Preparation and properties of 2′(or 3′)-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate, an analog of adenosine triphosphate

Binding and Hydrolysis of TNP-ATP by Escherichia coli F1-ATPase

“…Before hydrolysis, the P i content was 0.02 mol/mol TNP. Concentration determinations for TNP-ATP were based on an extinction coefficient of 26,400 M Ϫ1 cm Ϫ1 at 408 nm (Hiratsuka and Uchida, 1973). All experiments were performed at 23°C in buffer containing 50 mM Tris/H 2 SO 4 , pH 8.0, with further additions as indicated.…”

“…Since their introduction (Hiratsuka and Uchida, 1973), the trinitrophenyl (TNP) 1 derivatives of adenine nucleotides have been widely used to characterize nucleotide binding sites of proteins and enzymes. These analogs have the advantages that they are fluorescent and often bind with much higher affinity than the natural nucleotides.…”

EEDQ probably reacts with the Mg2+‐ATP catalytic sites of mitochondrial and bacterial F1‐ATPases

“…TNP[y-32P]ATP was synthesized and purified using the procedure in [8]. The concentrations of the TNP-nucleotides were measured spectrophotometrically at pH 8.0, assuming a molar extinction coefficient of 26400 M-' .cm-' at 408 nm and 18500 M-'.cm-' at 470 nm [7,8). Efrapeptin was provided by Dr Hamill of Eli Lilly Co.…”

Differentiation of two states of F1‐ATPase by nucleotide analogs

“…3'-Esters of adenine nucleotides were synthesized as in [5,6]; 2'(3')-trinitrophenyl analogs were synthesized as in [9]; a first sample of TNP-ATP was a gift from M. Klingenberg (Munich). All chemicals and auxiliary enzymes were purchased from Merck, Boehringer and Sigma, respectively.…”

“…The newly applied analogs, TNP . ADP or TNP a ATP, have been used to establish the presence of >l catalytic site on isolated Fr-ATPase [lo] to which they exhibit extremely high affinity; in contrast to the 3'-esters they contain a 3'(2')ether bond [9]. Nevertheless, because CPK models of these mole- ptvl nucleotide analog [saP]ATP was separated from "Pi as in [5,6).…”