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1.
Malate saturation isotherms for the NAD+ malic enzyme determined at widely differing, but saturating, concentrations (8, 80, 160 mm) of magnesium show the same response to malate concentration only when velocity is plotted against the concentration of free malate2?. This identification of the ionized malic acid as the true substrate for this enzyme, together with the observation that the complex of Mg with malate has no influence on the reaction rate even at very high concentrations, indicates that the metal ion activator of the enzyme must also bind in the ionized form. A kinetic analysis shows that, with respect to magnesium and malate, the malic enzyme catalyzes a rapid equilibrium reaction of the intersecting type. Either Mg2+ or malate2? may bind first but the fact that the Km's for both Mg2+ and malate2? are smaller than the respective Ki's suggests that, when either metal ion or malate is present on the enzyme, the other is bound more tightly than when it binds to the free enzyme. This demonstration of the nature of the true substrates for this enzyme has implications for studies of the NAD+ malic enzyme in which conditions influencing the amount of free magnesium and malate, e.g., changes in pH, addition of weak acid effectors etc., are involved. 相似文献
2.
Effect of parathyroid hormone on phospholipid metabolism in osteoblast-like rat osteogenic sarcoma cells.
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Previous results have shown that 1,25-dihydroxycholecalciferol [1,25(OH)2D3] enhances the synthesis of phosphatidylserine (PS) and suppresses the synthesis of phosphatidylethanolamine (PE) in osteoblast-like rat osteogenic sarcoma UMR 106 cells [Matsumoto, Kawanobe, Morita & Ogata (1985) J. Biol. Chem. 260, 13704-13709]. In the present study, the effect of parathyroid hormone (PTH) on phospholipid metabolism is examined by using these cells. Treatment of UMR 106 cells with human PTH-(1-34)-peptide suppresses the synthesis of phosphatidylethanolamine in a dose- and time-dependent manner without affecting the synthesis of PS. The maximal effect on PE synthesis is obtained with 2.4 nM-human PTH-(1-34)-peptide when the cells are treated for 48 h or longer. In addition, when human PTH-(1-34)-peptide is added together with the maximal dose of 1,25(OH)2D3, there is a further decline in PE synthesis, whereas the stimulation of PS synthesis by 1,25(OH)2D3 is not altered. Because methylation of PE is suggested to affect hormone receptor-adenylate cyclase coupling, the observed change in PE metabolism by PTH and 1,25(OH)2D3 may be, at least in part, involved in the development of desensitization phenomenon to PTH in these cells. 相似文献
3.
T D Gelehrter R Sznycer-Laszuk R Zeheb B J Cwikel 《Molecular endocrinology (Baltimore, Md.)》1987,1(1):97-101
Incubation of HTC rat hepatoma cells with the synthetic glucocorticoid dexamethasone rapidly inhibits plasminogen activator (PA) activity and reveals the presence of a specific PA inhibitor (PAI-1). To determine whether the hormonal inhibition of PA activity reflects a decrease in the amount of PA or an increased amount of the inhibitor, or both, we have assayed PA and PAI-1 immunologically. HTC PA was determined to be entirely of the tissue type (tPA), and both free and complexed antigen was quantified by a RIA using rabbit antirat tPA, with rat insulinoma tPA as tracer and standard. PAI-1 was quantified by a Western blot assay using rabbit anti-HTC PAI-1 antibody and purified HTC PAI-1 as standard. Under conditions in which dexamethasone inhibited PA activity by 90%, there was no decrease in the cellular content of tPA antigen. Paradoxically, dexamethasone increased tPA antigen approximately 1.5-fold. Under these same conditions, dexamethasone increased PAI-1 antigen 4- to 5-fold. We conclude that the glucocorticoid inhibition of tPA activity in HTC cells is not secondary to a decrease in the amount of tPA but is secondary to the induction of a specific PA inhibitor. 相似文献
4.
