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1.
Protein S has an important anticoagulant function by acting as a cofactor for activated protein C (APC). We recently reported that the EGF1 domain residue Asp95 is critical for APC cofactor function. In the present study, we examined whether additional interaction sites within the Gla domain of protein S might contribute to its APC cofactor function. We examined 4 residues, composing the previously reported "Face1" (N33S/P35T/E36A/Y39V) variant, as single point substitutions. Of these protein S variants, protein S E36A was found to be almost completely inactive using calibrated automated thrombography. In factor Va inactivation assays, protein S E36A had 89% reduced cofactor activity compared with wild-type protein S and was almost completely inactive in factor VIIIa inactivation; phospholipid binding was, however, normal. Glu36 lies outside the ω-loop that mediates Ca(2+)-dependent phospholipid binding. Using mass spectrometry, it was nevertheless confirmed that Glu36 is γ-carboxylated. Our finding that Gla36 is important for APC cofactor function, but not for phospholipid binding, defines a novel function (other than Ca(2+) coordination/phospholipid binding) for a Gla residue in vitamin K-dependent proteins. It also suggests that residues within the Gla and EGF1 domains of protein S act cooperatively for its APC cofactor function.  相似文献   

2.
We have identified 2 PROS1 missense mutations in the exon that encodes the vitamin K-dependent Gla domain of protein S (Gly11Asp and Thr37Met) in kindred with phenotypic protein S deficiency and thrombosis. In studies using recombinant proteins, substitution of Gly11Asp did not affect production of protein S but resulted in 15.2-fold reduced protein S activity in a factor Va inactivation assay. Substitution of Thr37Met reduced expression by 33.2% (P <.001) and activity by 3.6-fold. The Gly11Asp variant had 5.4-fold reduced affinity for anionic phospholipid vesicles (P <.0001) and decreased affinity for an antibody specific for the Ca(2+)-dependent conformation of the protein S Gla domain (HPS21). Examination of a molecular model suggested that this could be due to repositioning of Gla29. In contrast, the Thr37Met variant had only a modest 1.5-fold (P <.001), reduced affinities for phospholipid and HPS21. This mutation seems to disrupt the aromatic stack region. The proposita was a compound heterozygote with free protein S antigen levels just below the lower limit of the normal range, and this is now attributed to the partial expression defect of the Thr37Met mutation. The activity levels were strongly reduced to 15% of normal, probably reflecting the functional deficit of both protein S variants. Her son (who was heterozygous only for Thr37Met) had borderline levels of protein S antigen and activity, reflecting the partial secretion and functional defect associated with this mutation. This first characterization of natural protein S Gla-domain variants highlights the importance of the high affinity protein S-phospholipid interaction for its anticoagulant role.  相似文献   

3.
We expressed 2 chimeras between human protein S (PS) and human prothrombin (FII) in which the prothrombin gamma-carboxyglutamic acid (Gla) domain replaced the PS Gla domain in native PS (Gla(FII)-PS) or in PS deleted of the thrombin-sensitive region (TSR) (Gla(FII)-DeltaTSR-PS). Neither PS/FII chimera had activated protein C (APC) cofactor activity in plasma clotting assays or purified systems, but both bound efficiently to phospholipids. This pointed to a direct involvement of the PS Gla domain in APC cofactor activity through molecular interaction with APC. Using computational methods, we identified 2 opposite faces of solvent-exposed residues on the PS Gla domain (designated faces 1 and 2) as potentially involved in this interaction. Their importance was supported by functional characterization of a PS mutant in which the face 1 and face 2 PS residues were reintroduced into Gla(FII)-PS, leading to significant APC cofactor activity, likely through restored interaction with APC. Furthermore, by characterizing PS mutants in which PS face 1 and PS face 2 were individually replaced by the corresponding prothrombin faces, we found that face 1 was necessary for efficient phospholipid binding but that face 2 residues were not strictly required for phospholipid binding and were involved in the interaction with APC.  相似文献   

