Site of freeze-thaw damage and cryoprotection by amino acids of the calcium ATPase of sarcoplasmic reticulum.
AuthorsLalonde RJ, Lepock JR, Kruuv J
JournalBiochim Biophys Acta
PubMed ID1832965
'The Ca2+,Mg(2+)-ATPase of skeletal muscle sarcoplasmic reticulum (SR) is irreversibly inactivated by a freeze-thaw (FT) cycle. The membrane does not become more permeable to calcium after a FT cycle, suggesting that the reduced uptake is due to damage to the Ca2+,Mg(2+)-ATPase. Several amino acids, in addition to standard cryoprotectants provide ... More
Dicyclohexylcarbodiimide inhibits proton pumping in ubiquinol:cytochrome c oxidoreductase of Rhodobacter sphaeroides and binds to aspartate-187 of cytochrome b.
AuthorsWang Y, Obungu V, Beattie DS
JournalArch Biochem Biophys
PubMed ID9587406
'In recent studies we reported that dicyclohexylcarbodiimide (DCCD) inhibited proton translocation in ubiquinol:cytochrome c oxidoreductase (cytochrome bc1 complex) from yeast mitochondria where it was bound to aspartate-160 of cytochrome b. In the current study, we report that DCCD and its fluorescent analogue, N-cyclohexyl-N''-[4-(dimethylamino)naphthyl]-carbodiimide (NCD-4), inhibit 50-60% proton pumping in the ... More
Fluorescence quenching of reconstituted NCD-4-labeled cytochrome c oxidase complex by DOXYL-stearic acids.
AuthorsMusser SM, Larsen RW, Chan SI
JournalBiophys J
PubMed ID8312474
'It has been known for some time that dicyclohexylcarbodiimide (DCCD) inhibits the proton translocation function of the cytochrome c oxidase complex (CcO) and that there is one major site in subunit III which is modified upon reaction with DCCD (Glu-90 for the bovine enzyme). We have examined the reaction of ... More
The effect of carboxyl group modification on the chromophore regeneration of archaeopsin-1 and bacterioopsin.
AuthorsSugiyama Y, Fujii K, Mukohata Y
JournalJ Biochem (Tokyo)
PubMed ID10348918
'Carboxyl group modification with DCCD and NCD-4 was employed to investigate the chemical environment of the side chains of archaeopsin-1 (aO-1) and bacterioopsin (bO). Some differences were observed between aO-1 and bO. Although DCCD or NCD-4 did not modify aO-1 in bleached membrane, they modified bO in bleached membrane and ... More
Molecular topography of the Ca2+-ATPase of sarcoplasmic reticulum.
AuthorsScott TL
JournalMol Cell Biochem
PubMed ID2972913
'The Ca2+ binding site region of the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum was labeled with several fluorescent analogs of dicyclohexylcarbodiimide. As has been shown by Chadwick and Thomas, in the absence of Ca2+ in the medium, labeling with the naphthyl carbodiimide results in the inhibition of enzyme activity. Further, ... More
Subunit 9 of the mitochondrial ATP synthase of Trypanosoma brucei is nuclearly encoded and developmentally regulated.
AuthorsChi TB, Brown B SV, Williams N
JournalMol Biochem Parasitol
PubMed ID9574907
'We have previously shown that the mitochondrial ATP synthase is developmentally regulated through the life cycle of Trypanosoma brucei. The mechanism of this regulation is as yet unknown. We are currently examining regulation of expression of several key subunits of the ATP synthase to investigate this mechanism. In the work ... More
Ca2+ occlusion in monomeric sarcoplasmic reticulum Ca-ATPase.
AuthorsVilsen B, Andersen JP
JournalProg Clin Biol Res
PubMed ID2971223
Inhibition of (Ca2+ + Mg2+)-ATPase by carbodiimides. A structure-activity study.
AuthorsChadwick CC, Thomas EW
JournalBiochim Biophys Acta
PubMed ID3155970
Nine hydrophobic carbodiimides were synthesized and their chemical reactivities (towards acetic acid) and inhibitory capacities (towards the (Ca2+ + Mg2+)-ATPase were measured. No correlation between chemical reactivity and inhibitory efficacy emerges, but a significant effect of molecular bulk on reactivity towards the calcium-protectable carboxyl groups of the ATPase is noted: ... More
Ligand binding properties of the sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase labelled with N-cyclohexyl-N'-(4-dimethylamino-alpha-naphthyl)carbodiimide.
