Functional role of proteolytic cleavage at arginine-275 of human tissue plasminogen activator as assessed by site-directed mutagenesis

Activation of the zymogen form of a serine protease is associated with a conformational change that follows proteolysis at a specific site. Tissue-type plasminogen activator (t-PA) is homologous to mammalian serine proteases and contains an apparent activation cleavage site at arginine-275. To clarify the functional consequences of cleavage at arginine-275 of t-PA, site-specific mutagenesis was performed to convert arginine-275 to a glutamic acid. The mutant enzyme (designated Arg-275----Glu t-PA) could be converted to the two-chain form by Staphylococcus aureus V8 protease but not by plasmin. The one-chain form was 8 times less active against the tripeptide substrate H-D-isoleucyl-L-prolyl-L-arginine-p-nitroanilide (S-2288), and the ability of the enzyme to activate plasminogen in the absence of fibrinogen was reduced 20-50 times compared to the two-chain form. In contrast, one-chain Arg-275----Glu t-PA has equal activity to the two-chain form when assayed in the presence of physiological levels of fibrinogen and plasminogen. Fibrin bound significantly more of the one-chain form of t-PA than the two-chain form for both the wild-type and mutated enzymes. One- and two-chain forms of the wild-type and mutated plasminogen activators slowly formed complexes with plasma protease inhibitors, although the one-chain forms showed decreased complex formation with alpha 2-macroglobulin. The one-chain form of t-PA therefore is fully functional under physiologic conditions and has an increased fibrin binding compared to the two-chain form.     

Plasminogen activators catalyse conversion of inhibitor from fibrosarcoma cells to an inactive form with a lower apparent molecular mass

Purified approximately 54 kDa plasminogen activator inhibitor from human fibrosarcoma cells was converted to an inactive form with slightly higher electrophoretic mobility by incubation with catalytic amounts of urokinase-type or tissue-type plasminogen activator. Serine proteinase inhibitors and a monoclonal antibody against urokinase-type plasminogen activator inhibited the conversion, indicating that it was caused by plasminogen activator-catalyzed proteolysis. These findings represent the first demonstration of a well-defined protein apart from plasminogen, constituting a substrate for plasminogen activators.     

One-chain urokinase-type plasminogen activator from human sarcoma cells is a proenzyme with little or no intrinsic activity

We have compared the plasminogen activating capacity of one- and two-chain urokinase-type plasminogen activator (u-PA). In a 125I-plasminogen conversion assay in the presence of high amounts of a plasmin inhibitor, one-chain u-PA pretreated with diisopropyl fluorophosphate had no detectable activity, the detection limit corresponding to the activity of a 400-fold lower amount of two-chain u-PA. In coupled assays in which generated plasmin was measured with a synthetic substrate, activity was clearly observed with the one-chain preparation, but the initial rate of plasminogen activation was lower than that of a 250-fold smaller concentration of two-chain u-PA. The coupled assays for one-chain u-PA are self-activating because plasmin catalyzes conversion of one- to two-chain u-PA, and it is not possible to decide whether the low activity of one-chain u-PA observed with this type of assay is intrinsic or due to contaminations. On the basis of these findings and a discussion of previous studies, it is concluded that one-chain u-PA has a variety of properties similar to the one-chain proenzyme forms of other serine proteases and that it should, therefore, be considered as a genuine proenzyme form of u-PA.     

Proteolytically induced variations in the enzymatic properties of tissue plasminogen activator. Activations, inactivations and reactivations