4.
Tie JK  Jin DY  Straight DL  Stafford DW 《Blood》2011,117(10):2967-2974
We describe a cell-based assay for studying vitamin K-cycle enzymes. A reporter protein consisting of the gla domain of factor IX (amino acids 1-46) and residues 47-420 of protein C was stably expressed in HEK293 and AV12 cells. Both cell lines secrete carboxylated reporter when fed vitamin K or vitamin K epoxide (KO). However, neither cell line carboxylated the reporter when fed KO in the presence of warfarin. In the presence of warfarin, vitamin K rescued carboxylation in HEK293 cells but not in AV12 cells. Dicoumarol, an NAD(P)H-dependent quinone oxidoreductase 1 (NQO1) inhibitor, behaved similarly to warfarin in both cell lines. Warfarin-resistant vitamin K epoxide reductase (VKOR-Y139F) supported carboxylation in HEK293 cells when fed KO in the presence of warfarin, but it did not in AV12 cells. These results suggest the following: (1) our cell system is a good model for studying the vitamin K cycle, (2) the warfarin-resistant enzyme reducing vitamin K to hydroquinone (KH?) is probably not NQO1, (3) there appears to be a warfarin-sensitive enzyme other than VKOR that reduces vitamin K to KH?, and (4) the primary function of VKOR is the reduction of KO to vitamin K.  相似文献   

5.
Sun YH  Shen L  Dahlbäck B 《Blood》2003,101(6):2277-2284
Protein C is a member of the vitamin K- dependent protein family. Proteins in this family have similar gamma-carboxyglutamic acid (Gla)-rich domains, but their affinities for negatively charged phospholipid membranes vary more than 1000-fold. We have shown that it is possible to enhance anticoagulant activity and membrane affinity of protein C by selective mutagenesis of the Gla domain. In this study, 3 new mutants, Q10G11N12 (QGN), S23E32D33Y44 (SEDY), and Q10G11N12S23E32D33Y44 (QGNSEDY), were created. In plasma-based coagulation assays, the activated form of QGNSEDY (QGNSEDY-APC) demonstrated approximately 20-fold higher anticoagulant activity than wild-type activated protein C (WT APC), while QGN-APC and SEDY-APC did not. Both normal activated factor V (FVa) and FVa Leiden (Arg506Gln) were degraded much more efficiently by QGNSEDY-APC than by WT APC in the presence as well as in the absence of protein S. Binding of protein C variants to negatively charged phospholipid membranes was investigated using light scattering and the BIAcore technique. QGNSEDY demonstrated 3- to 7-fold enhanced binding as compared with WT protein C, suggesting the membrane affinity to be influenced by several residues located at different parts of the Gla domain. The anticoagulant activity as well as phospholipid binding ability was only enhanced when multiple regions of the Gla domain were modified. The results provide insights into the molecular mechanisms that are involved in determining the binding affinity of the interaction between Gla domains and phospholipid membranes. The unique properties of QGNSEDY-APC suggest this APC variant possibly to have greater therapeutic potential than WT APC.  相似文献   

6.
Hermansky-Pudlak syndrome (HPS) is one of the few genetic disorders associated with severe pulmonary fibrosis. Fifty percent of affected patients die as a result of respiratory insufficiency. Fibrosis is thought to be caused by the accumulation of ceroid, an insoluble fluorescent lipoprotein, both extracellularly and in the lysosomes of alveolar macrophages. In addition to pulmonary fibrosis, HPS is characterized by oculocutaneous albinism and a reduction in the number of platelet dense bodies. CD63 is a protein that was described originally in platelet lysosomes. It localizes to the membranes of melanosomes and platelet dense bodies. CD63 is decreased dramatically in the lysosomes and dense bodies of patients with HPS. We theorized that CD63, a membrane protein common to lysosomes, melanosomes, and platelet dense bodies, may play a role in HPS. We sought to characterize the gene coding for this protein in HPS lymphoid cell lines. The coding region for CD63 was sequenced in control and HPS cell lines. Messenger RNA from HPS and normal cell lines was examined by Northern analysis. Genomic DNA from the same cell lines was examined by Southern analysis and polymerase chain reaction (PCR). CD63 protein in lymphoid cell lines and peripheral blood monocytes was compared by Western analysis. We found no mutations in the coding region of CD63 in an HPS cell line. We also found no diminution in the quantity of CD63 RNA by Northern analysis and no gross defects in the structural gene by PCR and Southern analysis, suggesting that the CD63 structural gene, promoter, and untranslated regions were normal. Western analysis showed that the 43-kDa protein was present in control and HPS lymphoid cell lines and peripheral blood monocytes in equivalent amounts. Although CD63 is an attractive candidate for the primary defect of HPS, the disease is probably not caused by a mutation in the CD63 gene. Accepted for publication: 18 September 1997  相似文献   