Interaction of tissue-type plasminogen activator and plasminogen activator inhibitor 1 on the surface of endothelial cells 总被引:7,自引:0,他引:7
The site of the reaction between plasminogen activators and plasminogen activator inhibitor 1 (PAI-1) was investigated in cultures of human umbilical vein endothelial cells. In conditioned medium from endothelial cells, two forms of a plasminogen activator-specific inhibitor can be demonstrated: an active form that readily binds to and inhibits plasminogen activators and an immunologically related quiescent form which has no anti-activator activity but which can be activated by denaturation. In conditioned medium, only a few percent of PAI-1 is the active form. However, the addition of increasing concentrations of tissue-type plasminogen activator (t-PA) or urokinase to confluent endothelial cells produced a saturable (3.0 pmol/5 x 10(5) cells), dose-dependent increase of the activator-PAI-1 complex in the conditioned medium even in the presence of actinomycin D or cycloheximide. This resulted also in a dose-dependent decrease of the residual PAI activity measured by reverse fibrin autography both in the conditioned medium and cell extracts. Short-time exposure of endothelial cells to a large amount of t-PA caused almost complete depletion of all cell-associated PAI activity. Although there was no detectable PAI activity even after activation of PAI by denaturants or antigen in the culture medium at 4 degrees C without the addition of t-PA, the addition of t-PA at 4 degrees C not only resulted in the formation of 70% of the amount of the t-PA.PAI complex in conditioned medium at 37 degrees C, but also induced PAI-1 antigen in a time and dose-dependent manner in the conditioned medium. Moreover, 125I-labeled t-PA immobilized on Sepharose added directly to endothelial cells formed a complex with PAI-1 in a dose-dependent manner. On the other hand, no detectable complex was formed with PAI-1 when Sepharose-immobilized 125I-labeled t-PA was added to endothelial cells under conditions in which the added t-PA could not contact the cells directly but other proteins could pass freely by the use of a Transwell. All these results suggest that a "storage pool" on the surface of endothelial cells or the extracellular matrix produced by endothelial cells contains almost all the active PAI-1, and reaction between PA and PAI-1 mainly occurs on the endothelial cell membranes, resulting in a decrease of the conversion of active PAI-1 to the quiescent form. 相似文献
5.
S Fukumoto E H Allan J A Yee T D Gelehrter T J Martin 《Journal of cellular physiology》1992,152(2):346-355
In order to determine the mechanism by which parathyroid hormone (PTH) stimulates plasminogen activator (PA) activity in rat osteoblasts, we investigated the effect of human PTH(1-34) [hPTH(1-34)] on the synthesis of mRNAs for tissue-type PA (tPA), urokinase-type PA (uPA), and PA inhibitor-1 (PAI-1), and on release of PA activity and PAI-1 protein in both normal rat calvarial osteoblasts and UMR 106-01 osteogenic sarcoma cells. hPTH(1-34) (0.25-25 nM) decreased PAI-1 mRNA and protein, and increased PA activity in both cell types in a dose-dependent manner with ED50 of about 1 nM for both responses. Forskolin and isobutylmethylxanthine also stimulated PA activity and decreased PAI-1 protein and mRNA in both cell types. hPTH(1-34) did not show any consistent effect on tPA and uPA mRNA in calvarial osteoblasts, but a modest (two-fold) increase of both mRNAs was observed in UMR 106-01 cells treated with 25 nM hPTH(1-34). However, when protein synthesis was inhibited with 100 microM cycloheximide, the increase of tPA and uPA mRNA by hPTH(1-34) was enhanced in UMR 106-01 cells and became evident in calvarial osteoblasts. Fibrin autography also revealed that hPTH(1-34) increases tPA and uPA activity, especially after cycloheximide treatment in UMR 106-01 cells. These results strongly suggest that PTH increases PA activity predominantly by decreasing PAI-1 protein production through a cyclic adenosine monophosphate (cAMP)-dependent mechanism in rat osteoblasts. The reduction of PAI-1 protein by PTH results in enhanced action of both tPA and uPA, and would contribute to the specific roles of these PAs in bone. 相似文献
6.
