If atp were replaced by a nonhydrolyzable atp analog in different cell types, which of the following processes would not be negatively affected? - transport of vesicles from golgi to the er - g-actin polymerization into f-actin - transport of vesicles from er to the golgi - cytoplasmic streaming

ATP has long been known to play a central role in the energetics of cells both in transduction mechanisms and in metabolic pathways, and is involved in regulation of enzyme, channel and receptor activities. Numerous ATP analogues have been synthesised to probe the role of ATP in biosystems (Yount, 1975; Jameson and Eccleston, 1997; Bagshaw, 1998). In general, two contrasting strategies are employed. Modifications may be introduced deliberately to change the properties of ATP (e.g. making it non-hydrolysable) so as to perturb the chemical steps involved in its action. Typically these involve modification of the phosphate chain. Alternatively, derivatives (e.g. fluorescent probes) are designed to report on the action of ATP but have a minimal effect on its properties. ATP-utilising systems vary enormously in their specificity; so what acts as a good analogue in one case may be very poor in another. The accompanying poster shows a representative selection of derivatives that have been synthesised and summarises their key properties.

In energy-transducing reactions, ATP is normally hydrolysed between the ß and γ phosphate groups, and modification of this region produces slowly hydrolysable or non-hydrolysable analogues (e.g. AP.PNP). These derivatives can be used to assess the role of binding energy in the transduction process. Non-hydrolysable analogues are also useful in crystallographic studies, as are the stable complexes formed between protein-bound ADP and phosphate analogues, such as vanadate. Another route to making a stable ATP state is the use of Co(III) or Cr(III) metal substitutes that display very slow ligand-exchange rates. ATPγS is hydrolysed in many systems but usually shows a much reduced rate compared with ATP. This has been exploited in kinase/phosphatase studies, because once an amino acid side chain has been thiophosphorylated it may be resistant to rapid dephosphorylation. Sulphur analogues in the ɑ and ß positions give rise to stereoisomers that can be used to probe the specificity of binding sites. Introduction of bulky organic probes on the phosphate chain generally gives poorly binding analogues, but this factor is exploited in caged-ATP derivatives that contain a photolabile derivative (McCray and Trentham, 1989). Flashes of 350-nm light release ATP within milliseconds and can be used to initiate reactions in vitro or within cells. Different caging groups have different absorption characteristics and photolysis rates.

Introduction of spectroscopic probes (absorption, fluorescent, EPR and NMR probes) is best done through the adenosine or ribose groups, depending on the specificity of the particular binding site. Although ATP absorbs strongly in the UV light (259 nm) range, this signal is usually masked by protein absorbance and cannot be exploited in spectroscopic studies. The adenine ring can be modified to shift the absorption to >300 nm (e.g. 2-SH-ATP), but, in general, fluorescent derivatives provide more-sensitive probes. Among the apparently subtlest of changes is the substitution of an adenosine with a fluorescent formycin ring. However, the slightly longer C-C bond that connects to the ribose results in this analogue preferentially existing in the syn conformation, in which the base is positioned over the ribose, rather than the extended anti conformation, which is required by most protein-binding sites. In any event, this naturally occurring nucleoside base has not been available from commercial sources for several years. Substitution of groups in the 8 position of adenine also tends to favour the syn conformation.

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