A new inhibitor of plasmin and trypsin from porcine leukocytes.

A new intracellular inhibitor of plasmin and trypsin was isolated from porcine leukocytes by ion exchange chromatography and affinity chromatography. In dodecyl sulphate gel electrophoresis a single protein band with an apparent molecular mass of 15 kDa was found under reducing conditions. On isoelectric focusing three protein bands with isoelectric points between pH 4.0 and 4.5 were found. The association rate constants and the inhibition constants were determined for porcine plasmin and bovine trypsin. The inhibitor shows no immunologic cross-reactivity with any of the tested leukocyte inhibitors. On the basis of its N-terminal amino-acid sequence a great degree of similarity to Kunitz-type inhibitors was observed.     

An inhibitor of plasminogen activation from human placenta. Purification and characterization

Placental extracts contain inhibitors of human urinary urokinase. These extracts form a heterogeneous population of complexes with 125I-urokinase that are recognizable by changes in gel filtration profile and mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment with reducing agents eliminated the size heterogeneity without loss of activity, thereby allowing the placental inhibitor to be purified. Active inhibitor has been isolated in apparently homogeneous form after an eight-step procedure that included salt extraction, ammonium sulfate fractionation, column chromatography on CM-cellulose, DEAE-Sepharose, and hydroxylapatite, chromatofocusing, preparative gel electrophoresis, and hydrophobic chromatography. The purified inhibitor has Mr = 47,000. The inhibitor is relatively specific for plasminogen activators since it does not inhibit the action of plasmin, factor XIIa, plasma kallikrein, or thrombin. The inhibitor forms complexes with 1:1 stoichiometry that block the active sites of urokinase (but not prourokinase) and both one- and two-chain forms of tissue plasminogen activator. The stability of these complexes in sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggest that they are based on covalently bonded structures. Although both types of plasminogen activator are inhibited, the rate of interaction is significantly faster with urokinase, tissue plasminogen activator being inhibited less efficiently. The complexes formed can be dissociated by mild alkali or hydroxylamine, thereby regenerating both enzymes and inhibitor at their original molecular weights. The results suggest that the complexes are stabilized by ester-like bonds; these might involve the hydroxyl of serine at the active site of the proteases and a carboxyl group in the inhibitor.     

Purification and characterization of a novel inhibitor of urokinase from human urine. Quantitation and preliminary characterization in plasma

Urokinase-related proteins in human urine occur mainly as a 1:1 complex of urokinase with an inhibitor (Stump, D. C., Thienpont, M., and Collen, D. (1986) J. Biol. Chem. 261, 1267-1273). BALB/c mice were immunized with this urokinase-urokinase inhibitor complex and spleen cells fused with mouse myeloma cells, resulting in hybridomas producing monoclonal antibodies. Three antibodies reacting with the complex but not with urokinase were utilized to develop a sensitive (0.5 ng/ml) enzyme-linked immunosorbent assay for the urokinase inhibitor, which was used for monitoring its purification by chromatography on zinc chelate-Sepharose, concanavalin A-Sepharose, SP-Sephadex C-50, and Sephadex G-100. A homogenous glycoprotein of apparent Mr 50,000 was obtained with a yield of 40 micrograms/liter urine and a purification factor of 320. One mg of the purified protein inhibited 35,000 IU of urokinase within 30 min at 37 degrees C. This protein was immunologically related to both the purified urokinase-urokinase inhibitor complex and to the inhibitor portion dissociated from it by nucleophilic dissociation. It was immunologically distinct from all known protease inhibitors, including the endothelial cell-derived fast-acting inhibitor of tissue-type plasminogen activator, the placental inhibitor of urokinase and protease nexin. In electrophoresis the protein migrated with beta-mobility. Inhibition of urokinase occurred with a second order rate constant (k) of 8 X 10(3) M-1 s-1 in the absence and of 9 X 10(4) M-1 s-1 in the presence of 50 IU of heparin/ml. The urokinase inhibitor was inactive towards single-chain urokinase-type plasminogen activator and plasmin, but it inhibited two-chain tissue-type plasminogen activator with a k below 10(3) M-1 s-1 and thrombin with a k of 4 X 10(4) M-1 s-1 in the absence and 2 X 10(5) M-1 s-1 in the presence of heparin. The concentration of this urokinase inhibitor in plasma from normal subjects determined by immunoassay was 2 +/- 0.7 micrograms/ml (mean +/- S.D., n = 25). The protein purified from plasma by immunoabsorption had the same Mr, amino acid composition, and immunoreactivity as the urinary protein. Furthermore, when urokinase was added to plasma, time-dependent urokinase-urokinase inhibitor complex formation was observed at a rate similar to that observed for the inhibition of urokinase by the purified inhibitor from urine. This urokinase inhibitor, purified from human urine, most probably represents a new plasma protease inhibitor.     

