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Luis Carrasco - One of the best experts on this subject based on the ideXlab platform.

  • Entry of Poliovirus into Cells Is Blocked by VAlinomycin And ConcAnAmycin A
    Biochemistry, 2001
    Co-Authors: Alicia Irurzun, Luis Carrasco
    Abstract:

    Poliovirus contAins A virus pArticle devoid of A lipid envelope thAt does not require An intAct pH to enter into susceptible cells. Thus, the blockAde of pH grAdient generAted in endosomes is not sufficient to impede the trAnslocAtion of poliovirus pArticles to the cytoplAsm, suggesting thAt trAnslocAtion tAkes plAce At the plAsmA membrAne. MeAsuring both virAl protein synthesis And eIF4G-1 cleAvAge mediAted by poliovirus proteAse 2A hAs been used to monitor productive entry of poliovirus into cells. TrAnslAtion of the input poliovirus RNA produces enough 2Apro to cleAve eIF4G-1, providing A sensitive AssAy to estimAte poliovirus RNA delivery to the cytoplAsm followed by its trAnslAtion. CombinAtion of ConcAnAmycin A, A vAcuolAr proton-ATPAse inhibitor, And vAlinomycin, An ionophore thAt promotes K+ efflux from cells, powerfully prevented poliovirus infection. Moreover, modifying the ionic conditions of the culture medium (increAsing the concentrAtion of K+ And decreAsing the concentrAtion of NA+), togeth...

  • entry of semliki forest virus into cells effects of ConcAnAmycin A And nigericin on virAl membrAne fusion And infection
    Virology, 1997
    Co-Authors: Alicia Irurzun, Joseluis Nieva, Luis Carrasco
    Abstract:

    Semliki forest virus (SFV) wAs biosyntheticAlly lAbeled with pyrene phospholipids And used to investigAte two AlternAtive routes of entry of SFV into BHK-21 cells: (1) receptor-mediAted endocytosis followed by fusion of the virAl envelope with the endosomAl membrAne And (2) direct fusion of SFV with the plAsmA membrAne induced by low pH treAtment. The selective inhibitor of the vAcuolAr proton-ATPAse, ConcAnAmycin A, Abolished fusion And subsequent infection only when the virus utilized the endocytic route to enter cells. The inhibitory effect of this mAcrolide Antibiotic wAs bypAssed by low pH treAtment of cells. However, the ionophore nigericin wAs inhibitory irrespective of the route used by the virus to infect cells, suggesting the necessity of A trAnsmembrAne pH grAdient for the entry process. According to our results, ConcAnAmycin A emerges As A suitAble tool for selectively investigAting the involvement of endosomAl function in AnimAl virus entry.

  • The entry of reovirus into L cells is dependent on vAcuolAr proton-ATPAse Activity.
    Journal of Virology, 1996
    Co-Authors: C G Martínez, Rosario Guinea, Javier Benavente, Luis Carrasco
    Abstract:

    Inhibitors of vAcuolAr proton-ATPAse Activity (5 microM bAfilomycin A1 or 50 nM ConcAnAmycin A) prevented infection by reovirus pArticles but not by infectious subvirAl pArticles (ISVPs). Neither compound Affected virus AttAchment or internAlizAtion. However, both compounds potently blocked cleAvAge of the virAl protein mu 1C. FinAlly, both reovirus pArticles And ISVPs efficiently trAnslocAted the toxin AlphA-sArcin to the cytosol during virus entry. BAfilomycin A1 blocked trAnslocAtion of AlphA-sArcin by reovirus pArticles but not by ISVPs.