Tissue plasminogen activator was treated with Sepharose-bound trypsin or chymotrypsin. Trypsin rapidly converted the one-chain activator to the two-chain form. This caused a marked increase in the amidolytic activity, while plasminogen activation initially increased but then decreased again. SDS/polyacrylamide gel electrophoresis in combination with [3H]diisopropylfluorophosphate active-site labeling revealed that after the conversion to the two-chain activator a minor cleavage occurred in the B chain, while the A chain was substantially degraded. Chymotrypsin caused a marked decrease in both amidolytic activity and plasminogen activation. SDS/polyacrylamide gel electrophoresis under reducing conditions revealed that two pairs of new bands had appeared, with Mr or about 50,000/52,000 and 17,000/20,000 respectively. N-terminal sequence analysis identified cleavage sites at peptide bonds 420-421 and 423-424. These bonds are located in a region of the activator which is homologues to the segments of trypsin and chymotrypsin, where autocatalytic cleavages occur during their activations. However, treatment of two-chain activator with chymotrypsin had markedly less effect on plasminogen activation and amidolytic activity. By treatment of samples of chymotrypsin-digested one-chain activator with plasmin, amidolytic activity could be largely restored. Thus, chymotrypsin may, by cleaving bonds 420-421 and 423-424, convert the active one-chain activator into an 'inactive' zymogen, which is again 'activated' by plasmin cleavage.     

Characterization of a modified human tissue plasminogen activator comprising a kringle-2 and a protease domain

To study structure/function relationships of tissue plasminogen activator (t-PA) activity, one of the simplest modified t-PA structures to activate plasminogen in a fibrin-dependent manner was obtained by constructing an expression vector that deleted amino acid residues 4-175 from the full-length sequence of t-PA. The expression plasmid was introduced into a Syrian hamster cell line, and stable recombinant transformants, producing high levels of the modified plasminogen activator, were isolated. The resulting molecule, mt-PA-6, comprising the second kringle and serine protease domains of t-PA, produced a doublet of plasminogen activator activity having molecular masses of 40 and 42 kDa. The one-chain mt-PA-6 produced by cultured Syrian hamster cells was purified in high yield by affinity and size exclusion chromatography. The purified mt-PA-6 displayed the same two types of microheterogeneity observed for t-PA. NH2-terminal amino acid sequencing demonstrated that one-chain mt-PA-6 existed in both a GAR and a des-GAR form. Purified mt-PA-6 also existed in two glycosylation forms that accounted for the 40- and 42-kDa doublet of activity produced by the cultured Syrian hamster cells. Separation of these two forms by hydrophobic interaction chromatography and subsequent tryptic peptide mapping demonstrated that both forms contained N-linked glycosylation at Asn448; in addition, some mt-PA-6 molecules were also glycosylated at Asn184. Plasmin treatment of one-chain mt-PA-6 converted it to a two-chain molecule by cleavage of the Arg275-Ile276 bond. This two-chain mt-PA-6, like t-PA, had increased amidolytic activity. The fibrinolytic specific activities of the one- and two-chain forms of mt-PA-6 were similar and twice that of t-PA. The plasminogen activator activity of one-chain mt-PA-6 was enhanced greater than 80-fold by CNBr fragments of fibrinogen, and the one-chain enzyme lysed human clots in vitro in a dose-dependent manner. The ability to produce and purify a structurally simple plasminogen activator with desirable fibrinolytic properties may aid in the development of a superior thrombolytic agent for the treatment of acute myocardial infarction.     

Proteolytic cleavage of single-chain pro-urokinase induces conformational change which follows activation of the zymogen and reduction of its high affinity for fibrin

A plasminogen activator secreted from human kidney cells was highly purified by affinity chromatography on an anti-urokinase IgG-Sepharose column. The purified plasminogen activator was inactive and had a single-chain structure and a Mr of 50,000. It not only did not incorporate diisopropyl fluorophosphate, which reacts with active site serine residue in urokinase, but also did not bind to p-aminobenzamidine-immobilized CH-Sepharose, to which urokinase bind via its side-chain binding pocket present in active center. The plasminogen activator was converted to the active two-chain form with the same Mr by catalytic amounts of plasmin. Its potential enzymatic activity was quenched completely by anti-urokinase IgG, but not by anti-tissue plasminogen activator Ig. These results indicate that the plasminogen activator is an inactive proenzyme form of human urokinase. Therefore, the plasminogen activator was termed single-chain pro-urokinase. The cleavage of single-chain pro-urokinase by plasmin induced conformational change which followed the generation of reactive serine residue at active site, the increase enzyme activity and the reduction of its high affinity for fibrin. These findings suggest that conformational change occurs in both regions responsible for enzyme activity and affinity for fibrin upon activation of single-chain pro-urokinase.     

