title: Fluorescence studies of ATP-diphosphohydrolase from Solanum tuberosum var. Desirée.
abstract: Chemical modification of potato apyrase suggests that tryptophan residues are close to the nucleotide binding site. Kd values (+/- Ca2+) for the complexes of apyrase with the non-hydrolysable phosphonate adenine nucleotide analogues, adenosine 5'-(beta,gamma-methylene) triphosphate and adenosine 5'-(alpha,beta-methylene) diphosphate, were obtained from quenching of the intrinsic enzyme fluorescence. Other fluorescent nucleotide analogues (2'(3')-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate, 2'(3')-O-(2,4,6-trinitrophenyl) adenosine 5'-diphosphate. 1,N6-ethenoadenosine triphosphate and 1,N6-ethenoadenosine diphosphate) were hydrolysed by apyrase in the presence of Ca2+, indicating binding to the active site. The dissociation constants for the binding of these analogues were calculated from both the decrease of the protein (tryptophan) fluorescence and enhancement of the nucleotide fluorescence. Using the sensitised acceptor (nucleotide analogue) fluorescence method, energy transfer was observed between enzyme tryptophans and ethene-derivatives. These results support the view that tryptophan residues are present in the nucleotide-binding region of the protein, appropriately oriented to allow the energy transfer process to occur.
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Santiago.
authors.forename: V
authors.initials: V
authors.lastname: Espinosa
authors.forename: A M
authors.initials: AM
authors.lastname: Kettlun
authors.forename: A
authors.initials: A
authors.lastname: Zanocco
authors.forename: E
authors.initials: E
authors.lastname: Cardemil
authors.forename: M A
authors.initials: MA
authors.lastname: Valenzuela
mesh_terms: D000227:Adenine Nucleotides / Q000378:metabolism; D001081:Apyrase / Q000737:chemistry* / Q000378:metabolism; D006868:Hydrolysis; D019657:Solanaceae / Q000201:enzymology*; D013050:Spectrometry, Fluorescence
chemical_list: D000227:Adenine Nucleotides; D001081:Apyrase