A new approach to time-resolved studies of ATP-requiring biological systems; laser flash photolysis of caged ATP.

Abstract

2-Nitrobenzyl derivatives have been used for several years as photolabile protecting groups in synthetic organic chemistry. Recently, P3-1(2-nitro)phenylethyladenosine 5'-triphosphate "caged ATP," was synthesized and its photolysis was shown to generate ATP in situ. This and related reactions have great potential for structural and kinetic studies of both intact and soluble biological systems and it is thus important to define the kinetic characteristics of the photolytic reaction. Caged ATP (2.5 mM) was photolyzed at 347 nm by a single 30-nsec pulse from a frequency-doubled ruby laser of 25 mJ energy to generate 500 .M ATP. The kinetics of the overall reaction were determined by monitoring the kinetics of ATP-induced dissociation of actomyosin, a reaction of known kinetic characteristics. Release of 500 IAM ATP was found to be controlled by a process having a rate constant of 2.2 X log0H+J sec-1 at 220C at pH 5.8-9.5, which corresponds to 220 sec-at pH 7. This process is believed to be the breakdown of an aci-nitro compound, which was identified on the basis of its spectral properties and the photochromicity of related 2-nitrobenzyl compounds. Photodissociation of carbon monoxide and dioxygen from hemoglobin (1, 2) is an important tool for analysis of hemoglobin functions. It would be valuable if a similar approach could be developed to encompass biological reactions in general. In principle, it should be possible to generate photochemically reactants such as metabolites from biologically inert photolyzable compounds. This would allow us to study intact macromolecular systems in which limitations arise because of the difficulty of introducing reactants to their sites of reaction in a time shorter than the characteristic time of the reaction under investigation.First steps toward this goal of photochemical deprotection have been reviewed by Knowles (3). These include the generation of inorganic phosphate from 3-nitrophenyl phosphate by Havinga et al. (4) (5) and Engels and Schlaeger (6), who described the synthesis of photolabile amino acid, peptide, and cyclic AMP precursors and suggested possible applications of their methods. Alternatively, inhibitors can be photogenerated, as has been shown by Lester et al. (7) in their studies of ion-channel blockade mechanisms. These ideas were taken further by Kaplan et al. (8), who synthesized a y-phosphate ester of ATP, P3-1-(2-nitro)phenylethyladenosine 5'-triphosphate (I), which they called caged ATP. They showed that caged ATP was inert toward the sodium pump, could be incorporated into vesicles and, on continuous irradiation (on the time scale of seconds), formed ATP. In the work described here, the amount and rate of ATP generation after laser flash photolysis of caged ATP was measured to determine the potential of caged ATP as an ATP precursor for use in kinetic studies of intact biological systems. It is shown that the energy per pulse from a frequency doubled ruby laser generates ATP in sufficient concentration for biological studies. By using a well ...

The kinetics of magnesium adenosine triphosphate cleavage in skinned muscle fibres of the rabbit.

“…Even when a fibre has its sarcolemma removed the rate of diffusion of Mg ATP from the bathing medium to the hydrolytic sites is slow compared to the rate of the elementary processes (Godt, 1974;Goldman, Hibberd & Trentham, 1984a). However, the synthesis of P3-1-(2-nitro)phenylethyladenosine 5'-triphosphate (caged ATP) by Kaplan, Forbush & Hoffman (1978) and the use of laser flash photolysis to release ATP from caged ATP rapidly (McCray, Herbette, Kihara & Trentham, 1980) have made the study of the mechanical response as a function of ATP concentration in the organized muscle lattice possible (Goldman, Hibberd, McCray & Trentham, 1982b). …”

The associative nature of adenylyl transfer catalyzed by T4 DNA ligase

“…Illumination of DM-nitrophen triggers the intramolecular redox reaction, in which the 2-nitro group oxidizes a benzylic carbon, leading to a scission of the amino bond with the concomitant increase of K d Mg2ϩ from 2.5 M to 3 mM (26). The rate of Mg 2ϩ release in our light setup was estimated by using 31 P cryo-MAS NMR and caged ATP, a 2-nitrophenylethyl ester at the ␥-phosphate (26,(29)(30)(31). At 240 K, the apparent photoreaction rate of caged ATP is 1.2 Ϯ 0.2 h Ϫ1 , which is faster than the observed pseudo-first-order DNA ligase reaction events.…”

Caged agonist of P2Y1 and P2Y12 receptors for light-directed facilitation of platelet aggregation

“…A caged-ATP derivative (P3-1-(2-nitrophenylethyladenosine 5′-triphosphate) has been used to probe the fast kinetics of muscle contraction, etc. [9]. This principle of photocleavage has also been effectively applied to selective deprotection for synthesis in a spatially addressable fashion on a surface array [3], masking of intracellular and extracellular signaling molecules for biological studies [4][5][6], and the light-reversible rapid activation of proteins [7,8].…”