Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.68 (tissue plasminogen activator)
 11,311 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Apolipoprotein(a) [apo(a)] is a glycoprotein with Mr approximately equal to 280,000 that is disulfide linked to apolipoprotein B in lipoprotein(a) particles. Elevated plasma levels of lipoprotein(a) are correlated with atherosclerosis. Partial amino acid sequence of apo(a) shows that it has striking homology to plasminogen. Plasminogen is a plasma serine protease zymogen that consists of five homologous and tandemly repeated domains called kringles and a trypsin-like protease domain. The amino-terminal sequence obtained for apo(a) is homologous to the beginning of kringle 4 but not the amino terminus of plasminogen. Apo(a) was subjected to limited proteolysis by trypsin or V8 protease, and fragments generated were isolated and sequenced. Sequences obtained from several of these fragments are highly (77-100%) homologous to plasminogen residues 391-421, which reside within kringle 4. Analysis of these internal apo(a) sequences revealed that apo(a) may contain at least two kringle 4-like domains. A sequence obtained from another tryptic fragment also shows homology to the end of kringle 4 and the beginning of kringle 5. Sequence data obtained from two tryptic fragments show homology with the protease domain of plasminogen. One of these sequences is homologous to the sequences surrounding the activation site of plasminogen. Plasminogen is activated by the cleavage of a specific arginine residue by urokinase and tissue plasminogen activator; however, the corresponding site in apo(a) is a serine that would not be cleaved by tissue plasminogen activator or urokinase. Using a plasmin-specific assay, no proteolytic activity could be demonstrated for lipoprotein(a) particles. These results suggest that apo(a) contains kringle-like domains and an inactive protease domain.
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PMID:Partial amino acid sequence of apolipoprotein(a) shows that it is homologous to plasminogen. 347 6

Lipoprotein lipases from human, bovine or guinea-pig milk were purified, judged for domain relationships by characterization of sites sensitive to proteases, and structurally compared. The subunit of human lipoprotein lipase migrated slightly slower than those of bovine or guinea-pig lipoprotein lipases on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Bovine lipoprotein lipase is known to be a dimer of two non-covalently linked subunits of equal size, and the lipases from all three sources now yielded homogeneous N-terminal amino acid sequences (followed for 15-27 residues). The results indicate that the two subunits are identical. Bovine lipoprotein lipase had two additional N-terminal residues, Asp-Arg, compared to the human and guinea-pig enzymes, and the next two positions revealed residue differences, but further on homologies were extensive between all three enzymes as far as presently traced. Exposure of bovine lipoprotein lipase to trypsin led to production of three fragments (T1, T2a, and T2b), suggesting cleavage at exposed segments delineating domain borders. Time studies gave no evidence for precursor-product relationships between the fragments, and prolonged digestion did not lead to further cleavage. Fragments T2a and T2b had the same N-terminal sequence as intact lipase. Fragment T1 revealed a new sequence, and represents the C-terminal half of the molecule. Plasmin caused a similar cleavage as trypsin, whereas thrombin, factor Xa, and tissue plasminogen activator did not cleave the enzyme. Chymotrypsin cleaved off a relatively small fragment from the C-terminal of the molecule, after which exposure to trypsin still resulted in cleavage at the same sites as in intact lipase. Tryptic cleavage of guinea-pig lipoprotein lipase yielded two fragments. One had a similar size as bovine fragment T2b; the other had a similar size as bovine fragment T1 and an N-terminal sequence homologous with that of T1. Thus, trypsin recognizes the same unique site in guinea-pig lipoprotein lipase as in the bovine enzyme. This confirms the conclusion that this segment is the border between two domains in the subunit. The binding site for heparin was retained after both tryptic and chymotryptic cleavages and was identified as localized in the C-terminal part of the molecule.
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PMID:Lipoprotein lipases from cow, guinea-pig and man. Structural characterization and identification of protease-sensitive internal regions. 353 11

