RESUMEN
Two cell surface proteins (nectin-1/HveC and nectin-2/HveB) shown previously to serve as receptors for the entry of herpes simplex virus 1 (HSV-1) wild-type and/or mutant strains were found to serve also as receptors for HSV-1-induced cell fusion. Transfection with genomic DNA from a syncytial HSV-1 strain encoding wild-type gD resulted in fusion of Chinese hamster ovary (CHO) cells expressing nectin-1 but not of cells expressing nectin-2. In contrast, transfection with DNA from a related HSV-1 strain encoding the mutant Rid1 form of gD resulted in fusion of CHO cells expressing either receptor but not of control cells. These results are consistent with the ability of each receptor to mediate entry of viruses expressing wild-type or Rid1 gD and with results obtained previously with HVEM (HveA), a third HSV-l entry receptor. Undersulfation of GAGs in receptor-expressing cell lines predictably reduced susceptibility to HSV-l infection. In contrast, susceptibility to cell fusion mediated by HVEM or nectin-1 was not reduced. Undersulfation of GAGs partially inhibited cell fusion mediated by nectin-2. We conclude that HSV-1-induced cell fusion requires a gD-binding entry receptor, that ability of an HSV-1 strain to use HVEM, nectin-2 or nectin-1 for cell fusion depends on the allele of gD expressed and that GAGs may influence cell fusion, dependent on the gD-binding receptor used, but are less important for cell fusion mediated by HVEM, nectin-2 or nectin-l than for viral entry.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Fusión de Membrana , Receptores Virales/metabolismo , Simplexvirus/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Proteínas Virales de Fusión/metabolismo , Animales , Células CHO , Moléculas de Adhesión Celular/genética , Cricetinae , ADN Viral/efectos de los fármacos , Glicosaminoglicanos/química , Glicosaminoglicanos/metabolismo , Mutación , Nectinas , Receptores Virales/genética , Simplexvirus/genética , Ésteres del Ácido Sulfúrico/química , Ésteres del Ácido Sulfúrico/metabolismo , Transfección/métodos , Proteínas del Envoltorio Viral/genética , Proteínas Virales de Fusión/químicaRESUMEN
With the exception of hyaluronic acid, all mammalian saccharides assemble while attached to a lipid or protein primer. Several cases are now known in which oligosaccharide synthesis will occur on synthetic glycoside primers added to cells. A protocol is described in this unit in which beta-D-xylosides initiate glycosaminoglycan (GAG) synthesis by substituting for endogenous xylosylated core proteins. At high concentration xylosides will also prime oligosaccharides that resemble glycolipids. N-acetyl-alpha-D-galactosaminides initiate the synthesis of O-linked oligosaccharides found on mucins and other glycoproteins in an analogous manner. Even disaccharides, such as peracetylated N-acetyllactosaminide, can act as primers. Because these primers compete with endogenous substrates, they also act as inhibitors of proteoglycan (PG) and glycoprotein synthesis. Thus, primers have utility for studying the biological activity of glycoconjugates in cells, tissues, and animals. This unit describes procedures for using glycoside primers in cell culture.
Asunto(s)
Glicoproteínas/antagonistas & inhibidores , Glicoproteínas/metabolismo , Glicósidos/síntesis química , Oligosacáridos/biosíntesis , Proteoglicanos/antagonistas & inhibidores , Proteoglicanos/metabolismo , Acetilación , Animales , Glicósidos/química , Humanos , Oligosacáridos/químicaRESUMEN
The herpesvirus entry mediator A (HveA) is a recently characterized member of the tumor necrosis factor receptor family that mediates the entry of most herpes simplex virus type 1 (HSV-1) strains into mammalian cells. Studies on the interaction of HSV-1 with HveA have shown that of all the viral proteins involved in uptake, only gD has been shown to bind directly to HveA, and this binding mediates viral entry into cells. In addition to gD binding to HveA, the latter has been shown to interact with proteins of tumor necrosis factor receptor-associated factor family, lymphotoxin-alpha (LT-alpha), and a membrane-associated protein referred to as LIGHT. To study the relationship between HveA, its natural ligands, and the viral proteins involved in HSV entry into cells, we have screened two phage-displayed combinatorial peptide libraries for peptide ligands of a recombinant form of HveA. Affinity selection experiments yielded two peptide ligands, BP-1 and BP-2, which could block the interaction between gD and HveA. Of the two peptides, only BP-2 inhibited HSV entry into CHO cells transfected with an HveA-expressing plasmid. When we analyzed these peptides for the ability to interfere with HveA binding to its natural ligand LT-alpha, we found that BP-1 inhibited the interaction of cellular LT-alpha with HveA. Thus, we have dissected the sites of interaction between the cell receptor, its natural ligand LT-alpha and gD, the virus-specific protein involved in HSV entry into cells.
