Addition Of Phenylmethylsulfonyl Fluoride Research Articles

Low Concentration of the Neutrophil Proteases Cathepsin G, Cathepsin B, Proteinase-3 and Metalloproteinase-9 Induce Biofilm Formation in Non-Biofilm-Forming Staphylococcus epidermidis Isolates.

Neutrophils play a crucial role in eliminating bacteria that invade the human body; however, cathepsin G can induce biofilm formation in a non-biofilm-forming Staphylococcus epidermidis 1457 strain, suggesting that neutrophil proteases may be involved in biofilm formation. Cathepsin G, cathepsin B, proteinase-3, and metalloproteinase-9 (MMP-9) from neutrophils were tested on the biofilm induction in commensal (skin isolated) and clinical non-biofilm-forming S. epidermidis isolates. From 81 isolates, 53 (74%) were aap+, icaA−, icaD− genotype, and without the capacity of biofilm formation under conditions of 1% glucose, 4% ethanol or 4% NaCl, but these 53 non-biofilm-forming isolates induced biofilm by the use of different neutrophil proteases. Of these, 62.3% induced biofilm with proteinase-3, 15% with cathepsin G, 10% with cathepsin B and 5% with MMP -9, where most of the protease-induced biofilm isolates were commensal strains (skin). In the biofilm formation kinetics analysis, the addition of phenylmethylsulfonyl fluoride (PMSF; a proteinase-3 inhibitor) showed that proteinase-3 participates in the cell aggregation stage of biofilm formation. A biofilm induced with proteinase-3 and DNAse-treated significantly reduced biofilm formation at an early time (initial adhesion stage of biofilm formation) compared to untreated proteinase-3-induced biofilm (p < 0.05). A catheter inoculated with a commensal (skin) non-biofilm-forming S. epidermidis isolate treated with proteinase-3 and another one without the enzyme were inserted into the back of a mouse. After 7 days of incubation period, the catheters were recovered and the number of grown bacteria was quantified, finding a higher amount of adhered proteinase-3-treated bacteria in the catheter than non-proteinase-3-treated bacteria (p < 0.05). Commensal non-biofilm-forming S. epidermidis in the presence of neutrophil cells significantly induced the biofilm formation when multiplicity of infection (MOI) 1:0.01 (neutrophil:bacteria) was used, but the addition of a cocktail of protease inhibitors impeded biofilm formation. A neutrophil:bacteria assay did not induce neutrophil extracellular traps (NETs). Our results suggest that neutrophils, in the presence of commensal non-biofilm-forming S. epidermidis, do not generate NETs formation. The effect of neutrophils is the production of proteases, and proteinase-3 releases bacterial DNA at the initial adhesion, favoring cell aggregation and subsequently leading to biofilm formation.

The effects of PMSF and cysteine addition into partially purified bromelain from pineapple (Ananas comosus [L] Merr) cores to its kinetics behaviour

Bromelain found in various tissues in the pineapple plant (Ananas Comusus [L] Merr. The bromelain will be taken from pineapple cores for kinetic study. The effect of addition organic compounds, such as phenylmethylsulfonyl fluoride (PMSF) and cysteine on its kinetics behaviour will be determined by proteolytic activities of the bromelain. The stages of this study were started from isolation of crude enzyme, fractionation with ammonium sulphate salt, dialysis, and ion-exchange column chromatography with CM sephadex C-50 for enzyme purification. The obtained enzyme fractions from crude enzyme, ammonium sulphate, dialysis, and CM-sephadex C-50 ion exchange chromatography have specific activities of 45.39, 238.31, 311.16, and 427.13 U/mg, respectively. The purification stages result in increasing of the bromelain specific activities by 9 times compared to the crude enzyme. The Michaelis-Menten constant (Km) value for the purified bromelain was 0.85 % casein and the Vmax value was 0.41 U/min. Addition of PMSF and cysteine compound effect on its Vmax and Km with various trends. The Vmax value will be decreased by addition of PMSF and increased by addition of cysteine. Thus, proteolytic activity of bromelain from pineapple cores can be significantly increased by the addition of cysteine compounds and inhibited by the addition of PMSF.

