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)

The heavy chain of tissue plasminogen activator (t-PA) consists of four domains [finger, epidermal-growth-factor (EGF)-like, kringle 1 and kringle 2] that are homologous to similar domains present in other proteins. To assess the contribution of each of the domains to the biological properties of the enzyme, site-directed mutagenesis was used to generate a set of mutants lacking sequences corresponding to the axons encoding the individual structural domains. The mutant proteins were assayed for their ability to hydrolyze artificial and natural substrates in the presence and absence of fibrin, to bind to lysine-Sepharose and to be inhibited by plasminogen activator inhibitor-1. All the deletion mutants exhibit levels of basal enzymatic activity very similar to that of wild-type t-PA assayed in the absence of fibrin. A mutant protein lacking the finger domain has a 2-fold higher affinity for plasminogen than wild-type t-PA, while the mutant that lacks both finger and EGF-like domains is less active at low concentrations of plasminogen. Mutants lacking both kringles neither bind to lysine-Sepharose nor are stimulated by fibrin. However, mutants containing only one kringle (either kringle 1 or kringle 2) behave indistinguishably from one another and from the wild-type protein. We conclude that kringle 1 and kringle 2 are equivalent in their ability to mediate stimulation of catalytic activity by fibrin.
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PMID:Variants of human tissue-type plasminogen activator that lack specific structural domains of the heavy chain. 284 82

Factor I is a serine proteinase of complement which together with one of several specific cofactors cleaves activation products of the third and fourth components of complement (C3b and C4b) and modulates the activity of C3 convertase. A heterodimer glycoprotein (Mr = 88,000), factor I is synthesized as a single-chain precursor, prepro-I, which undergoes intracellular proteolytic processing. The human hepatoma line HepG2, however, secretes predominantly the single-chain precursor pro-I. In order to determine the molecular basis for this apparent processing defect, factor I cDNA clones were isolated from a HepG2 mRNA-derived library. Sequencing of the largest insert, HI1971, revealed that it contains 14 base pairs of 5' untranslated region, the complete coding sequence for the 583-residue prepro-I (NH2-signal peptide-heavy chain-linking peptide-light chain-COOH), two polyadenylation signals within the 200-base pair 3' untranslated region, and a portion of poly(A) tail. Analysis of the derived protein structure 1) reveals a mosaic multidomain structure of the heavy chain; 2) demonstrates structural similarity between intracellular conversion of pro-I and activation of other serine proteinase zymogens; and 3) indicates that the light chain of factor I resembles most closely the active subunit of tissue plasminogen activator among all serine proteinases and factor D among complement proteinases. Furthermore, this protein sequence was compared to the sequences of factor I cDNA clones isolated from normal human liver libraries and found to be identical. By exclusion, this defines as cellular the basis for the inefficient processing of pro-I by the HepG2 line. Chromosomal localization by the somatic cell hybrid method maps the factor I gene to chromosome 4.
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PMID:Human complement factor I: analysis of cDNA-derived primary structure and assignment of its gene to chromosome 4. 295 52

Two-chain tissue-type plasminogen activator (t-PA), which consists of a heavy chain (Mr congruent to 38,000) and a light chain (Mr congruent to 31,000) connected by a disulfide bridge, was reduced with 2-mercaptoethanol and then air-reoxidized at a low protein concentration and carboxamidomethylated. The two chains were separated by means of zinc chelate-agarose, which was found to bind the light chain selectively. The light chain was fully active on the tripeptide substrate H-D-isoleucyl-L-prolyl-L-arginine p-nitroanilide (S-2288) and partially active on plasminogen. The plasminogen activator activity of the light chain was, in contrast to that of two-chain t-PA, not stimulated by fibrin or fibrinogen fragments. Fibrin-agarose chromatography of radiolabeled chains showed that only the heavy chain bound to fibrin. These results indicate that the active site-containing light chain in t-PA needs the heavy chain for fibrin stimulation of its plasminogen activator activity.
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PMID:Isolation and functional characterization of the heavy and light chains of human tissue-type plasminogen activator. 308 1

