Serine Protease Inhibitor Diisopropylfluorophosphate Research Articles

Detection and characterization of endogenous protease associated with desquamation of stratum corneum.

In order to identify the endogenous protease associated with stratum corneum (SC) desquamation, we examined properties of proteases in the stratum corneum of normal human skin. SC were obtained by tape stripping, washed in toluene and then dried. The proteolytic activity in SC was measured using peptidyl 4-methyl-coumaryl-7-amides (MCAs). The SC was dispersed uniformly in the reaction mixture with dimethylformamide and Triton X-100 and incubated with the peptidyl MCAs. The protease in the SC hydrolysed both Boc-Phe-Ser-Arg-MCA and Boc-Gln-Ala-Arg-MCA (substrates for trypsin) very effectively. The hydrolytic activity was inhibited by the serine protease inhibitors diisopropyl fluorophosphate (DFP), aprotinin, antipain and leupeptin, but not by chymostatin, a chymotrypsin inhibitor. These results show that one or more trypsin-like serine protease is present in the SC of normal human skin. Casein-acrylamide electrophoresis showed that the molecular weight of this serine protease was about 30 kDa. We have previously shown that cells dissociate from human SC sheets in a detergent mixture (N,N-dimethyldodecylamine oxide and sodium lauryl sulphate). This cell dissociation was inhibited by aprotinin and leupeptin. In addition, the proteolytic activity in the outer SC was higher than that in the inner SC, and the activity in the SC of scaly skin induced by SLS treatment was higher than that of untreated skin. These results strongly suggest that the trypsin-like serine protease described here is involved in SC desquamation.

Inactivation of interleukin‐6 in vitro by monoblastic U937 cell plasma membranes involves both protease and peptidyl‐transferase activities

Human promonocytic U937 cells have previously been shown to possess at their cell surface specific transmembrane serine proteases and N-terminal amino acid proteases as well as associated enzymes including elastase and cathepsin G. In this study, purified plasma membranes from U937 cells are reported to degrade the recombinant 21-kDa 125I-interleukin-6 (125I-IL-6) into 8-kDa products with loss of biological activity, as monitored by polyacrylamide gel electrophoresis and a cell-proliferation bioassay. Degradation of 125I-IL-6 by plasma membranes was completely prevented by the serine-protease inhibitor diisopropyl fluorophosphate, but was only partially impaired by alpha 1-protease inhibitor and antibody against cathepsin G. A similar incubation of 125I-IL-6 with cathepsin G purified from U937 cells caused hydrolysis of the cytokine into similar inactive 8-kDa fragments, whereas incubation with purified U937 cell elastase failed to degrade the peptide. These findings indicate that U937 cells hydrolyze IL-6 using cell-associated serine-protease activity and that cathepsin G partially participates in this degradation. Prolonged incubation of 8-kDa 125I-IL-6 fragments with purified U937 plasma membranes, led to a complete loss of IL-6 activity related to the transformation of the 8-kDa forms into a higher-molecular-mass complex (16 kDa). This complex was stable in SDS and 2-mercaptoethanol at 100 degrees C and was not dissociated by hydroxylamine treatment, indicating the formation of a covalent non-ester bond between the 8-kDa 125I-IL-6-derived peptide and an undetermined acceptor. An initial oxidative treatment of 125I-IL-6 partially prevented complex formation, suggesting the presence of one or more oxidizable methionine residues at the binding site of 8-kDa 125I-IL-6 peptide. The kinetics of complex formation (time dependence and plasma-membrane-concentration dependence), as well as its inhibition by a specific inhibitor of N-amino-peptidase activity, bestatin, suggest the participation of peptidyl-transferase activity in complex formation. Finally, a plasma-membrane fraction, corresponding to a molecular mass > or = 30 kDa, was able to convert the 8-kDa 125I-IL-6 forms into the 125I-labeled 16-kDa complex, suggesting that a > or = 30-kDa peptidyl-transferase enzyme catalyzes the reaction and provides the 125I-labeled 16-kDa peptide by dimerization of 8-kDa 125I-IL-6-derived intermediates. Further identification of the plasma-membrane-associated peptidyl transferase as a regulator of IL-6 proteolysis may be of physiological relevance for the control of IL-6 biological activity.