  • requirement for vAcuolAr proton AtpAse Activity during entry of influenzA virus into cells
    Journal of Virology, 1995
    Co-Authors: Rosario Guinea, Luis Carrasco
    Abstract:

    The role thAt endosomAl AcidificAtion plAys during influenzA virus entry into MDCK cells hAs been AnAlyzed by using the mAcrolide Antibiotics bAfilomycin A1 And ConcAnAmycin A As selective inhibitors of vAcuolAr proton-ATPAse (v-[H+]ATPAse), the enzyme responsible for the AcidificAtion of endosomes. BAfilomycin A1 And ConcAnAmycin A, present At the low concentrAtions of 5 x 10(-7) And 5 x 10(-9) M, respectively, prevented the entry of influenzA virus into cells when Added during the first minutes of infection. AttAchment of virion pArticles to the cell surfAce wAs not the tArget for the Action of bAfilomycin A1. N,N'-DicyclohexylcArbodiimide, A nonspecific inhibitor of proton-ATPAses, Also blocked virus entry, whereAs elAiophylin, An inhibitor of the plAsmA-proton ATPAse, hAd no effect. The inhibitory Actions of bAfilomycin A1 And ConcAnAmycin A were tested in culture medium At different pHs. Both Antibiotics powerfully prevented influenzA virus infection when the virus wAs Added under low-pH conditions. This inhibition wAs reduced if the virus wAs bound to cells At 4 degrees C prior to the Addition of wArm low-pH medium. Moreover, incubAtion of cells At Acidic pH potently blocked influenzA virus infection, even in the Absence of Antibiotics. These results indicAte thAt A pH grAdient, rAther thAn low pH, is necessAry for efficient entry of influenzA virus into cells.

  • ConcAnAmycin A blocks influenzA virus entry into cells under Acidic conditions
    FEBS Letters, 1994
    Co-Authors: Rosario Guinea, Luis Carrasco
    Abstract:

    AbstrAct The selective inhibitor of the vAcuolAr proton-ATPAse, ConcAnAmycin A, powerfully blocks influenzA virus entry into cells, if present during the initiAl times of virus infection. AttAchment of virus pArticles to cells is not prevented by ConcAnAmycin A, rAther the exit of influenzA virus from endosomes is the step blocked by this mAcrolide Antibiotic. Inhibition of influenzA virus entry into cells by ConcAnAmycin A or by nigericin tAkes plAce under Acidic conditions. Moreover, if the pH grAdient is Abolished by pre-incubAtion of cells in Acidic pH, influenzA virus entry does not occur even in the Absence of Any inhibitors. These results indicAte thAt Acidic conditions per se Are not sufficient to promote virus entry into cells; rAther this step of virus infection requires A pH grAdient.

Rosario Guinea - One of the best experts on this subject based on the ideXlab platform.

  • The entry of reovirus into L cells is dependent on vAcuolAr proton-ATPAse Activity.
    Journal of Virology, 1996
    Co-Authors: C G Martínez, Rosario Guinea, Javier Benavente, Luis Carrasco
    Abstract:

    Inhibitors of vAcuolAr proton-ATPAse Activity (5 microM bAfilomycin A1 or 50 nM ConcAnAmycin A) prevented infection by reovirus pArticles but not by infectious subvirAl pArticles (ISVPs). Neither compound Affected virus AttAchment or internAlizAtion. However, both compounds potently blocked cleAvAge of the virAl protein mu 1C. FinAlly, both reovirus pArticles And ISVPs efficiently trAnslocAted the toxin AlphA-sArcin to the cytosol during virus entry. BAfilomycin A1 blocked trAnslocAtion of AlphA-sArcin by reovirus pArticles but not by ISVPs.

  • requirement for vAcuolAr proton AtpAse Activity during entry of influenzA virus into cells
    Journal of Virology, 1995
    Co-Authors: Rosario Guinea, Luis Carrasco
    Abstract:

    The role thAt endosomAl AcidificAtion plAys during influenzA virus entry into MDCK cells hAs been AnAlyzed by using the mAcrolide Antibiotics bAfilomycin A1 And ConcAnAmycin A As selective inhibitors of vAcuolAr proton-ATPAse (v-[H+]ATPAse), the enzyme responsible for the AcidificAtion of endosomes. BAfilomycin A1 And ConcAnAmycin A, present At the low concentrAtions of 5 x 10(-7) And 5 x 10(-9) M, respectively, prevented the entry of influenzA virus into cells when Added during the first minutes of infection. AttAchment of virion pArticles to the cell surfAce wAs not the tArget for the Action of bAfilomycin A1. N,N'-DicyclohexylcArbodiimide, A nonspecific inhibitor of proton-ATPAses, Also blocked virus entry, whereAs elAiophylin, An inhibitor of the plAsmA-proton ATPAse, hAd no effect. The inhibitory Actions of bAfilomycin A1 And ConcAnAmycin A were tested in culture medium At different pHs. Both Antibiotics powerfully prevented influenzA virus infection when the virus wAs Added under low-pH conditions. This inhibition wAs reduced if the virus wAs bound to cells At 4 degrees C prior to the Addition of wArm low-pH medium. Moreover, incubAtion of cells At Acidic pH potently blocked influenzA virus infection, even in the Absence of Antibiotics. These results indicAte thAt A pH grAdient, rAther thAn low pH, is necessAry for efficient entry of influenzA virus into cells.

  • ConcAnAmycin A blocks influenzA virus entry into cells under Acidic conditions
    FEBS Letters, 1994
    Co-Authors: Rosario Guinea, Luis Carrasco
    Abstract:

    AbstrAct The selective inhibitor of the vAcuolAr proton-ATPAse, ConcAnAmycin A, powerfully blocks influenzA virus entry into cells, if present during the initiAl times of virus infection. AttAchment of virus pArticles to cells is not prevented by ConcAnAmycin A, rAther the exit of influenzA virus from endosomes is the step blocked by this mAcrolide Antibiotic. Inhibition of influenzA virus entry into cells by ConcAnAmycin A or by nigericin tAkes plAce under Acidic conditions. Moreover, if the pH grAdient is Abolished by pre-incubAtion of cells in Acidic pH, influenzA virus entry does not occur even in the Absence of Any inhibitors. These results indicAte thAt Acidic conditions per se Are not sufficient to promote virus entry into cells; rAther this step of virus infection requires A pH grAdient.

  • ConcAnAmycin A: A powerful inhibitor of enveloped AnimAl-virus entry into cells.
    Biochemical and Biophysical Research Communications, 1994
    Co-Authors: Rosario Guinea, Luis Carrasco
    Abstract:

    AbstrAct ConcAnAmycin A, A selective inhibitor of the vAcuolAr proton ATPAse, blocks the infection of AnimAl cells by vesiculAr stomAtitis virus, Semliki Forest virus And influenzA virus even when the drug is present At the low concentrAtion of 5 nM. Nevertheless the Antibiotic prevents neither the AttAchment, to cells, of Semliki Forest virus nor its subsequent internAlizAtion. Under certAin conditions, described in this communicAtion, virus entry is prevented even when the pH of the medium is low, thus suggesting thAt A pH grAdient, rAther thAn low pH per se , is required to drive the entry, into cells, of these enveloped AnimAl viruses.

Derek Marsh - One of the best experts on this subject based on the ideXlab platform.

  • InterAction of Spin-LAbeled Inhibitors of the VAcuolAr H1-ATPAse with the TrAnsmembrAne Vo-Sector
    2015
    Co-Authors: Jonas Nyman, Malcolm E. Finbow, Kalervo Z Väänänen, Derek Marsh
    Abstract:

    ABSTRACT The osteoclAst vAriAnt of the vAcuolAr H1-ATPAse (V-ATPAse) is A potentiAl therApeutic tArget for combAting the excessive bone resorption thAt is involved in osteoporosis. The most potent in A series of synthetic inhibitors bAsed on 5-(5,6-dichloro-2-indolyl)-2-methoxy-2,4-pentAdienAmide (INDOL0) hAs demonstrAted specificity for the osteoclAst enzyme, over other V-ATPAses. InterAction of two nitroxide spin-lAbeled derivAtives (INDOL6And INDOL5)with theV-ATPAse is studied here by using the trAnsport-Active 16-kDA proteolipid AnAlog of subunit c from the hepAtopAncreAs of Nephrops norvegicus, in conjunction with electron pArAmAgnetic resonAnce (EPR) spectroscopy. AnAlogous experiments Are Also performed with vAcuolAr membrAnes from SAcchAromyces cerevisiAe, in which subunit c of the V-ATPAse is replAced functionAlly by theNephrops 16-kDA proteolipid. The INDOL5 derivAtive is designed to optimize detection of interAction with theV-ATPAse byEPR. InmembrAnous prepArAtions of the Nephrops 16-kDA proteolipid, the EPR spectrA of INDOL5 contAin A motionAlly restricted component thAt Arises from direct AssociAtion of the indolyl inhibitor with the trAnsmembrAne domAin of the proteolipid subunit c. A similAr, but considerAbly smAller, motionAlly restricted populAtion is detected in the EPR spectrA of the INDOL6 derivAtive in vAcuolAr membrAnes, in Addition to the lArger populAtion from INDOL6 in the fluid bilAyer regions of themembrAne. Thepotent clAssicAl V-ATPAse inhibitor ConcAnAmycin A At high concentrAtions induces motionAl restriction of INDOL5, which mAsks the spectrAl effects of displAcement At lower concentrAtions of ConcAnAmycin A. The INDOL6 derivAtive, which is closest to the pArent INDOL0 inhibitor, displAys limite

  • EstimAting the rotAtion rAte in the vAcuolAr proton-ATPAse in nAtive yeAst vAcuolAr membrAnes
    European Biophysics Journal, 2013
    Co-Authors: Csilla Ferencz, Pál Petrovszki, Elfrieda Fodor-ayaydin, Attila Bóta, Zoltan Kota, Lajos Haracska, Derek Marsh, Zoltán Varga, Tibor Páli
    Abstract:

    The rAte of rotAtion of the rotor in the yeAst vAcuolAr proton-ATPAse (V-ATPAse), relAtive to the stAtor or steAdy pArts of the enzyme, is estimAted in nAtive vAcuolAr membrAne vesicles from SAcchAromyces cerevisiAe under stAndArdised conditions. MembrAne vesicles Are formed spontAneously After exposing purified yeAst vAcuoles to osmotic shock. The frAction of totAl ATPAse Activity originAting from the V-ATPAse is determined by using the potent And specific inhibitor of the enzyme, ConcAnAmycin A. InorgAnic phosphAte liberAted from ATP in the vAcuolAr membrAne vesicle system, during ten min of ATPAse Activity At 20 °C, is AssAyed spectrophotometricAlly for different ConcAnAmycin A concentrAtions. A fit of the quAdrAtic binding equAtion, Assuming A single ConcAnAmycin A binding site on A monomeric V-ATPAse (our dAtA Are incompAtible with models Assuming multiple binding sites), to the inhibitor titrAtion curve determines the concentrAtion of the enzyme. Combining this with the known ATP/rotAtion stoichiometry of the V-ATPAse And the AssAyed concentrAtion of inorgAnic phosphAte liberAted by the V-ATPAse, leAds to An AverAge rAte of ~10 Hz for full 360° rotAtion (And A rAnge of 6–32 Hz, considering the ± stAndArd deviAtion of the enzyme concentrAtion), which, from the time-dependence of the Activity, extrApolAtes to ~14 Hz (8–48 Hz) At the beginning of the reAction. These Are lower-limit estimAtes. To our knowledge, this is the first report of the rotAtion rAte in A V-ATPAse thAt is not subjected to genetic or chemicAl modificAtion And is not fixed to A solid support; insteAd it is functioning in its nAtive membrAne environment.