Asunto(s)
Proteínas Portadoras/metabolismo , Linfotoxina-alfa/metabolismo , Péptidos/metabolismo , Receptores del Factor de Necrosis Tumoral/antagonistas & inhibidores , Receptores Virales/antagonistas & inhibidores , Proteínas del Envoltorio Viral/metabolismo , Secuencia de Aminoácidos , Animales , Bacteriófagos , Unión Competitiva , Células CHO , Línea Celular , Cricetinae , Ligandos , Datos de Secuencia Molecular , Péptidos/síntesis química , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/metabolismo , Miembro 14 de Receptores del Factor de Necrosis Tumoral , Receptores Virales/genética , Receptores Virales/metabolismo , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Spodoptera/citologíaRESUMEN
Certain mutant strains of herpes simplex virus type 1 (HSV-1) are unable to infect cells in which entry is dependent on HVEM, the previously described herpesvirus entry mediator designated here as herpesvirus entry protein A (HveA). These mutant viruses can infect other cells where entry is apparently dependent on other co-receptors. The mutant virus HSV-1(KOS)Rid1 was used to screen a human cDNA expression library for ability of transfected plasmids to convert resistant Chinese hamster ovary cells to susceptibility to virus entry. A plasmid expressing the previously described poliovirus receptor-related protein 2 (Prr2) was isolated on the basis of this activity. This protein, designated here as HveB, was shown to mediate the entry of three mutant HSV-1 strains that cannot use HVEM as co-receptor, but not wild-type HSV-1 strains. HveB also mediated the entry of HSV-2 and pseudorabies virus but not bovine herpesvirus type 1. HveB was expressed in some human neuronal cell lines, fibroblastic cells, keratinocytes, and primary activated T lymphocytes. Antibodies specific for HveB blocked infection of HveB-expressing CHO cells and a human fibroblastic cell strain HEL299. Differences in ability of HSV-1 and HSV-2 strains to use HveB for entry should influence the types of cells that can be infected and thereby account in part for serotype and strain differences in tissue tropism and pathogenicity.
