cDNA cloning and expression in Escherichia coli of a plasminogen activator inhibitor from human placenta

Two nearly full-length cDNAs for placental plasminogen activator inhibitor (PAI) have been isolated from a human placenta lambda gt11 cDNA library. One positive (lambda PAI-75.1) expressed a protein that could adsorb and purify anti-PAI antibodies. The expressed protein inhibited the activity of human urokinase in a fibrin autography assay, and formed a 79-kDa (reduced) covalent complex with 125I-urokinase that could be immunoprecipitated with anti-PAI. The cDNA insert of the longer isolate (lambda PAI-75.15) consisted of 1909 base pairs, including a 5'-noncoding region of 55 base pairs, an open reading frame of 1245 base pairs, a stop codon, a 3'-noncoding region of 581 base pairs, and a poly(A) tail. The size of the mRNA was estimated to be 2.0 kilobases by Northern blot analysis. The translated amino acid sequence consisted of 415 amino acids, corresponding to a 46.6-kDa protein. The sequence was related to members of the serpin gene family, particularly ovalbumin and the chicken gene Y protein. Like these avian proteins, placental PAI appears to lack a cleavable NH2-terminal signal peptide. Residues 347-376 were identical to the partial sequence reported recently for a PAI isolated from the human monocytic U-937 cell line. Placental PAI mRNA was apparently expressed at low levels in human umbilical vein endothelial cells, but was not detectable in HepG2 hepatoma cells. It was present in U-937 cells and was inducible at least 10-fold by phorbol 12-myristate 13-acetate. Thus placental PAI is a unique member of the serpin gene family, distinct from endothelial-type PAI. It is probably identical to monocyte-macrophage PAI.     

Characterization of the dexamethasone-induced inhibitor of plasminogen activator in HTC hepatoma cells

Incubation of HTC rat hepatoma cells with the synthetic glucocorticoid dexamethasone rapidly inhibits plasminogen activator (PA) activity secondary to the induction of a specific acid-stable inhibitor of plasminogen activation (Cwikel, B. J., Barouski-Miller, P.A., Coleman, P.L., and Gelehrter, T.D. (1984) J. Biol. Chem. 259, 6847-6851). We have further characterized this inhibitor with respect to its interaction with both urokinase and tissue plasminogen activator, and its protease specificity. The HTC PA inhibitor rapidly inhibits urokinase and tissue plasminogen activator with an apparent second-order rate constant of 3-5 x 10(7) M-1 X s-1. The inhibitor forms stable covalent complexes with both urokinase and tissue plasminogen activator, with which plasmin, trypsin, and factor Xa apparently do not compete. Complex formation is saturable and requires the active site of the PA. The mass of the inhibitor-PA complex is 50,000 daltons greater than that of PA alone, consistent with an Mr for the PA inhibitor of 50,000 as demonstrated directly by reverse fibrin autography. The HTC PA inhibitor does not inhibit thrombin and differs in its kinetic and biochemical properties from protease nexin.     

Inhibition of receptor-bound urokinase by plasminogen-activator inhibitors

Urokinase-type plasminogen activator (uPA) binds to a specific receptor on various cell types, the bound molecule retaining its enzymatic activity against plasminogen. We have now investigated whether receptor-bound uPA also retains the ability to react with and be inhibited by plasminogen activator inhibitors (PAI-1 and PAI-2). uPA bound to its receptor on human U937 monocyte-like cells was inhibited by PAI-1 (in its active form in the presence of vitronectin fragments) with an association rate constant of 4.5 x 10(6) M-1 s-1, which was 40% lower than that obtained for uPA in solution (7.9 x 10(6) M-1 s-1). The inhibition of uPA by PAI-2 was decreased to a similar extent by receptor binding, falling from 5.3 x 10(5) to 3.3 x 10(5) M-1 s-1. Stimulation of U937 cells with phorbol 12-myristate 13-acetate was accompanied by a further reduction in receptor-bound uPA inhibition by PAI-1 and PAI-2 to 1.7 x 10(6) and 1.1 x 10(5) M-1 s-1, respectively. These constants although lower than those for uPA in solution still represent rather rapid inhibition of the enzyme, and demonstrate that uPA bound to its specific cellular receptor remains available for efficient inhibition by PAI's, which may therefore play a major role in controlling cell-surface plasminogen activation and extracellular proteolytic activity.     