Transfected mouse Ltk- cells were employed for transient expression of recombinant human tissue-type plasminogen activator (t-PA; EC 3.4.21.31) or of recombinant-t-PA deletion proteins, encoded by SV40-pBR322-derived t-PA cDNA plasmids. The t-PA cDNA deletion mutants have two features in common, i.e., cDNA programming the signal peptide and the coding region for the light chain. Consequently, recombinant t-PA mutant proteins are efficiently secreted and display plasminogen activator activity. The gene encoding the amino-terminal heavy chain [an array of structural domains homologous to other plasma proteins (finger, epidermal growth factor, and kringle domains)] was mutated using restriction endonucleases to delete one or more structural domains. The stimulatory effect of fibrinogen fragments on the plasminogen activator activity of t-PA was demonstrated to be mediated by the kringle K2 domain and to a lesser extent by the finger/epidermal growth factor region but not by the kringle K1 domain. These data correlate well with the fibrin-binding properties of the recombinant t-PA deletion proteins, indicating that the stimulation of the activity by fibrinogen fragments is based on aligning the substrate plasminogen and t-PA on the fibrin matrix. Our results support the evolutionary concept of exon shuffling, arranging structural domains that constitute autonomous functions of the protein.
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PMID:Autonomous functions of structural domains on human tissue-type plasminogen activator. 308 64

In order to assess which part of the tissue-type plasminogen activator (t-PA) molecule should be (genetically) modified to obtain more-slowly-clearing mutants, two-chain t-PA and its isolated heavy and light chains were radiolabelled and injected into rats. The vast majority of t-PA and the heavy chain disappeared from the blood circulation with half-lives of 2.3 and 1.0 min respectively. The clearance of the light chain was biphasic, owing to complex-formation with plasma proteinase inhibitors. The disappearance of di-isopropylphospho-light chain, which has a blocked active site, was nearly monophasic, with a half-life of 5.7 min. Organ distribution studies showed that hepatic clearance constituted the major pathway in all cases. These results strongly suggest that t-PA is recognized by the liver primarily through the heavy chain.
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PMID:Clearance of the heavy and light polypeptide chains of human tissue-type plasminogen activator in rats. 309 71

An antibody population that reacted with the high-affinity lysine binding site of human plasminogen was elicited by immunizing rabbits with an elastase degradation product containing kringles 1-3 (EDP I). This antibody was immunopurified by affinity chromatography on plasminogen-Sepharose and elution with 0.2 M 6-aminohexanoic acid. The eluted antibodies bound [125I]EDP I, [125I]Glu-plasminogen, and [125I]Lys-plasminogen in radioimmunoassays, and binding of each ligand was at least 99% inhibited by 0.2 M 6-aminohexanoic acid. The concentrations for 50% inhibition of [125I]EDP I binding by tranexamic acid, 6-aminohexanoic acid, and lysine were 2.6, 46, and 1730 microM, respectively. Similar values were obtained with plasminogen and suggested that an unoccupied high-affinity lysine binding site was required for antibody recognition. The antiserum reacted exclusively with plasminogen derivatives containing the EDP I region (EDP I, Glu-plasminogen, Lys-plasminogen, and the plasmin heavy chain) and did not react with those lacking an EDP I region [miniplasminogen, the plasmin light chain or EDP II (kringle 4)] or with tissue plasminogen activator or prothrombin, which also contain kringles. By immunoblotting analyses, a chymotryptic degradation product of Mr 20,000 was derived from EDP I that retained reactivity with the antibody. The high-affinity lysine binding site was equally available to the antibody probe in Glu- and Lys-plasminogen and also appeared to be unoccupied in the plasmin-alpha 2-antiplasmin complex. alpha 2-Antiplasmin inhibited the binding of radiolabeled EDP I, Glu-plasminogen, or Lys-plasminogen by the antiserum, suggesting that the recognized site is involved in the noncovalent interaction of the inhibitor with plasminogen.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Topography of the high-affinity lysine binding site of plasminogen as defined with a specific antibody probe. 309 30