Asunto(s)
Alphaherpesvirinae/fisiología , Glicoproteínas de Membrana/fisiología , Mutación/fisiología , Receptores del Factor de Necrosis Tumoral , Receptores Virales , Alphaherpesvirinae/genética , Animales , Especificidad de Anticuerpos , Células CHO , Moléculas de Adhesión Celular/análisis , Línea Celular , Clonación Molecular , Cricetinae , ADN Recombinante , Fibroblastos , Células HeLa , Humanos , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Nectinas , ARN Mensajero/análisis , Miembro 14 de Receptores del Factor de Necrosis Tumoral , Replicación ViralRESUMEN
The purpose of this study was to determine whether a cell surface protein that can serve as coreceptor for herpes simplex virus type 1 (HSV-1) entry, herpesvirus entry mediator (previously designated HVEM but renamed HveA), also mediates HSV-1-induced cell-cell fusion. We found that transfection of DNA from KOS-804, a previously described HSV-1 syncytial (Syn) strain whose Syn mutation was mapped to an amino acid substitution in gK, induced numerous large syncytia on HveA-expressing Chinese hamster ovary cells (CHO-HVEM12) but not on control cells (CHO-C8). Antibodies specific for gD as well as for HveA were effective inhibitors of KOS-804-induced fusion, consistent with previously described direct interactions between gD and HveA. Since mutations in gD determine the ability of HSV-1 to utilize HveA for entry, we examined whether the form of virally expressed gD also influenced the ability of HveA to mediate fusion. We produced a recombinant virus carrying the KOS-804 Syn mutation and the KOS-Rid1 gD mutation, which significantly reduces viral entry via HveA, and designated it KOS-SR1. KOS-SR1 DNA had a markedly reduced ability to induce syncytia on CHO-HVEM12 cells and a somewhat enhanced ability to induce syncytia on CHO-C8 cells. These results support previous findings concerning the relative abilities of KOS and KOS-Rid1 to infect CHO-HVEM12 and CHO-C8 cells. Thus, HveA mediates cell-cell fusion as well as viral entry and both activities of HveA are contingent upon the form of gD expressed by the virus.
Asunto(s)
Fusión Celular , Herpesvirus Humano 1/fisiología , Receptores del Factor de Necrosis Tumoral , Receptores Virales/fisiología , Animales , Células CHO , Chlorocebus aethiops , Cricetinae , Mutación , Miembro 14 de Receptores del Factor de Necrosis Tumoral , Células VeroRESUMEN
HVEM (for herpesvirus entry mediator) is a member of the tumor necrosis factor receptor superfamily and mediates entry of many strains of herpes simplex virus (HSV) into normally nonpermissive Chinese hamster ovary (CHO) cells. We used sucrose density centrifugation to demonstrate that purified HSV-1 KOS virions bind directly to a soluble, truncated form of HVEM (HVEMt) in the absence of any other cell-associated components. Therefore, HVEM mediates HSV entry by serving as a receptor for the virus. We previously showed that soluble, truncated forms of HSV glycoprotein D (gDt) bind to HVEMt in vitro. Here we show that antibodies specific for gD, but not the other entry glycoproteins gB, gC, or the gH/gL complex, completely block HSV binding to HVEM. Thus, virion gD is the principal mediator of HSV binding to HVEM. To map sites on virion gD which are necessary for its interaction with HVEM, we preincubated virions with gD-specific monoclonal antibodies (MAbs). MAbs that recognize antigenic sites Ib and VII of gD were the only MAbs which blocked the HSV-HVEM interaction. MAbs from these two groups failed to coprecipitate HVEMt in the presence of soluble gDt, whereas the other anti-gD MAbs coprecipitated HVEMt and gDt. Previous mapping data indicated that site VII includes amino acids 11 to 19 and site Ib includes 222 to 252. The current experiments indicate that these sites contain residues important for HSV binding to HVEM. Group Ib and VII MAbs also blocked HSV entry into HVEM-expressing CHO cells. These results suggest that the mechanism of neutralization by these MAbs is via interference with the interaction between gD in the virus and HVEM on the cell. Group Ia and II MAbs failed to block HSV binding to HVEM yet still neutralized HVEM-mediated entry, suggesting that these MAbs block entry at a step other than HVEM binding.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Anticuerpos Antivirales/metabolismo , Herpesvirus Humano 1/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Receptores Virales/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Animales , Sitios de Unión , Células CHO , Línea Celular , Chlorocebus aethiops , Cricetinae , Humanos , Modelos Moleculares , Pruebas de Neutralización , Pruebas de Precipitina , Conejos , Spodoptera , Células Vero , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
Herpes simplex virus (HSV) 1 and 2 infect activated T lymphocytes by attachment of the HSV envelope glycoprotein D (gD) to the cellular herpesvirus entry mediator (HVEM), an orphan member of the tumor necrosis factor receptor superfamily. Here, we demonstrate that HVEM binds two cellular ligands, secreted lymphotoxin alpha (LTalpha) and LIGHT, a new member of the TNF superfamily. LIGHT is a 29 kDa type II transmembrane protein produced by activated T cells that also engages the receptor for the LTalphabeta heterotrimer but does not form complexes with either LTalpha or LTbeta. HSV1 gD inhibits the interaction of HVEM with LIGHT, and LIGHT and gD interfere with HVEM-dependent cell entry by HSV1. This characterizes herpesvirus gD as a membrane-bound viokine and establishes LIGHT-HVEM as integral components of the lymphotoxin cytokine-receptor system.