An inhibitor from placenta specifically binds urokinase and inhibits plasminogen activator released from ovarian carcinoma in tissue culture.

An inhibitor present in placenta and released in placental tissue culture forms specific complexes with each of two molecular forms of urokinase. Autoradiography demonstrated that the inhibitor shifted the electrophoretic position of 125I-labelled urokinase. It did not change the migration of diisopropyl-fluorophosphate-inactivated 125I-labelled urokinase, thereby indicating complex formation dependent on active serine site in urokinase. The inhibitor had a strong neutralizing effect on the plasminogen activators released from human ovarian carcinoma in tissue culture. The placental inhibitor might prove useful in inhibiting the fibrinolytic process necessary for proliferation of tumour vessels.     

An inhibitor from placenta specifically binds urokinase and inhibits plasminogen activator released from ovarian carcinoma in tissue culture

An inhibitor present in placenta and released in placental tissue culture forms specific complexes with each of two molecular forms of urokinase. Audoradiography demonstrated that the inhibitor shifted the electrophoretic position of ¹²⁵I-labelled urokinase. It did not change the migration of diisopropyl-fluorophosphate-inactivated ¹²⁵I-labelled urokinase, thereby indicating complex formation dependent on active serine site in urokinase. The inhibitor had a strong neutralizing effect on the plasminogen activators released from human ovarian carcinoma in tissue culture. The placental inhibitor might prove useful in inhibiting the fibrinolytic process necessary for proliferation of tumour vessels.     

Purification and characterization of a plasminogen activator inhibitor from the histiocytic lymphoma cell line U-937

We report the production, purification, characterization, and partial amino acid sequence of a plasminogen inhibitor (PA-I). The starting material is culture fluid from phorbol myristate 13-acetate-treated U-937 cells and the isolation steps consist of preparative isoelectric focusing followed by affinity chromatography on Cibacron Blue-Sepharose. PA-I migrates as a closely spaced doublet of 47-kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and forms covalent complexes with urokinase and two-chain tissue-type plasminogen activator, displaying second order rate constants of 0.9 X 10(6) M-1 s-1 and 0.2 X 10(6) M-1 s-1, respectively. Upon treatment with 1 M NH4OH, the covalent complexes were hydrolyzed, yielding a 35-kDa inhibitor fragment. A partial amino acid sequence of PA-I showed that it belongs to the antithrombin III family of inhibitors. PA-I is immunologically related to a PA-inhibitor from human placenta. mRNA from phorbol myristate 13-acetate-treated U-937 cells directed, in a rabbit reticulocyte derived cell-free system, the biosynthesis of only one 47-kDa protein that could be immunoprecipitated with anti-PA-I IgG, indicating that the two molecular forms of PA-I are the products of post-translational processing.     

Complex-formation and inhibition of urokinase by blood plasma proteins.

We have studied the formation of covalent complexes between 125I-urokinase (125I-UK) and proteins in human plasma. Although 125I-UK reacts with many proteinase inhibitors in purified systems, the predominant complexes formed in plasma are with antithrombin III (ATIII) and alpha 2-macroglobulin (alpha 2M). 125I-UK interacts with purified alpha 2M or alpha 2M in plasma to form a characteristic pattern of multiple complexes whose Mr values by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis are in the range of 380 000-720 000, under non-reducing conditions, and 180 000-430 000 after reduction. We also examined the inhibition of UK amidolytic activity by plasma and by purified ATIII. In the presence of saturating concentrations of ATIII and heparin, an apparent first-order rate constant of 6.8 X 10(-1) s-1 was calculated for the inhibition of urokinase. In contrast, the rate constant for the formation of covalent ATIII-UK complexes was lower, suggesting the inhibition of UK proceeds first via the formation of transient non-covalent intermediates that are then transformed more slowly into covalent end products. The observed rate constants for enzyme inhibition or complex-formation with plasma or purified inhibitors are insufficient to account for the reported clearance rate of injected UK in vivo.     