Monoclonal antibody to human 66,000 molecular weight plasminogen activator from melanoma cells. Specific enzyme inhibition and one-step affinity purification

Hybridomas producing a monoclonal IgG1 antibody to a human plasminogen-activating enzyme with an apparent mol. wt. of 66,000 (66 K, HPA66) from human melanoma cells were obtained by fusion of NSI-Ag 4/1 mouse myeloma cells with spleen cells from a mouse immunized with a partially purified preparation of the enzyme. Screening for clones of hybridomas producing antibodies to HPA66 was performed with the impure enzyme preparation. A preliminary screening included enzyme-linked immunosorbent assay and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting; the final identification was based on inhibition of the enzymatic activity of HPA66 which was complete at high antibody concentrations. No inhibition of three other human and murine plasminogen activators or of plasmin was observed. Employing a one-step affinity procedure with the antibody coupled to Sepharose, HPA66 was purified approximately 200-fold from conditioned medium from the melanoma cells with a yield of 79%. The purified HPA66 was homogeneous as evaluated by SDS-PAGE. Electrophoresis under reducing conditions indicated that it consisted of one polypeptide chain. The binding constant between the antibody and 125I-labelled HPA66 was approximately 2.5 x 10(9) l/mol. The antibody did not bind to a variety of other plasminogen activators, including 52-K and 36-K human enzymes and 48-K and 75-K murine enzymes. Previously, a monoclonal antibody against another enzyme was derived by the sole use of enzyme inhibition for screening. The present study represents a modification of this procedure that can be used when antibody-unrelated inhibitors of the enzyme are present in hybridoma culture fluid.     

The effect of polymerised fibrin on the catalytic activities of one-chain tissue-type plasminogen activator as revealed by an analogue resistant to plasmin cleavage

A one-chain recombinant tissue-type plasminogen activator (EC 2.4.31.-) (tPA) analogue was constructed in which Arg-275 of the activation site was changed to Gly by site-directed mutagenesis. This analogue, tPA-Gly275, was very resistant to plasmin (EC 2.4.21.5) cleavage. It has been used to gain information about the activity of the uncleaved one-chain tPA form, also when plasmin is generated as a result of a plasminogen activation reaction. The amidolytic activity of tPA-Gly275 with less than Glu-Gly-Arg-pNA was investigated and compared to that of one-chain and two-chain wild-type recombinant tPA. A small but significant intrinsic amidolytic activity was observed with the analogue as well as the wild-type one-chain tPA form. However, it was much lower than that of two-chain tPA. Polymerised fibrin enhanced the amidolytic activity of both one-chain tPA forms but not of two-chain tPA. Measurements of the plasminogen activation kinetics in the absence of fibrin revealed that tPA-Gly275 possessed a significant intrinsic activity. However, it was 30-fold lower than that of two-chain tPA. Addition of polymerised fibrin profoundly enhanced the plasminogen activation rate of both tPA-Gly275 and wild-type one- and two-chain tPA to approximately the same maximal level. The results were interpreted to mean that fibrin binding can induce an activated state of the intact tPA one-chain form.     

Effects of intact fibrin and partially plasmin-degraded fibrin on kinetic properties of one-chain tissue-type plasminogen activator

A comparative kinetic analysis of the enzymatic activities of one-chain and two-chain tissue-type plasminogen activator (t-PA) demonstrates that two-chain t-PA catalyzes the hydrolysis of the peptide substrate D-Val-Leu-Arg-pNA about 4-fold more effectively than one-chain t-PA. The difference is accounted for almost entirely by a corresponding difference is the kcat values of the enzymes, whereas the Km values are similar. The amidolytic activity of two-chain t-PA is not enhanced by intact or partially plasmin-degraded fibrin. In contrast, the activity of one-chain t-PA is stimulated up to 3.7-fold by intact fibrin and up to 4.7-fold by plasmin-degraded fibrin (fibrin X-fragment). The stimulatory effects are realized via increases in the kcat values. It appears thus that in the presence of fibrin the intrinsically inferior catalytic properties of one-chain t-PA become similar to the properties of two-chain t-PA. The dependency of the activity of one-chain t-PA on the concentration of fibrin monomer is consistent with a single association site of both proteins and an association constant of Kass = 6.25 x 10(6) l/mol. Stimulation of one-chain t-PA by plasmin-degraded fibrin is more complex and appears to involve two different binding sites with association constants of Kass = 0.67 x 10(9) l/mol and Kass = 3.85 x 10(6) l/mol, respectively. The stimulatory effects of fibrin and partially plasmin-degraded fibrin on one-chain t-PA are suppressed by epsilon-aminocaproic acid and by a monoclonal antibody directed against the lysine binding site of t-PA. The latter findings support the notion that fibrin activation of one-chain t-PA is mediated by the lysine binding site on kringel domains of the enzyme.     

