Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P00750 (PLA)
 16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Functional role of proteolytic cleavage at arginine-275 of human tissue plasminogen activator as assessed by site-directed mutagenesis. 310 80

Pemphigus IgG induces acantholysis in skin organ culture without the involvement of complement. Urokinase-type plasminogen activator, a proteolytic enzyme, has been implicated in the development of acantholysis. To test this hypothesis, we prepared a rabbit anti-urokinase antibody, which inhibited the plasminogen activator activity in normal human epidermis and in cultured keratinocytes. When added to skin organ cultures along with pemphigus IgG, anti-urokinase IgG completely prevented the development of acantholysis. Normal or preimmune rabbit IgG had no effect on pemphigus IgG-induced acantholysis. Plasminogen activator converts the zymogen plasminogen to its active form plasmin, a broad specificity serine proteinase. When high concentrations of plasminogen alone were added to skin organ culture, acantholysis of the pemphigus foliaceous type was induced. Anti-urokinase antibody also inhibited plasminogen-induced acantholysis. These results strongly support a pivotal role for plasminogen activator in the development of acantholysis.
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PMID:Involvement of urokinase-type plasminogen activator in acantholysis induced by pemphigus IgG. 311 5

Tissue-type plasminogen activator (t-pa) is a serine protease comprising four different putative structural domains with homologies to fibronectin finger-like structures (finger), epidermal growth factor, kringle structures, and the active site of serine proteases. Only the finger and epidermal growth factor domain are each entirely encoded by unique single exons. We assessed the functional contribution of these two structural domains by making mutants precisely deleted for one or both of the relevant exons. The three mutant genes were expressed in monkey cells, and the variant proteins, purified from the culture medium, were characterized for their fibrinolytic activity, fibrinogenolytic potential, and affinity for fibrin. No significant difference in any biochemical property was observed among the variants. All three variants retained a catalytic dependence on cyanogen bromide fragments of fibrinogen which could not be distinguished from the wild-type enzyme. The activities of the variants were also very similar to that of wild-type t-pa, showing no detectable fibrinogenolytic potential in human plasma at activator concentrations of 500 IU/ml, or when their fibrinolytic activity was tested in human plasma using the 125I-labeled fibrin clot lysis assay at activator concentrations of 150 IU/ml or greater. However, the variants were markedly defective in fibrinolysis at low activator concentrations such that essentially no fibrinolysis was detected at 15 IU/ml. Measurement of fibrin binding showed that the variants lacked the high fibrin binding characteristic of wild-type t-pa. These results demonstrate that the fibrin specificity and fibrin-dependent activity of t-pa are independent of the protein's high affinity for fibrin. The implication of these results is that the t-pa variants would be ineffective activators at a physiological concentration of approximately 2 IU/ml but would be expected to behave similarly to wild-type t-pa at the steady-state plasma concentrations of 0.75-1.25 micrograms/ml (approximately 500 IU/ml) currently required for coronary reperfusion in patients receiving t-pa for acute myocardial infarction (Garabedian, H.D., Gold, H.K., Leinbach, R.C., Yasuda, T., Johns, J.A., and Collen, D. (1986) Am. J. Cardiol. 58, 673-679).
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PMID:Variants of human tissue-type plasminogen activator. Fibrin binding, fibrinolytic, and fibrinogenolytic characterization of genetic variants lacking the fibronectin finger-like and/or the epidermal growth factor domains. 312 18