AuthorsChadwick CC, Thomas EW
JournalBiochim Biophys Acta
PubMed ID6141803
The (Ca2+ + Mg2+)-ATPase of rabbit sarcoplasmic reticulum, when labelled at two Ca2+-protected sites with N-cyclohexyl-N'-(4-dimethylamino-alpha-naphthyl)carbodiimide (NCD-4) retains Ca2+ binding capacity at the sites with Kd values of approx. 3 microM and 0.12 mM as assessed by fluorescence titration. The sites correspond to the two high-affinity Ca2+ binding sites present ... More
Ultraviolet and fluorescence derivatization reagents for carboxylic acids suitable for high performance liquid chromatography: a review.
AuthorsMukherjee PS, Karnes HT
JournalBiomed Chromatogr
PubMed ID8879524
Pre- and postcolumn derivatization with a suitable chromophore or fluorophore have often been utilized to enhance the sensitivity and selectivity of detection for analytes possessing a carboxyl group. The major classes of UV and fluorescent derivatization reagents include the coumarin analogues, alkyl halides, diazoalkanes, and amines. The derivatization reaction conditions, ... More
Mg(2+)-ADP protects against inactivation of sarcoplasmic reticulum Ca2+,Mg(2+)-ATPase by N-cyclohexyl-N'-(4-dimethylamino-alpha-naphthyl) carbodiimide.
AuthorsMerino JM, Gutiérrez-Merino C
JournalBiochem Biophys Res Commun
PubMed ID7857279
N-cyclohexyl-N'-(4-dimethylamino-alpha-naphthyl) carbodiimide (NCD-4) inactivates the sarcoplasmic reticulum Ca(2+)-ATPase by covalent labelling at or near the high affinity (transport) Ca2+ sites. Mg(2+)-ADP protects against the inactivation of the Ca(2+)-ATPase produced by NCD-4, with a K0.5 of Mg(2+)-ADP of 28 +/- 6 microM for purified Ca(2+)-ATPase. With native and solubilized sarcoplasmic reticulum ... More
Localization of Cys-344 on the (Ca(2+)-Mg(2+)-ATPase of sarcoplasmic reticulum using resonance energy transfer.
AuthorsMata AM, Stefanova HI, Gore MG, Khan YM, East JM, Lee AG
JournalBiochim Biophys Acta
PubMed ID8466933
4-Bromomethyl-6,7-dimethoxy-coumarin labels the (Ca(2+)-Mg(2+)-ATPase of skeletal muscle sarcoplasmic reticulum at Cys-344. Resonance energy transfer has been used to measure the distance between this site and Lys-515 labelled with fluorescein isothiocyanate as about 37 A. The height of Cys-344 above the phospholipid/water interface has been measured by resonance energy transfer for ... More
Cowpea mosaic virus for material fabrication: addressable carboxylate groups on a programmable nanoscaffold.
AuthorsSteinmetz NF, Lomonossoff GP, Evans DJ
JournalLangmuir
PubMed ID16584217
For the first time, decoration of surface-exposed carboxylate groups on Cowpea mosaic virus particles is reported, thus increasing the number and types of addressable surface groups on this nanoscaffold. First, the addressabilty of carboxylates was demonstrated using a carboxylate-selective fluorescent dye, N-cyclohexyl-N'-(4-(dimethylamino)naphthyl)carbodiimide. Second, it was shown that the virions can ... More
Inactivation of sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase by N-cyclohexyl-N'-(4-dimethylamino-alpha-naphthyl)carbodiimide.
AuthorsChadwick CC, Thomas EW
JournalBiochim Biophys Acta
PubMed ID6133553
The synthesis and characterisation of N-cyclohexyl-N'-(4-dimethylamino-alpha-naphthyl)carbodiimide (NCD-4) is described. Only the N-acetylurea and urea corresponding to NCD-4 are appreciably fluorescent: the O-phenylisourea and S-ethylisothiourea derivatives have negligible fluorescence. NCD-4 inhibits the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum irreversibly: Ca2+ protects against inhibition. Covalent incorporation of NCD-4 occurs into the Ca2+-protected ... More
Positions of the sites labeled by N-cyclohexyl-N'-(4-dimethylamino-1-naphthyl)carbodiimide on the (Ca2+ + Mg2+)-ATPase.