Preparative electrophoresis of living cells has been considered for some time as a potential tool for isolating, from heterogeneous mixtures, subpopulations of cells according to function. Such a purification depends upon the retention of electrophoretic heterogeneity and the retention of function. Human embryonic kidney cells that had been in monolayer culture for 1-5 subcultivations were resuspended by treatment with trypsin and/or EDTA and suspended in a variety of electrophoresis buffers, ranging in ionic strength from 0.0015 to 0.15 M. Analytical electrophoresis with a Zeiss Cytopherometer or Pen Kem 3000 automated light-scattering electrophoretic analyzer indicated that electrophoretic heterogeneity was retained under the full range of conditions tested. Preparative electrophoresis by three methods--in a density gradient, with continuous flow, and in microgravity--indicated that electrophoretic heterogeneity coincided with functional heterogeneity; for example, some electrophoretically isolated subpopulations produced increased levels of urokinase while others produced increased level of tissue plasminogen activator.
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PMID:Electrophoretic separation and analysis of living cells from solid tissues by several methods. Human embryonic kidney cell cultures as a model. 377 95

A human liver cDNA library was screened by colony hybridization with two mixtures of synthetic oligodeoxyribonucleotides as probes. These oligonucleotides encoded regions of beta-factor XIIa as predicted from the amino acid sequence. Four positive clones were isolated that contained DNA coding for most of factor XII mRNA. DNA sequence analysis of these overlapping clones showed that they contained DNA coding for part of an amino-terminal extension, the complete amino acid sequence of plasma factor XII, a TGA stop codon, a 3' untranslated region of 150 nucleotides, and a poly(A)+ tail. The cDNA sequence predicts that plasma factor XII consists of 596 amino acid residues. Within the predicted amino acid sequence of factor XII, we have identified three peptide bonds that are cleaved by kallikrein during the formation of beta-factor XIIa. Comparison of the structure of factor XII with other proteins revealed extensive sequence identity with regions of tissue-type plasminogen activator (the epidermal growth factor-like region and the kringle region) and fibronectin (type I and type II homologies). As the type II region of fibronectin contains a collagen-binding site, the homologous region in factor XII may be responsible for the binding of factor XII to collagen. The carboxyl-terminal region of factor XII shares considerable amino acid sequence homology with other serine proteases including trypsin and many clotting factors. A preliminary structural model of beta-factor XIIa is proposed based on the known high resolution x-ray diffraction structures of trypsin, chymotrypsin, and elastase.
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PMID:Characterization of human blood coagulation factor XII cDNA. Prediction of the primary structure of factor XII and the tertiary structure of beta-factor XIIa. 387 53

A highly active angiotensin-producing enzyme (enzyme II) was obtained from dog serum by acid treatment and fractionation to remove angiotensinase and converting enzyme, separate an inhibitor, and convert an inactive precursor (proenzyme II) to enzyme II. Proenzyme II was found to be converted to enzyme II by an endogenous activating enzyme identified as plasmin. Conversion was also caused by the interaction of bacterial streptokinase with human proactivator, by trypsin, and by an activator formed from liver tissue extract and dog serum. Neither plasma kallikrein nor the labile, human extrinsic tissue-type plasminogen activator induced activation. The inhibitor, which normally blocks the activation of proenzyme II, was unusually stable against high temperatures and extremes of pH, and it was not identical to any of the six known protease inhibitors of serum. Enzyme II was not identical to other angiotensin-producing enzymes such as enzyme I, renin, cathepsin D, pepsin, plasmin, tonin, or cathepsin G. Enzyme II reacted maximally at pH 4.7 and produced up to 2250 ng of angiotensin I/ml serum/hr from the substrate of dog serum (i.e., amounts 3200-fold higher than that produced by endogenous renin of normal dog serum). Since at pH 7.2, angiotensin I formation is still about 30 times higher than that of renin, enzyme II may be physiologically active under some conditions.
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PMID:Angiotensin-producing serum enzyme II. Formation by inhibitor removal and proenzyme activation. 390 15

Tripeptide derivatives of lysyl or arginyl chloromethylketone inhibit the trypsin-like serine proteases trypsin, thrombin, plasmin, Factor Xa, urokinase, tissue-type plasminogen activator and protein Ca following the reaction scheme: (formula; see text) Extremely potent tripeptide inhibitors were obtained for thrombin and trypsin (k2/Ki greater than 10(6) M-1s-1), moderate inhibitors for plasmin and Factor Xa (10(6) M-1s-1 greater than k2/Ki greater than 10(4) M-1s-1) and only weak inhibitors for urokinase, tissue-type plasminogen activator and protein Ca (k2/Ki less than 10(4) M-1s-1). Thrombin and Factor Xa as well as urokinase and tissue-type plasminogen activator can be discriminated on the basis of their inhibitory spectrum towards some of these inhibitors.
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PMID:Inhibition of trypsin-like serine proteinases by tripeptide arginyl and lysyl chloromethylketones. 623 78