It has recently been reported that the fluorogenic serine proteolytic active site titrant, 4-methyl-umbelliferyl-p-guanidinobenzoate (MUGB), cannot be employed in this capacity for tissue-type plasminogen activator (TPA) [Geiger, M., and Binder, B.R. (1987) Biochim. Biophys. Acta 912, 34-40]. Since this observation has such important ramifications in this area of research, we have studied the reaction of MUGB with recombinant (rec)TPA under a variety of experimental conditions and find that MUGB is indeed an effective titrant of rec-two chain TPA (recTCTPA) at 4 degrees, a condition under which the deacylation rate constant is diminished to the point that acylation can be readily observed. The KS for the interaction of MUGB with recTCTPA is 43 microM-46 microM, the acylation rate constant, k2, is approximately 3.6 min-1-4.2 min-1, and the rate constant for deacylation of p-guanidinobenzoyl-recTCTPA is 0.084 min-1-0.110 min-1. This same recTCTPA, after treatment with diisopropylfluorophosphate, does not react with MUGB. Single-chain TPA (recSCTPA) has been found to acylate more slowly than its two-chain counterpart and to exhibit a higher degree of turnover of the acyl-enzyme with this reagent. These results demonstrate that the active site concentration of TCTPA can be accurately determined by titration with MUGB, a consideration which is essential to the proper kinetic evaluation of this agent and its genetic variants. On the other hand, the presteady state kinetic characteristics for MUGB toward SCTPA are not favorable for its use as a titrant with this form of the enzyme.
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PMID:Reaction of tissue-type plasminogen activator with 4-methylumbelliferyl-p-guanidinobenzoate hydrochloride. 312 57

This mini-review deals with structure-function relationships of human tissue-type plasminogen activator. The enzyme consists of a single polypeptide chain of 527 amino acids. A two-chain form is produced by proteolytic cleavage of the Arg 275-Ile 276 peptide bond. The aminoterminal heavy or A-chain consists of a finger domain, a growth factor domain and two kringle domains. The carboxyterminal light or B-chain contains the active site and is homologous to the catalytic chains of other serine proteases. The light chain is able to activate plasminogen, but requires the heavy chain for fibrin-binding and fibrin-stimulation. Particularly, the finger domain and kringle 2 of the heavy chain are involved in the interaction with fibrin. Other specific properties of the plasminogen activator, such as its rapid hepatic clearance and its inhibition by plasminogen activator inhibitors have not yet been related to specific domains in the protein structure.
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PMID:Relationships between structure and function of tissue-type plasminogen activator. 312 46

Fibrin-specific thrombolytic agents are being developed based on a better understanding of the molecular mechanisms that regulate in vivo fibrinolysis. These agents may be more effective than those available currently and may induce less systemic fibrinolysis. In this respect, t-PA has been extensively investigated. Another fibrinolytic substance with anticipated fibrin-selectivity is scu-PA. Although scu-PA has been much less extensively investigated than t-PA, sufficient knowledge is available to evaluate its potential as a fibrin-specific thrombolytic agent. Both t-PA and scu-PA are single-chain glycoproteins with a catalytic mechanism common to all serine proteases (active site composed of the charge relay system). Both molecules occur as single-chain forms but are converted easily to two-chain disulfide bonded molecules by digestion with plasmin. Unlike most other serine proteases for which the single-chain molecular form is a zymogen with little or no activity toward its natural substrate, both single-chain t-PA and scu-PA have inherent plasminogen-activating potential. Both t-PA and scu-PA induce fibrin-specific thrombolysis in a plasma environment and in animal models of thrombosis. However, the fibrin specificity of t-PA- and scu-PA-induced thrombolysis is based on different molecular mechanisms. The effectiveness and fibrin specificity of t-PA obtained by recombinant DNA technology recently were established by three randomized multicenter trials in patients with acute myocardial infarction. For scu-PA, clinical results are presently more limited.
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PMID:Potential approaches for therapeutic intervention of thrombosis by fibrinolytic agents. 312 86