Molecular cloning and characterization of a thermostable lipase from deep-sea thermophile Geobacillus sp. EPT9.

A gene (1,254 bp) encoding a lipase was identified from a deep-sea hydrothermal field thermophile Geobacillus sp. EPT9. The open reading frame of this gene encoded 417 amino acid residues. The gene was cloned, overexpressed in Escherichia coli, and the target protein was purified to homogeneity. The purified recombinant enzyme presented a molecular mass of 44.8 kDa. When p-nitrophenyl palmitate was used as a substrate, the recombinant lipase was optimally active at 55 °C and pH 8.5. The recombinant enzyme retained 44 % residual activity after incubation at 80 °C for 1 h, which indicated that Geobacillus sp. EPT9 lipase was thermostable. Homology modeling of strain EPT9 lipase was developed with the lipase from Bacillus sp. L2 as a template. The core structure exhibits an α/β-hydrolase fold and the typical catalytic triad might consist of Ser142, Asp346, and His387. The enzymatic activity of EPT9 lipase was inhibited by addition of phenylmethylsulfonyl fluoride, indicating that it contains serine residue, which plays an important role in the catalytic mechanism.

Characterization of a new and thermostable esterase from a metagenomic library

A new gene encoding an esterase (designated as EstEP16) was identified from a metagenomic library prepared from a sediment sample collected from a deep-sea hydrothermal field in east Pacific. The open reading frame of this gene encoded 249 amino acid residues. It was cloned, overexpressed in Escherichia coli, and the recombinant protein was purified to homogeneity. The monomeric EstEP16 presented a molecular mass of 51.7kDa. Enzyme assays using p-nitrophenyl esters with different acyl chain lengths as the substrates confirmed its esterase activity, yielding highest specific activity with p-nitrophenyl acetate. When p-nitrophenyl butyrate was used as a substrate, recombinant EstEP16 exhibited highest activity at pH 8.0 and 60°C. The recombinant enzyme retained about 80% residual activity after incubation at 90°C for 6h, which indicated that EstEP16 was thermostable. Homology modeling of EstEP16 was developed with the monoacylglycerol lipase from Bacillus sp. H-257 as a template. The structure showed an α/β-hydrolase fold and indicated the presence of a typical catalytic triad. The activity of EstEP16 was inhibited by addition of phenylmethylsulfonyl fluoride, indicating that it contains serine residue, which plays a key role in the catalytic mechanism.

Cloning and characterization of a thermostable carboxylesterase from inshore hot spring thermophile Geobacillus sp. ZH1

The gene (741 bp) encoding carboxylesterase from the thermophilic bacterium Geobacillus sp. ZHl was cloned and overexpressed in Escherichia coli. The purified recombinant protein presented a molecular mass of about 40 kDa by SDS-PAGE analysis. Enzyme assays using p-nitrophenyl esters with different acyl chain lengths as the substrates confirmed its esterase activity, yielding highest specific activity with p-nitrophenyl acetate. Among the p-nitrophenyl esters tested, the carboxylesterase presented preference for p-nitrophenyl caprylate, but hydrolyzed p-nitrophenyl butyrate more efficiently. When p-nitrophenyl butyrate was used as a substrate, the recombinant carboxylesterase exhibited highest activity at pH 8.0 and 60°C. Almost no decrease in esterase activity was observed at 60°C for 3 h, and over 40% of activity was still maintained after incubation at 90°C for 3 h. These results indicate that Geobacillus sp. ZH1 recombinant esterase was thermostable. The enzymatic activity was inhibited by the addition of phenylmethylsulfonyl fluoride, indicating that it contains serine residue, which plays a key role in the catalytic mechanism. Except SDS and xylene, this esterase showed stability toward other tested detergents and organic solvents. Cloning, expression, and biochemical characterization of Geobacillus sp. ZH1 carboxylesterase lay a good foundation for its structural characterization and industrial application.