7.
心室电风暴的机制与起搏作用的实验观察   总被引:13,自引:5,他引:13  
目的观察心室电风暴与室房逆传的关系和起搏的作用。方法以扎、松冠状动脉左前降支的方法制作25只犬缺血-再灌注室性心律失常模型,以针电极探查希氏-浦肯野系统(HPS)的电冲动。结果16只犬自发3次以上室性心动过速/心室颤动(简称室速/室颤),符合电风暴。电风暴时室房传导呈4种类型:Ⅰ~Ⅲ型HPS异位电冲动皆连续逆传,Ⅰ型间断夺获心房;Ⅱ型未夺获心房,但连续抑制前向房波下传,产生假性Ⅲ度房室阻滞;Ⅲ型连续夺获心房;IV型HPS逆向与前向传导交替。快速起搏心房可重建房室前传和稳定的血压。快速起搏心室作用有:①抑制异位电冲动形成,防止触发室颤,但不终止自律性异常室速;②拖带和终止折返性室速,显现室速的拖带变形现象和双向折返;③多不夺获快速室速/室颤,偶见HPS起搏和串刺激夺获心室,快速室速/室颤频率减慢后自发终止。结论HPS异位电冲动逆向传导,阻滞窦性心律下传,促使室速/室颤反复发作而呈现电风暴现象。起搏重建房室传导和抑制异位电冲动形成,有预防电风暴的作用。  相似文献   

8.
Breast tumor cell lines have been shown to secrete several distinct polypeptide growth factors, although conflicting results exist for the insulin-like growth factors (IGFs). In contrast a limited number of breast tumor cell lines have definitely been shown to secrete the high affinity IGF binding proteins (IGFBPs) that modify IGF actions. To characterize the types of IGFBPs that are secreted by breast tumor cell lines, conditioned medium was collected from seven separate tumor cell lines, three of which were estrogen receptor (ER) negative, and four of which were ER positive. All three of the ER negative cell lines, MDA-231, MDA-330, and HS578T, secreted binding proteins of 49,000 and 43,000 Mr (IGFBP-3) as well as 29,000 (IGFBP-1) and 24,000 Mr. In contrast, all four ER positive cell lines secreted 34,000 (IGFBP-2) or 24,000 Mr forms, and none secreted the 49,000 and 43,000 or 29,000 Mr forms. BT-20, a cell line that is positive for ER messenger RNA (mRNA) but negative for ER protein, secreted predominantly a 34,000 Mr protein. The amount of total IGFBP activity released in 24 h ranged between 0.4 and 5.6 nM equivalents of IGFBP-1, and there was no significant difference between the ER positive and negative cell lines. The MCF-7 cells that produced predominantly 34,000 and 24,000 Mr forms showed a 1.8-fold increase in IGFBP secretion after estrogen stimulation. Immunoblotting and a specific RIA for IGFBP-1 showed that only the ER negative lines MDA-330, MDA-231, and HS578T secreted this form. Northern blotting analysis for the mRNA encoding this protein showed that both MDA-330 and MDA-231 contained a single 1.6 kilobase mRNA species that hybridized with an IGFBP-1 complementary DNA (cDNA) probe. Immunoblotting analysis of the other cell lines showed that only the 34,000 Mr form secreted by the ER positive cell lines reacted with IGFBP-2 antisera. Exposure of the conditioned media from the three ER negative cell lines to N-glycanase revealed that the 49,000 and 43,000 Mr forms of IGFBP were glycosylated and therefore probably represent IGFBP-3. We conclude that ER negative cell lines secrete three forms of IGFBPs, IGFBP-1, IGFBP-3, and a 24,000 Mr form. In contrast, the ER positive cell lines secrete predominantly IGFBP-2 and the 24,000 Mr form but do not secrete IGFBP-3 or 1.(ABSTRACT TRUNCATED AT 400 WORDS)  相似文献   

9.
A human osteosarcoma cell line derived from cells obtained from a patient with Paget's disease is shown to synthesize and secrete bone Gla protein (BGP); (osteocalcin), a noncollagenous bone matrix protein. Using a human BGP-specific RIA, we show that the human osteosarcoma cells synthesize significant amounts of BGP without any prior induction of BGP synthesis by 1,25-dihydroxyvitamin D. After specific immunoprecipitation of poly-A+ RNA in vitro translation products with antibodies to BGP, we found that BGP is synthesized as a precursor with an apparent mol wt of 13.5K, as demonstrated on 15% sodium dodecyl sulfate-polyacrylamide gels. Finally, pulse labeling of the osteosarcoma cells with [3H]proline reveals that the cells synthesize mature BGP of 12,000 mol wt as well as a higher mol wt precursor (13,500) of the protein.  相似文献   

