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

Cell lines established from the lepidopteran insect Spodoptera frugiperda (fall armyworm; Sf9) are used routinely as hosts for the expression of foreign proteins by recombinant baculovirus vectors. We have examined the pathway of protein glycosylation and secretion in these cells, using human tissue plasminogen activator (t-PA) as a model. t-PA expressed in Sf9 cells was both N glycosylated and secreted. At least a subset of the N-linked oligosaccharides in extracellular t-PA was resistant to endo-beta-N-acetyl-D-glucosaminidase H, which removes immature, high-mannose-type oligosaccharides. This refutes the general conclusion from previous studies that Sf9 cells cannot process immature N-linked oligosaccharides to an endo-beta-N-acetyl-D-glucosaminidase H-resistant form. A nonglycosylated t-PA precursor was not detected in Sf9 cells, even with very short pulse-labeling times. This suggests that the mammalian signal sequence of t-PA is efficiently recognized in Sf9 cells and that it can mediate rapid translocation across the membrane of the rough endoplasmic reticulum, where cotranslational N glycosylation takes place. However, t-PA was secreted rather slowly, with a half-time of about 1.6 h. Thus, a rate-limiting step(s) in secretion occurs subsequent to translocation and N glycosylation of the t-PA polypeptide. Treatment of Sf9 cells with tunicamycin, but not with inhibitors of oligosaccharide processing, prevented the appearance of t-PA in the extracellular medium. This suggests that N glycosylation per se, but not processing of the N-linked oligosaccharides, is required directly or indirectly in baculovirus-infected Sf9 cells for the secretion of t-PA. Finally, the relative efficiency of secretion decreased dramatically with time of infection, suggesting that the Sf9 host cell secretory pathway is compromised during the later stages of baculovirus infection.
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PMID:Glycosylation and secretion of human tissue plasminogen activator in recombinant baculovirus-infected insect cells. 249 30

Tissue-type plasminogen activator (t-PA), the serine protease responsible for catalyzing the production of plasmin from plasminogen at the site of blood clots, is synthesized as a single-chain polypeptide precursor. Proteolytic cleavage at the C-terminal side of Arg275 generates a two-chain form of the enzyme whose subunits are held together by a single disulfide bond. We have measured the activities of both forms of the wild-type enzyme, as well as that of a mutant enzyme (Arg275----Gly), created by oligonucleotide-directed mutagenesis, that cannot be cleaved into a two-chain form. Both types of single-chain t-PAs are enzymatically active and exhibit identical Vmax and Km values when assayed with synthetic peptide substrates, indicating that the single amino acid change had no effect on the amidolytic activity of the enzyme. However, cleavage of wild-type t-PA into the two-chain form results in increased activity both on a peptide substrate and on the natural substrates Lys- and Glu-plasminogen in the absence or presence of stimulation by soluble fibrin. The enhanced activity is due to a 3-5-fold increase in the Vmax of the cleaved enzyme, rather than to any change in the Km values for the various substrates. During incubation with plasminogen, the single-chain form of wild-type t-PA is converted to the two-chain form by plasmin generated during the reaction. This conversion, from the less active form of the enzyme, results in a reaction that displays biphasic kinetics.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The single-chain form of tissue-type plasminogen activator has catalytic activity: studies with a mutant enzyme that lacks the cleavage site. 249 49

Human tissue-type plasminogen activator (t-PA) is a glycoprotein used currently in thrombolytic therapy. Because of its rapid half-life (T1/2) of approximately five minutes, intravenous (IV) infusion of large doses (approximately 100 mg) are required in patients treated for myocardial infarction. To identify the determinant(s) on t-PA responsible for such rapid clearance, metabolically labeled forms of recombinant t-PA were analyzed in rats following IV administration. The following seven forms of t-PA were tested: (a) natural or glycosylated wild-type t-PA; (b) nonglycosylated wild-type t-PA; (c) delta F t-PA, which lacks the fibronectin fingerlike domain; (d) delta E t-PA, which lacks the epidermal growth factor (EGF) domain; (e) delta FE t-PA, which lacks both the finger and EGF domains; (f) delta FE3X t-PA, a form of delta FE t-PA in which Asn-linked glycosylation is prevented at all known glycosylation sites (Asn-117, 184, and 448; replaced by Gln); and (f) delta FE1X t-PA, a form of delta FE t-PA in which high-mannose-type glycosylation is prevented at Asn-117. Both glycosylated and nonglycosylated wild-type t-PA cleared in an exponential biphasic manner, with an initial alpha-phase T1/2 of 0.8 and 1.9 minutes, respectively. This result demonstrates that carbohydrate is not the primary mediator of the rapid clearance of t-PA. The liver was the primary organ responsible for uptake of these molecules. All other proteins tested, except for delta E t-PA, demonstrated primarily monophasic clearance patterns with T1/2 ranging between 12 and 27 minutes, and reduced uptake in the liver. delta E t-PA however, cleared in a biphasic manner with an alpha-phase T1/2 of 2.1 minutes. Results presented suggest that the clearance of t-PA is mediated by two distinct mechanisms. The primary determinant(s) responsible for modulating the rapid clearance of t-PA appears to be resident within the polypeptide sequence encoding the finger and/or EGF domains, with emphasis on the finger domain. A second and less significant contribution to clearance is defined by the presence and type of glycosylation.
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PMID:Pharmacokinetic and distribution analysis of variant forms of tissue-type plasminogen activator with prolonged clearance in rat. 249 74

