UNIPROT:P00750 - FACTA Search

Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Target Concepts:

Query: UNIPROT:P00750 (PLA)
16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human tissue-type plasminogen activator (t-PA) is cleared rapidly from the circulation by hepatic receptors, one of which recognizes a site in the epidermal growth factor-like domain of the molecule. To define this site more precisely, we have used oligonucleotide-mediated mutagenesis to introduce amino acid substitutions at specific positions located in turns that connect antiparallel beta-sheets in the epidermal growth factor-like domain. Mutated t-PA proteins with amino acid substitutions of the tyrosine residue at position 67 showed markedly lower rates of endocytosis and degradation by cultured cells of the rat hepatoma (H4) line that express a specific receptor for t-PA, and their half-life in the circulation of rats was extended significantly because of a reduction in the rate of the rapid alpha-phase of clearance. The enzymatic properties and fibrinolytic activity of these mutants in vitro were not significantly different from those of wild-type t-PA. We conclude that tyrosine 67 comprises a key determinant in the clearance of t-PA by a specific hepatic receptor.
...
PMID:Tyrosine 67 in the epidermal growth factor-like domain of tissue-type plasminogen activator is important for clearance by a specific hepatic receptor. 131 65

The use of temperature-sensitive (ts) src mutants for studies of cell transformation and differentiation has been limited by the availability of cloned ts-src genes that are inactivated at temperatures compatible with growth of mammalian cells. In this report, we describe the cloning and characterization of the tsLA90src gene, which displays tight thermal sensitivity at 39.5 degrees C. Nucleotide sequence comparison of tsLA90 and wild-type src genes from the Schmidt-Ruppin subgroup A and D strains of Rous sarcoma virus (RSV) revealed four amino acid differences in tsLA90src. Substitution of one of these residues (Lys-280) from tsLA90src with its wild-type homolog (Glu-280) caused a reversion to a wild-type src phenotype. The cloned tsLA90 gene, designated tsUP1, was introduced into avian and mammalian retroviral vectors. Chicken embryo fibroblasts and immortalized mouse 3T3 cells infected with these viral vectors displayed a temperature-dependent transformed phenotype as assessed by cell morphology, secretion of plasminogen activator, transcriptional activation of the primary response genes, Egr-1 and TIS 10, and stimulation of tyrosine phosphorylation. In addition, chicken myoblasts (infected with RSVtsUP1) showed a temperature-dependent differentiation into myotubes. Thus, this cloned src gene should be ideally suited for inducing reversible transformation and differentiation of mammalian cells in culture.
...
PMID:Cloning and characterization of a thermolabile v-src gene for use in reversible transformation of mammalian cells. 137 18

A recombinant deletion mutant of the 155-amino acid form of human basic fibroblast growth factor (bFGF), lacking amino acid residues 27-32 (Lys-Asp-Pro-Lys-Arg-Leu), was expressed in Escherichia coli and purified to homogeneity by heparin-Sepharose affinity chromatography. When maintained in the presence of an equimolar concentration of soluble heparin, the bFGF mutant (M1-bFGF) is as potent as bFGF in stimulating cell proliferation in normal and transformed fetal bovine aortic endothelial cells, in adult bovine aortic endothelial cells, and in NIH 3T3 fibroblasts. However, under the same experimental conditions, M1-bFGF is at least 100 times less efficient than bFGF in stimulating plasminogen activator (PA) production in endothelial cells, as assayed by chromogenic PA assay, SDS/PAGE zymography, and Northern blot analysis of urokinase-type PA mRNA. In the presence of heparin, M1-bFGF binds to bFGF plasma membrane receptors present on endothelial cells in a manner undistinguishable from bFGF. It also induces the same tyrosine phosphorylation pattern when added to NIH 3T3 cells. The data suggest that the PA-inducing activity of bFGF may depend upon a functional domain that differs from those involved in the mitogenic activity of the growth factor and that the binding of bFGF to its plasma membrane receptor may not be sufficient to induce urokinase-type PA production in endothelial cells.
...
PMID:A six-amino acid deletion in basic fibroblast growth factor dissociates its mitogenic activity from its plasminogen activator-inducing capacity. 184 69