Asunto(s)
Linfotoxina-alfa/metabolismo , Proteínas de la Membrana/metabolismo , Receptores del Factor de Necrosis Tumoral , Receptores Virales/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Herpesvirus Humano 1/inmunología , Herpesvirus Humano 1/metabolismo , Herpesvirus Humano 2/inmunología , Herpesvirus Humano 2/metabolismo , Humanos , Ligandos , Activación de Linfocitos , Linfotoxina-alfa/genética , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Miembro 14 de Receptores del Factor de Necrosis Tumoral , Receptores Virales/genética , Sensibilidad y Especificidad , Homología de Secuencia de Aminoácido , Linfocitos T/metabolismo , Linfocitos T/ultraestructura , Miembro 14 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral , Factor de Necrosis Tumoral alfa/genética , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Glycoprotein D (gD) is a structural component of the herpes simplex virus (HSV) envelope which is essential for virus entry into host cells. Chinese hamster ovary (CHO-K1) cells are one of the few cell types which are nonpermissive for the entry of many HSV strains. However, when these cells are transformed with the gene for the herpesvirus entry mediator (HVEM), the resulting cells, CHO-HVEM12, are permissive for many HSV strains, such as HSV-1(KOS). By virtue of its four cysteine-rich pseudorepeats, HVEM is a member of the tumor necrosis factor receptor superfamily of proteins. Recombinant forms of gD and HVEM, gD-1(306t) and HVEM(200t), respectively, were used to demonstrate a specific physical interaction between these two proteins. This interaction was dependent on native gD conformation but independent of its N-linked oligosaccharides, as expected from previous structure-function studies. Recombinant forms of gD derived from HSV-1(KOS)rid1 and HSV-1(ANG) did not bind to HVEM(200t), explaining the inability of these viruses to infect CHO-HVEM12 cells. A variant gD protein, gD-1(delta290-299t), showed enhanced binding to HVEM(200t) relative to the binding of gD-1(306t). Competition studies showed that gD-1(delta290-299t) and gD-1(306t) bound to the same region of HVEM(200t), suggesting that the differences in binding to HVEM are due to differences in affinity. These differences were also reflected in the ability of gD-1(delta290-299t) but not gD-1(306t) to block HSV type 1 infection of CHO-HVEM12 cells. By gel filtration chromatography, the complex between gD-1(delta290-299t) and HVEM(200t) had a molecular mass of 113 kDa and a molar ratio of 1:2. We conclude that HVEM interacts directly with gD, suggesting that HVEM is a receptor for virion gD and that the interaction between these proteins is a step in HSV entry into HVEM-expressing cells.
Asunto(s)
Receptores del Factor de Necrosis Tumoral/metabolismo , Receptores Virales/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Animales , Células CHO , Chlorocebus aethiops , Cromatografía en Gel , Cricetinae , Conformación Proteica , Conejos , Miembro 14 de Receptores del Factor de Necrosis Tumoral , Células Vero , Proteínas del Envoltorio Viral/químicaRESUMEN
We identified and cloned a cellular mediator of herpes simplex virus (HSV) entry. Hamster and swine cells resistant to viral entry became susceptible upon expression of a human cDNA encoding this protein, designated HVEM (for herpesvirus entry mediator). HVEM was shown to mediate the entry of several wild-type HSV strains of both serotypes. Anti-HVEM antibodies and a soluble hybrid protein containing the HVEM ectodomain inhibited HVEM-dependent infection but not virus binding to cells. Mutations in the HSV envelope glycoprotein gD significantly reduced HVEM-mediated entry. The contribution of HVEM to HSV entry into human cells was demonstrable in activated T cells. HVEM, the first identified mediator of HSV entry, is a new member of the TNF/NGF receptor family.