AuthorsMunkonge F, East JM, Lee AG
JournalBiochim Biophys Acta
PubMed ID2521797
N-Cyclohexyl-N'-(4-dimethylamino-1-naphthyl)carbodiimide (NCD-4) labels (Ca2+ + Mg2+)-ATPase at Ca2+-protectable sites, believed to be at or near the two Ca2+ binding sites on the ATPase, and at nonspecific sites. The labeled ATPase has been reconstituted into lipid bilayers containing phosphatidylethanolamine labeled with fluorescein isothiocyanate. The distance between NCD-4 and fluorescein groups was ... More
The subunit location of magnesium in cytochrome c oxidase.
AuthorsLin J, Pan LP, Chan SI
JournalJ Biol Chem
PubMed ID8408083
The magnesium ion in bovine heart cytochrome c oxidase can be depleted up to 75% by heat treatment of the enzyme at 43 degrees C followed by dialysis against EDTA buffer solution. The magnesium-depleted enzyme so obtained retains 40% of the activity of the native enzyme. This is the first ... More
Spectral and catalytical properties of the sarcoplasmic reticulum Ca-ATPase labeled with N-cyclohexyl-N'-(4-dimethylamino-1-naphthyl)-carbodiimide.
AuthorsPick U, Weiss M
JournalEur J Biochem
PubMed ID2931277
N-Cyclohexyl-N'-(dimethylamino)-carbodiimide (NCD-4) labels three sites in the sarcoplasmic reticulum Ca-ATPase which can be resolved by their spectral properties and by their effects on the catalytical activity of the enzyme. One site is not protectable by Ca2+ ions or by dicyclohexylcarbodiimide and is not essential for catalytical activity. Two Ca2+-protectable sites, ... More
Fluorescent analogues of N,N'-dicyclohexylcarbodiimide as structural probes of the bovine mitochondrial proton channel.
AuthorsPringle MJ, Taber M
JournalBiochemistry
PubMed ID2417624
N-Cyclohexyl-N'-[4-(dimethylamino)-alpha-naphthyl]carbodiimide (NCD-4) and N-cyclohexyl-N'-(1-pyrenyl)carbodiimide (NCP) are two novel fluorescent analogues of the mitochondrial inhibitor dicyclohexylcarbodiimide (DCCD). Although nonfluorescent in aqueous media, both compounds form fluorescent conjugates with mitochondrial electron transport particles (ETPH) or purified H+-ATPase (F1-F0) vesicles. DCCD prevents the reaction of ETPH with both NCD-4 and NCP. The fluorescent ... More
Identification of proteolipid from an extremely halophilic archaeon Halobacterium salinarum as an N,N'-dicyclohexyl-carbodiimide binding subunit of ATP synthase.
AuthorsIhara K, Watanabe S, Sugimura K, Katagiri I, Mukohata Y
JournalArch Biochem Biophys
PubMed ID9169014
ATP synthesis in an extremely halophilic archaeon, Halobacterium salinarum, was inhibited by N-cyclohexyl-N'-[4-(dimethylamino)-alpha-naphthyl]carbodiimide (NCD-4), a fluorescent analog of N,N'-dicyclohexylcarbodiimide (DCCD). By tracing the fluorescent signal, a hydrophobic 8-kDa protein (proteolipid) was purified from the halobacterial membrane as one of the most DCCD-reactive proteins and its N-terminal amino acid sequence was ... More
A proposed pathway of proton translocation through the bc complexes of mitochondria and chloroplasts.
AuthorsBeattie DS
JournalJ Bioenerg Biomembr
PubMed ID8394319
The cytochrome bc complexes of the electron transport chain from a wide variety of organisms generate an electrochemical proton gradient which is used for the synthesis of ATP. Proton translocation studies with radiolabeled N,N'-dicyclohexylcarbodiimide (DCCD), the well-established carboxyl-modifying reagent, inhibited proton-translocation 50-70% with minimal effect on electron transfer in the ... More
Location of N-cyclohexyl-N'-(4-dimethyl-amino-alpha-naphthyl)carbodiimide- binding site in sarcoplasmic reticulum Ca2+-transporting ATPase.