The sequences of urokinase (UK) and tissue-type plasminogen activator (TPA) were aligned with those of chymotrypsin, trypsin, and elastase according to their 'structurally conserved regions'. In spite of its trypsin-like specificity UK was model-built on the basis of the chymotrypsin structure because of a corresponding disulfide pattern. The extra disulfide bond falls to cysteines 50 and 111d. Insertions can easily be accommodated at the surface. As they occur similarly in both, UK and TPA, a role in plasminogen recognition may be possible. Of the functional positions known to be involved in substrate or inhibitor binding, Asp 97, Lys 143 and Arg 217 (Leu in TPA) may contribute to plasminogen activating specificity. PTI binding may in part be impaired by structural differences at the edge of the binding pocket.
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PMID:Adaptation of plasminogen activator sequences to known protease structures. 634 97

Recently we presented evidence that normal human foreskin fibroblasts (HF cells) limit the activity of secreted urokinase by secreting it as a proenzyme and by secreting protease nexin , an inhibitor of urokinase and certain other serine proteases (Scott, R.W., Eaton, D. L. Duran , N., and Baker, J.B. (1983) J. Biol. Chem. 258, 4397-4403). Using immunoaffinity chromatography we have now purified the HF cell urokinase proenzyme. It is a single 52-kDa polypeptide chain that is inactive toward both plasminogen and low molecular weight substrates. After proteolytic activation, this material (specific activity of 3 X 10(4) Committee on Thrombolytic Agents units/mg) is composed of two disulfide-bridged 33- and 19-kDa chains, and is thus similar to the predominant form of urokinase found in urine. Plasmin at 2 X 10(-10) M causes 50% activation of the proenzyme (1 X 10(-9) M) in 30 min at 37 degrees C. Thrombin and trypsin are one-twentieth as effective as plasmin. Activated HF cell 125I-urokinase forms sodium dodecyl sulfate stable complexes with purified protease nexin or protease nexin present in medium conditioned by HF cells. Purified protease nexin inhibits purified HF cell urokinase action on both plasminogen and low molecular weight substrates. The association rate constant for the reaction between protease nexin and HF cell urokinase is approximately 1.7 X 10(5) M-1 S-1. In contrast, the association rate constants for reactions between protease nexin and the one- and two-chain forms of tissue-type plasminogen activator are approximately 2 X 10(3) and approximately 3 X 10(4) M-1 S-1, respectively. The importance of protease nexin as a regulator of HF cell urokinase is supported by the finding that anti-protease nexin antibody potentiates the fibrinolytic activity of HF cell-conditioned medium incubated with plasminogen.
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PMID:Purification of human fibroblast urokinase proenzyme and analysis of its regulation by proteases and protease nexin. 637 53

Plasminogen activator that is associated with the development of hypersensitivity granulomas (gPA) was partially purified from a saline soluble fraction of murine lepromas elicited in "resistant" mice, C57BL/6N. The gPA was shown to consist of two subspecies (23,000 and 48,000 in molecular weight) with essentially identical enzymologic properties. The gPA was found to be a relatively heat stable weakly alkaline serine proteinase with trypsin-like characteristics in the specificity for synthetic substrates and proteinase inhibitors. It showed a high affinity for H-D-Ile-Pro-Arg-pNA (Km = 1.4 X 10(-4) M) H-D-Val-Leu-Lys-pNA (Km = 5.2 X 10(-4) M), and L-pyroGlu-Gly-Arg-pNA (Km = 9.3 X 10(-4) M). The gPA did not demonstrate antigenic cross reaction with urokinase-type or tissue-type plasminogen activator. Two distinct enzymatic regulators of the gPA were also demonstrated in the saline soluble fraction of the hypersensitivity granulomas. The gPA and its regulation are assumed to be correlated with macrophage activation in the hypersensitivity granulomas.
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PMID:Plasminogen activator and plasminogen activator inhibitor associated with granulomatous inflammation: a study with murine leprosy. 639 70

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.
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PMID:Purification of human tissue plasminogen activator with Erythrina trypsin inhibitor. 654 Dec 21


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