10.
Human gastric carcinoma cell line STKM-1 secretes a large protein that induces scattering of a rat liver epithelial cell line (BRL) into disconnected individual cells in monolayer culture. This cell-scattering factor was purified from serum-free conditioned medium of STKM-1 cells and found to be composed of three disulfide-linked subunits of 140, 150, and 160 kDa. The 140-kDa peptide contains an amino acid sequence homologous to that of the laminin B2t chain. The native protein has an apparent molecular mass of > 1000 kDa and a pI of 5.0. In addition to the cell-scattering activity, the purified protein stimulates attachment of BRL cells to substrate and their migration. Similar effects have been observed toward various cell lines, including nontumorigenic epithelial, endothelial, and fibroblastic cell lines and human cancer cell lines. Similar cell-scattering activity was secreted by human squamous carcinoma and gastric carcinoma cell lines and nontumorigenic epithelial and endothelial cell lines. These results indicate that the protein, named "ladsin," is probably an extracellular matrix protein that regulates cell-cell and cell-substrate interactions and cell migration.  相似文献   

11.
Platelet-derived endothelial cell growth factor (PD-ECGF) is a 45-kDa endothelial cell mitogen which has angiogenic properties in vivo. We report here that human foreskin fibroblasts, a human squamous cell carcinoma cell line, and 2 out of the 3 human thyroid carcinoma cell lines investigated produce PD-ECGF, whereas 21 other cell lines examined do not. The positive cell lines contained a 1.8-kilobase PD-ECGF mRNA, and a 45-kDa protein could be demonstrated in lysates of the cell lines by immunoblotting and immunoprecipitation using a specific antiserum against PD-ECGF. Furthermore, the cell lysates contained mitogenic activity for endothelial cells that was neutralized by the PD-ECGF antiserum. PD-ECGF was found to be secreted only slowly from the producer cells, consistent with the previous finding that the primary translation product lacks a signal sequence. The restricted expression and intracellular sequestration of PD-ECGF imply a strictly controlled function in endothelial cell proliferation and angiogenesis. Aberrant production of PD-ECGF may play a role in tumor angiogenesis.  相似文献   

12.
Growth hormone releasing hormone (GHRH) receptors are members of the G-protein receptor family that use cAMP as a second messenger. A human fetal kidney 293-derived cell line stably expressing the porcine GHRH receptor (pGHRHr/293 cells) and a cAMP-responsive reporter system were used to develop a bioassay for human GHRH. The reporter system (ph180SEAP) was constructed by subcloning the tandem cAMP response elements from the human glycoprotein hormone subunit gene promoter (h180) upstream from the secreted alkaline phosphatase cDNA of reporter plasmid pSEAP-Basic. To generate a stable cell line expressing both the GHRH receptor and SEAP reporter system, a DNA fragment from pPUR that confers puromycin resistance was subcloned downstream from the reporter construct of ph180SEAP. Tranfection of ph180SEAPpur into pGHRHr/293 cells yielded pGHRHr/SEAP/293 cell lines that responded to recombinant GHRH with dose-dependent increases in SEAP activity. The GHRH receptor-SEAP reporter bioassay was compared to a conventional bioassay using cultured rat anterior pituitary cells. Synthetic and recombinant GHRH induced a 3.1-fold increase in growth hormone release by rat pituitary cells with ED50's of 3.6 and 2.2×10−10 M, respectively. Recombinant GHRH was 1.7±0.7 times more potent than synthetic GHRH in the pituitary cell bioassay. In an analogous experiment, pGHRHr/SEAP/293 cells responded to synthetic and recombinant GHRH with a 9.1-fold increase in SEAP activity. The ED50's were 7.8 and 4.3×10−11 M, respectively, with recombinant GHRH being 1.8±0.1 times more potent than the synthetic preparation. Thus, the GHRH receptor-SEAP reporter bioassay is a sensitive, accurate, precise and efficient method for measuring GHRH biological activity.  相似文献   