Photodamage to photosystem II - primary and secondary events

High light stress results in a reduction in the photosynthetic capacity of plants. This photoinhibition is targeted to photosystem II and seems to be an inevitable consequence of the complicated redox chemistry involved in the light-driven water-plastoquinone oxidoreduction reaction. Photoinactivation leads to irreversible damage of the reaction centre of photosystem II, in particular the D1 protein. In this paper, we present evidence which indicates that the inhibition of electron transport can be initiated at both the acceptor and donor sides of photosystem II. In the former case, there is a sequential array of events leading to overreduction of the primary quinone acceptor Q A. This, in turn, leads to the formation of a P 680 chlorophyll triplet. The donor-side inhibition is thought to involve the accumulation of the highly oxidizing species P + 680 and Tyr + z. These species will lead to oxidative damage of the D1 protein, triggering it for degradation. During acceptor-side inhibition, the protein is damaged by singlet oxygen formed via a reaction between P 680 triplet and oxygen. Damaged D1 protein is degraded by a serine protease located in the photosystem II complex. Preliminary inhibition and binding studies using the classical serine protease inhibitor diisopropylfluorophosphate suggest that the active serine is located on a 43 kDa polypeptide. Several D1 protein digestion fragments have been identified and a cleavage site between the stromally exposed loop connecting transmembrane helices IV and V is suggested.

On the molecular mechanism of light-induced D1 protein degradation in photosystem II core particles.

The mechanism of D1 protein degradation was investigated during photoinhibitory illumination of isolated photosystem II core preparations. The studies revealed that a proteolytic activity resides within the photosystem II core complex. A relationship between the inhibition of D1 protein degradation and the binding of the highly specific serine protease inhibitor diisopropyl fluorophosphate to isolated complexes of photosystem II was observed, evidence that this protease is of the serine type. Using radiolabeled inhibitor, it was shown that the binding site, representing the active serine of the catalytic site, is located on a 43-kDa polypeptide, probably the chlorophyll a protein CP43. The protease is apparently active in darkness, with the initiation of breakdown being dependent on high light-induced substrate activation. The proteolysis, which has an optimum at pH 7.5, gives rise to primary degradation fragments of 23 and 16 kDa. In addition, D1 protein fragments of 14, 13, and 10 kDa were identified. Experiments with phosphate-labeled D1 protein and sequence-specific antisera showed that the 23- and 16-kDa fragments originate from the N- and C-termini, respectively, suggesting a primary cleavage of the D1 protein at the outer thylakoid surface in the region between transmembrane helices D and E.

Characterization of an endopeptidase of Trypanosoma brucei brucei

A soluble 80-kDa endopeptidase has been isolated from Trypanosoma brucei brucei. The enzyme, which has a p I 5.1, is optimally active at about pH 8.2 and has apparent p K a values of 6.0 and ⩾10. It is inhibited by the serine protease inhibitor diisopropylfluorophosphate and by the serine protease mechanism-based inhibitor 3,4-dichloroisocoumarin. Unexpectedly, the enzyme is inhibited by the cysteine protease inhibitor benzyloxycarbonyl-LeuLysCHN 2 but not by the related diazomethane, butoxycarbonyl-ValLeuGlyLysCHN 2, nor by other cysteine protease specific compounds. Specificity studies with a variety of amidomethylcoumaryl (AMC) derivatives of small peptides show that the enzyme has a highly restricted trypsin-like specificity. The best substrate, based on the magnitude of k cat K m , was benzyloxycarbonyl-ArgArgAMC; other good substrates were benzyloxycarbonyl-PheArgAMC, benzoyl-ArgAMC, and compounds with Arg at P 1 and Ala or Gly at P 2. The hydrolysis of most substrates obeyed classical Michaelis—Menton kinetics but several exhibited pronounced substrate inhibition. The enzyme did not activate plasminogen nor decrease blood clotting time; it was inhibited by aprotinin but not by chicken ovomucoid. We conclude that the enzyme is a trypsin-like serine endopeptidase with unusually restricted subsite specificities.