Glial-derived neurite-promoting factor is a slow-binding inhibitor of trypsin, thrombin, and urokinase

Glial-derived neurite-promoting factor was found to be a slow-binding inhibitor of trypsin, urokinase, and thrombin. The kinetic mechanism of the inhibition differs among the three proteases. With trypsin and urokinase, an initial protease-factor complex formed which isomerized to a tighter complex. For thrombin, however, no initial complex was kinetically observed. The dissociation constants of the equilibrium complexes of the factor with trypsin, urokinase, and thrombin were 17, 280, and 18 pM, respectively, and the apparent second-order rate constants for the interaction of the factor with these enzymes were, respectively, 4.7 X 10(6), 1.2 X 10(5), and 2.1 X 10(6) M-1S-1. Heparin increased the rate at which the factor reacted with thrombin by over 40-fold to 8.9 X 10(7) M-1S-1 and decreased the dissociation constant of the complex by over 80-fold to 0.3 pM. The values obtained for the apparent second-order rate constants when compared with the kinetics of neurite induction by the factor indicate that the neurite-promoting activity of the factor is not due to the inhibition of urokinase but could be due to the inhibition of an enzyme with a specificity similar to that of thrombin or trypsin. Comparison of the values of the apparent second-order rate constants obtained for the factor with those obtained for protease nexin suggests that these two molecules are very similar in their inhibitory properties.     

Detection and partial characterization of a specific plasminogen activator inhibitor in human chondrocyte cultures

Serum-free culture medium collected from primary monolayer cultures of human articular chondrocytes was found to inhibit human urokinase [EC 3.4.21.31] activity. Although chondrocyte culture medium contained a small amount of endothelial-type plasminogen activator inhibitor which could be demonstrated by reverse fibrin autography, most of the urokinase inhibitory activity of chondrocyte culture medium was shown to be due to a different molecule from endothelial-type inhibitor, since it did not react with a specific antibody to this type of inhibitor. The dominant urokinase inhibitor in chondrocyte culture medium was partially purified by concanavalin A-Sepharose affinity chromatography. The partially purified inhibitor inhibited high-Mr urokinase more effectively than low-Mr urokinase, but no obvious inhibition was detected against tissue-type plasminogen activator, plasmin, trypsin, and thrombin. The inhibitor had an apparent Mr of 43,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis, and it was unstable to sodium dodecyl sulfate, acid, and heat treatments. Inhibition of urokinase by the inhibitor was accompanied with the formation of a sodium dodecyl sulfate-stable high-Mr complex between them. Inhibition and complex formation required the active site of urokinase. The partially purified inhibitor was thought to be immunologically different from the known classes of plasminogen activator inhibitors, including endothelial-type inhibitor, macrophage/monocyte inhibitor, and protease nexin, since it did not react with specific antibodies to these inhibitors.     

Ribonuclease inhibitor from human placenta. Purification and properties

A soluble ribonuclease inhibitor from the human placenta has been purified 4000-fold by a combination of ion exchange and affinity chromatography. The inhibitor has been isolated in 45% yield (about 2 mg/placenta) as a protein that is homogeneous by sodium dodecyl sulfate-gel electrophoresis. In common with the inhibitors of pancreatic ribonuclease from other tissues that have been studied earlier, the placental inhibitor is an acidic protein of molecular weight near 50,000; it forms a 1:1 complex with bovine pancreatic RNase A and is a noncompetitive inhibitor of the pancreatic enzyme, with a Ki of 3 X 10(-10) M. The amino acid composition of the protein has been determined. The protein contains 30 half-cystine plus cysteine residues determined as cysteic acid after performic acid oxidation. At pH 8.6 the nondenatured protein alkylated with iodoacetic acid in the presence of free thiol has 8 free sulfhydryl groups. The inhibitor is irreversibly inactivated by sulfhydryl reagents and also by removal of free thiol from solutions of the protein. Inactivation by sulfhydryl reagents causes the dissociation of the RNase - inhibitor complex into active RNase and inactive inhibitor.     