13.
The gamma-carboxyglutamic acid (Gla) domain of blood coagulation factors is responsible for Ca2+-dependent phospholipid membrane binding. Factor X-binding protein (X-bp), an anticoagulant protein from snake venom, specifically binds to the Gla domain of factor X. The crystal structure of X-bp in complex with the Gla domain peptide of factor X at 2.3-A resolution showed that the anticoagulation is based on the fact that two patches of the Gla domain essential for membrane binding are buried in the complex formation. The Gla domain thus is expected to be a new target of anticoagulant drugs, and X-bp provides a basis for designing them. This structure also provides a membrane-bound model of factor X.  相似文献   

14.
Epstein-Barr virus (EBV) infects human B lymphocytes and epithelial cells. We have compared the requirements for EBV glycoprotein-induced cell fusion between Chinese hamster ovary effecter cells and human B lymphoblasts or epithelial cells by using a virus-free cell fusion assay. EBV-encoded gB, gH, gL, and gp42 glycoproteins were required for efficient B cell fusion, whereas EBV gB, gH, and gL glycoproteins were required for Chinese hamster ovary effecter cell fusion with epithelial cell lines (AGS and SCC68) or the human embryonic kidney cell line 293-P. Fusion with human embryonic kidney 293-P cells was greater than fusion observed with B cells, indicative of an important role for cell contact. An antibody directed against the gH and gL complex inhibited epithelial cell fusion. Increased surface expression of gB alone as a result of truncations or point mutants in the carboxyl-terminal tail allowed gB-mediated fusion with epithelial cells, albeit at a lower level than with coexpression of gB, gH, and gL. Overall, gB appears to be the critical component for EBV glycoprotein-mediated cell fusion.  相似文献   

15.
Virulence for BALB/c mice, infectivity for Phlebotomus papatasi, haemagglutination activity and expression of metacyclic lipophosphoglycan (LPG) were studied in four strains of Leishmania major (LV561, FV1, L119 and Neal) and various lines of the LV561 strain. Attenuated line LV561/AV was passaged five times through sandflies or mice and the resulting lines (AVS5 and AVM5, respectively) and two of the earlier sandfly passages (AVS1 and AVS2) were used for further study. The highly virulent line LV561/V served as a control. Virulence for mice was not regained during passaging of LV561/AV in sandflies or mice (none of the mice infected with AVM5, AVS1, AVS2 or AVS5 displayed overt lesions) and the success rate in cultivating parasites, from lymph-node samples from inoculated mice, was not significantly higher for any of these lines than for the original line (LV561/AV). However, AVM5 and AVS5 developed better than LV561/AV in P. papatasi and the intensity and localisation of their infections were similar to those of the virulent control. In smears of the infected guts of P. papatasi, the AVS5 parasites resembled the virulent line (LV561/V) morphologically whereas the AVM5 parasites were similar to the avirulent LV561/AV. Haemagglutination activity increased as a result of passaging, the most pronounced difference being observed in AVM5, which had 60-fold higher activity than LV561/AV. Expression of metacyclic LPG was not increased by passaging. The proportion of forms reacting positively with 3F12 antibodies was high (about 17%) in the virulent LV561/V but low (2%-3%) in the avirulent lines LV561/AV, AVS5 and AVM5. A defect in LPG is not, however, likely to be the only reason for the avirulence observed, as the avirulent lines of LV561 still produced about 10 times as many metacyclic promastigotes as the strain L119, which caused delayed lesions in mice.  相似文献   

16.
Injury and repair in chronic liver disease involve cell adhesion, migration, apoptosis, proliferation, and a wound healing response. In liver, fibroblast activation protein (FAP) has both collagenase and dipeptidyl peptidase IV (DPIV) activities and is expressed only by activated hepatic stellate cells (HSC) and myofibroblasts, which produce and degrade extracellular matrix (ECM). FAP was colocalized with collagen fibers, fibronectin, and collagen type I in human liver. FAP function was examined in vitro by expressing green fluorescent protein FAP fusion protein in cell lines cultured on collagen-I, fibronectin, and Matrigel. Glutamates at 203 and 204 as well as serine624 of FAP were essential for peptidase activity. Human embryonic kidney 293T cells overexpressing FAP showed reduced adhesion and migration. FAP overexpression in the human HSC line LX-2 caused increased cell adhesion and migration on ECM proteins as well as invasion across transwells in the absence or presence of transforming growth factor beta-1. FAP overexpression enhanced staurosporine streptomyces-stimulated apoptosis in both cell lines. Interestingly, the enzyme activity of FAP was not required for these functions. Overexpressing FAP increased the expression of matrix metalloproteinase-2 and CD44 and reduced integrin-beta1 expression in 293T cells, suggesting potential pathways of FAP-mediated impairment of cell adhesion and migration in this epithelial cell line. In conclusion, these findings further support a pro-fibrogenic role for FAP by indicating that, in addition to its enzymatic functions, FAP has important nonenzymatic functions that in chronic liver injury may facilitate tissue remodeling through FAP-mediated enhancement of HSC cell adhesion, migration, and apoptosis.  相似文献   