AuthorsVelasco-Guillén I, Corbalán-García S, Gómez-Fernández JC, Teruel JA
JournalEur J Biochem
PubMed ID9578493
The Ca2+-transporting ATPase has been labeled with N-cyclohexyl-N'-(4-dimethyl-amino-alpha-naphthyl)carbodiimide (NCD-4), a fluorescent carbodiimide which reacts with carboxyl groups of acidic residues. It has been reported that NCD-4 labels a transmembrane portion of the protein at the high-affinity calcium-binding sites. We have determined the depth of the calcium-sensitive probe by quenching the ... More
Beta2-agonist administration increases sarcoplasmic reticulum Ca2+-ATPase activity in aged rat skeletal muscle.
Aging is associated with a slowing of skeletal muscle contractile properties, including a decreased rate of relaxation. In rats, the age-related decrease in the maximal rate of relaxation is reversed after 4-wk administration with the beta2-adrenoceptor agonist (beta2-agonist) fenoterol. Given the critical role of the sarcoplasmic reticulum (SR) in regulating ... More
Covalent modification of the amine transporter with N,N'-dicyclohexylcarbodiimide.
AuthorsSuchi R, Stern-Bach Y, Gabay T, Schuldiner S
JournalBiochemistry
PubMed ID2054348
N,N'-Dicyclohexylcarbodiimide (DCC) has been previously shown to inhibit the amine transporter from chromaffin granules [Gasnier, B., Scherman, D., & Henry, J.P. (1985) Biochemistry 24, 3660-3667]. A study of the mechanism of inhibition is presented together with the demonstration of covalent modification of the protein. DCC inhibits binding of R1 (reserpine) ... More
Interdependence of Ca2+ occlusion sites in the unphosphorylated sarcoplasmic reticulum Ca(2+)-ATPase complex with CrATP.
AuthorsVilsen B, Andersen JP
JournalJ Biol Chem
PubMed ID1531342
The beta, gamma-bidentate chromium(III) complex of ATP (CrATP) was used as a substrate analog to stabilize a form of the Ca(2+)-ATPase of the sarcoplasmic reticulum containing both of the bound calcium ions in an occluded state without enzyme phosphorylation. The kinetics of dissociation of Ca2+ from the occlusion sites in ... More
Topographical organization of cytochrome b in the yeast mitochondrial membrane determined by fluorescence studies with N-cyclohexyl-N'-[4-(dimethylamino)naphthyl]carbodiimide.
AuthorsWang Y, Howton MM, Beattie DS
JournalBiochemistry
PubMed ID7779791
In previous studies, we reported that dicyclohexylcarbodiimide (DCCD) inhibited proton translocation in the cytochrome bc1 complex from yeast mitochondria and was bound selectively to cytochrome b. Extensive trypsin digestion of [14C]DCCD-labeled cytochrome b isolated from a cytochrome bc1 complex treated with DCCD yielded a single radiolabeled 7.0 kDa peptide with ... More
Structural studies of the vacuolar H(+)-pyrophosphatase: sequence analysis and identification of the residues modified by fluorescent cyclohexylcarbodiimide and maleimide.
AuthorsMaruyama C, Tanaka Y, Takeyasu K, Yoshida M, Sato MH
JournalPlant Cell Physiol
PubMed ID9871364
We determined the amino acid residues of the H(+)-translocating inorganic pyrophosphatase (H(+)-PPase) of pumpkin which are covalently labeled by two fluorescent labeling reagents; N-cyclohexyl-N'-[4-(dimethyl amino)-alpha-naphthyl] carbodiimide (NCD) and N-pyrenylmaleimide (NPM). NCD and NPM are fluorescent analogues of N,N-dicycrohexylcarbodiimide and N-ethylmaleimide, respectively, and inactivate H(+)-PPase activity. Excess Mg2+ protected the H(+)-PPase ... More
Ischemia-induced structural change in SR Ca2+-ATPase is associated with reduced enzyme activity in rat muscle.
AuthorsTupling R, Green H, Senisterra G, Lepock J, McKee N
JournalAm J Physiol Regul Integr Comp Physiol
PubMed ID11641141
In this study, we employed an in vivo model of prolonged ischemia in rat skeletal muscle to investigate the hypothesis that structural modifications to the sarcoplasmic reticulum (SR) Ca2+-ATPase can explain the alterations in Ca2+-ATPase activity that occur with ischemia. To induce total ischemia, a tourniquet was placed around the ... More
Inactivation of calcium uptake by EGTA is due to an irreversible thermotropic conformational change in the calcium binding domain of the Ca(2+)-ATPase.
