Trypsin action on the growth of Sendai virus in tissue culture cells. V. An activating enzyme for Sendai virus in the chorioallantoic fluid of the embryonated chicken egg

A trypsin-like protease which is responsible for activation of Sendai virus was found in the chorioallantoic fluid (CAF) of embryonated chicken eggs. Treatment of the inactive form of Sendai virus, grown in LLC-MK2 cells, with CAF enhanced both hemolytic activity and infectivity for the cells. Soybean trypsin inhibitor restrained the enhancing activity of CAF. These results indicate that CAF contains a trypsin-like protease which activates the inactive form of Sendai virus. The activation was strongly inhibited by phenylmethylsulfonylfluoride, ethylenediaminetetraacetate, antipain, and leupeptin but not by tosyllysylchloromethylketone, suggesting that the activating enzyme in CAF is a protease similar to but not identical with trypsin. The inactive form of the virion was produced in ovo when the seed virus was inoculated along with antipain or leupeptin. In deembryonated chicken eggs in which CAF was substituted for a culture medium, multiple cycle growth occurred, but not when soybean trypsin inhibitor was present. These observations indicate that some activating enzyme, possibly the same one as found in CAF, was secreted from the chorioallantoic membrane.     

Prophenoloxidase activation in the hemolymph of Sarcophaga bullata larvae

Phenoloxidase in the hemolymph of Sarcophaga bullata larvae is present as an inactive proenzyme form. Localization studies indicate that the majority of the prophenoloxidase is present in the plasma fraction whereas only a minor fraction (about 4%) is present in the cellular compartments (hemocytes). Inactive prophenoloxidase can be activated by zymosan, not by either endotoxin or laminarin. This activation process is inhibited by the serine protease inhibitors, benzamidine and p-nitrophenyl-p～-guanidobenzoate. Exogenously added proteases, such as chymotrypsin and subtilisin, also activated the prophenoloxidase in the whole hemolymph but failed to activate the partially purified proenzyme. However, an activating enzyme isolated from the larval cuticle, which exhibits trypsinlike specificity, activated the partially purified prophenoloxidase. Inhibition studies and activity measurements also revealed the presence of a similar activating enzyme in the hemolymph. Thus, the phenoloxidase system in Sarcophaga bullata larval hemolymph seems to be comprised of a cascade of reactions. An endogenous protease inhibitor isolated from the larvae inhibited chymotrypsin-mediated prophenoloxidase activation but failed to inhibit the cuticular activating enzyme-catalyzed activation. Based on these studies, the roles of prophenoloxidase, endogenous activating proteases, and protease inhibitor in insect immunity are discussed.     

Mechanism of activation of inactive renin in human plasma by puff adder venom

Venom of the puff adder (Bitis arietans) contains a potent, basic, Mr 24,000 metalloproteinase activity that can destroy all detectable trypsin and chymotrypsin inhibitory activity, when venom is incubated with human plasma. We have found that during such incubation, concomitant activation of inactive renin occurs. In an examination of the mechanism involved we now report the activation, in addition, of plasma prekallikrein and serine proteinase activity, but not plasminogen, when human plasma is incubated with venom. Furthermore, venom was not able to release active trypsin from its complex with alpha 1-proteinase inhibitor and human renin was not inhibited by alpha 1-proteinase inhibitor. The activities in venom and venom/plasma mixtures were analysed using Sephacryl S-200 gel filtration and the effect of 10 mM EDTA and 5 mM phenylmethanesulphonyl fluoride on activities in column fractions was tested. The inactive-renin-activating, plasma prekallikrein-activating and serine proteinase-activating activities could be accounted for to a large extent by a venom metalloproteinase which was estimated to have a Mr of 24,000 by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. This enzyme activity appeared to complex with alpha 2-macroglobulin when venom was mixed with plasma. Since both EDTA and phenylmethanesulphonyl fluoride could inhibit the activation of inactive renin by this metalloproteinase, it is suggested that the enzyme activates serine proteinase(s), which then activate inactive renin. Plasma kallikrein may have a role in this process. Additional peaks of inactive-renin-activating activity eluted from Sephacryl S-200 at Mr 30,000 and 80,000 (minor) and an additional, minor peak of caseinolytic activity eluted at Mr 60,000. The Mr 24,000 metalloproteinase in venom may have considerable utility in activating inactive renin at physiological pH owing to its ability to destroy plasma proteinase inhibitors at the same time.     

