouse ENaC was activated and cleaved similar to that of the rat. We conclude that tissue kallikrein can be a physiologically relevant regulator of ENaC activity.     

Purification and Characterization of Albumin from Frog Skin of <Emphasis Type="Italic">Duttaphrynus melanostictus</Emphasis>

Following determination of trypsin inhibitory activity, a serine protease inhibitor was purified and characterized from frog Duttaphrynus melanostictus serum. It was identified as serum albumin, with molecular weight of 67 kDa (DmA-serum). Different from bovine serum albumin, DmA-serum potently inhibited trypsin with similar K _i values around 1.6 × 10⁻⁷ M. No inhibitory effect on thrombin, chymotrypsin, elastase and subtilisin was observed under the assay conditions. The N-terminal amino acid is EAEPHSRI. Subsequently, a protein with same N-terminal amino acid was purified from skin, termed as DmA-skin. However, DmA-skin is distinct from DmA-serum by binding of a haem b (0.5 mol/mol protein), and with low trypsin inhibitory activity. Frog albumin is distributed in frog skin and exhibited trypsin inhibitory activity, suggesting that it plays important roles in skin physiological functions, like water economy, metabolite exchange and osmoregulation, etc.     

A survey of the effects of proteases and glycosidases on culture of rabbit morulae to blastocysts

The effects of a range of commercially available proteases and glycosidases on blastocyst development and hatching were examined on rabbit embryos cultured from the morula stage in a defined medium supplemented with charcoal-treated bovine serum albumin. The proteases tested were trypsin, alpha-chymotrypsin, thrombin, elastase, plasmin, papain, clostripain, collagenase, Streptomyces griseus protease and cathepsin C. The glycosidases tested were neuraminidase, alpha-mannosidase, beta-galactosidase and hyaluronidase. None of these enzymes appeared to stimulate blastocyst growth. The only enzymes which digested the embryonic investments, the zona and mucin coat, sufficiently to cause complete blastocyst hatching were trypsin and Streptomyces griseus protease at relatively low concentrations (250 ng/ml) and chymotrypsin and elastase at higher concentrations.     

Purification and some properties of a neutral protease from human leukocyte granules and its comparison with pancreatic elastase.

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.     

Self‐sterility of eggs induced by exogenous and endogenous protease in the solitary ascidian,Halocynthia roretzi

The unfertilized eggs (UFE) of the solitary ascidian, Halocynthia roretzi, which are released naturally, are strictly self‐sterile. However, ovarian eggs isolated after spawning, which are expected to develop into UFE on the following day, are self‐fertile. Some exogenous proteases‐trypsin, chymotrypsin, papain and elastase‐induced self‐sterility in the self‐fertile ovarian eggs within an hour in vitro. The establishment of self‐sterility by the exogenous protease did not require the synthesis of new protein, or the participation of follicle cells. Some of the ovarian eggs were able to differentiate into self‐sterile eggs spontaneously in vitro. The protein synthesis inhibitors puromycin and cycloheximide had no effect on the spontaneous establishment of self‐sterility. However, several protease inhibitors such as leupeptin, soybean trypsin inhibitor (SBTI) and antipain, did inhibit the spontaneous establishment of self‐sterility. The possible participation of trypsin‐like protease in the establishment of self‐sterility in the ovary is discussed. Mol. Reprod. Dev. 52:99–106, 1999. © 1999 Wiley‐Liss, Inc.     

Ovalbumin is an elastase substrate

Ovalbumin is partially homologous in sequence with the proteinase inhibitors alpha 1-proteinase inhibitor and anti-thrombin III. The region of sequence in ovalbumin which corresponds to the reactive sites of these proteinase inhibitors is susceptible to attack by subtilisin, elastase, thermolysin, bromelain, and Bacillus cereus protease. The esterase activity of elastase is not inhibited by ovalbumin, but ovalbumin is efficiently cleaved by elastase. In contrast with these proteases, trypsin does not cleave ovalbumin.     

Effects of protease inhibitors and dietary protein level on the black field cricket Teleogryllus commodus

Growth and survival responses were determined for black field crickets Teleogryllus commodus (Walker) (Orthoptera: Gryllidae) on artificial diets containing a range of levels of dietary protein, and protease inhibitors (PI's) at 0.33% (weight volume, w:v). The effect on cricket gut enzyme activities of adding PI's to a high protein diet was measured. All PI's which had in vitro binding activity against either trypsin or elastase (the two major cricket gut endopeptidases) reduced growth, but those which bound to both enzymes had the greatest effect. None of the PI's acted as a source of nutritional protein. Cricket growth rate increased with the addition of casein up to 3% w:v, but not with a similar addition of wheatgerm. The impact of PI's on growth was greatest on a 1.5% casein diet. On a high protein (3% casein) diet, the gut activity of trypsin was increased by potato proteinase inhibitors 1 and 2 while the activity of elastase and leucine amino peptidase were increased by soybean trypsin inhibitor and potato proteinase inhibitor 2. Increasing dietary casein up to 3.3% improved cricket survival. The potential of PI's as plant resistance factors against crickets was confirmed.     

Neutrophil elastase activates near-silent epithelial Na+ channels and increases airway epithelial Na+ transport

Neutrophil elastase is a serine protease that is abundant in the airways of individuals with cystic fibrosis (CF), a genetic disease manifested by excessive airway Na(+) absorption and consequent depletion of the airway surface liquid layer. Although endogenous epithelium-derived serine proteases regulate epithelial Na(+) transport, the effects of neutrophil elastase on epithelial Na(+) transport and epithelial Na(+) channel (ENaC) activity are unknown. Low micromolar concentrations of human neutrophil elastase (hNE) applied to the apical surface of a human bronchial cell line (16HBE14o-/beta gamma) increased Na(+) transport about twofold. Similar effects were observed with trypsin, also a serine protease. Proteolytic inhibitors of hNE or trypsin selectively abolished the enzyme-induced increase of epithelial Na(+) transport. At the level of the single channel, submicromolar concentrations of hNE increased activity of near-silent ENaC approximately 108-fold in patches from NIH-3T3 cells expressing rat alpha-, beta-, and gamma-ENaC subunits. However, no enzyme effects were observed on basally active ENaCs. Trypsin exposure following hNE revealed no additional increase in amiloride-sensitive short-circuit current or in ENaC activity, suggesting these enzymes share a common mode of action for increasing Na(+) transport, likely through proteolytic activation of ENaC. The hNE-induced increase of near-silent ENaC activity in CF airways could contribute to Na(+) hyperabsorption, reduced airway surface liquid height, and dehydrated mucus culminating in inefficient mucociliary clearance.     

Production of specific monoclonal antibodies against the active sites of human pancreatic secretory trypsin inhibitor variants by in vitro immunization with synthetic peptides

Specific monoclonal antibodies against the active sites of two genetically engineered pancreatic secretory trypsin inhibitor (PSTI) variants (PSTI 0 and PSTI 4) were produced. The protease inhibitors PSTI 0 and PSTI 4 differ only by three amino acid substitution at their active sites. PSTI 0 inhibits trypsin, whereas PSTI 4 inhibits human granulocyte elastase and chymotrypsin. Immunization was performed in vitro with a synthetic heptapeptide that covers the mutated region of the protein. For this purpose in vitro culture conditions for the production of specific monoclonal antibodies against synthetic peptides were improved. The monoclonal antibodies obtained react specifically with the corresponding protease inhibitor variant. Competition experiments with trypsin and human elastase demonstrate that the protease displace the monoclonal antibody from the active site of PSTI 0 and PSTI 4 respectively.