Oncostatin M (OSM) is a glycoprotein cytokine that is produced by activated T-lymphocytes, monocytes, and macrophages. In a DNA synthesis assay, OSM reduced tritiated thymidine incorporation by 53% in Calu-1 lung carcinoma cells. Radiolabeled cDNAs from untreated Calu-1 cells and 30-h OSM-treated cells were used to probe duplicate nylon membrane cDNA expression arrays. This study revealed OSM-mediated expression of mRNAs encoding tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1). Northern blot analysis showed that the steady-state level of tPA mRNA is nearly undetectable in Calu-1 cells. Exposure of these cells to OSM for 30 h increased tPA mRNA expression by 20-fold and PAI-1 mRNA expression by 5-fold. Exposure of these cells to other gp130 receptor family cytokines, including leukemia inhibitory factor (LIF), interleukin-6 (IL-6), and IL-11, do not significantly affect DNA synthesis or induction of tPA/PAI-1. Western blot studies demonstrated that OSM mediates a marked increase in secretion of the tPA protein. Secreted tPA was present in the conditioned medium almost exclusively as tPA/PAI-1 complexes. Inhibitor studies demonstrated that OSM-mediated induction of tPA and PAI-1 mRNAs is largely dependent upon activation of the MEK1/2 pathway. The JAK3/STAT3 pathway potentially serves a secondary role in these regulatory events. 相似文献
7.
T Ny Y X Liu M Ohlsson P B Jones A J Hsueh 《The Journal of biological chemistry》1987,262(24):11790-11793
Gonadotropin-releasing hormone (GnRH) acts directly on the ovary to induce ovulation in hypophysectomized proestrous rats. Because plasminogen activators (PAs) are implicated in gonadotropin-induced ovulation, we have studied the effect of GnRH on ovarian PA synthesis. GnRH induced tissue-type PA (tPA) secretion by cultured rat granulosa cells, but inhibited the secretion of urokinase-type PA. These effects were blocked by co-treatment with a GnRH antagonist, suggesting that stereospecific GnRH receptors are involved. Follicle-stimulating hormone (FSH) also induced tPA in granulosa cells but with a different time course than GnRH; the combined effect of FSH and GnRH was additive. The GnRH effect was mimicked by the calcium- and phospholipid-dependent protein kinase C activator, phorbol myristate acetate. In isolated cumulus-oocyte complexes and cumulus cells, GnRH treatment also increased tPA activity. In contrast, treatment of denuded oocytes with GnRH did not increase enzyme activity. After GnRH stimulation of the cumulus-oocyte complexes, tPA content in the denuded oocyte was elevated, suggesting that the cumulus cells mediate the action of GnRH to increase the oocyte enzyme levels. Hybridization experiments using a labeled rat tPA-specific DNA probe showed that both FSH and GnRH increased the level of tPA mRNA in cultured granulosa cells; the stimulatory effect of GnRH was blocked by the GnRH antagonist. Our results indicate that GnRH treatment increases tPA secretion by cultured granulosa cells and cumulus-oocyte complexes. The stimulation of enzyme activity in the granulosa cells is accompanied by increases in tPA mRNA levels. 相似文献
8.
Regulation of tissue-type plasminogen activator and plasminogen activator inhibitor type-1 in cultured rat Sertoli and Leydig cells 总被引:3,自引:0,他引:3
New data are provided to show that (i) rat Sertoli cells produce two types of plasminogen activators, tissue type (tPA) and urokinase type (uPA), and a plasminogen activator inhibitor type-1 (PAI-1); (ii) both tPA (but not uPA) and PAI-1 secretion in the culture are modified by FSH, forskolin, dbcAMP, GnRH, PMA and growth factors (EGF and FGF), but not by hCG and androstenedione (△4); (iii) in vitro secretion of tPA and PA-PAI-1 complexes of Sertoli cells are greatly enhanced by presence of Leydig cells which produce negligible tPA but measurable PAI-1 activity;(iv) combination culture of Sertoli and Leydig cells remarkably increases FSH-induced PAI-1 activity and decreases hCG- and forskolin-induced inhibitor activity as compared with that of two cell types cultured alone. These data suggest that rat Sertoli cells, similar to ovarian granulosa cells, are capable of secreting both tPA and uPA, as well as PAI-1. The interaction of Sertoli cells and Leydig cells is essential for the cells to response to 相似文献
9.