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.     

Determination of intermediates, products and cleavage site in the reaction between plasminogen activator inhibitor type-2 and urokinases

Several specific inhibitors for plasminogen activators have been isolated from various organs and cell lines, those from human placenta and the human monocyte-like cell line U-937 being virtually identical. The reaction between this type of inhibitor, designated as type-2, and high-M_r and low-M_r urokinase-type plasminogen activators was followed by reversed-phase high-performance liquid chromatography and gel electrophoresis. The components, their stable complexes and their dissociation and cleavage products could be clearly identified in both systems. The amino acid sequence of the inhibitor at the cleavage site was determined to be -Met-Thr-Gly-Arg↓Thr-Gly-His-Gly-. A 35-residue carboxy-terminal fragment was found to be released.     

Opposite effects of thrombospondin-1 via CD36 and CD47 on homotypic aggregation of monocytic cells.

Thrombospondin-1 (TSP-1), an extracellular matrix protein, has a multimodular structure and each domain specifies a distinct biological function through interaction with a specific ligand. In this study we found that exogenously added TSP-1 inhibits phorbol myristate acetate (PMA)/LPS-induced homotypic aggregation of human monocytic U937 cells, whereas the 70-kDa fragment of TSP-1 generated by the proteolytic cleavage of the intact molecule promotes the homotypic aggregation. The aggregation was also inhibited by anti-CD47 mAb or the 4N1K peptide, of which sequence is derived from the CD47-binding site of TSP-1 and absent in the 70-kDa fragment. In contrast, the augmented cell aggregation by the 70-kDa fragment was hampered by anti-CD36 mAb or antibody against the CD36-binding site of TSP-1. The cell aggregation of U937 cells was completely blocked, even in the presence of the 70-kDa fragment, by mAb against leukocyte function associated antigen-1 (LFA-1) or intercellular adhesion molecule-1 (ICAM-1). We therefore propose that TSP-1 may regulate LFA-1/ICAM-1-mediated cell adhesion of monocytes/macrophages by either the inhibitory effect through CD47 or the promoting effect through CD36 depending on which domain/fragment is functional in a given biological setting.     

Platelet plasminogen activator inhibitor: purification and characterization of interaction with plasminogen activators and activated protein C

Plasminogen activator inhibitor (PAI) was purified in active form from porcine platelets under nondenaturing conditions. The purified inhibitor (Mr 47,000) reacts with tissue-type plasminogen activator (t-PA), urokinase (UK), and activated protein C (APC) to yield both SDS-stable complexes and a modified PAI of slightly reduced molecular weight. The second-order rate constants for the inhibition of t-PA and UK by PAI are 3.5 X 10(7) and 3.4 X 10(7) M-1 s-1, respectively. Activated protein C reacts with PAI with a second-order rate constant of 1.1 X 10(4) M-1 s-1. This rate is not accelerated by protein S, phospholipid, and calcium, or heparin. It is concluded that (1) PAI can function as both inhibitor and substrate of its target proteases, (2) if APC promotes fibrinolysis via inactivation of PAI, then APC must be present in concentrations several orders of magnitude greater than t-PA, or the interaction of APC and PAI must be accelerated by presently unknown mechanisms, and (3) in the absence of heparin, platelet PAI is the most rapid inhibitor of APC yet described.     

Vitronectin governs the interaction between plasminogen activator inhibitor 1 and tissue-type plasminogen activator.