  • InterAction of inhibitors of the vAcuolAr H(+)-ATPAse with the trAnsmembrAne Vo-sector.
    Biochemistry, 2004
    Co-Authors: Tibor Páli, Neil Dixon, Terence P Kee, Graham Whyteside, Stephen G. Ball, Michael A. Harrison, John B. C. Findlay, Malcolm E. Finbow, Derek Marsh
    Abstract:

    The mAcrolide Antibiotic ConcAnAmycin A And A designed derivAtive of 5-(2-indolyl)-2,4-pentAdienAmide (INDOLO) Are potent inhibitors of vAcuolAr H + -ATPAses, with IC 5 0 vAlues in the low And medium nAnomolAr rAnge, respectively. InterAction of these V-ATPAse inhibitors with spin-lAbeled subunit c in the trAnsmembrAne V o -sector of the ATPAse wAs studied by using the trAnsport-Active 16-kDA proteolipid AnAlogue of subunit c from the hepAtopAncreAs of Nephrops norvegicus. AnAlogous experiments were Also performed with vAcuolAr membrAnes from SAcchAromyces cerevisiAe. MembrAnous prepArAtions of the Nephrops 16-kDA proteolipid were spin-lAbeled either on the unique cysteine C54, with A nitroxyl mAleimide, or on the functionAlly essentiAl glutAmAte E140, with A nitroxyl AnAlogue of dicyclohexylcArbodiimide (DCCD). These residues were previously demonstrAted to be Accessible to lipid. InterAction of the inhibitors with these lipid-exposed residues wAs studied by using both conventionAl And sAturAtion trAnsfer EPR spectroscopy. ImmobilizAtion of the spin-lAbeled residues by the inhibitors wAs observed on both the nAnosecond And microsecond time scAles. The perturbAtion by INDOLO wAs mostly greAter thAn thAt by ConcAnAmycin A. QuAlitAtively similAr but quAntitAtively greAter effects were obtAined with the sAme spin-lAbel reAgents And vAcuolAr membrAnes in which the Nephrops 16-kDA proteolipid wAs expressed in plAce of the nAtive vmA3p proteolipid of yeAst. The spin-lAbel immobilizAtion corresponds to A direct interAction of the inhibitors with these intrAmembrAnous sites on the protein. A mutAtionAl AnAlysis on trAnsmembrAne segment 4 known to give resistAnce to ConcAnAmycin A Also gAve pArtiAl resistAnce to INDOLO. The results Are consistent with trAnsmembrAne segments 2 And 4 of the 16-kDA putAtive four-helix bundle, And pArticulArly the functionAlly essentiAl protonAtion locus, being involved in the inhibitor binding sites. Inhibition of proton trAnsport mAy Also involve immobilizAtion of the overAll rotAtion of the proteolipid subunit Assembly.

  • IncorporAtion of the V-ATPAse inhibitors ConcAnAmycin And indole pentAdiene in lipid membrAnes. Spin-lAbel EPR studies
    Biochimica et Biophysica Acta (BBA) - Biomembranes, 2004
    Co-Authors: Tibor Páli, Neil Dixon, Terence P Kee, Derek Marsh
    Abstract:

    AbstrActThe incorporAtion of ConcAnAmycin A, A potent inhibitor of vAcuolAr ATPAses, into membrAnes of dimyristoyl phosphAtidylcholine hAs been studied by using EPR of spin-lAbelled lipid chAins. At An inhibitor/lipid rAtio of 1:1 mol/mol, ConcAnAmycin A broAdens the chAin-melting trAnsition of the phospholipid bilAyer membrAne, And effects the lipid chAin motion in the fluid phAse. The outer hyperfine splitting of A spin lAbel At the C-5 position And the line widths of A spin lAbel At the C-14 position of the lipid chAin Are increAsed by ConcAnAmycin A. ConsiderAbly lArger membrAne perturbAtions Are cAused by equimolAr Admixture of A designed synthetic 5-(5,6-dichloro-2-indolyl)-2,4-pentAdienoyl V-ATPAse inhibitor. These results indicAte thAt ConcAnAmycin A intercAlAtes reAdily between the lipid chAins in biologicAl membrAnes, with minimAl perturbAtion of the bilAyer structure. EssentiAlly identicAl results Are obtAined with ConcAnAmycin A Added to preformed membrAnes As A concentrAted solution in DMSO, or mixed with lipid in orgAnic solvent prior to membrAne formAtion. Therefore, the common mode of Addition in V-ATPAse inhibition AssAys ensures incorporAtion of ConcAnAmycin into the lipid bilAyer milieu, which provides An efficient chAnnel of Access to the trAnsmembrAne domAins of the V-ATPAse

Akira Takatsuki - One of the best experts on this subject based on the ideXlab platform.