Mouse submaxillary renin has a protease activity and converts human plasma inactive prorenin to an active form

The activation of inactive prorenin by active renin was investigated. Inactive prorenin extensively purified from human plasma was activated by active renin which had been purified from mouse submaxillary glands by multiple chromatographic steps. The apparent lack of protease activity in renin was puzzling in view of the close similarity of its active site structure with that of acid proteases. After a series of affinity chromatographic steps designed to eliminate minute contaminants, renin was found to contain a very low but finite level of a neutral protease activity which was equivalent to 1/40,000 of that of cathepsin D tested by hemoglobinolytic activity. The protease activity was considered as intrinsic to renin since it co-purified with renin persistently at a constant ratio to the renin activity, was precipitated by a monoclonal antibody specific for renin, showed a neutral pH optimum of the enzyme activity in the same pH range as that of renin, and was inhibited by pepstatin. The neutral protease activity is likely to mediate the activation of inactive prorenin.     

Profiling the enzymatic properties and inhibition of human complement factor B

Human complement factor B is the crucial catalytic component of the C3 convertase enzyme that activates the alternative pathway of complement-mediated immunity. Although a serine protease in its own right, factor B circulates in human serum as an inactive zymogen and there is a crystal structure only for the inactive state of factor B and various fragments. To provide greater insight to the catalytic function and properties of factor B, we have used short para-nitroanilide derivatives of 4- to 15-residue peptides as substrates to profile the catalytic properties of factor B. Among factors found to influence catalytic activity of factor B was an unusual dependence on pH. Non-physiological alkaline conditions strongly promoted substrate cleavage by factor B, consistent with a pH-accessible conformation of the enzyme that may be critical for catalytic function. Small N-terminal extensions to conventional hexapeptide para-nitroanilide substrates significantly increased catalytic activity of factor B, which was more selective for its cleavage site than trypsin. The new chromogenic assay enabled optimization of catalysis conditions, the profiling of different substrate sequences, and the development of the first reversible and competitive substrate-based inhibitor of factor B. The inhibitor was also shown to prevent in vitro formation of C3a from C3 by factor B, by synthetic and by natural C3 convertase of the alternative complement activation pathway, and to block formation of membrane attack complex. The availability of a reversible substrate-based inhibitor that could stabilize the active conformation of factor B, in conjunction with a pH-promoted higher processing activity, may offer a new avenue to obtain crystal structures of factor B and C3 convertase in an active conformation.     

Binding of latent rheumatoid synovial collagenase to collagen fibrils.

Collagenase released from rheumatoid synovial cells in culture is in a latent form. Subsequently, it may be activated by limited proteolysis. This study was designed to determine whether latent enzyme could bind to collagen fibrils and await activation. The data showed that latent collagenase bound to fibrils equally well at 24 degrees C and 37 degrees C, but that this represented little more than half the binding achieved by active enzyme at temperatures lower than that at which fibrils can be degraded. Binding was not inhibited by the presence of alpha2 macroglobulin, the principal proteinase inhibitor of plasma which cannot complex with inactive or latent collagenase but readily complexes with active species of enzyme. The data support the hypotheses that inactive forms of collagenase accumulate in tissues by binding to substrate, and that activation by proteases such as plasmin initiates collagen breakdown.     

Nonproteolytic "activation" of prorenin by active site-directed renin inhibitors as demonstrated by renin-specific monoclonal antibody.

Incubation of human plasma prorenin (PR), the enzymatically inactive precursor of renin (EC 3.4.23.15), with a number of nonpeptide high-affinity active site-directed renin inhibitors induces a conformational change in PR, which was detected by a monoclonal antibody that reacts with active renin but not with native inactive PR. This conformational change also occurred when inactive PR was activated during exposure to low pH. Nonproteolytically acid-activated PR, and inhibitor-"activated" PR, as well as native PR, were retained on a blue Sepharose column, in contrast to proteolytically activated PR. Kinetic analysis of the activation of plasma prorenin by renin inhibitor (INH) indicated that native plasma contains an open intermediary form of prorenin, PRoi, in which the active site is exposed and which is in rapid equilibrium with the inactive closed form, PRc. PRoi reacts with inhibitor to form a reversible complex, PRoi.INH, which undergoes a conformational change resulting in a tight complex of a modified open form of prorenin, PRo, and the inhibitor, PRoi.INH-->PRo.INH. The PRoi-to-PRo conversion leads to the expression of an epitope on the renin part of the molecule that is recognized by a renin-specific monoclonal antibody. Presumably, PRo corresponds to the enzymatically active form of PR that is formed during exposure to low pH. Thus, it seems that the propeptide of PR interacts with the renin part of the molecule not only at or near the enzyme's active site but also at some distance from the active site. Interference with the first interaction by renin inhibitor leads to destabilization of the propeptide, by which the second interaction is disrupted and the enzyme assumes its active conformation. The results of this study may provide a model for substrate-mediated prorenin activation and increase the likelihood that enzymatically active prorenin is formed in vivo.     