We studied the effects of natural and recombinant human IL-2 (rIL-2) on secretion of prostacyclin (PGI2), vWf, and tissue-type plasminogen activator (tPA). IL-2 elicited a steady increase in PGI2 synthesis by cultured human umbilical vein endothelial cells (HUVECS) and bovine aortic endothelial cells but had no effect on vWf or tPA. Both purified natural IL-2 (nIL-2) and rIL-2 induced significant PGI2 synthesis. Substitution of the cysteine residue at position 125 of rIL-2 with serine or alanine led to loss of PGI2-stimulatory activity in HUVECS without affecting thymidine incorporation in lymphocytes. HPLC analysis of arachidonate metabolites detected predominantly 6 keto-PGF1 alpha (6KPGF1 alpha) peak. Treatment of cultured endothelial cells with cycloheximide and actinomycin D resulted in inhibition of 6KPGF1 alpha synthesis. The Western blot using a polyclonal antibody against PGH synthase revealed an increment in the 70-kD subunit of PGH synthase by nIL-2 and rIL-2, but not by alanine-substituted rIL-2. We conclude that IL-2 stimulated sustained PGI2 production by a mechanism that includes the de novo synthesis of PGH synthase. This mechanism for regulating AA metabolism probably has important physiologic implications.
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PMID:Influence of natural and recombinant interleukin 2 on endothelial cell arachidonate metabolism. Induction of de novo synthesis of prostaglandin H synthase. 314 45

Halomethylated derivatives of dihydrocoumarins are efficient enzyme-activated inhibitors ("suicide" substrates) of plasminogen activators. Kinetic analysis indicate that the one-chain and two-chain forms of the human plasminogen activator are inhibited by 3,4-dihydro-3-benzyl-6-chloromethylcoumarin through a mechanism-based inactivation characterized by the following kinetic parameters (4 degrees C, pH 6.8) : k2 equal to 0.02 s-1 and 0.03 s-1 (for one- and two-chain tissue plasminogen activators, respectively) and Ki equal to 0.16 mM for both forms. Human urokinase and human tissue-type plasminogen activator can be discriminated on the basis of their inhibition by this suicide substrate. The design of a new series of suicide substrates of serine proteases (functionalized cyclopeptides possessing a potential alkylating function closely related to that found in halomethylated derivatives of dihydrocoumarins) is described.
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PMID:Susceptibility of plasminogen activators to suicide inactivation. 314 67

Three cases of multicentric reticulohistiocytosis showing typical clinical, histologic, and ultrastructural findings are reported. In one, gastric cancer occurred; in the other two cases, severe polyarthritis was the only detectable internal involvement. The serine proteinases, urokinase and tissue-type plasminogen activator, were evaluated both with the autohistographic technique and spectrophotometric assay in lesional skin and synovia. Urokinase levels appeared grossly increased in the lesional synovia and moderately increased in the lesional skin. We suggest that urokinase, presumably released by the activated proliferating histiocytes, may play a major role in the extracellular matrix degradation leading to erosion of cartilage and adjacent bone in multicentric reticulohistiocytosis.
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PMID:Multicentric reticulohistiocytosis. Report of three cases with the evaluation of tissue proteinase activity. 314 26

A cDNA encoding rat plasminogen activator-inhibitor (PAI-1) has been isolated from an HTC rat hepatoma cell cDNA library constructed in phage lambda gt10. The cDNA contains 118 bp of 5'-untranslated sequence, 1206 bp encoding a 402-amino acid (aa) protein and 1747 bp of 3'-untranslated sequence. The protein-coding sequence and the derived amino acid sequence share 82% and 81% identity, respectively, with human PAI-1 cDNA and protein. The rat cDNA encodes a preprotein with a 23-aa leader peptide and a predicted N-terminal serine for the mature protein. Three of four potential N-glycosylation acceptor sites as well as the active site of rat PAI-1 are identical to the human protein. The 3'-untranslated region contains a number of unusual regions, including 80 bp of tandemly repeated GpA dinucleotides, a 115-bp stretch which shares greater than 90% sequence identity with a region within the 3'-untranslated cDNA of human PAI-1, and two 70-bp stretches of highly T-rich sequence located close to the 3'-terminus of the cDNA.
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PMID:Cloning and sequencing of cDNA for the rat plasminogen activator inhibitor-1. 314 11


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