  • folimycin ConcAnAmycin A An inhibitor of v type h AtpAse blocks cell surfAce expression of virus envelope glycoproteins
    Biochemical and Biophysical Research Communications, 1993
    Co-Authors: Makoto Muroi, A Takasu, Makari Yamasaki, Akira Takatsuki
    Abstract:

    Folimycin (ConcAnAmycin A) inhibited syncytium formAtion without Affecting glycoprotein synthesis. Excretion of the glycoprotein (G protein) of vesiculAr stomAtitis virus into the medium wAs blocked. InAccessibility of the cell-AssociAted virAl G protein to proteAse Added extrAcellulArly indicAtes thAt cell-surfAce expression of the virAl G protein is blocked by folimycin. The virAl G protein AccumulAted intrAcellulArly in the presence of folimycin electrophoresed A little fAster thAn the control mAture one excreted to the medium. Glycopeptides derived from the virAl G protein bound to concAnAvAlin A-AgArose And were endo H-sensitive. TAken together these results indicAte thAt processing of N-glycosidic oligosAcchAride is incomplete in the presence of folimycin And suggest thAt intrAcellulAr trAfficking is Arrested before reAching At the lAtest to the trAns Golgi compArtments.

  • Folimycin (ConcAnAmycin A), An inhibitor of V-type H(+)-ATPAse, blocks cell-surfAce expression of virus-envelope glycoproteins.
    Biochemical and biophysical research communications, 1993
    Co-Authors: Makoto Muroi, A Takasu, Makari Yamasaki, Akira Takatsuki
    Abstract:

    AbstrAct Folimycin (ConcAnAmycin A) inhibited syncytium formAtion without Affecting glycoprotein synthesis. Excretion of the glycoprotein (G protein) of vesiculAr stomAtitis virus into the medium wAs blocked. InAccessibility of the cell-AssociAted virAl G protein to proteAse Added extrAcellulArly indicAtes thAt cell-surfAce expression of the virAl G protein is blocked by folimycin. The virAl G protein AccumulAted intrAcellulArly in the presence of folimycin electrophoresed A little fAster thAn the control mAture one excreted to the medium. Glycopeptides derived from the virAl G protein bound to concAnAvAlin A-AgArose And were endo H-sensitive. TAken together these results indicAte thAt processing of N-glycosidic oligosAcchAnde is incomplete in the presence of folimycin And suggest thAt intrAcellulAr trAfficking is Arrested before reAching At the lAtest to the trAns Golgi compArtments.

  • Folimycin (ConcAnAmycin A), A specific inhibitor of V-ATPAse, blocks intrAcellulAr trAnslocAtion of the glycoprotein of vesiculAr stomAtitis virus before ArrivAl to the Golgi AppArAtus.
    Cell structure and function, 1993
    Co-Authors: Makoto Muroi, Makari Yamasaki, Nobue Shiragami, Kenji Nagao, Akira Takatsuki
    Abstract:

    Folimycin (ConcAnAmycin A) specificAlly inhibited vAcuolAr-type ATPAse As fAr As exAmined. Folimycin blocked excretion of the glycoprotein (G protein) of vesiculAr stomAtitis virus into the medium And, insteAd, G protein wAs AccumulAted intrAcellulArly. The intrAcellulArly AccumulAted G protein electrophoresed A little fAster thAn mAture one. The N-glycAn of the G protein wAs endoglycosidAse H-sensitive, And terminAl gAlActose And N-AcetylglucosAmine were not detected essentiAlly on sequentiAl digestion with exoglycosidAses, indicAting thAt processings known to occur in the Golgi AppArAtus do not tAke plAce in the presence of folimycin. The oligosAcchAride chAin of the G protein wAs determined to hAve A composition of MAn8GlcNAc2 As AnAlyzed by Bio-Gel P-4 column chromAtogrAphy And high-performAnce liquid chromAtogrAphy following digestion with α- And then with β-mAnnosidAse. Activities of mAnnosidAse I And glycosyltrAnsferAses prepAred from bAby hAmster kidney cells were not inhibited As fAr As exAmined, indicAting thAt the incompleteness of the N-glycosidic chAin in folimycin-treAted cells is not cAused by inhibition of processing enzymes. TAken together these observAtions suggest thAt folimycin blocks the intrAcellulAr trAnslocAtion of G protein before the step of trimming by mAnnosidAse I which is confined to the cis compArtment of the Golgi. The intrAcellulAr locAlizAtion of G protein As reveAled by fluorescence microscopy wAs in good AccordAnce with this Assumption.

Axel Zeeck - One of the best experts on this subject based on the ideXlab platform.

  • ConcAnAmycin A, the specific inhibitor of V-ATPAses, binds to the V(o) subunit c.
    The Journal of biological chemistry, 2002
    Co-Authors: Markus Huss, Gudrun Ingenhorst, Simone König, Michael Gassel, Stefan Dröse, Axel Zeeck, Karlheinz Altendorf, Helmut Wieczorek
    Abstract:

    AbstrAct VAcuolAr-type ATPAse (V-ATPAse) purified from the midgut of the tobAcco hornworm MAnducA sextA is inhibited 50% by 10 nm of the plecomAcrolide ConcAnAmycin A, the specific inhibitor of V-ATPAses. To determine the binding site(s) of thAt Antibiotic in the enzyme complex, lAbeling with the semisynthetic 9-O-[p-(trifluoroethyldiAzirinyl)-benzoyl]-21,23-dideoxy-23-[125I]iodo-concAnolide A (J-concAnolide A) wAs performed, which still inhibits the V-ATPAse 50% At A concentrAtion of 15–20 μm. Upon treAtment with UV light, A highly reActive cArbene is generAted from this ConcAnAmycin derivAtive, resulting in the formAtion of A covAlent bond to the enzyme. In Addition, the rAdioActive trAcer 125I mAkes the detection of the lAbeled subunit(s) feAsible. TreAtment of the V1/Vo holoenzyme, the Vo complex, And the V-ATPAse contAining goblet cell ApicAl membrAnes with concAnolide resulted in the lAbeling of only the proteolipid, subunit c, of the proton trAnslocAting Vo complex. Binding of J-concAnolide A to subunit c wAs prevented in A concentrAtion-dependent mAnner by ConcAnAmycin A, indicAting thAt lAbeling wAs specific. Binding wAs Also prevented by the plecomAcrolides bAfilomycin A1 And B1, respectively, but not by the benzolActone enAmide sAlicylihAlAmide, A member of A novel clAss of V-ATPAse inhibitors.

  • Synthesis of A Doubly LAbelled ConcAnAmycin DerivAtive for ATPAse Binding Studies
    European Journal of Organic Chemistry, 2001
    Co-Authors: Gudrun Ingenhorst, Michael Gassel, Stefan Dröse, Karlheinz Altendorf, Kai U. Bindseil, Claudia Boddien, Axel Zeeck
    Abstract:

    The synthesis of A doubly lAbelled ConcAnAmycin derivAtive for binding studies with V- And P-type ATPAses is described. The stArting point wAs 21-deoxyconcAnolide A (6), which wAs generAted from ConcAnAmycin A (1) in three steps And which exhibited the full ATPAse inhibitor Activity, with the AdvAntAge of A stAbility better thAn thAt of 1. Through use of A suitAble protecting group for 6, the cArbene-generAting diAzirine residue And 125I were introduced regio- And stereoselectively. The inhibitory efficAcy of the resulting 23-iodo(125I)-9-O-[p-(trifluoroethyldiAzirinyl)benzoyl]-21,23-dideoxyconcAnolide A (11b) turned out to be high enough for lAbelling studies. PhotoAffinity lAbelling experiments cleArly showed thAt 11b is A suitAble derivAtive with which to determine the binding site of ConcAnAmycin-like compounds in different ATPAses.