Covalent linkage of tissue-type plasminogen activator (t-PA) to a monoclonal antibody specific for the fibrin beta chain (anti-fibrin 59D8) results in a thrombolytic agent that is more specific and more potent than t-PA alone. To provide a ready source of this hybrid molecule and to allow tailoring of the active moieties for optimal activity, we have engineered a recombinant version of the 59D8-t-PA conjugate. The rearranged 59D8 heavy chain gene was cloned and combined in the expression vector pSV2gpt with sequence coding for a portion of the gamma 2b constant region and the catalytic beta chain of t-PA. This construct was transfected into heavy chain loss variant cells derived from the 59D8 hybridoma. Recombinant protein was purified by affinity chromatography and analyzed with electrophoretic transfer blots. These revealed a 65-kDa heavy chain-t-PA fusion protein that is secreted in association with the 59D8 light chain in the form of a 170-kDa disulfide-linked dimer. Chromogenic substrate assays showed the fusion protein to have 70% of the peptidolytic activity of native t-PA and to activate plasminogen as efficiently as t-PA. In a competitive binding assay, reconstituted antibody was shown to have a binding profile similar to that of native 59D8. Thus, by recombinant techniques, we have produced a hybrid protein capable of high-affinity fibrin binding and plasminogen activation.
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PMID:Construction and expression of a recombinant antibody-targeted plasminogen activator. 311 46

The relationship of N-linked glycosylation and association with heavy chain binding protein (BiP) to the secretion of Factor VIII (FVIII), von Willebrand Factor (vWF), and tissue plasminogen activator (tPA) was studied in Chinese hamster ovary (CHO) cells. FVIII has a heavily glycosylated region containing 20 clustered potential N-linked glycosylation sites. A significant proportion of FVIII was detected in a stable complex with BiP and not secreted. Deletion of the heavily glycosylated region resulted in reduced association with BiP and more efficient secretion. Tunicamycin treatment of cells producing this deleted form of FVIII resulted in stable association of unglycosylated FVIII with BiP and inhibition of efficient secretion. vWF contains 17 potential N-linked glycosylation sites scattered throughout the molecule. vWF was transiently associated with BiP and efficiently secreted demonstrating that CHO cells are competent to secrete a highly glycosylated protein. tPA, which has three utilized N-linked glycosylation sites, exhibited low level association with BiP and was efficiently secreted. Disruption of N-linked glycosylation of tPA by either site-directed mutagenesis or tunicamycin treatment resulted in reduced levels of secretion and increased association with BiP. This effect was enhanced by high levels of tPA expression. The glycosylation state and extent of association with BiP could be correlated with secretion efficiency.
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PMID:The relationship of N-linked glycosylation and heavy chain-binding protein association with the secretion of glycoproteins. 312 36

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

Deletion mutants of human tissue plasminogen activator (tPA) were expressed in Chinese hamster ovary cells. These cells had been transfected with genes that encoded tPA but included restriction sites that allowed the deletion of DNA encoding specific structural domains of the tPA molecule's heavy chain. Purified, two-chain mutant tPAs, or analogues of tPA, lacking one or several structural domains, along with Bowes melanoma tPA were studied in order to determine their susceptibility to inhibition by plasminogen activator inhibitor-1 (PAI-1). The full-length analogue of tPA, designated by its domains FGK1K2P, as well as analogues GK1K2P, FK2P, and FGK1P were treated with various amounts of PAI-1. When the amounts of added tPA and analogues were standardized so that each generated the same absorbance in a chromogenic assay containing S-2251, plasminogen, and fibrinogen fragments, there was a significant difference in the way in which the analogues were titrated by the inhibitor. The melanoma tPA and FGK1K2P were the most susceptible, FK2P slightly less sensitive, and GK1K2P and FGK1P the least sensitive to inhibition. In contrast, when the amounts of enzyme used were standardized on the basis of absorbance generated in a direct assay employing the chromogenic substrate S-2288 and then titrated with PAI-1, these differences in susceptibility to inhibition were not observed. Based on these data, the differential susceptibility to inhibition observed in the plasminogen-dependent assay was attributed to the extent to which the activity of a given analogue is enhanced by fibrinogen fragments, and thus reflected the different amounts of enzymes added in order to standardize the assay on the basis of absorbance.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Neutralization by plasminogen activator inhibitor-1 of mutants of tissue plasminogen activator. 313 1


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