Partial purification and characterization of a gelatin-specific protease from the culture media of human pulmonary alveolar macrophages

A gelatin-specific protease from the culture media of human pulmonary alveolar macrophages has been partial purified by gel filtration and characterized. The macrophages were obtained by bronchopulmonary lavage from the lungs of disease-free smoking volunteers. The gelatin-specific protease initially requires trypsin activation. After chromatographing the culture media on a Sephadex G-200 column, trypsin is no longer required for activation. The gelatin-specific protease reported here shares many properties of previously reported gelatinases. It is inhibited by EDTA, cysteine, dithiothreitol and serum. It is unaffected by other protease inhibitors: phenylmethylsulfonyl fluoride, tosyllysine chloromethyl ketone and p-chloromercuribenzoate. Of all substrates tested activity was observed only with gelatin. It was inactive toward collagen, elastin and methemoglobin. This enzyme may have a role in the digestion of collagen which has been cleaved by a mammalian collagenase.     

Intracellular proteolysis of pancreatic zymogens.

Activation of pancreatic digestive zymogens within the pancreatic acinar cell may be an early event in the development of pancreatitis. To detect such activation, an immunoblot assay has been developed that measures the relative amounts of inactive zymogens and their respective active enzyme forms. Using this assay, high doses of cholecystokinin or carbachol were found to stimulate the intracellular conversion of at least three zymogens (procarboxypeptidase A1, procarboxypeptidase B, and chymotrypsinogen 2) to their active forms. Thus, this conversion may be a generalized phenomenon of pancreatic zymogens. The conversion is detected within ten minutes of treatment and is not associated with changes in acinar cell morphology; it has been predicted that the lysosomal thiol protease, cathepsin B, may initiate this conversion. Small amounts of cathepsin B are found in the secretory pathway, and cathepsin B can activate trypsinogen in vitro; however, exposure of acini to a thiol protease inhibitor (E64) did not block this conversion. Conversion was inhibited by the serine protease inhibitor, benzamidine, and by raising the intracellular pH, using chloroquine or monensin. This limited proteolytic conversion appears to require a low pH compartment and a serine protease activity. After long periods of treatment (60 minutes), the amounts of the active enzyme forms began to decrease; this observation suggested that the active enzyme forms were being degraded. Treatment of acini with E64 reduced this late decrease in active enzyme forms, suggesting that thiol proteases, including lysosomal hydrolases, may be involved in the degradation of the active enzyme forms. These findings indicate that pathways for zymogen activation as well as degradation of active enzyme forms are present within the pancreatic acinar cell.     

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.     

Binding of latent rheumatoid synovial collagenase to collagen fibrils

Collagenase released from rheumatoid synivial cells in culture is in a latent form. Subsequently, it may be activated by limited proteolysis. This study was designed to determine whether latent enzyme could bind to collagen fibrils and await activation. The data showed that latent collagenase bound to fibrils equally well at 24°C and 37°C, but that this represented little more than half the binding achieved by active enzyme at temperatures lower than that at which fibril can be degraded. Binding was not inhibited by the presence of α₂ macroglobulin, the principal proteinase inhibitor of plasma which cannot complex with inactive or latent collagenase but readily complexes with active species of enzyme. The data support the hypotheses that inactive forms of collagenase accumulate in tissues by binding the substrate, and that activation by proteases such as plasmin intiates collagen breakdown.     

Isolation of a protease from sea urchin eggs before and after fertilization.

Upon fertilization, sea urchin eggs (Stronglyocentrotus pupuratus) release a protease into the surrounding sea water. This protease is in a particulate form which can be solubilized. The soluble form was purified by affinity chromatography on columns of immobilized soybean trypsin inhibitor. The purified enzyme is similar to bovine trypsin both in molecular weight (22500) and in susceptibility to inhibitors such as diisopropyl phosphofluoridate and soybean trypsin inhibitor. In contrast, extracts of unfertilized eggs appear to contain an inactive form of the enzyme which can be activated by dialysis at pH 4.6. The enzyme, as purified from extracts activated in this manner, was similar in its properties to that from fertilized eggs.     