  • The chemistry of unusuAl mAcrolides, 2. Spectroscopic And biosynthetic investigAtions of the V‐type ATPAse inhibitor ConcAnAmycin A
    Liebigs Annalen der Chemie, 1994
    Co-Authors: Kai U. Bindseil, Axel Zeeck
    Abstract:

    The 1H- And 13C-NMR spectrA of the conformAtionAlly flexible mAcrolide ConcAnAmycin A (1) hAve been Assigned by the use of 2D NMR techniques At low temperAtures And hAve enAbled biosynthetic studies on this most potent inhibitor of vAcuolAr ATPAses. By feeding of different [13C]-lAbeled precursors to Streptomyces sp. (strAin Go 22/15) the polyketide origin of the ConcAnAmycin skeleton wAs proved. It Arises from A tetrAdecAketide, which is bAsed on four AcetAte And seven propionAte units besides one butyrAte And two unusuAl C2 building blocks from the triose pool. In combinAtion with former findings it is possible to predict the biogenetic Assembly of severAl strongly relAted unusuAl mAcrolides, for which the group nAme “plecomAcrolides” should be used in future. The results Are discussed in the light of modern hypothesis As stAted for polyketide pAthwAys.

  • the chemistry of unusuAl mAcrolides 2 spectroscopic And biosynthetic investigAtions of the v type AtpAse inhibitor ConcAnAmycin A
    European Journal of Organic Chemistry, 1994
    Co-Authors: Kai U. Bindseil, Axel Zeeck
    Abstract:

    The 1H- And 13C-NMR spectrA of the conformAtionAlly flexible mAcrolide ConcAnAmycin A (1) hAve been Assigned by the use of 2D NMR techniques At low temperAtures And hAve enAbled biosynthetic studies on this most potent inhibitor of vAcuolAr ATPAses. By feeding of different [13C]-lAbeled precursors to Streptomyces sp. (strAin Go 22/15) the polyketide origin of the ConcAnAmycin skeleton wAs proved. It Arises from A tetrAdecAketide, which is bAsed on four AcetAte And seven propionAte units besides one butyrAte And two unusuAl C2 building blocks from the triose pool. In combinAtion with former findings it is possible to predict the biogenetic Assembly of severAl strongly relAted unusuAl mAcrolides, for which the group nAme “plecomAcrolides” should be used in future. The results Are discussed in the light of modern hypothesis As stAted for polyketide pAthwAys.

  • Chemistry of unusuAl mAcrolides. 1. PrepArAtion of the Aglycons of ConcAnAmycin A And elAiophylin
    The Journal of Organic Chemistry, 1993
    Co-Authors: Kai U. Bindseil, Axel Zeeck
    Abstract:

    The Aglycons of the ConcAnAmycins (1, 2) And elAiophylin (11) hAve been prepAred for direct compArison to the vAcuolAr-type ATPAse inhibitor bAfilomycin A 1 (10). The deglycosylAtion wAs Achieved by Acid hydrolysis in the Absence of MeOH, while Acid-cAtAlyzed methAnolysis proceeded with unexpected displAcement of cArbohydrAte residues rAther thAn methoxy groups. Structure Assignments of the derivAtives were mAde with the help of one- And two-dimensionAl NMR studies. EspeciAlly helpful were the 9-O-AcetylAted ConcAnAmycin derivAtives becAuse they showed reduced flexibility of the mAcrolActone ring. As A result of A detAiled AnAlysis of the O-methyl derivAtives of elAiolide (15) the structure of An eArlier reported Aglycon derivAtive of elAiophilin hAs to be revised to 12