The "serpin" plasminogen activator inhibitor 1 (PAI-1) is the fast acting inhibitor of plasminogen activators (tissue-type (t-PA) and urokinase type-PA) and is an essential regulatory protein of the fibrinolytic system. Its P1-P1' reactive center (R346 M347) acts as a "bait" for tight binding to t-PA/urokinase-type PA. In vivo, PAI-1 is encountered in complex with vitronectin, an interaction known to stabilize its activity but not to affect the second-order association rate constant (k1) between PAI-1 and t-PA. Nevertheless, by using PAI-1 reactive site variants (R346M, M347S, and R346M M347S), we show that the binding of vitronectin to the PAI-1 mutant proteins improves plasminogen activator inhibition. In the absence of vitronectin the PAI-1 R346M mutants are virtually inactive toward t-PA (k1 less than 1 x 10(3) M-1 s-1). In contrast, in the presence of vitronectin the rate of association increases about 1,000-fold (k1 of 6-8 x 10(5) M-1 s-1). This inhibition coincides with the formation of serpin-typical, sodium dodecyl sulfide-stable t-PA.PAI-1 R346M (R346M M347S) complexes. As evidenced by amino acid sequence analysis, the newly created M346-M/S347 peptide bond is susceptible to attack by t-PA, similar to the wild-type R346-M347 peptide bond, indicating that in the presence of vitronectin M346 functions as an efficient P1 residue. In addition, we show that the inhibition of t-PA and urokinase-type PA by PAI-1 mutant proteins is accelerated by the presence of the nonprotease A chains of the plasminogen activators.     

Inhibition of cysteine proteinases and dipeptidyl peptidase I by egg-white cystatin.

The interactions between egg-white cystatin and the cysteine proteinases papain, human cathepsin B and bovine dipeptidyl peptidase I were studied. Cystatin was shown to be a competitive reversible inhibitor of cathepsin B (Ki 1.7 nM, k-1 about 2.3 X 10(-3) s-1). The inhibition of dipeptidyl peptidase I was shown to be reversible (Ki(app.) 0.22 nM, k-1 about 2.2 X 10(-3) s-1). Cystatin bound papain too tightly for Ki to be determined, but an upper limit of 5 pM was estimated. The association was a second-order process, with k+1 1.0 X 10(7) M-1 X s-1. Papain was shown to form equimolar complexes with cystatin. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of complexes formed between papain or cathepsin B and an excess of cystatin showed no peptide bond cleavage after incubation for 72 h. The reaction of the active-site thiol group of papain with 5,5'-dithiobis-(2-nitrobenzoic acid) at pH 8 and 2,2'-dithiobispyridine at pH 4 was blocked by complex-formation. Dipeptidyl peptidase I and papain were found to compete for binding to cystatin, contrary to a previous report. The two major isoelectric forms of cystatin were found to have similar specific inhibitory activities for papain, and similar affinities for papain, cathepsin B and dipeptidyl peptidase I. This, together with specific oxidation of the N-terminal serine residue with periodate, showed the N-terminal amino group of cystatin 1 to be unimportant for inhibition. General citraconylation of amino groups resulted in a large decrease in the affinity of cystatin for dipeptidyl peptidase I. It is concluded that the interaction of cystatin with cysteine proteinases has many characteristics similar to those of an inhibitor such as aprotinin with serine proteinases.     

On the reversible interaction of plasminogen activator inhibitor-1 with tissue-type plasminogen activator and with urokinase-type plasminogen activator

The reaction between plasminogen activators and plasminogen activator inhibitor-1 is characterized by an initial rapid formation of an inactive reversible complex. The second-order association rate constant (k1) of complex formation of recombinant two-chain tissue-type plasminogen activator (rt-PA) or recombinant two-chain urokinase-type plasminogen activator (rtcu-PA) by recombinant plasminogen activator inhibitor-1 (rPAI-1) is 2.9 +/- 0.4 x 10(7) M-1 s-1 (mean +/- S.D., n = 30) and 2.0 +/- 0.6 x 10(7) M-1 s-1 (n = 12), respectively. Different molecular forms of tissue- or urokinase-type plasminogen activator which do not form covalent complexes with rPAI-1, including rt-PA-Ala478 (rt-PA with the active-site Ser478 mutagenized to Ala) and anhydro-urokinase (rtcu-PA with the active-site Ser356 converted to dehydroalanine) reduced k1 in a concentration-dependent manner, compatible with 1:1 stoichiometric complex formation between rPAI-1 and these ligands. The apparent dissociation constant (KD) of the complex between rPAI-1 and rt-PA-Ala478, determined as the concentration of rt-PA-Ala478 which reduced k1 to 50% of its control value, was 3-5 nM. Corresponding concentrations of active-site-blocked two-chain rt-PA were 150-250-fold higher. The concentration of anhydro-urokinase which reduced k1 to 50% was 4-6 nM, whereas that of active-site-blocked rtcu-PA was 100-250-fold higher. Recombinant single-chain urokinase-type plasminogen activator had an apparent KD of about 2 microM. These results suggest that inhibition of rt-PA or rtcu-PA by rPAI-1 proceeds via a reversible high affinity interaction which does not require a functional active site but which is markedly reduced following inactivation of the enzymes with active-site titrants.     