Structure of the Novel C-terminal Domain of Vacuolar Protein Sorting 30/Autophagy-related Protein 6 and Its Specific Role in Autophagy

Vacuolar protein sorting 30 (Vps30)/autophagy-related protein 6 (Atg6) is a common component of two distinct phosphatidylinositol 3-kinase complexes. In complex I, Atg14 links Vps30 to Vps34 lipid kinase and exerts its specific role in autophagy, whereas in complex II, Vps38 links Vps30 to Vps34 and plays a crucial role in vacuolar protein sorting. However, the molecular role of Vps30 in each pathway remains unclear. Here, we report the crystal structure of the carboxyl-terminal domain of Vps30. The structure is a novel globular fold comprised of three β-sheet-α-helix repeats. Truncation analyses showed that the domain is dispensable for the construction of both complexes, but is specifically required for autophagy through the targeting of complex I to the pre-autophagosomal structure. Thus, the domain is named the β-α repeated, autophagy-specific (BARA) domain. On the other hand, the N-terminal region of Vps30 was shown to be specifically required for vacuolar protein sorting. These structural and functional investigations of Vps30 domains, which are also conserved in the mammalian ortholog, Beclin 1, will form the basis for studying the molecular functions of this protein family in various biological processes.

A Survey of Antiprion Compounds Reveals the Prevalence of Non-PrP Molecular Targets

Prion diseases are fatal neurodegenerative diseases caused by the accumulation of the misfolded isoform (PrP(Sc)) of the prion protein (PrP(C)). Cell-based screens have identified several compounds that induce a reduction in PrP(Sc) levels in infected cultured cells. However, the molecular targets of most antiprion compounds remain unknown. We undertook a large-scale, unbiased, cell-based screen for antiprion compounds and then investigated whether a representative subset of the active molecules had measurable affinity for PrP, increased the susceptibility of PrP(Sc) to proteolysis, or altered the cellular localization or expression level of PrP(C). None of the antiprion compounds showed in vitro affinity for PrP or had the ability to disaggregate PrP(Sc) in infected brain homogenates. These observations suggest that most antiprion compounds identified in cell-based screens deploy their activity via non-PrP targets in the cell. Our findings indicate that in comparison to PrP conformers themselves, proteins that play auxiliary roles in prion propagation may be more effective targets for future drug discovery efforts.

Stability of Pro-Gastrin-Releasing Peptide in Serum versus Plasma

Background/Aims: Although serum assays for pro-gastrin-releasing peptide (ProGRP) assays have been commercially available in Japan for several years, the stability of ProGRP in serum and plasma has not been well documented. We investigated the stability of ProGRP in serum and plasma with fresh and stored (frozen) specimens, as well as the cause of the observed instability in serum. Methods/Results: ProGRP concentrations in fresh serum were decreased by 6–28% after room temperature storage for 2 h and by 8–32% after 2–8°C storage for 24 h. The average change in ProGRP concentrations in fresh plasma was within ±10% of baseline for more than 4 h at room temperature and for more than 24 h at 2–8°C. The incubation of a serine protease, thrombin (activated blood coagulation factor II), in a buffer solution containing ProGRP caused decreases in ProGRP concentrations. Following the addition of phenylmethylsulfonyl fluoride, a serine protease inhibitor, to serum, the serum stability for ProGRP was similar to that in plasma. ProGRP is significantly more stable in plasma than in serum. We speculate that thrombin in serum is one of the factors that inactivate ProGRP in serum by proteolysis of the ProGRP antigen. Conclusion: The use of plasma samples for ProGRP may improve the clinical reliability of this marker by minimizing preanalytical changes in ProGRP concentrations.