The contribution of His64 to the function and stability of tissue plasminogen activator (t-PA) kringle-2 domain (His244 in t-PA numbering) has been studied by using microcalorimetric methods to compare the ligand binding and thermal denaturation behavior of wild-type kringle-2 and mutants having His64 replaced with Tyr or Phe. This site was examined because modeling studies suggested that the His64 side chain could play an important role in ligand binding by forming an ion-pair with the carboxylate of the ligand, L-lysine. Kringle-2 domains were expressed by secretion of the 174-263 portion of t-PA in E. coli and purified as previously described for the wild-type domain. Both mutant proteins retain affinity for L-lysine, although reduced three- to four-fold relative to wild-type, demonstrating that His64 does not interact with the ligand carboxylate through an ion-pair interaction or by hydrogen bonding. The H64Y substitution does result in an altered specificity of the lysine binding site with the mutant domain having greatest affinity for a ligand of 6.8 A chain length, whereas the wild-type domain prefers an 8.8 A long ligand. For both wild-type and mutant, the binding of the optimal chain length ligand is dominated by enthalpic effects (delta H = -6,000 to -7,000 cal/mol) and T delta S accounts for less than 15% of delta G. In addition, the H64Y mutant differs from wild-type in the effect of ligand alpha-amino group modification on binding affinity. Based on examination of the x-ray structure recently determined for wild-type kringle-2, the specificity changes accompanying the H64Y substitution probably result from changes in side chain interactions in the lysine binding site. Thermal denaturation experiments show that the H64Y mutant is also more stable than the wild-type protein with the difference in stabilization free energy (delta delta G) equal to 2.7 kcal/mol at 25 degrees C and pH 3. The increased stability of the mutant appears to be related to the difference in hydrophobicity between His and Tyr.
...
PMID:Thermodynamics of ligand binding and denaturation for His64 mutants of tissue plasminogen activator kringle-2 domain. 196

The cDNA encoding full-length human tissue-type plasminogen activator (t-PA) and five variant cDNAs, constructed by in vitro site-directed mutagenesis, were cloned and expressed in Chinese hamster ovary cells. The variant cDNAs were designed to increase the fibrin affinity of t-PA by mutagenesis in the kringle domains of specific amino acids which are assumed to constitute the lysine-binding site. These amino acids were replaced with the corresponding amino acids present in kringle 1 of plasminogen, which has a high affinity for lysine analogues. The mutants included: rt-PA-Arg125 with a Pro125----Arg mutation; rt-PA-Arg164,Tyr165 with Ser164,Ser165----Arg,Tyr; rt-PA-Arg125,Arg164,Tyr165 with Pro125,Ser164,Ser165----Arg,Arg,Tyr; rt-PA-Arg213 with Val213----Arg; and rt-PA-Arg252 with Thr252----Arg. Compared to wild-type recombinant t-PA (rt-PA), the catalytic efficiency for plasminogen activation was enhanced 4-fold for rt-PA-Arg125, and 3-fold for rt-PA-Arg252 while stimulation of plasminogen activation by CNBr-digested fibrinogen was comparable to wild-type rt-PA for rt-PA-Arg125 and 2-fold enhanced for rt-PA-Arg252. All rt-PA moieties showed a similar concentration-dependent and nearly quantitative binding to fibrin as well as to lysine-Sepharose and induced a similar time- and concentration-dependent lysis of a 125I-fibrin-labeled plasma clot immersed in human plasma. Equieffective concentrations (causing 50% clot lysis in 2 h) were 0.17 micrograms/ml for rt-PA-Arg125, and 0.31 micrograms/ml for rt-PA-Arg252 as compared to 0.55 micrograms/ml for rt-PA. The initial plasma half-life following intravenous bolus injection of 0.25 mg/kg in hamsters was 1.2-2.6 min, not significantly different from wild-type rt-PA (2.4 min). Continuous infusion over 60 min in hamsters with a 125I-fibrin-labeled pulmonary embolus produced 50% clot lysis over background with a dose of 0.9-1.8 mg/kg, which is not markedly superior to wild-type rt-PA (2.1 mg/kg). It is concluded that these variants, designed to mimic the high affinity fibrin-binding site of plasminogen, are not endowed with a markedly improved thrombolytic potency.
...
PMID:Biochemical and functional characterization of human tissue-type plasminogen activator variants with mutagenized kringle domains. 211 13