A single-step separation of the one- and two-chain forms of tissue plasminogen activator

Preparations of one-chain tissue plasminogen activator (tPA), usually a mixture of 65- and 63-kDa differentially glycosylated forms, contain variable amounts of two-chain tPA. There is no effective procedure currently available for removal of the two-chain contaminant from one-chain tPA preparations. In this report, affinity chromatography on benzamidine-Sepharose was investigated for the separation of two-chain from one-chain tPA. Activase, a preparation of recombinant tPA containing 80% one-chain tPA, a mixture of 65- and 63-kDa variants, and 20% two-chain tPA, was applied to a column of benzamidine-Sepharose, equilibrated with 1 M ammonium bicarbonate. Under this condition, both one-chain and two-chain forms of tPA were adsorbed by the column. Addition of 0.1 M arginine to the equilibration buffer led to elution of two-peaks, corresponding to the 65- and 63-kDa variants of one-chain tPA. Two-chain tPA remained bound to the column, but could be eluted with sodium acetate buffer, pH 4.0, containing 0.1 M arginine. The present procedure allows rapid and effective removal of two-chain tPA with concomitant separation of 65- and 63-kDa one-chain glycoforms from preparations of one-chain tPA. Kinetic analysis for the hydrolysis of D-Ile-Pro-Arg-p-nitroanilide (S-2288) by the highly purified molecular forms of tPA suggests that 63-kDa one-chain tPA possesses 30% higher catalytic efficiency than the 65-kDa variant, while two-chain tPA is 9- or 12-fold more efficient than 63- or 65-kDa one-chain tPA, respectively.     

The single-chain form of tissue-type plasminogen activator has catalytic activity: studies with a mutant enzyme that lacks the cleavage site

Tissue-type plasminogen activator (t-PA), the serine protease responsible for catalyzing the production of plasmin from plasminogen at the site of blood clots, is synthesized as a single-chain polypeptide precursor. Proteolytic cleavage at the C-terminal side of Arg275 generates a two-chain form of the enzyme whose subunits are held together by a single disulfide bond. We have measured the activities of both forms of the wild-type enzyme, as well as that of a mutant enzyme (Arg275----Gly), created by oligonucleotide-directed mutagenesis, that cannot be cleaved into a two-chain form. Both types of single-chain t-PAs are enzymatically active and exhibit identical Vmax and Km values when assayed with synthetic peptide substrates, indicating that the single amino acid change had no effect on the amidolytic activity of the enzyme. However, cleavage of wild-type t-PA into the two-chain form results in increased activity both on a peptide substrate and on the natural substrates Lys- and Glu-plasminogen in the absence or presence of stimulation by soluble fibrin. The enhanced activity is due to a 3-5-fold increase in the Vmax of the cleaved enzyme, rather than to any change in the Km values for the various substrates. During incubation with plasminogen, the single-chain form of wild-type t-PA is converted to the two-chain form by plasmin generated during the reaction. This conversion, from the less active form of the enzyme, results in a reaction that displays biphasic kinetics.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and interrelationships of the multiple molecular tissue-type and urokinase-type plasminogen activator forms produced by cultured human umbilical vein endothelial cells