Kinetic analysis of the interactions between plasminogen activator inhibitor 1 and both urokinase and tissue plasminogen activator

Plasminogen activator inhibitor 1 (PAI-1) was purified from medium conditioned by cultured bovine aortic endothelial cells by successive chromatography on concanavalin A Sepharose, Sephacryl S-200, Blue B agarose, and Bio-Gel P-60. As shown previously for conditioned media (C. M. Hekman and D. J. Loskutoff (1985) J. Biol. Chem. 260, 11581-11587) the purified PAI-1 preparation contained latent inhibitory activity which could be stimulated 9.4-fold by sodium dodecyl sulfate and 45-fold by guanidine-HCl. The specific activity of the preparation following treatment with 0.1% sodium dodecyl sulfate was 2.5 X 10(3) IU/mg. The reaction between purified, guanidine-activated PAI-1 and both urokinase and tissue plasminogen activator (tPA) was studied. The second-order rate constants (pH 7.2, 35 degrees C) for the interaction between guanidine-activated PAI-1 and urokinase (UK), and one- and two-chain tPA are 1.6 X 10(8), 4.0 X 10(7), and 1.5 X 10(8) M-1 S-1, respectively. The presence of CNBr fibrinogen fragments had no affect on the rate constants of either one- or two-chain tPA. Steady-state kinetic analysis of the effect of PAI-1 on the rate of plasminogen activation revealed that the initial UK/PAI-1 interaction can be competed with plasminogen suggesting that the UK/PAI-1 interaction may involve a competitive type of inhibition. In contrast, the initial tPA/PAI-1 interaction can be competed only partially with plasminogen, suggesting that the tPA/PAI-1 interaction may involve a mixed type of inhibition. The results indicate that PAI-1 interacts more rapidly with UK and tPA than any PAI reported to date and suggest that PAI-1 is the primary physiological inhibitor of single-chain tPA. Moreover, the interaction of PAI-1 with tPA differs from its interaction with UK, and may involve two sites on the tPA molecule.     

The major plasma kallikrein inhibitor of guinea pig plasma.

A plasma kallikrein inhibitor in guinea pig plasma (KIP) was purified to homogeneity. KIP is a single chain protein and the apparent molecular weight is estimated to be 59,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In amino acid composition, KIP is similar to human and mouse alpha 1-proteinase inhibitors and mouse contrapsin. KIP forms an equimolar complex with plasma kallikrein in a dose- and time-dependent fashion. The association rate constants for the inhibition of guinea pig plasma kallikrein by KIP, alpha 2-macroglobulin, C1-inactivator and antithrombin III were 2.5 +/- 0.3.10(4), 2.4 +/- 0.4.10(4), 6.6 +/- 0.5.10(4) and 9.1 +/- 0.6.10(2), respectively. Comparison of the association rate constants and the normal plasma concentrations of the four inhibitors demonstrates that KIP is ten-times as effective as alpha 2-MG and other two inhibitors are marginally effective in the inhibition of kallikrein. KIP inhibits trypsin and elastase rapidly, and thrombin and plasmin slowly, but is inactive for chymotrypsin and gland kallikrein. These results suggest that KIP is the major kallikrein inhibitor in guinea pig plasma and the proteinase inhibitory spectrum is unique to KIP in spite of the molecular similarity to alpha 1-proteinase inhibitor.     

Thrombin interaction with platelets. Influence of a platelet protease nexin

A fraction of the 125I-thrombin that binds to human platelets is taken into a sodium dodecyl sulfate-resistant 77 kDa complex with a platelet factor (Bennett, W. F., and Glenn, K. C. (1980) Cell 22, 621-627). Here we show that this platelet factor is in several respects similar to protease nexin I (PNI), a fibroblast thrombin inhibitor. The complexes are of the appropriate size, bind to Sepharose that has been derivatized with anti-PNI antibody, do not form when the thrombin active site has been blocked with diisopropylphosphofluoridate, and do not appear on platelets when heparin is present. However, the platelet factor does not bind urokinase, indicating that this "platelet PN" may be distinct from PNI. Following brief incubation with 125I-thrombin, platelet PN X 125I X thrombin complexes are found both associated with the platelets and free in the binding medium. 125I-Thrombin has a higher affinity for platelet PN than for platelet receptors. In 30-s binding incubations carried out with thrombin at concentrations below 0.3 nM, formation of the 77-kDa complex accounts for most of the platelet specific binding of 125I-thrombin. Subtracting this large contribution to 125I-thrombin-specific binding reveals that the reversible binding of 125I-thrombin to platelet receptors exhibits sigmoidal thrombin dose-dependence. Thrombin stimulation of platelet [14C]serotonin release exhibits similar thrombin dose dependence. These results indicate that platelets may possess a mechanism for suppressing their interaction with active thrombin at thrombin doses below 0.3 nM. It is possible that platelet PN carries out this function by capturing thrombin before thrombin binds to its signal-transmitting receptors.     