The v-fms oncogene of the McDonough strain of feline sarcoma virus (SM-FeSV) encodes a plasma-membrane-associated tyrosine kinase (gp140v-fms) which is closely related, both structurally and functionally, to the c-fms-specified receptor for the macrophage colony stimulating factor (CSF-1). In mammalian fibroblasts, the natural producers of CSF-1, expression of v-fms leads to cell transformation. To study the interaction between CSF-1 and gp140v-fms molecules in a cell system that does not produce endogenous cross-reactive CSF-1, we have expressed the entire v-fms gene as well as a nontransforming deletion mutant (SC2) in chicken embryo cells (CEC). For this purpose the avian retroviral vectors pDS3 and pREP, based on Rous sarcoma virus, were used to isolate recombinant virus particles. CEC infected with virus that carried the entire v-fms gene expressed high amounts of gp140v-fms, comparable to those in SM-FeSV transformed NRK cells. However, these CEC remained flat, retained their fibronectin network, and did not produce enhanced levels of plasminogen activator. The cells grew faster than control CEC for more than 8 weeks but failed to form colonies in soft agar. Within 2 days after addition of CSF-1 to the growth medium, a transformed cell phenotype was induced, as judged by loss of the fibronectin network, again with a growth rate fourfold faster than that of the parental cells and with colony formation in soft agar. Moreover, human CSF-1 caused a rapid tyrosine phosphorylation of v-fms molecules detectable within 5 min after addition of the growth factor. In contrast, CSF-1 had none of the above effects on cells that expressed the SC2 v-fms deletion mutant.
...
PMID:Transformation of chicken fibroblasts by the v-fms oncogene. 217 Nov 88

1. Regulatory mechanism of cell growth of endometriosis in comparison with endometrium. Estradiol alone has no growth-promoting effect on both endometriotic and endometrial cells. Epidermal growth factor (EGF) stimulates cell growth of both cell types. Endometrial cells but not endometriotic cells produce and release EGF into culture media so that stimulatory effect of exogenous addition of EGF is blunted in endometrial cells. Estradiol exerts its mitogenic action by enhancing the mitogenic effect of EGF in endometrium. By contrast, the effect of estradiol is minimal in endometriotic cells, showing less dependency on estradiol for their proliferation. Progesterone inhibits cell growth of the both cell types in the same manner. 2. A biological role of EGF in endometriosis. Endometriotic cells possess EGF receptors. The affinity of the receptor is the same as that of endometrial cells. However, the number of receptor per cell is about half of that for endometrium. Estradiol increases the number of EGF receptors in endometrial cells which may explain the mitogenic effect of estradiol in the face of EGF. However, stimulatory effect of estradiol for EGF receptors is less pronounced in endometriotic cells. Mitogenic action of EGF is suggested to be mediated by phosphorylation of tyrosine residues of 170 kd protein in the tissues. EGF increases the production of tissue plasminogen activator (t-PA) and activates the aromatase activity of the both cell types. However, the stimulatory action of EGF on progestin receptor is observed only in endometrial cells. 3. Biochemical characterization of endometriotic cells in comparison with endometrial cells. Endometriotic tissues accumulate less amount of glycogen and XIII factor of blood coagulation as compared to endometrial tissues. The ability of endometriotic cells to release prostaglandin is also weaker, suggesting suppressed differentiated function of endometriotic cell. Endometriotic cells produce the same amount of CA125 as endometrial cells. Danazol and EGF inhibit the release of CA125 into culture media when standardized per cell. Therefore, normalization of CA125 levels during the treatment dose not always mean the reduction of the lesions but reflect the suppressed function of the endometriotic tissues. 4. Altered microenvironment of endometriotic tissues. An analysis of peritoneal fluid. The amount of peritoneal fluid (PF) with endometriosis increased throughout the menstrual cycle. A number of macrophage is reported to increase in PF with endometriosis.(ABSTRACT TRUNCATED AT 400 WORDS)
...
PMID:[Fundamental and clinical studies on biochemical properties of endometriosis in comparison with endometrium]. 250 2