Protease nexin. Properties and a modified purification procedure

The present paper describes chemical and functional properties of protease nexin, a serine protease inhibitor released from cultured human fibroblasts. It is shown that protease nexin is actually synthesized by fibroblasts and represents about 1% of their secreted protein. Analysis of the amino acid composition of purified protease nexin indicates that it is evolutionarily related to antithrombin III and heparin cofactor II. Protease nexin contains approximately 6% carbohydrate, with 2.3% amino sugar, 1.1% neutral sugar, and 3.0% sialic acid. The Mr calculated from equilibrium sedimentation analysis is 43,000. Protease nexin is a broad specificity inhibitor of trypsin-like serine proteases. It reacts rapidly with trypsin (kassoc = 4.2 +/- 0.4 X 10(6) M-1 s-1), thrombin (kassoc = 6.0 +/- 1.3 X 10(5) M-1 s-1), urokinase (kassoc = 1.5 +/- 0.1 X 10(5) M-1 s-1), and plasmin (kassoc = 1.3 +/- 0.1 X 10(5) M-1 s-1), and slowly inhibits Factor Xa and the gamma subunit of nerve growth factor but does not inhibit chymotrypsin-like proteases or leukocyte elastase. In the presence of heparin, protease nexin inhibits thrombin at a nearly diffusion-controlled rate. Two heparin affinity classes of protease nexin can be detected. The present characterization pertains to the fraction of protease nexin having the higher affinity for heparin. The low affinity material, which is the minor fraction, is lost during purification.     

Lateral diffusion of wheat germ agglutinin-labeled glycoconjugates in the membrane of differentiating HL-60 and U-937 cells assessed with fluorescence recovery after photobleaching (FRAP)

The promyelocytic leukemia cell line HL-60 and the histiocytic cell line U-937 were grown in suspension culture. They were induced to differentiate during 5-d cultivation in the presence of dimethylsulfoxide (DMSO; 1.3% w/v) or phorbol-12-myristate-acetate (PMA; 10(-7) M), which yields granulocyte- and macrophage-like cells, respectively. Differentiation was evidenced by increased capacity to recognize and phagocytize IgG- or complement-coated yeast particles. Aliquots taken from the cultures with and without DMSO (or PMA) were spun down directly on glass microscope slides, washed, labeled with fluoresceinated wheat germ agglutinin (WGA), and directly examined at room temperature for the rate of fluorescence recovery after photobleaching (FRAP). It was found that cultivation of the HL-60 and the U-937 cells in the presence of DMSO, which yields granulocyte-like cells, reduced the average value of lateral diffusion coefficient D (X 10(10] from 1.72 +/- 0.13 cm2s-1 to 0.97 +/- 0.13 cm2s-1 and from 1.77 +/- 0.11 cm2s-1 to 0.82 +/- 0.13 cm2s-1, respectively. U-937 cells grown with PMA also showed a reduction of D(X 10(10] to 0.88 +/- 0.10 cm2s-1. There was a larger immobile fraction of fluorescence in the HL-60 cells than in the U-937 cells, viz., 70-80% compared to 10-50%. The total number of binding sites for WGA was not altered, but the surface density changed, since the HL-60 and the U-937 cells became smaller and larger, respectively, when grown in the presence of DMSO. It is concluded that differentiation reduces the average lateral mobility of the WGA-binding membrane component by a factor around 2.     