AuthorsCheng KH, Lepock JR
JournalBiochemistry
PubMed ID1533156
Calcium uptake by rabbit skeletal sarcoplasmic reticulum (SR) is inhibited with an effective inactivation temperature (TI) of 37 degrees C in EGTA with no effect on ATPase activity. Since the Ca-ATPase denatures at a much higher temperature (49 degrees C) in EGTA, this suggests that a small or localized conformational ... More
Topographical organization of the N-terminal segment of lung pulmonary surfactant protein B (SP-B(1-25)) in phospholipid bilayers.
AuthorsWang Y, Rao KM, Demchuk E
JournalBiochemistry
PubMed ID12680754
The location and depth of each residue of lung pulmonary surfactant protein B (SP-B(1-25)) in a phospholipid bilayer (PB) was determined by fluorescence quenching using synthesized single-residue-substituted peptides that were reconstituted into 1,2-dipalmitoyl phosphatidylcholine (DPPC)-enriched liposomes. The single-residue substitutions in peptides were either aspartate or tryptophan. The aspartate was subsequently ... More
Structural perturbation of the transmembrane region interferes with calcium binding by the Ca2+ transport ATPase.
The Ca(2+)-ATPase of sarcoplasmic reticulum reacts with N-cyclohexyl-N'-(4-dimethylamino-1-naphthyl) carbodiimide (NCD4) yielding a fluorescence labeling that interferes with calcium binding to activating and transport sites of the enzyme and, thereby, with Ca(2+)-dependent ATPase activity. On the other hand, the catalytic site does not appear altered, as revealed by the normal occurrence ... More
Clotrimazole, an antimycotic drug, inhibits the sarcoplasmic reticulum calcium pump and contractile function in heart muscle.
AuthorsSnajdrova L, Xu A, Narayanan N
JournalJ Biol Chem
PubMed ID9774419
Clotrimazole (CLT), an antimycotic drug, has been shown to inhibit proliferation of normal and cancer cell lines and its systemic use as a new tool in the treatment of proliferative disorders is presently under scrutiny (Benzaquen, L. R., Brugnara, C., Byers, H. R., Gattoni-Celli, S., and Halperin, J. A. (1995) ... More
Topographical organization of cytochrome b6 in the thylakoid membrane of spinach chloroplasts determined by fluorescence studies with N-cyclohexyl-N'-[4-(dimethylamino)naphthyl]carbodiimide.
AuthorsWang Y, Beattie DS
JournalBiochemistry
PubMed ID8373766
In a recent study [Wang & Beattie (1992) Biochemistry 31, 8445-8459], we reported that dicyclohexylcarbodiimide (DCCD) was bound to either aspartate-155 or glutamate-166 localized in an amphiphilic, non-membrane-spanning, helix of cytochrome. Moreover, DCCD inhibits proton translocation in a cytochrome bf complex reconstituted into proteoliposomes without significant inhibition of electron transfer, ... More
Exogenous ubiquinol analogues affect the fluorescence of NCD-4 bound to aspartate-160 of yeast cytochrome b.
AuthorsWang Y, Bruel C, Yan L, Beattie DS
JournalJ Bioenerg Biomembr
PubMed ID9932648
Previously, we reported that the carboxyl-reacting reagent DCCD, and its fluorescent derivative NCD-4 binds covalently to aspartate-160 localized in amphipathic helix cd of the CD loop connecting membrane-spanning helices C and D of cytochrome b (Wang et al., 1995). We have investigated the fluorescent properties of NCD-4 to probe possible ... More
Mechanisms underlying increases in SR Ca2+-ATPase activity after exercise in rat skeletal muscle.
AuthorsSchertzer JD, Green HJ, Duhamel TA, Tupling AR
JournalAm J Physiol Endocrinol Metab
PubMed ID12409282
Prolonged exercise followed by a brief period of reduced activity has been shown to result in an overshoot in maximal sarcoplasmic reticulum (SR) Ca(2+)-ATPase activity [maximal velocity (V(max))] in rat locomoter muscles (Ferrington DA, Reijneveld JC, Bär PR, and Bigelow DJ. Biochim Biophys Acta 1279: 203-213, 1996). To investigate the ... More