IsdA ProtectsStaphylococcus aureusagainst the Bactericidal Protease Activity of Apolactoferrin

An important facet of the Staphylococcus aureus host-pathogen interaction is the ability of the invading bacterium to evade host innate defenses, particularly the cocktail of host antimicrobial peptides. In this work, we showed that IsdA, a surface protein of S. aureus which is required for nasal colonization, binds to lactoferrin, the most abundant antistaphylococcal polypeptide in human nasal secretions. The presence of IsdA on the surface of S. aureus confers resistance to killing by lactoferrin. In addition, the bactericidal activity of lactoferrin was inhibited by addition of phenylmethylsulfonyl fluoride, implicating the serine protease activity of lactoferrin in the killing of S. aureus. Recombinant IsdA was a competitive inhibitor of lactoferrin protease activity. Reciprocally, antibody reactive to IsdA enhanced killing of S. aureus. Thus, IsdA can protect S. aureus against lactoferrin and acts as a protease inhibitor.

Characterization of a thermostable carboxylesterase from the hyperthermophilic bacterium Thermotoga maritima

The gene encoding carboxylesterase from the hyperthermophilic bacterium Thermotoga maritima (tm0053) was cloned. The recombinant protein (EST53) was overexpressed in Escherichia coli without its NH(2)-terminal hydrophobic region, and with a C-terminal hexahistidine sequence. The enzyme was purified to homogeneity by heat treatment, followed by Ni(2+) affinity chromatography, and then characterized. Among the p-nitrophenyl esters tested, the best substrate was p-nitrophenyl decanoate with K (m) and k (cat) values of 3.1 muM and 10.8 s(-1), respectively, at 60 degrees C and pH 7.5. The addition of O,O'-bis(2-aminoethyl)ethyleneglycol-N,N,N',N'-tetraacetic acid decreased the esterase activity, indicating that EST53 is dependent on the presence of Ca(2+) ion. In addition, its activity was inhibited by the addition of phenylmethylsulfonyl fluoride and diethyl pyrocarbonate, indicating that it contains serine and histidine residues, which play key roles in the catalytic mechanism. EST53 shows a relatively high degree of similarity to Burkholderia lipases that belong to family I.2 of the lipolytic enzymes, whereas the local sequence around the pentapeptide of EST53 is most similar to those of Bacillus lipases belonging to family I.4.

Heparin Binding Induces a Conformational Change in Pigment Epithelium-derived Factor

Pigment epithelium-derived factor (PEDF) is a noninhibitory serpin found in plasma and in the extracellular space. The protein is involved in different biological processes including cell differentiation and survival. In addition, it is a potent inhibitor of angiogenesis. The function is likely associated with binding to cell surface receptors in a heparin-dependent way (Alberdi, E. M., Weldon, J. E., and Becerra, S. P. (2003) BMC Biochem. 4, 1). We have investigated the structural basis for this observation and show that heparin induces a conformational change in the vicinity of Lys(178). This structural change was evident both when binding to intact heparin and specific heparin-derived oligosaccharides at physiological conditions or simply when exposing PEDF to low ionic strength. Binding to other glycosaminoglycans, heparin-derived oligosaccharides smaller than hexadecasaccharides (dp16), or type I collagen did not affect the structure of PEDF. The conformational change is likely to expose the epitope involved in binding to the receptor and thus regulates the interactions with cell surface receptors.

Identification of a Novel Mitochondrial Complex Containing Mitofusin 2 and Stomatin-like Protein 2