Tissue-type plasminogen activator (t-PA), an important component of the fibrinolytic system, is now available as a biotechnologically manufactured recombinant protein for therapeutic use. It has proved highly effective in the clinical therapy of acute thromboembolic diseases such as myocardial infarction and pulmonary embolism. t-PA activates plasminogen to plasmin, which subsequently dissolves the fibrin network of a blood clot. This activation by t-PA occurs selectively on the clot surface, with negligible systemic side effects. The half-life of t-PA in vivo is in the order of minutes due to rapid hepatic elimination. t-PA is a glycoprotein with serine protease activity and consists of a polypeptide chain with 527 amino acids. Recently, intensive research efforts have been devoted to modification of the molecular structure of t-PA, with the objective of further increasing fibrin specificity and catalytic activity, or reducing the rate of elimination. As a result, considerable insights into structure-function relationships within the t-PA molecule have been gained, but as yet no clinically utilizable variant has been constructed which is in all respects superior to naturally occurring t-PA.
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PMID:Properties of molecular variants of tissue-type plasminogen activator. 250 92

To probe the effects of N-glycosylation on the fibrin-dependent plasminogenolytic activity of tissue-type plasminogen activator (t-PA), we have expressed a human recombinant t-PA (rt-PA) gene in Chinese hamster ovary (CHO) cells and in a murine C127 cell line. The resulting rt-PA glycoproteins were isolated and their associated N-linked oligosaccharide structures determined by using a combination of high-resolution Bio-Gel P-4 gel filtration chromatography, sequential exoglycosidase digestion, and methylation analysis. The results show that CHO rt-PA is N-glycosylated differently from murine C127 derived rt-PA. Further, both rt-PA's are N-glycosylated differently from t-PA derived from a human colon fibroblast and the Bowes melanoma cell line (Parekh et al., 1989), confirming that N-glycosylation of the human t-PA polypeptide is cell-type-specific. Both CHO and murine rt-PA were fractionated on lysine-Sepharose chromatography. The N-glycosylation of the major forms was analyzed and their fibrin-dependent plasminogenolytic activity determined by using an indirect amidolytic assay with Glu-plasminogen and a chromogenic plasmin substrate. The results suggest that the various forms of rt-PA differ from one another with respect to the kinetics of their fibrin-dependent activation of plasminogen. Together, these data support the notion (Wittwer et al., 1989) that N-glycosylation influences the fibrin-dependent catalytic activity of t-PA and that t-PA when expressed in different cell lines may consist of kinetically and structurally distinct glycoforms.
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PMID:N-glycosylation and in vitro enzymatic activity of human recombinant tissue plasminogen activator expressed in Chinese hamster ovary cells and a murine cell line. 251 93

Cells from gliomas induced by N-ethyl-N-nitrosourea have a high basal level of plasminogen activator activity compared with cells from normal tissue. Plasminogen activator activity is known to be affected by many substances but whether inhibition or stimulation occurs depends on the cell and agent involved. It is not clear whether tumour and control cells from the same type of tissue respond similarly. A comparison has been made of the effect of several factors on both cell associated and secreted enzyme activity of cloned lines from a glioma and normal tissue. The effect of two cAMP elevating compounds was stimulatory while that of the steroid, dexamethasone, was generally inhibitory for both cells. However, the polypeptide hormone, epidermal growth factor, had a differential effect. It caused an increase in secreted enzyme activity in the tumour line but had no such effect on the control clone. The precise mechanism by which this occurs is unknown. Co-operative effects of the enzyme and growth hormone could result in more aggressive behaviour of the tumour cells.
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PMID:A comparison of the effect of several factors on the plasminogen activator activity of cloned lines from an ethylnitrosourea-induced glioma and from normal tissue. 262 4

