PubMed Health⌕ Search

Biomedical subjects

S C Tu

Publications and source records attributed to S C Tu.

69 records · Page 4Linked to original sources
PubMed1980

Cyclophosphamide potentiation and aldehyde oxidase inhibition by phosphorylated aldehydes and acetals.

Fourteen phosphorylated acetals and aldehydes were synthesized for testing in vitro as inhibitors or substrates of aldehyde oxidase, an enzyme involved in the conversion of aldophosphamide to inactive carboxyphosphamide, and for concurrent in vivo administration with cyclophosphamide to mice bearing L1210 ascites tumor cells. Five phosphorus derivatives gave Ki values of 0.1--0.3 mM compared to 0.03 mM for pyridoxal, as determined in aldehyde oxidase assays using N-methylnicotinamide as the substrate. The most active phosphorus inhibitor, ethyl phenyl(2-formylethyl)phosphinate (2b), and pyridoxal were further shown to give competitive and mixed inhibition, respectively. Three aldehydes, administered concurrently with cyclophosphamide, produced greater increases in life span of L1210-implanted mice than did pyridoxal. All four agents gave an average increase in life span greater than 50% over that shown by cyclophosphamide alone.

Acetals↗
PubMed1980

Physical interaction and activity coupling between two enzymes induced by immobilization of one.

Flavin reductase and bacterial luciferase are believed to be coupled in the in vivo light emitting reaction. In extracts, however, they are both soluble enzymes that exhibit little or no association. Immobilized luciferase, covalently attached to Sepharose, was found to bind the soluble reductase and to exhibit activity in the coupled reaction reaction with an enhanced efficiency of electron transfer.

Catalysis↗
PubMed1979

Isolation and properties of bacterial luciferase-oxygenated flavin intermediate complexed with long-chain alcohols.

Nonsubstrate long-chain aliphatic alcohols, carboxylic acids, and their methyl esters were found to complex reversibly with and stabilize an oxygenated flavin-luciferase intermediate, with alcohols being more effective in stabilizing the intermediate. Dissociation constants for the binding of alcohols to luciferase intermediate are in the order of K8 greater than K10 greater than K12 congruent to K14 where the subscripts represent the numbers of carbon atoms of various alcohols. Thermodynamic activation parameters for the decay of oxygenated flavin-luciferase intermediate complexed with alcohols or aldehydes were determined, and similarities were noted between alcohol and aldehyde complexes. Luciferase intermediate complexes formed with 1-decanol and 1-tetradecanol were isolated at 0 degrees C in neutral phosphate buffer, and both showed absorption properties characteristic of 4a-substituted dihydroflavins. The 1-tetradecanol-intermediate species contained one favin per luciferase molecule. Initially this complex was weakly fluorescent, but upon exposure to 370-nm light it was transformed to a highly fluorescent species. The latter shows a fluorescence excitation peak at 370 nm, and its fluorescence emission (lambda max 505 nm) and quantum yield (0.17) closely correspond to that of bioluminescence in vitro. Both the weakly and the highly fluorescent species exhibit full bioluminescence activities when reacted with decanal.

Alcohols↗
PubMed1978

Activity and stability of the luciferase--flavin intermediate.

A luciferase intermediate in the bacterial bioluminescence system, which is formed by reaction of enzyme with reduced flavin mononucleotide (FMNH2) and oxygen, is shown to emit light with added aldehyde under anaerobic conditions. The reaction with oxygen is thus effectively irreversible under the conditions used. The flavin chromophore has an absorption maximum at about 370 nm and the potential activity (bioluminescence yield) in the further reaction of the isolated intermediate with aldehyde is strictly proportional to the amount of this flavin chromophore.

Anaerobiosis↗
PubMed1978

Structural studies on bacterial luciferase using energy transfer and emission anisotropy.