Asunto(s)
Genes , Receptores del Factor de Necrosis Tumoral , Receptores Virales/fisiología , Simplexvirus/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células CHO/metabolismo , Células CHO/virología , Línea Celular , Chlorocebus aethiops , Clonación Molecular , Cricetinae , Cricetulus , ADN Complementario/genética , Células HeLa/metabolismo , Células HeLa/virología , Humanos , Datos de Secuencia Molecular , Miembro 14 de Receptores del Factor de Necrosis Tumoral , Receptores Virales/genética , Receptores Virales/aislamiento & purificación , Porcinos , Linfocitos T/metabolismo , Linfocitos T/virología , Transfección , Células Vero/metabolismo , Células Vero/virologíaRESUMEN
Stable cell lines that synthesize heparin have been established from the Furth murine mastocytoma. The parental line (MST) divides in suspension every 14-18 h in growth medium supplemented with fetal bovine serum or defined growth factors. Adherent subclones were selected by adhesion to plastic culture vessels. Both adherent and nonadherent cells contain about 0.4 micrograms of glycosaminoglycan hexuronic acid per 10(6) cells, composed of 80% heparin and 20% chondroitin sulfate E. Deaminative cleavage of MST heparin by HNO2 at pH 1.5 released disaccharides that were similar in composition to those obtained from commercial heparin, except that disaccharides containing 3,6-O-desulfated GlcN units were not found. Greater than 90% of the glycosaminoglycans were stored in cytoplasmic granules, and challenge of the cells with dinitrophenylated bovine serum albumin and anti-dinitrophenyl IgE released a portion of the stored material. Growth studies of subclones showed that MST cells tolerate a 10-fold variation in glycosaminoglycan content. Incubation of cells with sodium chlorate reduced glycosaminoglycan sulfation by > 95% without affecting cell growth. Thus, granule glycosaminoglycans appear to be nonessential for growth of MST cells.
Asunto(s)
Sulfatos de Condroitina/biosíntesis , Heparina/biosíntesis , Mastocitos/metabolismo , Sarcoma de Mastocitos/patología , Animales , División Celular , Gránulos Citoplasmáticos/metabolismo , Glicosaminoglicanos/metabolismo , Técnicas In Vitro , Mastocitos/citología , Ratones , Microscopía Electrónica , Células Tumorales CultivadasRESUMEN
The role of cell surface heparan sulfate in herpes simplex virus (HSV) infection was investigated using CHO cell mutants defective in various aspects of glycosaminoglycan synthesis. Binding of radiolabeled virus to the cells and infection were assessed in mutant and wild-type cells. Virus bound efficiently to wild-type cells and initiated an abortive infection in which immediate-early or alpha viral genes were expressed, despite limited production of late viral proteins and progeny virus. Binding of virus to heparan sulfate-deficient mutant cells was severely impaired and mutant cells were resistant to HSV infection. Intermediate levels of binding and infection were observed for a CHO cell mutant that produced undersulfated heparan sulfate. These results show that heparan sulfate moieties of cell surface proteoglycans serve as receptors for HSV.
Asunto(s)
Heparitina Sulfato/metabolismo , Receptores Virales/metabolismo , Simplexvirus/metabolismo , Animales , Células CHO/metabolismo , Células CHO/microbiología , Cricetinae , Expresión Génica/genética , Heparitina Sulfato/biosíntesis , Heparitina Sulfato/genética , Mutación/genética , Unión Proteica , Proteoglicanos/metabolismo , Virus de la Estomatitis Vesicular IndianaRESUMEN
The sera of 37 patients with IgA nephropathy (IgA NP) were assayed for levels of antibodies specific for the Fab fragment of homologous IgA, and the values obtained were compared to antibody levels in a panel of 26 normal volunteers. IgG antibody levels in IgA NP patients were significantly elevated over those of the controls (P less than 0.01); at the same time IgM anti-Fab alpha levels were significantly decreased when compared to the control panel (P less than 0.01). There was no correlation of antibody levels of either isotype with levels of circulating immune complexes; however, IgM antibody levels of IgA NP patients showed a significant negative correlation with severity of renal insufficiency.