The interaction between some serine proteinases and horse leucocyte inhibitor

Horse blood leucocyte cytosol exhibits a broad inhibitory activity against serine proteinases. The purified inhibitor was exposed to investigated enzymes (trypsin, chymotrypsin, elastases and serine proteinase from S. aureus) for variable time and the products were analyzed by gradient polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The molar ratio I:E, association rate constants k on and inhibition constants Ki for the enzymes and inhibitor were determined. The examined elastases form stable, stoichiometric complexes with the inhibitor (Ki less than 10(-10) M), and do not undergo proteolytic degradation during 30 min incubation at 20 degrees C even at the 2-fold molar excess of the proteinases. The reactions with elastases are extremely rapid (k on greater than 10(7) M-1 s-1) and are completed within one second whereas similar reactions with chymotrypsin and trypsin are much slower (k on = 3 X 10(5) M-1 s-5 and 5 X 10(2) M-1 s-1, respectively). Serine proteinase from S. aureus neither react nor inactivates the investigated inhibitor. The complexes of the inhibitor with trypsin and chymotrypsin are digested even at a molar ratio I:E = 2:1. All these observations point out that the inhibitor from horse leucocyte cytosol is a specific and effective inhibitor of elastases.     

Detection of both type 1 and type 2 plasminogen activator inhibitors in human cells

This report describes the development and use of functional immunoradiometric assays that distinguish the activity of beta-migrating endothelial-type plasminogen activator inhibitor (PAI-1) from that of placental-type plasminogen activator inhibitor (PAI-2). These assays are based upon the binding of PAI-1 and PAI-2 to immobilized single-chain tissue-type plasminogen activator (tPA) and to immobilized urokinase (UK), respectively. The extent of binding of each PAI is quantified by incubating the PAI-PA complex first with rabbit antiserum specific for the individual PAI and then with 125I-labeled goat antirabbit IgG. In control experiments, the assays were shown to be sensitive, dose-dependent over a wide range, and specific for each PAI. These assays were employed to establish the PAI profile of a variety of human cells. Neither PAI-1 nor PAI-2 could be detected in Bowes melanoma cells or in a renal adenocarcinoma cell line (ACHN), while the histiocytic lymphoma cell (U-937) produced only PAI-2. Five cell lines, including two that were previously shown to contain one or the other PAI (e.g., umbilical vein endothelial cells and a fibrosarcoma cell line, HT-1080) in fact contained both PAIs. The cells containing both PAIs were studied in more detail. In each case, SDS treatment of CM was shown to enhance PAI-1 activity (by converting the latent form of this inhibitor into its active form) and to destroy PAI-2 activity. Various compounds including interleukin 1, dexamethasone, and phorbol myristate acetate were found to selectively influence the cellular production of one PAI without concomitantly affecting the production of the other, suggesting that the synthesis of these inhibitors is not coordinately regulated.     

Dexamethasone induces the expression of the mRNA of lipocortin 1 and 2 and the release of lipocortin 1 and 5 in differentiated, but not undifferentiated U-937 cells.

The effect of dexamethasone on mRNA and protein synthesis of lipocortins (LCT) 1, 2 and 5 has been investigated in U-937 cells. A constitutive expression of both mRNAs and proteins was detected in undifferentiated U-937 cells. This constitutive level was increased time- and dose-dependently by incubation with phorbol myristate acetate (PMA). In U-937 cells differentiated by 24 h incubation with 6 ng/ml PMA, dexamethasone (DEX) (1 microM for 16 h) caused an increased synthesis of the mRNA level of LCT-1 and 2, but not of LCT-5, over the level induced by PMA. DEX had no effect in undifferentiated cells. Moreover, DEX stimulated the extracellular release of LCT-1 and 5, but not of LCT-2, and inhibited the release of PGE2 and TXB2 only in the differentiated U-937 cells. These results suggest that the responsiveness of these cells to glucocorticoids is dependent on the phase of cell differentiation. The selective release of lipocortins by differentiated U-937 cells may explain, at least in part, the inhibition by DEX of the prostanoid release.     

cDNA for Mo3, a monocyte activation antigen, encodes the human receptor for urokinase plasminogen activator.

