Fluorescent Nucleotide Analogs Research Articles

Affinity labeling of the myosin ATPase with ribose-modified fluorescent nucleotides and vanadate.

Ribose-modified fluorescent nucleotide analogs, 3'-O-anthraniloyl and 3'-O-(N-methylanthraniloyl) derivatives of AT(D)P, dAT(D)P, CT(D)P, UT(D)P, IT(D)P, and GT(D)P, were synthesized for use as substrates and affinity labels for the myosin ATPase [Hiratsuka, T. (1983) Biochim. Biophys. Acta 742, 496-508]. None of the fluorescent nucleoside triphosphate (NTP) analogs was significantly different from the corresponding natural NTP in its ability to support superprecipitation of actomyosin. When fluorescent and natural NTPs were used as substrates for the myosin subfragment-1(S-1) ATPase in the presence of 1mM vanadate ion (V1), a slight initial inhibition of the S-1 NTPase was followed by progressive inhibition to more than 60% over a period of 1 h. The apparent second-order rate constants were 0.14-0.44M-1 . s-1, suggesting the formation of the inactive fluorescent NDP-labeled S-1. After incubation of S-1 with the nucleoside diphosphate (NDP) analog in the presence of Vi, the resultant fluorescent NDP-labeled S-1 was isolated free of unbound Vi and the analog by gel filtration. The isolated complexes had stoichiometries of 0.6-1.1 NDP analog per S-1 active site. Native polyacrylamide gel electrophoresis revealed conveniently that the NDP analog is associated with S-1 as indicated by two intense fluorescent bands corresponding to S-1 isozymes. On dissociating gels, the analog was released from S-1, suggesting that the labeled S-1 is held together by strong secondary forces rather than covalent bonds.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and properties of fluorescent nucleotide substrates for DNA-dependent RNA polymerases.

A new class of fluorescent nucleotide analogs which contain the fluorophore 1-aminonaphthalene-5-sulfonate attached via a gamma-phosphoamidate bond has been synthesized. Both the purine and pyrimidine analogs have fluorescence emission maxima at 460 nm. Cleavage of the alpha-beta-phosphoryl bond produces change in both the absorption and fluorescence emission spectra. The fluorescence of the pyrimidine analogs is quenched; cleavage of the alpha-beta-phosphoryl bond of the UTP analog produces about a 14-fold increase in fluorescence intensity at 500 nm. Under the same conditions the fluorescence of the CTP analog increases about 8-fold, whereas the fluorescence of the purine analogs shows only a slight change. These derivatives are good substrates for Escherichia coli RNA polymerase with only slightly increased Km values and with Vmax values about 50 to 70% that of the normal nucleotides. They are used less efficiently by wheat germ RNA polymerase II. The ATP analog can be used by E. coli RNA polymerase to initiate RNA chains.

Synthesis of fluorescent nucleotide analogues: 5'-mono-, di-, and triphosphates of linear-benzoguanosine, linear-benzoinosine, and linear-benzoxanthosine.

The fluorescent nucleotide analogues (the 5'-mono-, di-, and triphosphates of lin-benzoguanosine, lin-benzoxanthosine, and lin-benzoinosine) have been prepared for use as dimensional probes of enzyme binding sites. They have quantum yields in aqueous solution of 0.39, 0.55, and 0.04 and fluorescent lifetimes of 6, 9, and approximately equal to 1.5 nsec, respectively. lin-Benzoinosine 5'-monophosphate is a substrate for xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2), providing lin-benzoxanthosine 5'-monophosphate, and lin-benzoinosine 5'-diphosphate is a substrate for polynucleotide phosphorylase (polyribonucleotide:orthophosphate nucleotidyltransferase. EC 2.7.7.8), giving poly(lin-benzoinosinic acid). The benzologues of the purine diphosphates are substrates for pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40), which is used to prepare the triphosphates.

Allosteric activation of aspartate transcarbamylase with a fluorescent nucleotide analogue: linear-benzo-ATP.

The interaction of Escherichia coli aspartate transcarbamylase with linear-benzo-ATP has been investigated by means of fluorescence spectroscopy. The fluorescent nucleotide analogue activates the enzyme to the same extent as ATP. Fluorescence polarization has been used to determine the association constant of lin-benzo-ATP with aspartate transcarbamylase (ATCase) which is 5 X 10(-3) M-1 at pH 8.7, at 4 degrees C, assuming six binding sites. This association constant is similar to those previously obtained for ATP at a variety of temperatures, buffers, and pH. The fluorescence emission of lin-benzo-ATP is not quenched when bound to ATCase, which indicates absence of pi interactions between the activator and tyrosyl residues in the protein. These residues have been implicated in the stereochemical mechanism of allosteric interactions in ATCase. Furthermore, this fluorescence behavior implicates hydrogen bond formation between the amino group of lin-benzo-ATP and a nucleophilic center at the enzyme binding site. The fact that lin-benzo-ATP activates ATCase is consistent with a previously published model for nucleotide regulation of the enzyme.

Fluorescence energy transfer between ligand binding sites on chloroplast coupling factor 1.

The method of fluorescence energy transfer is used to measure the distance from the tight nucleotide binding sites to the 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole reactive sites on solubilized spinach chloroplast coupling factor 1 (CF1). The fluorescent adenine nucleotide analogs 1,N-6-ethenoadenosine diphosphate and 1,N-6-ethenoadenylyl imidodiphosphate were used as donors and 4-nitrobenzo-2-oxa-1,3-diazole bound to a tyrosine group and to an amino group were used as acceptors of energy transfer. Using three different donor-acceptor pairs, the distance measured varied from 38 to 43 A assuming both donor sites are equidistant from the acceptor site. A model is proposed for the location of the tight nucleotide binding sites and the active site on the alpha and beta subunits of CF1.

Photophosphorylation studies with fluorescent adenine nucleotide analogs

Photophosphorylation studies with fluorescent adenine nucleotide analogs