The kringle 2 domain of human tissue-type plasminogen activator (t-PA) has been characterized via 1H NMR spectroscopy at 300 and 620 MHz. The experiments were performed on the isolated domain obtained by expression of the 174-263 portion of t-PA in Escherichia coli [Cleary et al. (1989) Biochemistry 28, 1884-1891]. The spectrum of t-PA kringle 2 is characteristic of a globular structure and shows overall similarity to that of the plasminogen (PGN) kringle 4. Spectral comparison with human and bovine PGN kringle 4 identifies side-chain resonances from Leu46, which afford a fingerprint of kringle folding, and from most of the aromatic ring spin systems. Assignment of signals arising from the His13, His48a, and His64 side chains, which are unique to t-PA kringle 2, was assisted by the availability of a His64----Tyr mutant. Ligand-binding studies confirm that t-PA kringle 2 binds L-lysine with an association constant Ka approximately 11.9 mM-1. The data indicate that homologous or conserved residues relative to those that compose the lysine-binding sites of PGN kringles 1 and 4 are involved in the binding of L-lysine to t-PA kringle 2. These include Tyr36 and, within the kringle inner loop, Trp62, His64, Trp72, and Tyr74. Acid/base titration of aromatic singlets in the presence of L-lysine yields pKa* approximately 6.25 and approximately 4.41 for His13 and His64, respectively, and shows that the His48a imidazole group does not protonate down to pH* approximately 4.3. Thus, the His48a and His64 side chains are in solvent-shielded locations. As observed for the PGN kringles, the Trp62 indole group titrates with pKa* approximately 4.60, which indicates proximity of the side chain to a titratable carboxyl group, most likely that of Asp57 at the binding site. Several labile NH protons of t-PA kringle 2 exhibit retarded H-exchange kinetics, requiring more than a week in 2H2O for full deuteration in the presence of L-lysine at 37 degrees C. This reveals that kringle 2 is endowed with a compact, dynamically stable conformation. Proton Overhauser experiments in 1H2O, centered on well-resolved NH resonances between 9.8 and 12 ppm, identify signals arising from the His48a imidazole NH3 proton and the three Trp indole NH1 protons. A strong dipolar interaction was observed among the Trp25 indole NH1, the Tyr50 amide NH, and the His48a imidazole CH2 protons, which affords evidence for an aromatic cluster in t-PA kringle 2 similar to that found at the hydrophobic kernel of PGN kringles.(ABSTRACT TRUNCATED AT 400 WORDS)
...
PMID:1H NMR structural characterization of a recombinant kringle 2 domain from human tissue-type plasminogen activator. 255 18

Differentiated clonal cell lines were isolated from pluripotent P19 embryonal carcinoma (EC) cells treated as aggregates with retinoic acid. Two were characterized in detail. The lines differ in morphology, proliferation rate, the production of plasminogen activator, and in their mitogenic response to insulin but both produce extracellular matrix proteins and can be serially passaged over extended periods, in contrast to differentiated derivatives of many other EC lines. Further, both lines have receptors for and respond mitogenically to epidermal growth factor (EGF). Endogenous phosphorylation of several proteins, including the EGF receptor (150 kDa) and a 38-kDa protein, is induced by EGF in membranes isolated from these cells. Preincubation of membranes with EGF renders them able to catalyze phosphorylation of tyrosine residues in exogenously added peptide substrates. High voltage electrophoresis confirmed the tyrosine specificity of the phosphorylation on the 150- and 38-kDa bands. By contrast, similar experiments in undifferentiated cells showed that intact P19 EC neither bind nor respond to EGF mitogenically and EGF induces no changes in phosphorylation in isolated membranes.
...
PMID:Clonal variants of differentiated P19 embryonal carcinoma cells exhibit epidermal growth factor receptor kinase activity. 298 69

We have examined the phosphorylation state of five proteins known to become phosphorylated on tyrosine during transformation by Rous sarcoma virus by using cells infected with a panel of partially transforming mutant viruses. Situations of viral mutant and growth temperature were found in which phosphorylation of some proteins occurred more extensively than that of others, indicating that mutations in the src gene had affected the specificity of pp60src for some of its substrates as well as affecting the activity of the enzyme. To obtain insight into the biological functions of these phosphorylations, comparisons were made between the degree of phosphorylation of these proteins and the expression of various indicators of the transformed phenotype. The data suggest that phosphorylation of proteins l, p, and q (Mr of 46,000, 39,000 and 28,000, respectively) is not sufficient to induce changes in adhesiveness, hexose transport or morphology. The phosphorylation of protein p or l or total phosphotyrosine content correlated well with the production of plasminogen activator, and the phosphorylation of proteins l and q correlated well with increased hexose transport. However, even when good correlations were observed, significant exceptions were sometimes noted. It thus remains possible that some phosphorylations on tyrosine observed in Rous sarcoma virus-transformed cells are not causally related to the expression of the measured parameters of transformation.
...
PMID:Phosphotyrosine-containing proteins and expression of transformation parameters in cells infected with partial transformation mutants of Rous sarcoma virus. 618 22

1 2 3 4 5 6 7 8 9 10 Next >>