Primary and early subcultures (1st- to 3rd passage) of human umbilical vein endothelial cells produce tissue-type plasminogen activator (t-PA) antigen, consisting only of a major Mr 110,000 t-PA form. Later subcultures (greater than 4th passage) produce increasing amounts of t-PA antigen, consisting of a major Mr 110,000 and a minor Mr 68,000 form as well as increasing amounts of urokinase-type plasminogen activator (u-PA) antigen, consisting of a minor Mr 95,000 and major Mr 54,000 form. All of the major plasminogen activator forms were purified to homogeneity from 72 h serum-free conditioned media (3 liters, 1-1.8 x 10(9) cells) by a combination of immunoaffinity and gel filtration chromatography. Typically, 4th to 6th passage cultures produced/secreted t-PA-type proteins consisting of an inactive Mr 110,000 (220 IU/mg) and active Mr 68,000 (76,500 IU/mg) form representing about 39 and 8%, respectively, of the total starting sodium dodecyl sulfate stable t-PA activity, and u-PA-type proteins consisting of an inactive Mr 95,000 (700 IU/mg) and active Mr 54,000 (81,000 IU/mg) form representing about 9 and 38%, respectively, of the total starting sodium dodecyl sulfate stable u-PA activity. The isolated Mr 68,000 t-PA and Mr 54,000 u-PA proteins, exist only as two-chain forms in the absence of aprotinin and as mixtures of single- and two-chain proteins in the presence of aprotinin. Treatment with nucleophilic agents completely dissociated the Mr 110,000 t-PA and Mr 95,000 u-PA proteins into their respective Mr 68,000 t-PA and Mr 54,000 u-PA activity forms and a common Mr 46,000 protein, confirming the enzyme-inhibitor complex nature of these inactive plasminogen activator forms.     

Activation with plasmin of tow-chain urokinase-type plasminogen activator derived from single-chain urokinase-type plasminogen activator by treatment with thrombin

Thrombin converts single-chain urokinase-type plasminogen activator (scu-PA) to an inactive two-chain derivative (thrombin-derived tcu-PA) by hydrolysis of the Arg-156--Phe-157 peptide bond. In the present study, we show that inactive thrombin-derived tcu-PA (specific activity 1000 IU/mg) can be converted with plasmin to active two-chain urokinase-type plasminogen activator (specific activity 43,000 IU/mg) by hydrolysis of the Lys-158--Ile-159 peptide bond. This conversion follows Michaelis-Menten kinetics with a Michaelis constant Km of 37 microM and a catalytic rate constant k2 of 0.013 s-1. The catalytic efficiency (k2/Km) for the activation of thrombin-derived tcu-PA by plasmin is about 500-fold lower than that for the conversion of intact scu-PA to tcu-PA. tcu-PA, generated by plasmin treatment of thrombin-derived tcu-PA, has similar properties to tcu-PA obtained by digestion of intact scu-PA with plasmin (plasmin-derived tcu-PA); its plasminogen activating potential and fibrinolytic activity in an in vitro plasma clot lysis system appear to be unaltered. These observations confirm that the structure of the NH2-terminal region of the B chain of u-PA is an important determinant for its enzymatic activity, whereas that of the COOH-terminal region of the A chain is not.     

Plasminogen and tissue-type plasminogen activator bind to immobilized fibronectin

Fibronectin immobilized onto polystyrene surface was found to bind plasminogen and tissue-type plasminogen activator (t-PA) but only slightly the urokinase type as determined using mono- and polyclonal antibodies against the activators. Of the defined fibronectin fragments tested, the Mr 120,000-140,000 fragment was found to bind both plasminogen and t-PA. Proteolytically modified plasminogen (Lys-plasminogen) bound considerably better than the native form (Glu-plasminogen). Experiments with 125I-plasminogen yielded Kd = 9.1 X 10(-8) M for the binding to immobilized fibronectin. The partially or completely inactive single-chain form of t-PA (pro-t-PA) bound considerably better than the activated two-chain form. Lysine at greater than 3 mM inhibited the binding of plasminogen. The interaction was independent of calcium ions. CaCl2 (greater than 0.5 mM) and NaCl (greater than 0.2 M) inhibited the binding of pro-t-PA and of t-PA. Fibronectin-bound t-PA retained its ability to activate plasminogen. The observed interactions may operate in directional proteolysis localizing plasminogen and plasminogen activator to degrade fibronectin-containing extracellular matrix including fibrin clots.     

