title: Comparative study of recombinant rat nucleoside diphosphate kinases alpha and beta by intrinsic protein fluorescence.
abstract: Nucleoside diphosphate (NDP) kinases of mammals are hexamers of two sorts of randomly associated highly homologous subunits of 152 residues each and, therefore exist in cell as NDP kinase isoforms. The catalytic properties and three-dimensional structures of the isoforms are very similar. The physiological meaning of the existence of the isoforms in cells remained unclear, but studying recombinant rat NDP kinases alpha and beta, each containing only one sort of subunits, we discovered that, in contrast to the isoenzyme beta, NDP kinase alpha is able to interact with the complex between bleached rhodopsin and G-protein transducin in retinal rod membranes at lowered pH values (Orlov et al. FEBS Lett. 389, 186-190, 1996). In order to search for possible molecular basis of such differences between these isoenzymes, a detailed comparative study of their intrinsic fluorescence properties in a large range of solvent conditions was performed in this work. The isoenzymes alpha and beta both contain the same three tryptophan (Trp78, 133, Ind 149) and four tyrosine (Tyr 52, 67, 147, and 151) residues per subunit, but exhibit pronounced differences in their fluorescence properties (both in spectral positions and shape and quantum yield values) and behave differently under pH titration. Whereas NDP kinase alpha undergoes spectral changes in the pH range 5-7 with the mid-point at 6.2, no unequivocal indication of a structural change of NDP kinase beta under pH titration from 9 to 5 was obtained. Since the pH dependencies obtained for fluorescence of isoenzyme alpha resembles the dependence of its binding to the rhodopsin-transducin complex it was suggested that the differences between the NDP kinase isoenzymes alpha and beta in the pH-induced behavior, revealed by the fluorescence spectroscopy, and the differences in their ability to interact with rhodopsin-transducin complex may have the same physical nature, that would be a physico-chemical reason of possible functional dissimilarity of NDP kinase isoforms in cell. An additional analysis of three-dimensional structure of homologous NDP kinases revealed that the source of the differences in fluorescence properties and pH-titration behavior between the isoenzymes alpha and beta may be due to the difference in their global electrostatic charges, rather than to any structural differences between them at neutral pH. The unusually high positive electrostatic potential at he deeply buried active site Tyr52 makes possible that it exists in deprotonated tyrosinate form at neutral and moderately acidic solution. Such a possibility may account for rather unusual fluorescence properties of NDP kinase alpha: (i) rather long-wavelength emission of NDP kinase alpha at ca. 340 nm at pH ca. 8 at extremely low accessibility to external quenchers and, possibly, (ii) an unusually high quantum yield value (ca. 0.42).
authors.affiliation: Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino. orlov@fluor.iteb.serpukhov.su
authors.forename: N Y
authors.initials: NY
authors.lastname: Orlov
authors.forename: T G
authors.initials: TG
authors.lastname: Orlova
authors.forename: Y K
authors.initials: YK
authors.lastname: Reshetnyak
authors.forename: E A
authors.initials: EA
authors.lastname: Burstein
authors.forename: N
authors.initials: N
authors.lastname: Kimura
mesh_terms: D000818:Animals; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry*; D007700:Kinetics; D008958:Models, Molecular; D020559:Monomeric GTP-Binding Proteins; D054778:NM23 Nucleoside Diphosphate Kinases; D009701:Nucleoside-Diphosphate Kinase; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D014157:Transcription Factors / Q000737:chemistry*
chemical_list: D007527:Isoenzymes; D054778:NM23 Nucleoside Diphosphate Kinases; D011994:Recombinant Proteins; D014157:Transcription Factors; D009701:Nucleoside-Diphosphate Kinase; D020559:Monomeric GTP-Binding Proteins