Effect of retinoic acid on the synthesis of tissue-type plasminogen activator and plasminogen activator inhibitor-1 in human endothelial cells 总被引:2,自引:0,他引:2
The synthesis of plasminogen activators and inhibitors in endothelial cells is highly regulated by hormones, drugs and growth factors. The present study evaluates the effect of retinoic acid on the synthesis of tissue-type plasminogen activator (t-PA) and of plasminogen activator inhibitor-1 (PAI-1) by cultured human umbilical vein endothelial cells (HUVEC). Retinoic acid produced a time- and concentration-dependent increase in the secretion of t-PA-related antigen but not of PAI-1 related antigen into the culture medium. A maximal sevenfold increase of t-PA antigen after 24 h was observed with 10 microM and a half-maximal increase with 0.1 microM retinoic acid. Retinoic acid induced a time-dependent increase of the t-PA mRNA, with a maximum at 8 h and returning to normal at 24 h. The protein kinase inhibitor H7 decreased the t-PA antigen induced by both retinoic acid and phorbol 12-myristate 13-acetate. These results suggest that treatment of HUVEC with retinoic acid increases t-PA production by a pathway which, at some level, involves protein kinases. Thus, retinoic acid induces t-PA synthesis in the absence of altered PAI-1 synthesis, which may enhance the fibrinolytic potential of the endothelium. 相似文献
10.
The occurrence of plasminogen activators of the urokinase-type (u-PA) and tissue-type (t-PA) at various stages of the epithelial cycle was studied immunohistochemically in rat seminiferous tubule segments. u-PA immunoreactivity was detected exclusively at stages VII and VIII in Sertoli cells, displaying a distinct granular cytoplasmic staining. t-PA immunoreactivity was found during mid- and late pachytene and diakinesis (stages VII-XIII) in spermatogenic cells, displaying a granular cytoplasmic staining with maximal intensity in stages IX-XIII. The specificity of the stainings was supported by staining controls, including absorption of the antibodies with purified preparations of the activators. It was also supported by zymographic studies of the occurrence of u-PA and t-PA in extracts of tubular segments at different stages of the cycle, isolated by transillumination-assisted microdissection. The possible functions of the two types of plasminogen activators in the seminiferous epithelium are discussed. 相似文献
11.
We have employed parathyroid hormone (PTH) responsive human cells cultured from dermis or giant cell tumors of bone (GT) to evaluate the biological properties of a newly developed in vivo PTH inhibitor, [Tyr34]bPTH-(7-34)-amide (PTH-Inh). Short periods of incubation of cells from dermis or GT with maximal stimulatory concentrations of PTH in the presence of increasing concentrations of PTH-Inh resulted in a dose-dependent inhibition of the adenosine cyclic 3',5'-phosphate (cAMP) response (Ki = 3 X 10(-7) M and 4.2 X 10(-7) M for GT and dermal cells, respectively). In both cell cultures, PTH-Inh alone did not increase cAMP levels, and in desensitization experiments, preincubation with PTH-Inh alone did not desensitize cells to PTH. Hence, the analogue displayed no agonist properties. Unexpectedly, when PTH-Inh was incubated with dermal cells in the presence of PTH, the PTH-Inh failed to block desensitization, suggesting a loss of biological effectiveness of the inhibitor. When medium containing PTH-Inh alone was removed from dermal cells and tested for inhibition of the acute PTH response in untreated cells, there was apparent loss of inhibitory efficacy (t1/2 = 20 h). In contrast, incubation of native PTH or bPTH-(1-34) with cells did not affect the biological activity of these ligands. Unlike the dermal cells, the PTH-Inh did block desensitization to PTH in GT, and there was no loss of inhibitor efficacy when medium containing PTH-Inh was incubated with GT (48 h) and then tested in untreated cells.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
12.