Partial purification and characterization of a neutral protease which cleaves type IV collagen

A neutral protease has been extracted from the media of cultured metastatic tumor cells and purified approximately 1000 times after sequential ammonium sulfate fractionization, concanavalin A column chromatography, and molecular sieve chromatography. The protease has an apparent molecular weight of 70 000--80 000, is inactive at acid pH, requires trypsin activation, and is inhibited by ethylene-diaminetetraacetic acid but not by phenylmethanesulfonyl fluoride, N-ethylmaleimide, or soybean trypsin inhibitor. The enzyme produces specific cleavage products for both chains of pro type IV collagen isolated without pepsinization and apparently cleaves at one point in a major pepsin-extracted chain of placenta type IV collagen. The partially purified enzyme fails to significantly degrade other collagens or fibronectin under digestion conditions in which specific reaction products are produced for type IV collagen. The existence of this enzyme is significant since previously described animal collagenases fail to degrade type IV collagen. Such a type IV specific collagenase could play a role in tumor invasion and may be secreted by other cells such as endothelial cells, epithelial cells, and immune cells.     

Collagenase production by human skin fibroblasts.

Normal human skin fibroblasts, when cultured in serum free medium, produce collagenase in an inactive form. The enzyme in the crude medium can be activated by a brief preincubation with trypsin or by autoactivation. Once activated, the fibroblast collagenase is identical in its mechanism of action to human skin collagenase obtained from organ cultures. In addition, an inhibitor of collagenase is also present in the medium of fibroblast cultures. The inhibitor appears to be produced by the cells and its molecular weight is slightly higher than that of the enzyme. The presence of this inhibitor may account for previous inability to detect collagenase in human skin fibroblast cultures. It is also possible that some of the inactive enzyme exists in the medium in the form of a proenzyme.     

The activation of matriptase requires its noncatalytic domains,serine protease domain,and its cognate inhibitor

The activation of matriptase requires proteolytic cleavage at a canonical activation motif that converts the enzyme from a one-chain zymogen to an active, two-chain protease. In this study, matriptase bearing a mutation in its catalytic triad was unable to undergo this activational cleavage, suggesting that the activating cleavage occurs via a transactivation mechanism where interaction between matriptase zymogen molecules leads to activation of the protease. Using additional point and deletion mutants, we showed that activation of matriptase requires proteolytic processing at Gly-149 in the SEA domain of the protease, glycosylation of the first CUB domain and the serine protease domain, and intact low density lipoprotein receptor class A domains. Its cognate inhibitor, hepatocyte growth factor activator inhibitor-1, may also participate in the activation of matriptase, based on the observation that matriptase activation did not occur when the protease was co-expressed with hepatocyte growth factor activator inhibitor-1 mutated in its low density lipoprotein receptor class A domain. These results suggest that besides matriptase catalytic activity, matriptase activation requires post-translational modification of the protease, intact noncatalytic domains, and its cognate inhibitor.     

Testing for diffusion limitations in salt-activated enzyme catalysts operating in organic solvents

The dramatic activation of serine proteases in nonaqueous media resulting from lyophilization in the presence of KCl is shown to be unrelated to relaxation of potential substrate diffusional limitations. Specifically, lyophilizing subtilisin Carlsberg in the presence of KCl and phosphate buffer in different proportions, ranging from 99% (w/w) enzyme to 1% (w/w) enzyme in the final lyophilized solids, resulted in biocatalyst preparations that were not influenced by substrate diffusion. This result was made evident through use of a classical analysis whereby initial catalytic rates, normalized per weight of total enzyme in the catalyst material, were measured as a function of active enzyme for biocatalyst preparations containing different ratios of active to inactive enzyme. The active enzyme content of a given biocatalyst preparation was controlled by mixing native subtilisin with subtilisin preinactivated with PMSF, a serine protease inhibitor, and lyophilizing the enzyme mixture in the presence of different fractions of KCl and phosphate buffer. Plots of initial reaction rates as a function of percent active subtilisin in the biocatalyst were linear for all biocatalyst preparations. Thus, enzyme activation (reported elsewhere to be as high as 3750-fold in hexane for the transesterification of N-Ac-L-Phe-OEt with n-PrOH) is a manifestation of intrinsic enzyme activation and not relaxation of diffusional limitations resulting from diluted enzyme preparations. Similar activation is reported herein for thermolysin, a nonserine protease, thereby demonstrating that enzyme activation due to lyophilization in the presence of KCl may be a general phenomenon for proteolytic enzymes.     