Macrophage fibrinolytic activity: identification of two pathways of plasmin formation by intact cells and of a plasminogen activator inhibitor

Endotoxin-stimulated macrophages hydrolyze fibrin by a plasmin-mediated process in the absence of detectable soluble plasminogen activator (PAs). The data show that macrophages also activate plasmin by a membrane-associated plasminogen activator (PAm). In the presence of endotoxin, PAm activity increases, and plasmin is formed only by PAm. In addition, endotoxin stimulates macrophages to secrete a proteinase inhibitor that blocks PAs activity but not PAm or plasmin activity. The increased PAm activity and the PA inhibitor secretion in response to endotoxin explains the ability of intact macrophages to hydrolyze fibrin in the absence of detectable PAs. Endotoxin, 100 ng/ml, induced an intracellular PA inhibitor in cultured macrophages, and this correlated with accumulation of inhibitor in medium over the cells. The intracellular PA inhibitor was found to be 50--60 kilodaltons by gel chromatography, to be of anionic charge at pH 7.4 and to inhibit urokinase esterolytic and proteolytic activity but not preformed plasmin. These results define two pathways of plasmin formation by intact macrophages and identify the macrophage cell surface as a site of PA activity relatively protected from soluble proteinase inhibitors.     

Kinetics of the inhibition of plasminogen activators by the plasminogen-activator inhibitor. Evidence for ''second-site'' interactions.

The reactions between plasminogen-activator inhibitor (PAI) and different plasminogen activators were studied in the presence of chromogenic peptide substrates for the enzymes. Our findings suggest that the rate constants for the reactions of PAI with single-chain tissue plasminogen activator (tPA), two-chain tPA, high-Mr urokinase and low-Mr urokinase are high and quite similar (1.6 X 10(7)-3.9 X 10(7) M-1.s-1). A free active site in the enzymes seems to be necessary for their reaction with PAI. Amino acids with antifibrinolytic properties did not interfere with the reactions. However, di-isopropyl phosphorofluoridate-inactivated tPA inhibited the reaction between PAI and all plasminogen activators in a similar way. These findings clearly demonstrated that a 'second-site' interaction, in addition to that between the enzyme active site and the inhibitor 'bait' peptide bond, is of importance for the high reaction rate. The reaction rate between PAI and single-chain tPA in the presence of an activator substrate (D-Ile-Pro-Arg p-nitroanilide) was decreased in the presence of fibrin. Fibrin caused a decrease in the Km for the single-chain tPA-substrate reaction. As a consequence, the 'free' concentration of single-chain tPA in the system decreased in the presence of fibrin, affecting the reaction rate between PAI and single-chain tPA. The phenomenon might be of physiological relevance, in the sense that single-chain tPA bound to fibrin in the presence of plasminogen would be protected against inactivation by PAI.     

Endogenous inhibitor of cysteine proteases from the human kidney

A thermo- and acid stable inhibitor of cysteine proteinases was isolated from the human kidney by successive procedures--acid fractionation, gel-filtration on Sephadex G-75, affinity chromatography on papain-sepharose. The final purification factor was 650 fold. The inhibitor molecular weight was equal to 12 kDa. The values of Ki measured by different methods are (7.9-9.4) X 10(-4) M for papain and (7.1-8.0) X 10(-10) M for purified human kidney cathepsin B. In experiments with papain, inhibitor kass and kd were 1.1 X 10(6) M-1 s-1 and 9.0 X 10(-4) s-1, respectively. The inhibitor did not influence the trypsin activity, its properties being similar to those of related thermo- and acid-stable inhibitors from other human and animal tissues.     