Kinetics of inhibition of tissue-type and urokinase-type plasminogen activator by plasminogen-activator inhibitor type 1 and type 2

Highly purified plasminogen-activator inhibitors of type 1 (PAI-1) and type 2 (PAI-2), low-Mr form, were compared with respect to their kinetics of inhibition of tissue-type (t-PA) and urokinase-type plasminogen activator (u-PA). The time course of inhibition of plasminogen activator was studied under second-order or pseudo-first-order conditions. Residual enzyme activity was measured by the initial rate of hydrolysis of a chromogenic t-PA or u-PA substrate or by an immunosorbent assay for t-PA activity. PAI-1 rapidly reacted with single-chain t-PA as well as with two-chain forms of t-PA and u-PA. The second-order rate constant k for inhibition of single-chain t-PA (5.5 x 10(6) M-1 s-1) was about three times lower than k for inhibition of the two-chain activators. PAI-2 reacted slowly with single-chain t-PA, k = 4.6 x 10(3) M-1 s-1. The association rate was 26 times higher with two-chain t-PA and 435 times higher with two-chain u-PA. The k values for inhibition of single-chain t-PA, two-chain t-PA and two-chain u-PA were respectively, 1200, 150 and 8.5 times higher with PAI-1 than with PAI-2. The removal of the epidermal growth factor domain and the kringle domain from two-chain u-PA did not affect the kinetics of inhibition of the enzyme, suggesting that the C-terminal proteinase part of u-PA (B chain) is responsible for both the primary and the secondary interactions with PAI-1 and PAI-2. The k values for inhibition of single-chain t-PA and endogenous t-PA in plasma by PAI-1 or PAI-2 were identical indicating that t-PA in blood consists mainly in its single-chain form.     

The incorporation of a fluorescent probe into the active sites of one- and two-chain tissue-type plasminogen activator

Dansyl-glutamyl-glycyl-arginyl chloromethyl ketone (DEGR-CK) was shown to inactivate both one- and two-chain human, recombinant tissue-type plasminogen activator (t-PA). The interaction of DEGR-CK with both forms of t-PA was accompanied by an identical increase in the fluorescence intensity and a blue shift in the wavelength of maximum emission, which suggests that the environment of the incorporated DEGR is similar in both one- and two-chain t-PA. The kinetics of the interaction of t-PA with DEGR-CK could be followed by both loss of activity and increase in fluorescence. The second order rate constants (k2/Ki) obtained with these two methods agreed quite well. With two-chain t-PA the values were 42 X 10(4) M-1 min-1 and 46 X 10(4) M-1 min-1 by the activity loss and fluorescence methods, respectively. With one-chain t-PA the results were 2.5 X 10(4) M-1 min-1 and 3.1 X 10(4) M-1 min-1. The rate at which one-chain t-PA is inactivated by DEGR-CK is 15 times lower than the rate with two-chain t-PA. The results demonstrated, however, that the cleavage of the one-chain protein to the two-chain form is not required for reactivity with DEGR-CK. This fluorescently labeled t-PA should be useful in probing the interactions of one- and two-chain t-PA with other proteins.     

The mechanism of the reaction between human plasminogen-activator inhibitor 1 and tissue plasminogen activator.

The structural events taking place during the reaction between PAI-1 (plasminogen-activator inhibitor 1) and the plasminogen activators sc-tPA (single-chain tissue plasminogen activator) and tc-tPA (two-chain tissue plasminogen activator) were studied. Complexes were formed by mixing sc-tPA or tc-tPA with PAI-1 in slight excess (on an activity basis). The complexes were purified from excess PAI-1 by affinity chromatography on fibrin-Sepharose. Examination of the purified complexes by SDS/polyacrylamide-gel electrophoresis (SDS/PAGE) and N-terminal amino acid sequence analysis demonstrated that a stoichiometric 1:1 complex is formed between PAI-1 and both forms of tPA. Data obtained from both complexes revealed the amino acid sequences of the parent molecules and, in addition, a new sequence: Met-Ala-Pro-Glu-Glu-. This sequence is found in the C-terminal portion of the intact PAI-1 molecule and thus locates the reactive centre of PAI-1 to Arg346-Met347. The proteolytic activity of sc-tPA is demonstrated by its capacity to cleave the 'bait' peptide bond in PAI-1. The complexes were inactive and dissociated slowly at physiological pH and ionic strength, but rapidly in aq. NH3 (0.1 mol/l). Amidolytic tPA activity was generated on dissociation of the complexes, corresponding to 0.4 mol of tPA/mol of complex. SDS/PAGE of the dissociated complexes indicated a small decrease in the molecular mass of PAI-1, in agreement with proteolytic cleavage of the 'bait' peptide bond during complex-formation.     