A reverse genetics approach was utilized to discover new proteins that interact with the mitochondrial fusion mediator mitofusin 2 (Mfn2) and that may participate in mitochondrial fusion. In particular, in vivo formaldehyde cross-linking of whole HeLa cells and immunoprecipitation with purified Mfn2 antibodies of SDS cell lysates were used to detect an approximately 42-kDa protein. This protein was identified by liquid chromatography and tandem mass spectrometry as stomatin-like protein 2 (Stoml2), previously described as a peripheral plasma membrane protein of unknown function associated with the cytoskeleton of erythrocytes (Wang, Y., and Morrow, J. S. (2000) J. Biol. Chem. 275, 8062-8071). Immunoblot analysis with anti-Stoml2 antibodies showed that Stoml2 could be immunoprecipitated specifically with Mfn2 antibody either from formaldehyde-cross-linked and SDS-lysed cells or from cells lysed with digitonin. Subsequent immunocytochemistry and cell fractionation experiments fully supported the conclusion that Stoml2 is indeed a mitochondrial protein. Furthermore, demonstration of mitochondrial membrane potential-dependent import of Stoml2 accompanied by proteolytic processing, together with the results of sublocalization experiments, suggested that Stoml2 is associated with the inner mitochondrial membrane and faces the intermembrane space. Notably, formaldehyde cross-linking revealed a "ladder" of high molecular weight protein species, indicating the presence of high molecular weight Stoml2-Mfn2 hetero-oligomers. Knockdown of Stoml2 by the short interfering RNA approach showed a reduction of the mitochondrial membrane potential, without, however, any obvious changes in mitochondrial morphology.

Topology and Membrane Association of Lecithin: Retinol Acyltransferase

Fatty acid retinyl esters are the storage form of vitamin A (all-trans-retinol) and serve as metabolic intermediates in the formation of the visual chromophore 11-cis-retinal. Lecithin:retinol acyltransferase (LRAT), the main enzyme responsible for retinyl ester formation, acts by transferring an acyl group from the sn-1 position of phosphatidylcholine to retinol. To define the membrane association and localization of LRAT, we produced an LRAT-specific monoclonal antibody, which we used to study enzyme partition under different experimental conditions. Furthermore, we examined the membrane topology of LRAT through an N-linked glycosylation scanning approach and protease protection assays. We show that LRAT is localized to the membrane of the endoplasmic reticulum (ER) and assumes a single membrane-spanning topology with an N-terminal cytoplasmic/C-terminal luminal orientation. In eukaryotic cells, the C-terminal transmembrane domain is essential for the activity and ER membrane targeting of LRAT. In contrast, the N-terminal hydrophobic region is not required for ER membrane targeting or enzymatic activity, and its amino acid sequence is not conserved in other species examined. We present experimental evidence of the topology and subcellular localization of LRAT, a critical enzyme in vitamin A metabolism.

Nontoxic crystal protein from Bacillus thuringiensis demonstrates a remarkable structural similarity to β‐pore‐forming toxins

Nontoxic crystal protein from <i>Bacillus thuringiensis</i> demonstrates a remarkable structural similarity to β‐pore‐forming toxins

Chemical Modification of Cysteine Residues Is a Misleading Indicator of Their Status as Active Site Residues in the Vitamin K-dependent γ-Glutamyl Carboxylation Reaction

The enzymatic activity of the vitamin K-dependent proteins requires the post-translational conversion of specific glutamic acids to gamma-carboxy-glutamic acid by the integral membrane enzyme, gamma-glutamyl carboxylase. Whether or not cysteine residues are important for carboxylase activity has been the subject of a number of studies. In the present study we used carboxylase with point mutations at cysteines, chemical modification, and mass spectrometry to examine this question. Mutation of any of the free cysteine residues to alanine or serine had little effect on carboxylase activity, although C343A mutant carboxylase had only 38% activity compared with that of wild type. In contrast, treatment with either thiol-reactive reagent 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, disodium salt, or sodium tetrathionate, caused complete loss of activity. We identified the residues modified, using matrix-assisted laser desorption/ionization time of flight mass spectrometry, as Cys(323) and Cys(343). According to our results, these residues are on the cytoplasmic side of the microsomal membrane, whereas catalytic residues are expected to be on the lumenal side of the membrane. Carboxylase was partially protected from chemical modification by factor IXs propeptide. Although all mutant carboxylases bound propeptide with normal affinity, chemical modification caused a >100-fold decrease in carboxylase affinity for the consensus propeptide. We conclude that cysteine residues are not directly involved in carboxylase catalysis, but chemical modification of Cys(323) and Cys(343) may disrupt the three-dimensional structure, resulting in inactivation.