17.
Yet  MG; Jones  SS 《Blood》1993,82(6):1713-1719
We have generated a truncated form of the erythropoietin receptor (EPO- R), the extracytoplasmic ligand-binding domain, that is secreted from a transfected Chinese hamster ovary (CHO) cell line. The truncated receptor is readily purified from CHO conditioned media as a 33-Kd glycosylated protein, which is converted to a 25-Kd species upon treatment with protein N-glycan glycosidase F. Cross-linking of radioiodinated EPO to the secreted receptor yielded a complex of 72 Kd. Also, the growth of the EPO-dependent cell line, FDCPE, was inhibited in a dose-responsive manner by the truncated receptor. The complex of the secreted receptor and EPO was isolated by gel filtration and shown to be a one-to-one complex of the receptor and growth factor by quantitative amino terminal sequencing. Finally, analysis of the interaction of the receptor and growth factor by gel filtration indicated an apparent dissociation constant of 1.1 nmol/L for the truncated receptor.  相似文献   

18.
Protein-S is a vitamin K (Vit K)-dependent protein synthesized by hepatocytes, megakaryocytes, and endothelial cells and plays an important role in the regulation of hemostasis. Two cases of free protein-S congenital deficiency were recently reported to be associated with osteopenia. We hypothesized that this osteopenia could be the result of a bone deficit of protein-S synthesized by bone cells. Using enzyme-linked immunoassay, immunocytochemistry, immunoblotting, and immunoprecipitation after labeling with [35S]methionine, we have shown that this protein is secreted by three human osteosarcoma cell lines and by human adult osteoblast-like cells. In addition, protein-S was present in protein extracts of human bone matrix. Protein-S secreted by MG 63 cells increased linearly from 1-7 days of culture, was biologically active, and was regulated by warfarin, as previously described for the other cell types secreting protein-S. Vit K had no direct effect on protein-S secretion or activity, but could overcome the effects of warfarin. In conclusion, in addition to osteocalcin and matrix gamma-carboxyglutamic acid (Gla) protein, osteoblasts secrete another Vit K-dependent protein, which is a constituent of the bone matrix. Our data suggest that osteopenia occurring in patients with congenital protein-S deficiency might be related to a deficiency of protein-S secretion by the osteoblasts. This finding raises the intriguing possibility that protein-S might play a role in bone turnover and bone mass.  相似文献   

19.
20.
The murine mast cell line PB-3c is dependent on interleukin 3 (IL-3) with respect to survival and proliferation. These cells also require IL-3 to display antigen-mediated serotonin release, which is coupled to a transient increase of cytosolic free calcium ([Ca2+]i). The antigen-mediated exocytosis is inhibited by phorbol 12-tetradecanoate 13-acetate (PTA), an activator of phospholipid/Ca2+-sensitive protein kinase. In contrast, the malignant mast cell variant PB-1 is IL-3 independent with respect to proliferation but is unable to undergo antigen-mediated exocytosis. Yet this cell line exhibits basal levels of [Ca2+]i, serotonin content, and numbers of IgE receptors comparable to those of PB-3c cells. Subcellular distribution studies revealed that the specific activity of cytosolic protein kinase C of PB-1 cells was only 40% of that found in PB-3c cells. Furthermore, the PB-1 cells showed a significantly higher specific activity of membrane-bound protein kinase C than PB-3c cells. Scatchard plot analysis of [3H]-phorbol 12,13-dibutyrate binding to intact PB-1 cells demonstrated the presence of 20% high-affinity (Kd = 6 nM) and 80% low-affinity (Kd = 60 nM) phorbol ester receptors, whereas PB-3c cells displayed only the low-affinity phorbol ester binding. Immunological characterization of protein kinase C from both cell lines revealed the presence of a normal 77-kDa protein kinase C holoenzyme in both cell lines. In addition, a 72-kDa protein kinase C-related protein band was found mainly in the membrane fraction of the PB-1 variant. It is suggested that this altered and membrane-bound form of protein kinase C may be involved in the blockage of the antigen-mediated exocytosis of PB-1 cells.  相似文献   

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