Purification and characterization of a developmentally regulated carboxypeptidase from Mucor racemosus

A developmentally regulated carboxypeptidase was purified from hyphae of the dimorphic fungus Mucor racemosus. The enzyme, designated carboxypeptidase 3 (CP3), has been purified greater than 900-fold to homogeneity and characterized. The carboxypeptidase migrated as a single electrophoretic band in isoelectric focusing polyacrylamide gel electrophoresis (PAGE), with an isoelectric point of pH 4.4. The apparent molecular mass of the native enzyme was estimated by gel filtration to be 52 kDa. Sodium dodecyl sulfate (SDS)-PAGE under nonreducing conditions revealed the presence of a single polypeptide of 51 kDa. SDS-PAGE of CP3 reacted with 2-mercaptoethanol revealed the presence of two polypeptides of 31 and 18 kDa, indicating a dimer structure (alpha 1 beta 1) of the enzyme with disulfide-linked subunits. By using [1,3-3H]diisopropylfluorophosphate as an active-site labeling reagent, it was determined that the catalytic site resides on the small subunit of the carboxypeptidase. With N-carboben zoxy-L-phenylalanyl-L-leucine (N-CBZ-Phe-Leu) as the substrate, the Km, kcat, and Vmax values were 1.7 x 10(-4) M, 490 s-1, and 588 mumol of Leu released per min per mg of protein, respectively. CP3 was determined to be a serine protease, since its catalytic activity was blocked by the serine protease inhibitors diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, and 3,4-dichloroi Socoumarin (DCI). The enzyme was strongly inhibited by the mercurial compound p-chloromercuribenzoate. The carboxypeptidase readily hydrolyzed peptides with aliphatic or aromatic side chains, whereas most of the peptides which contained glycine in the penultimate position did not serve as substrates for the enzyme. Although CP3 activity was undetectable in Mucor yeast cells, antisera revealed the presence of the enzyme in the yeast form of the fungus. The partial amino acid sequence of the carboxypeptidase was determined.

Molecular characterization of the Enterococcus faecalis cytolysin activator

The gene encoding component A (cylA), the activator protein of the Enterococcus faecalis cytolysin, has been localized on pAD1, and the nucleotide sequence was determined. cylA consists of a 1,236-bp open reading frame encoding a 412-amino-acid polypeptide. A search of the National Biomedical Research Foundation data base revealed significant homology between the inferred amino acid sequence of component A and subtilisin BPN'. Component A activation of the cytolysin precursor (component L) was observed to be inhibited by the serine protease inhibitor diisopropylfluorophosphate. Mature component A exhibits a molecular weight of approximately 30,000 and an isoelectric point of 4.5. Differences between the size of the primary translation product (45,625 daltons) and the mature enzyme suggest that, as for subtilisin, component A is secreted as a proenzyme. These results provide the basis for a model of component A activation of component L and a role for component A in protecting the cytolysin-producing cell from lysis.

Reaction of LexA repressor with diisopropyl fluorophosphate. A test of the serine protease model.

The LexA repressor of Escherichia coli modulates the expression of the SOS regulon. In the presence of DNA damaging agents in vivo, the 202-amino acid LexA repressor is inactivated by specific RecA-mediated cleavage of the Ala-84/Gly-85 peptide bond. In vitro. LexA cleavage requires activated RecA at neutral pH, and proceeds spontaneously at high pH in an intramolecular reaction termed autodigestion. A model has been proposed for the mechanism of autodigestion in which serine 119 serves as the reactive nucleophile that attacks the Ala-84/Gly-85 peptide bond in a manner analogous to a serine protease, while uncharged lysine 156 activates the serine 119 hydroxyl group. In this work, we have tested this model by examining the effect of the serine protease inhibitor diisopropyl fluorophosphate (DFP) on autodigestion. We found that DFP inhibited autodigestion and that serine 119 was the only serine residue to react with DFP. We also examined [3H]DFP incorporation by a number of cleavage-impaired LexA mutant proteins and found that mutations in the proposed active site, but not in the cleavage site, significantly reduced the rate of [3H]DFP incorporation. Finally, we showed that the purified carboxyl-terminal domain, which contains the proposed catalytic residues, incorporated [3H]DFP at a rate indistinguishable from the intact protein. These data further support our current model for the mechanism of autodigestion and the organization of LexA.

