2--3--o-(2-4-6-trinitrophenyl)adenosine-5--triphosphate and 8-azidoadenosine-5--triphosphate

The human erythrocyte glucose transport protein (GluT1) is an adenine nucleotide binding protein. When complexed with cytosolic ATP, GluT1 exhibits increased affinity for the sugar export site ligand cytochalasin B, prolonged substrate occlusion, reduced net sugar import capacity, and diminished reactivity with carboxyl terminal peptide-directed antibodies. The present study examines the kinetics of nucleotide interaction with GluT1. When incorporated into resealed human red blood cell ghosts, (2,3)-trinitrophenyl-adenosine-triphosphate (TNP-ATP) mimics the ability of cytosolic ATP to promote high-affinity 3-O-methylglucose uptake. TNP-ATP fluorescence increases upon interaction with purified human red cell GluT1. TNP-ATP binding to GluT1 is rapid (t(1/2) approximately 0.5 s at 50 microM TNP-ATP), cooperative, and pH-sensitive and is stimulated by ATP and by the exit site ligand cytochalasin B. Dithiothreitol inhibits TNP-ATP binding to GluT1. GluT1 preirradiation with saturating, unlabeled azidoATP enhances subsequent GluT1 photoincorporation of [gamma-32P]azidoATP. Reduced pH enhances azidoATP photoincorporation into isolated red cell GluT1 but inhibits ATP modulation of sugar transport in resealed red cell ghosts and in GluT1 proteoliposomes. We propose that cooperative nucleotide binding to reductant-sensitive, oligomeric GluT1 is modulated by a proton-sensitive saltbridge. The effects of ATP on GluT1-mediated sugar transport may be determined by the number of ATP molecules complexed with the transporter.

Topics: 3-O-Methylglucose; Adenosine Triphosphate; Azides; Binding Sites; Biological Transport, Active; Erythrocyte Membrane; Glucose Transporter Type 1; Humans; Hydrogen-Ion Concentration; Models, Chemical; Monosaccharide Transport Proteins; Photoaffinity Labels; Proteolipids; Spectrometry, Fluorescence

Dinitrophenyl S-glutathione (DNP-SG) ATPase is a 38 kDa membrane protein expressed in erythrocytes and other tissues. Although stimulation of ATP hydrolysis catalyzed by DNP-SG ATPase has been demonstrated in the presence of several structurally unrelated amphiphilic ions, structural and functional properties of this protein have not been well-defined. In the present study, we have developed an improved protocol for the purification of DNP-SG ATPase and investigated its kinetic and substrate-binding properties. The purification procedure was based on highly specific elution of the 38 kDa protein from DNP-SG affinity resin in the presence of ATP. The protein could not be eluted using either ADP or adenosine-5'-[beta,gamma-methylene]triphosphate (methylene-ATP), a nonhydrolyzable analogue of ATP. Doxorubicin (DOX), a weakly basic anthracycline chemotherapy agent, was found to be the preferred activator for stimulation of ATP hydrolysis by the enzyme. ATP binding to the enzyme was demonstrated using 8-azido-ATP photoaffinity labeling and binding of trinitrophenyl (TNP)-ATP, a fluorescent analogue of ATP. The photoaffinity labeling of DNP-SG ATPase (38 kDa) was saturable with respect to 8-azido ATP (Kd = 2 microM), indicating that the enzyme was capable of specific and saturable binding to ATP. DNP-SG binding was evident from the purification procedure itself and was also demonstrable by quenching of tryptophan fluorescence. Results of quenching of tryptophan fluorescence as well as radioactive isotope-binding studies indicated that DOX was bound to the purified protein as well.

Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Azides; Carrier Proteins; Doxorubicin; Erythrocyte Membrane; Glutathione; Humans; Membrane Transport Proteins; Photoaffinity Labels; Spectrometry, Fluorescence

Porcine liver annexin VI (AnxVI) of Mr 68.000 is an ATP-binding protein as evidenced by specific and saturable UV-dependent labelling with 8-azido-[gamma-32P]ATP or the fluorescent analog of ATP, 2'-(or 3')-O-(2,4,6-trinitrophenyl)adenosine triphosphate and by binding of AnxVI to ATP-agarose. These characteristics of purified AnxVI were used to identify and characterize preliminary nucleotide-binding domain of the protein. AnxVI labelled with 8-azido-ATP was subjected to limited proteolysis and the proteolytic fragments of AnxVI that retained the covalently-bound nucleotide were separated by means of gel electrophoresis and visualized by exposure of the gel to a phosphor storage screen. It was found that the AnxVI proteolytic fragments of Mr 34-36.000 and smaller retained the nucleotide. In a reciprocal experiment, AnxVI was digested with proteolytic enzymes and in an ATP eluate from an ATP-agarose column protein fragments of similar Mr to these labelled with 8-azido-ATP were identified. The extent of AnxVI labelling with 8-azido-ATP and the distribution of proteolytic fragments varied upon calcium concentration. These results lead to the conclusion that there is a nucleotide-binding domain within the AnxVI molecule that is functionally similar to the nucleotide-binding domains of other nucleotide-binding proteins. The nucleotide-binding domain is located close to the tryptophan residue 343 of AnxVI and in close vicinity to the Ca2+- and phospholipid-binding sites of the protein. This is confirmed by the observation that the tryptophan fluorescence intensity of AnxVI decreases in the presence of a fluorescence analog of ATP in a calcium-dependent manner, due to the quenching properties of the nucleotide and/or fluorescence energy transfer from AnxVI tryptophan to fluorophore. Both processes were modulated by the presence of phospholipid molecules.