We have cloned the cDNA for Mo3, an activation Ag expressed by human monocytes and myelomonocytic cell lines after stimulation by PMA, LPS, muramyl dipeptide, certain cytokines, and cAMP agonists. We have previously shown that Mo3 expression in vivo is associated predominantly with macrophages in inflammatory sites. Mo3 is a highly glycosylated protein of about 50 kDa in monocytes and U-937 cells and is anchored to the plasma membrane by glycosyl-phosphatidylinositol linkage. We purified Mo3 protein by cleavage from the U-937 cell surface with phosphatidylinositol-specific phospholipase C, followed by affinity chromatography using a mAb. An internal peptide sequence was determined and used to design oligonucleotide probes for screening an expression cDNA library. Nucleotide sequencing indicated that the complete coding sequence encodes 335 amino acids, including a predicted signal peptide of 22 residues and a hydrophobic C-terminal portion that is probably cleaved during formation of the GPI linkage. The resulting mature protein of about 290 amino acids is consistent with the 29-kDa molecular mass of deglycosylated Mo3. A Northern blot of RNA from U-937 cells revealed a 1.5-kb band that was induced by PMA treatment. Mo3 cDNA was transfected into Cos cells and surface expression of Mo3 was detected by ELISA using various anti-Mo3 mAb. We performed a computer search of the National Biomedical Research Foundation database and found that Mo3 is identical to the human receptor for the urokinase plasminogen activator (uPA-R). Purified soluble Mo3, as well as anti-Mo3 antibodies, were able to block uPA binding to its receptor on U-937 cells, indicating that Mo3 is indeed uPA-R. The use of these anti-Mo3 antibodies may be helpful in assessing the role of uPA-R in processes such as inflammation and tumor invasion.     

pH-regulated anion antiport in nucleated mammalian cells

The uptake of 36Cl- into cells was measured after preincubation in medium containing nigericin and KCl to allow control of the intracellular pH. When the pH was increased from pH 7.0 to pH 7.3 there was a 10-fold increase in the rate of 36Cl- uptake. The increase was half maximal at pH 7.15 in Vero and L-cells, whereas in phorbol 12-myristate 13-acetate-treated Vero cells the increase was half maximal at pH 6.9. Kinetic studies showed that in cells preincubated with nigericin and isotonic KCl, both at pH 7.0 and at pH 8.0, the Km for Cl- was 7 mM. In the two cases the Jmax was 1.7 X 10(8) Cl- ions X cell-1 X s-1 and 1.6 X 10(9) Cl- ions X cell-1 X s-1, respectively. Bicarbonate inhibited 36Cl- uptake with a Ki of 5-6 mM. Probably, the anion antiporter plays a role in the regulation of the intracellular pH.     

Structure-function studies of the SERPIN plasminogen activator inhibitor type 1. Analysis of chimeric strained loop mutants.

Three chimeric mutants of plasminogen activator inhibitor 1 (PAI-1) have been constructed where the strained loop of wild type PAI-1 (wtPAI-1) has been replaced with a 19-amino acid region from either plasminogen activator inhibitor 2 (PAI-2), antithrombin III, or with an artificial serine protease inhibitor superfamily consensus strained loop. The inhibitors were expressed in Escherichia coli, and the purified proteins had specific activities toward urokinase-type plasminogen activator (uPA) or the single- and two-chain forms of tissue type plasminogen activator (tPA) that were similar to wtPAI-1. Experiments suggest that the strained loop of PAI-1 is not responsible for the transition between the latent and the active conformations or for binding to vitronectin. Second-order rate constants for the interactions with uPA and single- or two-chain tPA were similar to those of wtPAI-1. Values range from a low of 1.8 x 10(5) M-1 s-1 for the interaction of the PAI-2 chimera with single-chain tPA to a high value of 1.6 x 10(7) M-1 s-1 for the consensus mutant with two-chain tPA. This former value is 200 times higher than the reported rate constant for the interaction between PAI-2 and single-chain tPA, suggesting that structures outside of the strained loop are responsible for the major differences in specificity between PAI-1 and PAI-2.