Isolation and characterization of an inter-alpha-trypsin inhibitor-IgG complex from human serum

Inter-alpha-trypsin inhibitor is a human serum protease inhibitor of Mr 180 000 which may release physiological derivatives. A complex between IgG and an inter-alpha-trypsin inhibitor derivative of Mr 30 000 has been recently detected in human serum and was found to be inactive against trypsin, in contrast with the known inhibitory activity of the free 30-kDa derivative. The present study deals with detailed characterization of an inter-alpha-trypsin inhibitor-IgG complex following its purification by affinity chromatography techniques (anti-inter-alpha-trypsin inhibitor immunoadsorbent and Protein A-Sepharose) in mild conditions. The resulting product reacted simultaneously with anti-IgG and anti-inter-alpha-trypsin inhibitor antibodies. This complex contained Mr 180 000 inhibitor at least to some extent. It migrated in the beta-gamma zone in agarose; its molecular weight was estimated to be 1 500 000 or more; part of it displayed covalent bonding between inter-alpha-trypsin inhibitor and IgG; it had a trypsin inhibitor activity. Immunoelectrophoresis allowed one to demonstrate the native complex in serum owing to the use of anti-inter-alpha-trypsin inhibitor and anti-gamma radioactively labelled antibodies. The double immunoreactivity thus evidenced proved to be heterogeneous with respect to its level and location in the native as well as in the purified complex.     

Characterization and purification of a protease in serum that cleaves proatrial natriuretic factor (ProANF) to its circulating forms

Atrial natriuretic factor (ANF) is synthesized and stored in atrial cardiocytes as a 17-kilodalton (kDa), 126 amino acid polypeptide, proANF, but circulates as smaller, 24 and 28 amino acid peptide fragments of the carboxy terminus of proANF. It has previously been shown that proANF is secreted intact from cultured atrial cardiocytes and can be cleaved by a serum protease to smaller, 3-kDa peptides believed to be the circulating forms. This report describes the purification and characterization of this proANF-cleaving protease from rat serum. The cleavages both of 35S-labeled proANF derived from rat atrial cell cultures, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/autoradiography, and of a synthetic p-nitroanilide-containing substrate were used as assays for the detection of enzyme activity. ProANF-cleaving activity was found in rat serum, with no such activity detectable in rat plasma. Cleavage in serum was not dependent on the presence of platelets or other cellular elements. Complete inhibition of proANF cleavage was obtained with the protease inhibitors benzamidine, leupeptin, phenylmethanesulfonyl fluoride, and diisopropyl fluorophosphate (DFP) but not with aprotinin, soybean trypsin inhibitor, pepstatin, or hirudin. Unlike the vitamin K dependent plasma proteins, the proANF-cleaving protease did not adsorb to barium sulfate. With the sequential application of ion-exchange, hydroxylapatite, lectin affinity, and gel filtration chromatography, a 5000-6000-fold purification of the enzyme from rat serum was achieved. Fractionation of either whole serum or the purified enzyme by gel filtration chromatography revealed a single peak of activity corresponding to a protein with a Stokes radius of 45 A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of urinary inactive kallikrein by an extract from the rat kidney cortex

Activation of purified urinary inactive kallikrein by an extract from the rat kidney cortex was investigated. The extract produced a dose-dependent activation of the inactive kallikrein and the optimum pH for this activation was 5.0. Marked depression of the activation was observed when the extract was pre-incubated with E-64, p-CMB and iodoacetate, but not with DFP, PMSF or pepstatin A. The molecular weight of the inactive kallikrein (Mr 44,000) was reduced to 38,000 by treatment with the extract, this molecular weight value being identical with that of urinary active kallikrein. These results indicate that the rat kidney cortex contains a protease catalyzing conversion of urinary inactive kallikrein into its active form, and that the protease has properties compatible with those of a thiol protease, but not of trypsin which has been used as a tool for the activation of urinary inactive kallikrein. The thiol protease is probably one of regulators of the kallikrein-kinin system in the kidney.