Functional effects of asparagine-linked oligosaccharide on natural and variant human tissue-type plasminogen activator 总被引:5,自引:0,他引:5
L Hansen Y Blue K Barone D Collen G R Larsen 《The Journal of biological chemistry》1988,263(30):15713-15719
The role of Asn-linked oligosaccharide in the functional properties of both human tissue-type plasminogen activator (t-PA) and a genetic variant of t-PA was studied. Nonglycosylated and glycosylated wild-type t-PA were produced in mammalian cells which express recombinant t-PA. These proteins were compared in fibrin binding and 125I-labeled fibrin clot lysis assays, using purified components. The nonglycosylated form showed higher fibrin binding, as well as higher fibrinolytic potency than the glycosylated form. Subsequently, prevention of glycosylation of a t-PA variant which lacked the finger and epidermal growth factor domains (delta FE), was carried out in an attempt to enhance its fibrinolytic activity. Glycosylation was prevented by changing Asn to Gln; at Asn-117 to produce delta FE1X t-PA, and at Asn-117, -184, and -448 to produce delta FE3X t-PA. All variants were similar to wild-type t-PA in their catalytic dependence on fibrinogen fragments, fibrinolytic activity in fibrin autography analysis, and plasminogen activator activity. In a clot lysis assay, using citrated human plasma, the fibrinolytic potency of the variants were comparable to that of wild-type t-PA at activator concentrations of 17-51 nM (approximately 1-3 micrograms/ml). At 0.5-5.1 nM (approximately 0.03-0.3 micrograms/ml), however, the variant proteins had lower fibrinolytic potency than wild-type t-PA. Fifty percent lysis in 1.5 h for wild-type, delta FE, delta FE1X, and delta FE3X t-PA, required 2.5, 10, 7.5, and 5.5 nM t-PA, respectively. The fibrinogenolytic activity in human plasma was measured for wild-type, delta FE, delta FE1X, and delta FE3X t-PA, and showed significant fibrinogen depletion after 3 h of incubation at 51 nM, decreasing to 11, 11, 50, and 72% of basal levels, respectively. These data indicate that partial or total nonglycosylated t-PA variants have a higher fibrinolytic versus fibrinogenolytic ratio than their fully glycosylated counterparts. 相似文献
13.
The Leydig I-10 tumor cell line was synchronized by the double thymidine block method using 1.0 mM thymidine. Protein phosphokinase activity of subcellular fractions was determined at various times throughout the cell cycle. Microsomal cAMP-independent kinase activity increased in G2 and decreased during the S and G1 phases. Except for relatively small increases during the G1 and late S phases, microsomal cAMP-dependent kinase activity remained unchanged throughout most of the cycle. In the lysosomal-mitochondrial fraction, cAMP-dependent and cAMP-independent protein kinase activity increased during the S phase. Independent kinase activity peaked again during G1, while the dependent kinase became depressed. Phosphokinase activity increased in the nuclear fraction in late G2 and during mitosis, and was due to increases in both cAMP-independent and cAMP-dependent kinase activity. Cytosol cAMP-dependent kinase activity increased in G2 and during mitosis; cAMP-independent kinase activity showed some increased activity during late G2 and mitosis. These temporal variations in the subcellular kinase activities throughout the cell cycle may act to phosphorylate subcellular protein substrates in a cell cycle-specific fashion. 相似文献
14.
15.