Identification of the primary antimicrobial domains in human neutrophil cathepsin G.

Lysosomal cathepsin G from human neutrophils is a chymotrypsin-like protease which also possesses antimicrobial activity. The antimicrobial activity, however, is independent of protease activity, because treatment of this enzyme with the irreversible serine protease inhibitor diisopropylfluorophosphate has no effect on its antimicrobial action. In this study, we found that digestion of cathepsin G with clostripain caused a loss of proteolytic activity in this neutrophil proteinase. However, bactericidal activity in in vitro assays against Staphylococcus aureus and Neisseria gonorrhoeae was retained. Fractionation of the clostripain-digested cathepsin G mixture yielded two distinct antimicrobial peptides. The sequences of these peptides were IIGGR and HPQYNQR (residues 1-5 and 77-83 in cathepsin G, respectively). Synthetic peptides corresponding to these sequences were also prepared and found to exert broad-spectrum antimicrobial activity in vitro, displaying conditions of temperature- and pH-dependent optima for antimicrobial action resembling that of the full-length enzyme. Depending on the target bacterial strain, these peptides exhibited antimicrobial activity between 5.0 x 10(-5) and 4.0 x 10(-4) M. Significantly, replacement of certain residues within these peptides with either alanine or valine significantly reduced their antibacterial capacities. Our studies suggest that cathepsin G has two antimicrobial sequences, either or both of which may contribute to its bactericidal activity.

A novel ATP-requiring protease from skeletal muscle that hydrolyzes non-ubiquitinated proteins

Previously, we isolated an ATP-dependent proteolytic pathway in muscle, liver, and reticulocytes that requires ubiquitin and the enzymes which conjugate ubiquitin to proteins. We report here that skeletal muscle contains another soluble alkaline energy-dependent (but ubiquitin-independent) proteolytic activity. The cleavage of non-ubiquitinated protein substrates by the partially purified protease requires ATP hydrolysis since ATP in the absence of Mg2+, nonhydrolyzable ATP analogs, and pyrophosphate all fail to stimulate proteolysis. Proteolytic activity is also stimulated by UTP, CTP, and GTP, although not as effectively as by ATP (Km(ATP) = 0.027 mM). The enzyme is inactivated by the serine protease inhibitors diisopropyl fluorophosphate and 3,4-dichloroisocoumarin, but not by specific inhibitors of aspartic, thiol, or metalloproteases. It is maximally active at pH 8 and has a molecular weight of approximately 600,000. This new activity differs from the 720-kDa multicatalytic proteinase, but resembles the soluble ATP-dependent proteolytic system that we previously isolated from murine erythroleukemia cells.

Purification of an 80,000-Mr glycylprolyl peptidase from Bacteroides gingivalis.

An enzyme from Bacteroides gingivalis SUNYAB A7A1-28 that hydrolyzes the synthetic peptide glycyl-L-proline 4-methoxy-beta-naphthylamide was purified 1,040-fold by urea extraction, gel filtration, ion-exchange chromatography, and fast protein liquid chromatography. The molecular weight of the enzyme was 80,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 75,000 as determined by gel filtration. The optimum pH for the hydrolysis of glycyl-L-proline 4-methoxy-beta-naphthylamide was 7.5 to 8.5. The enzyme activity was inhibited by the serine protease inhibitors diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride by 82.5 and 78%, respectively. The activity was also inhibited by Hg2+ (55.6%) and Zn2+ (45%).

The effect of serine esterase inhibitors on ionophore-induced histamine release from human pulmonary mast cells

The serine proteases tryptase and chymase are present in human pulmonary mast cells. About 10-100 times more tryptase than chymase is found in these cells. However, a clear physiological role for both enzymes remains to be elucidated; angiotensin processing has been proposed as one possible function of chymase. A dose-dependent inhibition of A23187-induced histamine release from dispersed human lung mast cells was observed after pretreatment with the serine protease inhibitor diisopropylfluorophosphate (DFP) or the chymotrypsin-like enzyme inhibitor N-tosyl-L-phenylalanine chloromethylketone (TPCK) but not with the trypsin-like enzyme inhibitor N-tosyl-L-lysine chloromethylketone (TLCK). These results indicate that a chymase is probably an important factor in a late phase of human lung mast cell activation.