Up-regulation of multiple serine proteinases during earthworm tail regeneration

Summary Previous studies have shown that spatiotemporal regulation of extracellular matrix (ECM) by proteinases is implicated in the initial step of regeneration. In amphibian regeneration, the up-regulation of proteinases such as metalloproteinases (MMPs) and cathepsin D, and proteinase-related proteins such as proteinase tissue inhibitors and activators has been demonstrated. Since the earthworm could provide a unique and valuable model to investigate the mechanism of regeneration, we studied the developmental change in proteinase expression during earthworm tail regeneration. Zymographic analysis revealed that proteinase activities began to increase within 1 h after amputation and reached a maximum at 7 days post-amputation. This peak in activity was approximately 22-fold greater than the unamputated controls. Thereafter, the proteinase activities tended to decrease followed by another peak at 30 days before returning to control levels. At least four types of proteinase were distinguishable at 7 and 30 days post-amputation, with molecular weights of 25, 28, 38, and 44 kDa, respectively. All proteinase activities were strongly inhibited by addition of phenylmethylsulfonyl fluoride (PMSF) and aprotinin, specific inhibitors for serine proteinase. Pepstatin A, E-64, iodoacetamide and a metal ion-free medium were not effective inhibitors, indicating that proteinases expressed during earthworm tail regeneration would be serine proteinases. In addition, we were able to detect two types of plasminogen activator (PA) with molecular weights of 40 and 47 kDa, respectively. PA activities were predominantly expressed at 1, 5, and 25 days post-amputation, which preceded two peaks of serine proteinase activities appearing at approximately 7 and 30 days after amputation, respectively. This fact supports the view that serine proteinases expressed in respond to tail amputation may be plasmin-like proteinases activated by PA.

Tissue plasminogen activator requires plasminogen to modulate amyloid-beta neurotoxicity and deposition.

Tissue plasminogen (plgn) activator (tPA) modulates neuronal death in models of stroke, excitotoxicity, and oxidative stress. Amyloid-beta (Abeta) appears central to Alzheimer's disease and is neurotoxic to neurons in vitro. Here, we evaluate tPA effects on Abeta toxicity. We report that tPA alone had no effect on Abeta toxicity. However, in combination with plgn, tPA reduced Abeta toxicity in a robust fashion. Moreover, the combined tPA and plgn treatment markedly inhibited Abeta accumulation. The addition of phenylmethylsulfonyl fluoride, a serine protease inhibitor, to a sample of tPA, plgn, and Abeta resulted in a marked reduction of Abeta degradation. We interpret the actions of tPA and plgn within the context of the ability of plasmin to degrade Abeta.

In Vitro Skin Penetration and Degradation of Peptides and Their Analysis Using a Kinetic Model.

The main purpose of this study was to estimate the net percutaneous absorption of physiologically active peptides in vitro. The degradation of two peptides, Leu-enkephalin (Enk) and Tyr-Pro-Leu-Gly amide (TPLG), during skin penetration and on the dermal side following penetration, and the prevention of degradation by some protease inhibitors, were investigated using rat skin in vitro. In addition, these permeation and degradation data were analyzed using a kinetic model. These peptides were rapidly degraded in the receptor fluid of a Franz diffusion cell (rate constant: 0.977 h(-1) for Enk and 0.250 h(-1) for TPLG). The addition of phenylmethylsulfonyl fluoride (PMSF) and phenanthroline and the pretreatment of skin with these inhibitors prevented almost completely any degradation in the receptor fluid and skin, respectively. The pretreatment of skin with PMSF and phenanthroline had no effect on the penetration of dextran (1000 Da). The degradation rate constant during skin penetration, calculated from the difference in the penetration rate constants via pretreated and untreated skins, was also high (0.037 h(-1) for Enk and 0.050 h(-1) for TPLG). A kinetic model including an input rate (zero-order), the permeation rate across the viable skin (first-order) and the degradation rate in skin (first-order) was sufficient to describe the apparent steady-state flux of the peptides through skin. We have, thus, established a method for measuring the true flux of peptides across skin in vitro and a kinetic model which simply describes the skin penetration of peptides.