immunocytochemical localization of urokinase-type plasminogen activator in lewis lung carcinoma

The invasively growing and metasizing Lewis lung carcinoma consistently contained urokinase-type plasminogen activator (u-PA) enzyme activity. When investigated immunocytochemically with antibodies against u-PA, different parts of individual tumors showed a pronounced heterogeneity in staining intensity. Strong staining was found in areas with invasive growth and degradation of surrounding normal tissue, while other areas were completely devoid of staining. Immunoreactivity occurred both with a perinuclear cytoplasmic localization in tumor cells and associated with apparently extracellular material. SDS PAGE of tumor extracts, under both reducing and nonreducing conditions, followed by immunoblotting, showed only one immunocytochemically stainable band with an electrophoretic mobility corresponding to that of purified proenzyme to u-PA, while no two-chain u-PA was detected. This indicates that the major part of the activator in Lewis lung carcinoma is present as one-chain pro-u-PA.     

Novel properties of human monocyte plasminogen activator

Human peripheral monocytes stimulated by either muramyl dipeptide [N-acetyl-muramoyl-L-alanyl-D-isoglutamine], bacterial lipopolysaccharide or lymphokine-containing supernatants of human lymphocytes, could be shown to produce and secrete appreciable activities of a 52 000-Mr plasminogen activator. This enzyme was suppressed in control and stimulated cultures by dexamethasone (0.1 microM). Monocyte plasminogen activator could only be assayed under conditions of low ionic strength and had no detectable activity at 0.15 M NaCl. Intracellular enzyme was present as a proenzyme, requiring activation by preincubation with plasminogen containing traces of plasmin, before its activity could be seen on sodium dodecyl sulphate/polyacrylamide gel electrophoresis by a fibrin overlay method. Secreted enzyme was in the active form. Further incubation of lysate or supernatant plasminogen activator with plasminogen did not produce any active enzyme species of Mr 36 000, unlike incubations of urokinase with plasminogen. Moreover, comparisons with other plasminogen activators of Mr 52 000 from transformed cell lines showed that the monocyte activator was unique in its resistance to monocyte minactivin, a specific inactivator of urokinase-type plasminogen activators, and in its sensitivity to human alpha 2-macroglobulin. It was therefore concluded that human monocyte plasminogen activator, although sharing an Mr of 52 000 in common with other such activators, is not identical to the high Mr form of urokinase or the plasminogen activators of transformed cells. On present evidence it is the least likely of these enzymes to be active extracellularly under normal physiological conditions.     

Purification of human tissue plasminogen activator with Erythrina trypsin inhibitor

Seeds of the legume Erythrina latissima contain a 20,000-dalton, single-chain protein that has been shown to inhibit the amidolytic activity of trypsin and tissue plasminogen activator. It had no comparable effect on urokinase. IC50 values of 1.1 X 10(-7) M for tissue plasminogen activator and 6.9 X 10(-10) M for trypsin were determined by titration. When coupled to agarose, the Erythrina inhibitor provided an effective reagent for affinity purification of tissue plasminogen activator from melanoma cell-conditioned tissue culture medium. Using this as a single-step procedure, 270-fold purified enzyme was reproducibly obtained with yields of 90% or greater. Both one- and two-chain forms of tissue plasminogen activator were purified. The enzyme migrated, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as a predominant 72,000-dalton doublet with lesser amounts of immunochemically similar, 115,000- and 68,000-dalton components.