Lysosomal and non-lysosomal peptidyl hydrolases of the bloodstream forms of Trypanosoma brucei brucei.

African trypanosomes have thiol-dependent proteolytic activity that resembles some of the cathepsin-like activity found in mammalian lysosomes [Lonsdale-Eccles, J. D. & Mpimbaza, G. W. N. (1986) Eur. J. Biochem. 155, 469-473]. Here we show that this activity is found in lysosome-like organelles which we have isolated (density = 1.082 g/cm3 in Percoll) from bloodstream forms of Trypanosoma brucei brucei. They are approximately 250 nm in diameter, are bounded by a single limiting membrane, and contain acid phosphatase. The predominant proteolytic and peptidolytic activity of these organelles has a pH optimum about 6.0, exhibits latency, and has the characteristics of mammalian cathepsin L (and possibly cathepsin H) with respect to its hydrolysis of small fluorogenic peptidyl substrates such as benzyloxycarbonyl-phenylalanyl-arginyl-7-amido-4-methylcoumarin. This substrate appears to be a good marker for trypanosomal lysosomes. The cathepsin-L-like activity is inhibited by the thiol-protease inhibitors, E-64, cystatin, leupeptin and mercurial compounds. The proteolytic activity of the lysosome-like fraction is observed as a single band of activity with an approximate molecular mass of 27 kDa when measured after electrophoresis in the fibrinogen-containing sodium dodecyl sulphate/polyacrylamide gels. The addition of mammalian serum to this purified fraction, or to whole trypanosome homogenates, results in the appearance of additional bands of activity, with a concomitant increase in the total observed proteolytic activity. The serum of some species of animal (e.g. goat and guinea pig) appear to lack the ability to generate this new and increased activity, while rat, rabbit, human and bovine sera exhibit varying capacities to generate the new activity, the cow being the most effective. The apparent molecular masses of the new bands of activity are different for each mammalian species, suggesting that the activator is a species-specific molecule or class of molecules. We also show that Trypanosoma brucei contains soluble peptidolytic activity with an alkaline pH optimum. It is inhibited by the serine-protease inhibitor diisopropylfluorophosphate, but not by inhibitors such as phenylmethylsulphonyl fluoride, alpha 1-antitrypsin, or aprotinin. Nor is it inhibited by the thiol-protease-specific inhibitors E-64 or cystatin, although it is susceptible to inhibition by tosyllysylchloromethane, leupeptin, HgCl2 and p-chloromercuribenzoate. This enzymic activity has a preference for arginyl residues in the primary binding site (the P1 position), as also does the activity from the lysosomes.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochemical and functional properties of serine esterases in acidic cytoplasmic granules of cytotoxic T lymphocytes.

Percoll gradient fractions of homogenates of murine cloned cytotoxic T lymphocytes (CTL) were analyzed for the trypsin-like enzyme alpha-N-benzyloxy-carbonyl-L-lysinethiobenzyl ester (BLT) esterase recently described in CTL homogenates. Enzymatic activity was found in three areas of the gradient: the dense cytolysin containing granules; a light granule fraction; and a variable amount in the soluble fraction at the top of the gradient. Gel filtration columns showed a major peak of BLT esterase activity eluted at the position of a 60-kDa protein, and an additional, minor BLT esterase peak eluting at about 27 kDa. The separated enzymes were both significantly inhibited by the serine protease inhibitors diisopropylfluorophosphate and phenylmethyl sulfonyl fluoride (PMSF), indicating they are both serine proteases, but showed different patterns of inhibition by a series of inhibitors, suggesting the larger enzyme is not a simple dimer of the smaller. pH activity profiles of both CTL BLT esterases showed an optimum at about pH 8. PMSF inactivation of BLT esterase in detergent extracts of CTL diminished sharply as the pH was dropped below 7. Agents which raise the pH of acidic intracellular compartments were found to markedly enhance the PMSF inactivation of BLT esterase in intact CTL, showing that the granules have a low internal pH. Similarly, [3H]diisopropylfluorophosphate labeling of intact CTL gave four protein bands on non-reduced gels, of which two were labeled threefold more effectively in the presence of chloroquine. In parallel studies of inactivation of CTL lytic activity, PMSF pretreatment caused a 50% reduction of the lytic activity under conditions where greater than 90% of the BLT esterase activity was inactivated. Addition of agents raising the intragranular pH dramatically enhanced the BLT esterase inactivation but did not concomitantly reduce CTL lytic activity. These results indicate that inactivation of lytic function by PMSF is unlikely to be due to its reaction with protease in acidic granules, and suggest that the activity of these enzymes may not be required for cytotoxicity.