The mechanism of the degradation of psaB gene product, one of the photosynthetic reaction center subunits of photosystem I, upon photoinhibition

The psaB gene product (PsaB protein), one of the reaction center subunits of Photosystem I (PS I), was specifically degraded by light illumination of spinach thylakoid membranes. The degradation of the protein yielded N-terminal fragments of molecular mass 51 kDa and 45 kDa. The formation of the 51 kDa fragment was i) partially suppressed by the addition of phenylmethylsulfonyl fluoride or 3,4-dichloroisocoumarin, which are inhibitors of serine proteases, and ii) enhanced in the presence of hydrogen peroxide during photoinhibitory treatment, but iii) not detected following hydrogen peroxide treatment in the dark. These results suggest that the hydroxyl radical produced at the reduced iron-sulfur centers in PS I triggers the conformational change of the PS I complex, which allows access of a serine-type protease to PsaB. This results in the formation of the 51 kDa N-terminal fragment, presumably by cleavage on the loop exposed to the stromal side, between putative helices 8 and 9. On the other hand, the formation of the 45 kDa fragment, which was enhanced in the presence of methyl viologen but did not accompany the photoinhibition of PS I, was not affected by the addition of hydrogen peroxide or protease inhibitors. Another fragment of 18 kDa was identified as a C-terminal counterpart of the 45 kDa fragment. N-terminal sequence analysis of the 18 kDa fragment revealed that the cleavage occurred between Ala500 and Val501 on the loop exposed to the lumenal side, between putative helices 7 and 8 of the PsaB protein.

Plasminogen-dependent and -independent Proteolytic Activity of Murine Endothelioma Cells with Targeted Inactivation of Fibrinolytic Genes

Plasminogen-dependent and -independent proteolytic activity of marine endothelioma (End) cells that were derived from mice with targeted inactivation of the tissue-type plasminogen activator (t-PA-/-), urokinase-type plasminogen activator (u-PA-/-) or plasminogen activator inhibitor-1 (PAI-1-/-) genes was studied with the use of fibrin and extracellular matrix degradation assays. In a buffer milieu, the activation rate of plasminogen (final concentration 0.25 microM) with wild-type and t-PA-/- End cells (3 x 10(4) to 4 x 10(6) cells/ml) was comparable, but it was about 4-fold reduced with u-PA-/- End cells and 3-fold enhanced with PAI-1-/- End cells. Plasminogen activation was markedly reduced by addition of amiloride or of anti-murine u-PA antibodies but not by addition of anti-murine t-PA antibodies, and it was not stimulated by addition of fibrin. Lysis of 125I-fibrin labeled matrix in the presence of plasminogen was comparable with wild-type, t-PA-/- and PAI-1-/- End cells (50% lysis in 3 h with 0.7 to 1.5 x 10(6) cells/ml), but was significantly reduced with u-PA-/- End cells (50% lysis in 20 h with 0.87 x 10(6) cells/ml). Lysis of 3H-proline labeled extracellular matrix in the presence of plasminogen with wild-type, t-PA-/- and PAI-1-/- End cells (20% lysis in 48 h with 3 to 5 x 10(6) cells/ml) was comparable, but it was virtually abolished with u-PA-/- End cells. In the absence of plasminogen, lysis of both the fibrin and the extracellular matrix by all four cell types was drastically reduced and was virtually abolished by addition of phenylmethylsulfonylfluoride or 1,10 phenanthroline. These data indicate that the proteolytic activity of the transformed murine endothelioma cells, measured in plasminogen activation or matrix degradation assays, is essentially u-PA-related and largely plasminogen-dependent.