G Y Shi J S Hau S J Wang I S Wu B I Chang M T Lin Y H Chow W C Chang L Y Wing C J Jen 《The Journal of biological chemistry》1992,267(27):19363-19368
Plasmin inhibited the biosynthesis of tissue-type plasminogen activator (tPA) antigen by human umbilical vein endothelial cells (HUVEC) in a dose-dependent manner. The amount of tPA antigen found in the 24-h conditioned medium of cells treated with 100 nM plasmin for 1 h was 20-30% of that in the control group. However, in contrast to tPA, such treatment led to a 3-fold increase in plasminogen activator inhibitor (PAI) activity, whereas the amount of PAI type 1 antigen was unchanged. The effects of plasmin on HUVEC were binding- and catalytic activity-dependent and were specifically blocked by epsilon-aminocaproic acid. Microplasmin, which has no kringle domains, was less effective in reducing tPA antigen biosynthesis or enhancing PAI activity in HUVEC. Kringle domains of plasmin affected neither tPA antigen nor PAI activity of the cells. Other proteases including chymotrypsin, trypsin, and collagenase at comparable concentrations did not have a significant effect on the biosynthesis of tPA antigen or PAI activity of HUVEC. Thrombin stimulated the biosynthesis of tPA and PAI-1 antigens by HUVEC. Thrombin also stimulated an increase in the protein kinase activity in HUVEC, whereas plasmin inhibited the protein kinase activity of the cells. It is possible that plasmin regulates the biosynthesis of tPA in HUVEC through the signal transduction pathway involving protein kinase. 相似文献
16.
One-chain urokinase-type plasminogen activator from human sarcoma cells is a proenzyme with little or no intrinsic activity 总被引:15,自引:0,他引:15
L C Petersen L R Lund L S Nielsen K Dan? L Skriver 《The Journal of biological chemistry》1988,263(23):11189-11195
We have compared the plasminogen activating capacity of one- and two-chain urokinase-type plasminogen activator (u-PA). In a 125I-plasminogen conversion assay in the presence of high amounts of a plasmin inhibitor, one-chain u-PA pretreated with diisopropyl fluorophosphate had no detectable activity, the detection limit corresponding to the activity of a 400-fold lower amount of two-chain u-PA. In coupled assays in which generated plasmin was measured with a synthetic substrate, activity was clearly observed with the one-chain preparation, but the initial rate of plasminogen activation was lower than that of a 250-fold smaller concentration of two-chain u-PA. The coupled assays for one-chain u-PA are self-activating because plasmin catalyzes conversion of one- to two-chain u-PA, and it is not possible to decide whether the low activity of one-chain u-PA observed with this type of assay is intrinsic or due to contaminations. On the basis of these findings and a discussion of previous studies, it is concluded that one-chain u-PA has a variety of properties similar to the one-chain proenzyme forms of other serine proteases and that it should, therefore, be considered as a genuine proenzyme form of u-PA. 相似文献
17.
G V Andreenko L V Podorol'skaia T N Serebriakova A V Liaginski? B B Egorov 《Biulleten' eksperimental'no? biologii i meditsiny》1988,106(10):424-426
The plasminogen activator 960 IU/mg protein activity isolated from cultured fluid of the calf kidney cells was introduced to albino rats (180-200 g) with experimental Heynmann nephritis every day during 4 days. Nephritis caused activation of haemostasis and inhibition of fibrinolysis in the blood. There was increased excretion of the fibrin, fibrinogen degradation products in urine as a results of the local fibrin deposition in diseased kidneys. The fibrinolytic activity of the cortical zone of kidney was markedly decreased. The plasminogen activator, infused to experimental animals, resulted in normalization of the altered indexes. 相似文献
18.
Plasma tissue-type plasminogen activator (t-PA) is cleared rapidly in vivo by the liver. Previous studies with the human hepatoma cell line HepG2 have identified a clearance system for t-PA modulated by plasminogen activator inhibitor type 1 (PAI-1). In the present study, a rat hepatoma cell line MH1C1 is shown to contain a PAI-1-independent t-PA clearance system. At 4 degrees C, binding of 125I-t-PA to MH1C1 cells was rapid, specific, and saturable. Scatchard analysis of the binding data yielded a mean estimate of 105,000 high affinity binding sites per cell (Kd = 4.1 nM). When the bound ligand was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the majority (about 90%) of the specific binding was in the form of uncomplexed 125I-t-PA. This is in contrast to HepG2 cells in which specific binding was mainly in the form of a sodium dodecyl sulfate-stable 125I-t-PA.PAI-1 complex. When availability of matrix-associated PAI-1 was blocked by preincubation with anti-PAI-1 antibody or removed by elastase treatment, specific 125I-t-PA binding to MH1C1 cells was unaffected, whereas most of the specific 125I-t-PA binding to HepG2 cells was abolished. Furthermore, when the active site of t-PA was inactivated with diisopropyl fluorophosphate, the diisopropyl fluorophosphate-t-PA specifically competed for binding of 125I-t-PA to MH1C1 cells, but failed to block specific 125I-t-PA binding to HepG2 cells. At 37 degrees C, PAI-1-independent t-PA binding to MH1C1 cells was followed by ligand uptake and degradation with kinetics similar to that seen in HepG2 cells. Chemical cross-linking of t-PA to MH1C1 cells revealed a specific t-PA binding protein with a molecular mass of about 500,000 daltons. Ligand-receptor complexes generated by chemical cross-linking were immunoprecipitable by anti-t-PA antibody but not by anti-PAI-1 antibody, further supporting the finding that binding of t-PA to MH1C1 cells is PAI-1-independent. 相似文献
19.