A novel form of factor VII in plasma from men at risk for cardiovascular disease.

In the Northwick Park Heart Study high activity of the coagulation factor VII was associated with cardiovascular deaths. We confirm here that men at high risk for such disease have increased levels of factor VII activity and demonstrate for the first time that this is due to a phospholipid-factor VII complex in their plasma. This complex increases the specific activity of its factor VII significantly. It is not inhibited by a neutralizing antiserum to thromboplastin and thus does not represent a factor VII-thromboplastin complex. Gel filtration data suggest an apparent Mr of 66-72 000 for the complex. Factor VII in this phospholipid complex is more sensitive than native factor VII to the serine protease inhibitor diisopropyl-fluorophosphate, consistent with a more accessible active site conformation (and a higher specific activity) for factor VII in the complex. The level of phospholipid-factor VII complex showed a significant positive correlation with plasma triglycerides and correlation of borderline significance with cholesterol.

Cationic antimicrobial proteins isolated from human neutrophil granulocytes in the presence of diisopropyl fluorophosphate.

Acid (0.2 M sodium acetate, pH 4.0) extracts of granules recovered from disrupted human polymorphonuclear granulocytes (PMNs) exhibited in vitro antimicrobial activity against Salmonella typhimurium. To minimize proteolytic destruction or modification of antimicrobial proteins derived from these granules, we pretreated the PMNs with the serine protease inhibitor diisopropyl fluorophosphate. Fractionation of such extracts by carboxymethyl Sephadex and Sephadex G-75 chromatography resulted in the recovery of at least two antimicrobial, cationic proteins. These proteins differed substantially in antimicrobial activity, amino acid composition, and molecular weight (Mr, 37,000 and 57,000). As we have shown before (Shafer et al., Infect. Immun. 43:834-858), with unfractionated proteins, these two proteins exhibited diminished activity against a polymyxin B-resistant (PBr) mutant of S. typhimurium compared with their activity against the isogenic parental polymyxin B-sensitive (PBs) strain. Expression of the relevant mutation (prmA) in the PBr mutant decreases the electronegativity of lipid A, owing to increased 4-amino-4-deoxy-L-arabinosylation at the 4' phosphate residue (Vaara et al., FEBS Lett. 129:145-149). The data suggest that at least two different cationic proteins account for the antimicrobial capacity of extracts from human PMN granules. Moreover, the availability of anionic charges in the outer membrane of S. typhimurium due to free lipid A phosphates apparently dictates phenotypic levels of resistance to both of the cationic proteins extracted from human PMN granules.

Isolation and characterization of an intracellular serine protease from Rhodococcus erythropolis