Asunto(s)
Anticuerpos Antiidiotipos/biosíntesis , Autoanticuerpos/biosíntesis , Glomerulonefritis por IGA/inmunología , Inmunoglobulina A/inmunología , Fragmentos Fab de Inmunoglobulinas/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Femenino , Humanos , Inmunoglobulina A/biosíntesis , Inmunoglobulina G/biosíntesis , Inmunoglobulina M/biosíntesis , Masculino , Persona de Mediana EdadRESUMEN
Liver mitochondria from rats fed ethanol chronically demonstrated a 35% decrease in mitochondrial ATPase activity. Moreover, the ATPase activity was inhibited only 61% by addition of oligomycin. Treatment of mitochondria from ethanol-fed rats with the detergent, Lubrol-WX, caused the release of 36% of the F1 from the resulting inner membrane particles. In comparison, only 5% of the F1 was dissociated when control mitochondria were subjected to the Lubrol treatment. However, when the units of ATPase activity from the supernatant and particles obtained after Lubrol treatment were added together, their sums were equivalent in preparations from control and ethanol-fed animals. Moreover, polyacrylamide gel electrophoresis analyses indicated equal amounts of the alpha + beta subunits of F1 in mitochondria from control and ethanol-fed rats. Reconstitution experiments with urea particles and F1 prepared from both control and ethanol mitochondria revealed a decrease in oligomycin sensitivity which could be attributed to an alteration in the functioning of either the oligomycin sensitivity conferring protein or a membrane sector subunit that interacts with oligomycin. Analysis by reconstitution also demonstrated that there were no ethanol-elicited alterations in the properties of the F1 portion of the ATP synthase complex. These observations indicate that the activity of the ATP synthase complex is altered significantly by ethanol-elicited changes in the functioning of those polypeptides involved in modulating both oligomycin sensitivity and the association of F1 with membrane sector subunits.
Asunto(s)
Etanol/farmacología , Mitocondrias Hepáticas/enzimología , Oligomicinas/farmacología , ATPasas de Translocación de Protón/metabolismo , Partículas Submitocóndricas/enzimología , Animales , Estabilidad de Enzimas , Membranas Intracelulares/enzimología , Masculino , Ratas , Ratas EndogámicasRESUMEN
Serum samples from 26 normal volunteers were evaluated by isotype-specific ELISA for the presence of IgG and IgM antibodies directed at IgA. Although there were wide variations in antibody levels, anti-IgA antibodies of both isotypes were found in all individuals tested. The anti-IgA activity was detected against a variety of polymeric and monomeric IgA1 and IgA2 myeloma proteins containing both kappa and lambda light chains. By using Fab and Fc fragments generated by incubation of an IgA1 myeloma protein with IgA1 protease, it was shown that the anti-IgA activity was specific for the Fab portion of the IgA molecule. It was also demonstrated that the serum of two individuals contained both IgG and IgM activity directed at autologous affinity-purified IgA. IgM antibody levels against both whole IgA and Fab of IgA were significantly higher than IgG antibody levels. Cells producing anti-IgA antibodies of both isotypes were detected in lipopolysaccharide-stimulated human spleen.