Intracellular plasminogen activator (PA) was examined in 3T3 and transformed 3T3 cells under various growth conditions to determine whether expression of this activity changes with the growth state. During exponential growth, SV40 and benzpyrene (BP) transformed 3T3 cells exhibited 3- to 5-fold more intracellular PA activity than untransformed 3T3 cells. This relationship changed as the cells exhausted serum factors and arrested in G1. The specific activity of intracellular PA in cells that have retained a serum-sensitive restriction point in G1 (G0) (3T3 and BP 3T3) increased 200- and 20-fold, respectively, at this time, while the level in cells that have lost most growth control mechanisms (SV3T3) remained constant. At confluency, 3T3 cells had considerably more PA than either of their transformed counterparts. Sparse cultures of 3T3 and BP3T3 cells arrest at G1 following serum depravation, and also accumulate high intracellular PA activity. The addition of serum or purified epidermal growth factor to these cultures initiated cell proliferation and resulted in a rapid, actinomycin D-sensitive loss of this activity. Less than 50% of the original activity remained 30 minutes after growth stimulation. This loss of intracellular PA activity did not appear to result from the presence of serum or cellular inhibitors. Intracellular PA activity remained low following growth stimulation. It increased again as the cells traversed through G1. These findings indicate that intracellular PA activity fluctuates with the growth state of cells, and may be related to the cell cycle. Culture conditions which place cells, whether normal or transformed, in G1 arrest lead to increased intracellular PA, while factors that initiate growth again result in a rapid loss of this activity. This behavior is lacking in cells not subject to density-dependent inhibition of growth. Like many other correlates of transformation, comparison of intracellular PA in normal and transformed cells must be defined in terms of the growth state of the cells in question. 相似文献
20.
M Hamaguchi I Takahashi T Takehara J Takamatsu H Saito 《Biochimica et biophysica acta》1989,1009(2):143-150
Tissue-type plasminogen activator produced by recombinant DNA technology (rt-PA) has now been recognized as a promising clot-selective thrombolytic agent. We have compared the properties of rt-PA expressed in mouse C127 cells with those of naturally occurring human vascular plasminogen activator (HV-PA). The molecular weight of HV-PA and rt-PA was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to be approx. 66,000. HV-PA and rt-PA were labile and rapidly lost their activities at pH values below 5.5. The optimum pH of HV-PA and rt-PA for plasminogen activation was around 8.5. HV-PA and rt-PA appeared to be very similar in amidolytic properties, amino-acid composition and carbohydrate composition. Moreover, the N-terminal amino-acid sequence of HV-PA was in good agreement with that of rt-PA. The purified preparations of HV-PA and rt-PA had specific activities of about 250,000 and 600,000 IU/mg, respectively. Both activators bound to fibrin clots to similar degree. In immunodiffusion as well as in the quenching experiments of the fibrinolytic activities, rt-PA appeared to be immunodiffusion as well as in the quenching experiments of the fibrinolytic activities, rt-PA appeared to be immunologically indistinguishable from HV-PA. All these findings indicate that rt-PA expressed in mouse C127 cells is identical with naturally occurring HV-PA in physical and chemical properties. 相似文献