Topics: Adenosine Triphosphate; Affinity Labels; Animals; Annexin A6; Azides; Calcium; Chromatography, Affinity; Endopeptidases; Fluorescent Dyes; Liver; Peptide Fragments; Sepharose; Spectrometry, Fluorescence; Swine

Annexin VI from porcine liver can be photoaffinity-labeled with 8-azido-[gamma-32P]ATP in a concentration-dependent, saturable manner. The extent of labeling varied with the concentration of calcium. The dissociation constant for the nucleotide was found to be in the range reported for ATP-binding proteins. The ATP analog, 2'-(or 3')-O-(2,4,6-trinitrophenyl)-adenosine 5'-triphosphate, also bound to AnxVI, as indicated by shift in its fluorescence spectra in the presence of protein. Any significant 8-azido-ATP or TNP-ATP binding was not observed with AnxIV. ATP modulated the binding of AnxVI to erythrocyte membrane and increased the Ca2+ concentration required for half-maximal binding of AnxVI to F-actin.

Topics: Actins; Adenosine Triphosphate; Affinity Labels; Animals; Annexin A4; Annexin A6; Azides; Calcium; Cell Membrane; Erythrocyte Membrane; Fluorescent Dyes; Humans; Sepharose; Swine

Adenosine tri- and diphosphate (ATP and ADP) and their structural analogues stimulate insulin secretion from the isolated perfused rat pancreas, an effect mediated by P2Y-purinoceptor activation. Concerning the base moiety of the nucleotide, it was previously shown that purine but not pyrimidine nucleoside triphosphates were active and that substitution on purine C2 with the 2-methylthio group greatly enhanced the potency. In this study, we further analyze the consequences of ribose and polyphosphate chain modifications. Modifications in 2' and 3' position on the ribose led to a decrease in insulin response when bulky substitutions were made: indeed, 2'-deoxy ATP was similar in activity to ATP, whereas arylazido-aminopropionyl ATP (ANAPP3) was weakly effective and trinitrophenyl ATP (TNP-ATP) was inactive. Substitution on the gamma phosphorus of the triphosphate chain led to a decrease (gamma-anilide ATP) or no change (gamma-azido ATP) in potency; the replacement of the bridging oxygen between beta and gamma phosphorus by a peroxide group did not significantly change the activity, whereas substitution by a methylene group completely abolished stimulation of insulin secretion. As for the phosphorothioate analogues, adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S) induced an insulin response similar to that produced by ATP, whereas adenosine-5'-O-(2-thiodiphosphate) (ADP beta S) was about 100-fold more potent than ATP, as previously shown. In conclusion, two structural features seem to have a strategic importance for increasing the insulin secretory activity of ATP analogues: substitution at the C2 position on the adenine ring of ATP and modifications of the polyphosphate chain at the level of the beta phosphorus.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Area Under Curve; Azides; Deoxyadenine Nucleotides; In Vitro Techniques; Insulin; Insulin Secretion; Islets of Langerhans; Pancreas; Polyphosphates; Rats; Receptors, Purinergic P2; Ribose; Structure-Activity Relationship; Thionucleotides

The binding of 2'(3')-O-(2,4,6-trinitrophenyl)-adenosine-5'-triphosphate (TNP-ATP), [35S]ATP alpha S and 8-azido-[gamma-32P]ATP to isolated cytochrome c oxidase of bovine heart and liver and to the two-subunit enzyme of Paracoccus dentrificans was studied by measuring the fluorescence change or bound radioactivity, respectively. With TNP-ATP three binding sites were determined at cytochrome c oxidase from bovine heart and liver, both with two dissociation constants Kd of about 0.2 and 0.9 microM. Trypsin treatment of the enzyme from bovine heart, resulted in one binding site with a Kd of 0.3 microM. The two-subunit enzyme of Paracoccus dentrificans had only one binding site with a Kd of 3.6 microM. The binding of [35S]ATP alpha S to cytochrome c oxidase was studied by equilibrium dialysis. With the enzyme of bovine heart seven and the enzyme of liver six high-affinity binding sites with apparent Kd's of 7.5 and 12 microM, respectively, were obtained. The two-subunit enzyme of Paracoccus denitrificans had one binding site with a Kd of 20 microM. The large number of binding sites at cytochrome c oxidase from bovine heart, mainly at nuclear coded subunits, was verified by photoaffinity labelling with 8-azido-[gamma-32P]ATP.

Topics: Adenine Nucleotides; Adenosine Triphosphate; Affinity Labels; Animals; Azides; Binding Sites; Cattle; Electron Transport Complex IV; Fluorescent Dyes; In Vitro Techniques; Kinetics; Liver; Myocardium; Paracoccus denitrificans; Protein Conformation; Spectrometry, Fluorescence; Thionucleotides; Trypsin