Iodinated fibroblast beta-glucuronidase as a ligand for receptor-mediated endocytosis.

Antibodies raised to human placental beta-glucuronidase were shown to cross-react with the beta-glucuronidase secreted by mouse 3T3 fibroblasts, but did not react with other lysosomal enzymes. The beta-glucuronidase secreted by 3T3 cells was purified 15000-fold by chromatography on an affinity column made from this antibody and resolved into a single component, of Mr 68000, by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Iodinated samples of purified enzyme were taken up into mouse peritoneal macrophages by receptor-mediated endocytosis at a rate similar to that calculated previously for unlabelled enzyme, and uptake was competitively inhibited by yeast mannan. Binding of beta-glucuronidase to macrophages was saturable, with a Kd of 7 X 10(-9)l/mol, an affinity comparable with that calculated for the binding of mannosylated bovine serum albumin (Kd 1.3 X 10(-9)l/mol), a ligand specific for mannose receptors. Four times as many molecules of mannosylated albumin (12000) as of beta-glucuronidase (3000), however, bound to each cell. This purification and iodination procedure did not therefore have any adverse effect on the uptake properties of secreted beta-glucuronidase, and provides a ligand with which to investigate binding and specific endocytosis into a range of different types of cell.     

Identity between the placental protein PP10 and the specific plasminogen activator inhibitor of placental type PAI-2

The highly specific plasminogen activator inhibitor of placental type, PAI-2, occurs in the placenta in a low molecular mass form of 46.6 kDa, and in pregnancy plasma in a (possibly glycosylated) high molecular mass form of 60 kDa. Extensive knowledge is available about the functional properties of PAI-2 as a plasminogen activator inhibitor and about its molecular biology and regulation. Of the several placenta proteins (PP) isolated, one of them, PP10, has a molecular mass of 48 kDa and its occurrence in malignancy and in complications during pregnancy has been the topic of a number of studies, though its properties and physiological significance are unknown. The present findings constitute evidence of immunological identity between PP10 and PAI-2. The sections of the amino acid sequence of PP10 analysed here were found to have identical counterparts in the sequence of the low molecular mass form of PA1-2, but in several preparations PP10 was found to occur in an inactive two-chain form due to cleavage of an Arg-Thr bond, the two peptide chains being linked to each other by a disulphide bridge. The cleavage site is identical to that observed in the reaction between PAI-2 and urokinase. The results make it possible to coordinate and correlate the findings of many separate studies and our own observations on PP10 and PAI-2.     

Mechanism of inhibition of activated protein C by protein C inhibitor

Protein C inhibitor isolated from human plasma inhibited thrombin, factor Xa, trypsin and chymotrypsin as well as activated protein C, but had very little effect on urokinase and plasmin. The inhibition constants (K1) of protein C inhibitor for activated protein C, thrombin and factor Xa were 5.6 X 10(-8) M, 6.7 X 10(-8) M and 3.1 X 10(-7) M, respectively. The second-order rate constant for inhibition of activated protein C by the inhibitor increased about 30-fold in the presence of an optimal heparin concentration (5-10 units/ml). The inhibition of activated protein C by plasma protein C inhibitor was also accelerated by heparin. When activated protein C (Mr = 62,000) was incubated with protein C inhibitor (Mr = 57,000), enzyme-inhibitor complexes with apparent Mr = 102,000 and 88,000 were observed in the nonreduced and the reduced samples, respectively, on SDS-polyacrylamide gel electrophoresis. In addition to these complexes, a band of unbound enzyme and a band with Mr = 54,000 were detected. When 125I-labeled protein C inhibitor was exposed to activated protein C, the inhibitor band was converted to bands with apparent Mr = 102,000 and 54,000 in the nonreduced samples, as determined by autoradiography after gel electrophoresis in SDS. The band with Mr = 54,000 also appeared when the inhibitor reacted with other serine proteases. The activated protein C was released from the inactive complex by treatment with 1 M ammonia or hydroxylamine. This phenomenon was found by SDS-polyacrylamide gel electrophoresis to represent the dissociation of the enzyme-inhibitor complex by ammonia or hydroxylamine into the free enzyme and the proteolytically modified inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)