The distance between specific sites on bacterial luciferase was estimated by energy transfer. Luciferase was fluorescently labeled by reaction of an essential sulfhydryl group with N-(1-pyrene)maleimide and N-[p-(2-benzoxazolyl)phenyl]meleimide. Both of the modified enzymes bind 8-anilino-1-naphthalenesulfonate (Ans) with affinities similar to that exhibited by the native luciferase. Using each of the two fluorescent probes as a donor and the bound Ans as an acceptor, the energy transfer efficiencies were determined by the resulting enhancement of fluorescence of the acceptor. The corresponding distance was calculated to be in the range of 21 to 37 A. Energy-transfer studies were also carried out using fluorescence lifetime measurements of bound ANS, acting as a donor with bound FMN as an acceptor. The corresponding distance was calculated to be between 30 and 58 A. Using samples of luciferase:Ans complex and luciferase modified with N-(1-pyrene)maleimide, the rotational correlation time of the enzyme-dye conjugate as awhole was found to be 47 +/- 2 ns. The observed rotational correlation time is much longer than that calculated for luciferase assuming a spherical structure, thus indicating an elongated form for the luciferase-dye conjugate.

Anilino Naphthalenesulfonates↗
PubMed1977

Bacterial luciferase subunits are synthesized in equal quantities.

Synthesis of luciferase, an alpha beta dimer, occurs during a relatively short period of time near the end of exponential growth of Beneckea harveyi. The rates of synthesis of the individual alpha and beta chains of luciferase are compared by quantitating the molar ratio of total cellular alpha to that of beta at different growth stages. The addition of exogenous alpha or beta subunit to crude lysates of cells taken prior to and during the luciferase induction produces no increase in luciferase activity. This result, together with previous evidence that there are no antigenically cross-reacting precursors, allows us to conclude that luciferase alpha and beta chains are synthesized pari passu, i.e. in equal proportions, and exist primarily in the alpha beta dimeric form. It is also shown that insoluble sedimentable cellular materials contain no detectable luciferase subunits.

Gram-Negative Anaerobic Bacteria↗
PubMed1975

Photoexcited bacterial bioluminescence. Identity and properties of the photoexcitable luciferase.

Properties of photoexcitable luciferase are compared with those of luciferase, both isolated from the bacterium Beneckea harveyi. The proteins have the same molecular weight, are similarly charged at pH 8, and can be inactivated, with comparable efficiencies, by antibodies against either pure luciferase (a heterodimeric protein) or individual subunits thereof. Compared with luciferase, photoexcitable luciferase has a broader pH range for optimal activity, is more stable under acidic conditions, is less stable under alkaline conditions, and is more resistant at neutral pH to inactivation by heat, urea, and trypsin; A flavine-like chromophore, designated B, can be isolated from photoexcitable luciferase. The binding of B to luciferase restores all the properties characteristic of photoexcitable luciferase. Moreover, photoexcitable luciferases from mutants selected to have heat labile luciferases are also thermally unstable. It is concluded that photoexcitable luciferase actually consists of a luciferase-B complex which is conformationally distinct from luciferase under certain conditions.

Animals↗
PubMed1975

Photoexcited bacterial luminescence. Spectral properties and mechanistic implication of a reduced flavine-like prosthetic group associated with photoexcitable luciferase.

A prosthetic group, designated B, has been isolated from bacterial photoexcitable luciferase and found to possess spectral and photochemical properties characteristic of substituted reduced flavines. Its fluorescence when bound to luciferase has an excitation maximum at 375 nm, correlating well with the absorption spectrum, and an emission peaking at 495 nm. However, free B is nonfluorescent in aqueous solution at ambient temperature. Both free and luciferase-bound B show similar negative circular dichroism in the region 330-475 nm with troughs at 375 and 380 nm, respectively. In the luciferase reaction initiated by FMNH2, B is an inhibitor competitive with FMNH2. Irradiation of photoexcitable luciferase converts B to FMN, the latter identified spectrally, enzymatically, and chromatographically. These findings lead to the suggestion that B is a substituted FMNH2. The luciferase-bound B resembles but is not identical with the normal flavine intermediate obtainable by reacting luciferase with reduced flavine mononucleotide and oxygen. It is hypothesized that B is a false intermediate of the bacterial bioluminescence reaction, and a mechanism for the photoexcited bioluminescence reaction is suggested.

Binding Sites↗