Interaction between cells of the immune system and of the synovial tissue may be contributing to the pathogenesis of rheumatoid arthritis. Cytokines, produced by PBMC, were tested for their effects on plasminogen activator activity and PGE2 levels of a number of human synovial fibroblast-like cell lines. Evidence was obtained for a human monocyte polypeptide, synovial activator, which can stimulate synovial cell plasminogen activator activity but not PGE2 levels. Purified human rIL-1 alpha and IL-1 beta increased the levels of both products in the supernatants of the synovial cells; TNF-alpha raised the PGE2 levels but raised the plasminogen activator activity only weakly and inconsistently. Synovial activator was further distinguished from IL-1 alpha and IL-1 beta (and TNF-alpha) on biochemical, immunologic, and functional criteria. No other purified native or recombinant human cytokine tested was active on the synovial cells when judged by the criteria in question. Synovial activator could therefore be a novel monokine. The differences in the type of response elicited by the various cytokines in the synovial fibroblast-like cells could have implications for the understanding of the cellular interactions occurring in rheumatoid lesions.
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PMID:Plasminogen activator and prostaglandin E2 levels in human synovial fibroblasts. Differential stimulation by synovial activator and other cytokines. 278 54

The iodinated Mr approximately equal to 15,000 amino-terminal fragment (ATF) of the urokinase-type plasminogen activator (u-PA) molecule bound specifically to the cell surface of all of seven cultured human tumor cell lines studied. Cross-linking of iodinated ATF to the cell surface using a bifunctional amino-reactive reagent followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed with the four cell lines studied the occurrence of a single band migrating with an Mr of 70,000-75,000, indicating complex formation with an Mr of 55,000-60,000 u-PA receptor protein (u-PA-R). In the human monocyte cell line U937 cultivated in the presence of phorbol ester, the amount of complex was strongly increased, and a fraction of the complex had a slower electrophoretic mobility. Comparison between autoradiograms of reduced and unreduced samples suggests that u-PA-R consists of one polypeptide chain. Two forms of u-PA-R, which differed with respect to affinity to concanavalin A, were identified. u-PA-R retained its ability to bind to ATF after cell lysis, and it was purified approximately 2,200-fold from biosynthetically labeled U937 cells by affinity chromatography with proenzyme u-PA coupled to Sepharose. The purified Mr 55,000-60,000 protein was specifically bound and cross-linked to u-PA, proenzyme u-PA, and ATF, but not to tissue-type plasminogen activator or other unrelated proteins.
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PMID:A 55,000-60,000 Mr receptor protein for urokinase-type plasminogen activator. Identification in human tumor cell lines and partial purification. 282 65

A urokinase-type plasminogen activator was purified from conditioned media of several human cell cultures, but preferably from the human lung adenocarcinoma line CALU-3 (ATCC, HTB-55), using a combination of chromatography on zinc chelate-Sepharose, SP-Sephadex C-50, and Sephadex G-100. Final yields of 65-100 micrograms/liter of starting material were obtained with a 290-fold purification factor and a recovery of 30%. The purified plasminogen activator consists of a single polypeptide chain with Mr 54,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and is very similar or identical to single-chain urokinase-type plasminogen activator on the basis of immunodiffusion, amino acid composition, and the lack of specific binding to fibrin. It has very low amidolytic activity on Pyroglu-Gly-Arg-rho-nitroanilide and is converted to two-chain urokinase by limited exposure to plasmin. It has a specific activity of 60,000 IU/mg on fibrin plates and directly activates plasminogen following Michaelis-Menten kinetics with Km = 1.1 microM and kappa cat = 0.0026 S-1. It is concluded that the plasminogen activator purified from CALU-3-conditioned media is physically and kinetically identical to single-chain urokinase-type plasminogen activator. With the present straightforward purification method and a readily available source, sufficient amounts of single-chain urokinase-type plasminogen activator can be obtained for more detailed investigations of its biochemical, biological, and thrombolytic properties.
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PMID:Purification and characterization of single-chain urokinase-type plasminogen activator from human cell cultures. 308 Apr 23

Purified 2-chain recombinant tissue-type plasminogen activator (t-PA) was reduced under mild conditions - 10 mM dithiothreitol/5 degrees C/1.5 h - and the two chains were separated by chromatography on lysine Sepharose. The t-PA B chain was fully active as determined by its activity towards the chromogenic substrate S-2288 (H-D-ile-pro-arg p-nitroanilide). Analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis under reducing or non-reducing conditions revealed a single polypeptide at Mr = 35,000 or 29,000 respectively. In addition, under non-reducing conditions a fibrinolytic band at apparent Mr = 29,000 was present after fibrin zymography. The N-terminal sequence was confirmed as ile-lys-gly. The t-PA B chain had a specific amidolytic activity, using S-2288, of 170,000 to 210,000 SU/mg protein. (This compares to a specific activity of the native 2-chain t-PA of 170,000 SU/mg). It resembles urokinase-type plasminogen activator in its inability to be stimulated by fibrin and its dose response on human fibrin plates. However, t-PA B-chain was stimulated to almost the same extent as t-PA by poly-D-lysine. The isoelectric points, at pH 5.6 and 5.7, fall outside the range generally quoted for t-PA preparations (pH 7.8-8.8).
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PMID:Isolation and preliminary characterisation of active B-chain of recombinant tissue-type plasminogen activator. 308 69


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