An intracellular serine protease from the bacterium Rhodococcus erythropolis was partially purified and characterized. The enzyme, which is present in the 100,000 g supernatant fraction of disrupted cells, was purified 160-fold by ammonium sulfate fractionation, and chromatography on DEAE-cellulose, Sephadex G-150, hydroxyapatite, and benzamidine-glycylglycine-Sepharose. The molecular weight of the protease estimated by gel filtration was 82,000. The subunit molecular weight of the protein was estimated to be 90,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Thus, the enzyme appears to be a monomer. The purified enzyme preparation hydrolyzed several arginine-containing synthetic substrates; benzoyl-arginine-4-methyl-7-coumarylamide was routinely used as substrate. Hemoglobin, casein, and azocasein were also hydrolyzed. No carboxypeptidase activity was detected. The pH optimum of the enzyme was 7.0–7.2. The protease was not affected by inhibitors of cysteine proteases (iodoacetate), aspartic proteases (pepstatin), or metallo-proteases (EDTA). Inhibition was observed with the serine protease inhibitor diisopropylfluorophosphate. Several trypsin inhibitors (tosyl-lysine chloromethyl ketone, antipain, leupeptin, 4-aminobenzamidine, ovomucoid, and gramicidin S) inhibited the Rhodococcus protease, but others did not (phenylmethylsulfonylfluoride, lima bean trypsin inhibitor, aprotinin, α-1-antitrypsin). Salts with monovalent cations (e.g., NaCl, KCl) activated the protease two-to threefold at ionic strengths of 0.2 to 0.5. MgCl 2 stimulated activity fourfold at ionic strengths of 0.05 to 0.15; CaCl 2 stimulated activity (1.5-fold) maximally at an ionic strength of 0.05 and inhibited above 0.15.

Detection and partial characterization of lymphoid cell surface proteases

Cultures of viable thymocytes and lymph node cells (LNC) were found to exhibit neutral protease activity toward radiolabeled protein substrates. Proteases were not actively secreted in serum-free culture. Thymocyte surface proteases were not affected by incubation of the cells in 1 m M ethylenediaminetetraacetic acid (EDTA) or 1 m M ethylene glycol bis(aminoethyl ether) N, N'-tetraacetic acid (EGTA); however, approximately 25% of lymph node cell surface protease activity was released from the cells by EDTA. It was concluded that the majority of protease activity displayed by both cell types was tightly associated with the cell surface. The inhibitor sensitivity of the cell surface proteases detected on hamster thymocytes and LNC and rat thymocytes was very similar. Cell surface protease activity was inhibited (85%) by the serine protease inhibitors diisopropylfluorophosphate (DFP) and phenylmethylsulfonylfluoride (PMSF) and was partially inhibited by l-1-tosylamide-2-phenylethylchloromethyl ketone(TPCK) and soybean trypsin inhibitor (SBTI), but not by N-α-p-tosyl- l-lysine-chloromethyl ketone (TLCK) or ϵ-aminocaproic acid (EACA). The bacterial protease inhibitor antipain was strongly inhibitory whereas leupeptin was less effective and elastinal did not inhibit cell surface protease activity. Thymocyte surface proteases were also inhibited (65%) by ZnCl 2, but not be several other divalent cations. In LNC, both ZnCl 2 and NiCl 2 were inhibitory to a lesser extent (32% inhibition). At least one surface protease in both thymocytes and LNC could function as a plasminogen activator.

Acetylcholinesterase of the human erythrocyte membrane

It has been shown that erythrocyte membrane proteins become susceptible to degradation by membrane-bound serine protease activity after oxidative modification of the membranes (M. Beppu, M. Inoue, T. Ishikawa, K. Kikugawa, Biochim. Biophys. Acta 1196 (1994) 81–87). The aim of the present study was to clarify the presence of the serine protease in oxidized erythrocyte membranes and to characterize the selectivity of the enzyme to oxidized proteins. Human erythrocytes were oxidized in vitro with xanthine/xanthine oxidase/Fe(III) and oxidized membranes isolated. Proteolytic activity of the membranes toward spectrin obtained from oxidized membranes and bovine serum albumin oxidized with H2O2/horseradish peroxidase was increased by membrane oxidation, and the degradability of the substrates was increased by substrate oxidation. The proteolytic activity was inhibited by the serine protease inhibitor diisopropyl fluorophosphate (DFP). The 72 kDa and 80 kDa proteins in the membranes were labeled by [3H]DFP when detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and subsequent fluorography. The 72 kDa protein was found to be a serine enzyme, acetylcholine esterase. The 80 kDa protein appeared to be responsible for the degradation of oxidatively damaged proteins. The 80 kDa protein was loosely bound to membranes and readily solubilized into a 0.1% NP-40 detergent solution. The presence of the same 80 kDa protease in intact erythrocyte cytosol was suggested. The increased serine protease activity in oxidized membranes can result from the increased adherence of the cytosolic 80 kDa serine protease to the membranes due to oxidation.