Asunto(s)
Anticuerpos Antiidiotipos/análisis , Autoanticuerpos/análisis , Inmunoglobulina A/inmunología , Fragmentos Fab de Inmunoglobulinas/inmunología , Formación de Anticuerpos , Especificidad de Anticuerpos , Sangre Fetal/inmunología , Humanos , Inmunoglobulina G/análisis , Inmunoglobulina M/análisis , Proteínas de Mieloma/inmunología , Bazo/citología , Bazo/inmunologíaRESUMEN
Beef heart mitochondrial F0.F1-ATPase was reconstituted into phospholipid liposomes using the octylglucoside solubilization, discontinuous sucrose gradient centrifugation procedure described in the preceding manuscript (Laird, D., Smith Eble, K., and Cunningham, C. (1986) J. Biol. Chem. 261, 14844-14850). The influence of individual phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), and diphosphatidylglycerol (DPG)) on the kinetic parameters related to ATPase activity were investigated. The specific activities for the PC, PE, and DPG reconstituted preparations were 9.8, 6.8, and 7.6 mumol of ATP hydrolyzed per min/mg of protein, respectively. The F0.F1-DPG complex demonstrated a 40% decrease in the Km for ATP. Both the F0.F1-PC and the F0.F1-PE complexes exhibited Ki values for adenyl-5'-yl imidodiphosphate and guanyl-5'-yl imidodiphosphate approximately 2.5 times lower than those obtained in the absence of exogenous phospholipid. The F0.F1-DPG complex displayed Ki values 11.7- and 1.8-fold lower for adenyl-5'-yl imidodiphosphate and guanyl-5'-yl imidodiphosphate, respectively, as compared to the lipid-depleted enzyme. The phospholipids with which F0.F1 were reconstituted also influenced the ATP-induced decrease in the fluorescence of enzyme-associated aurovertin. The rate of the ATP-elicited decrease in aurovertin fluorescence was accelerated in the presence of all three phospholipids with DPG having the most dramatic effect; the t1/2 for maximal decrease in aurovertin fluorescence was 4.3 s for lipid-deficient enzyme and 0.48 s with the F0.F1-DPG complex. The effects of phospholipids on these parameters associated with the catalytic center of the ATPase suggest that phospholipids can modulate catalytic events occurring in F1. In the intact mitochondrion the primary role of phospholipids may be to stabilize conformations of the enzyme consistent with its range of activities.
Asunto(s)
Liposomas , Mitocondrias Cardíacas/enzimología , Fosfolípidos/farmacología , ATPasas de Translocación de Protón/metabolismo , Animales , Aurovertinas/metabolismo , Bovinos , Cinética , Sustancias Macromoleculares , Unión Proteica , Relación Estructura-ActividadRESUMEN
The association of different phospholipids with a lipid-depleted oligomycin-sensitive ATPase from bovine cardiac mitochondria [Serrano, Kanner & Racker (1976) J. Biol. Chem. 251, 2453-2461] has been examined using three approaches. First, reconstitution of the ATPase with different synthetic diacyl phospholipids resulted in a 2-10-fold stimulation of ATPase specific activity depending upon the particular phospholipid employed. The phospholipid headgroup region displayed the following order of ATPase reactivation potential: dioleoylphosphatidylglycerol greater than dioleoylphosphatidic acid greater than dioleoylphosphatidylcholine. Furthermore, the ATPase showed higher levels of specific activity when reconstituted with dioleoyl phospholipid derivatives compared with dimyristoyl derivatives. Second, examination of the phospholipid remaining associated with the lipid-depleted ATPase upon purification showed that phosphatidylcholine, phosphatidylethanolamine, and diphosphatidylglycerol were present. No relative enrichment of any of these phospholipids (compared with their distribution in submitochondrial particles) was noted. Therefore, no preferential association between the ATPase and any one phospholipid could be found in the mitochondrial ATPase. Third, the sodium cholate-mediated phospholipid exchange procedure was employed for studying the phospholipid requirements of the ATPase. Replacement of about 50% of the mitochondrial phospholipid remaining with the lipid-depleted ATPase could be achieved utilizing either synthetic phosphatidic acid or phosphatidylcholine. Examination of the displaced mitochondrial phospholipid showed that phosphatidylcholine, phosphatidylethanolamine, and diphosphatidylglycerol were replaced with equal facility.