Chloroplast adenylate kinase from tobacco. Purification and partial characterization

Article abstract of DOI:10.1016/0031-9422(95)00913-2

A soluble isoform of adenylate kinase (AK, EC 2.7.4,3) from tobacco leaves (Nicotiana tabacum L.) was purified about 60-fold by a protocol using ammonium sulphate fractionation, anion exchange chromatography, affinity chromatography and gel filtration. The purified protein was homogeneous, as judged by SDS-PAGE, IEF-PAGE and Mono Q ion exchange chromatography, and had a specific activity of 500 nkat mg^-1. Its M(r) was determined as 28 000 and 30 000 by SDS-PAGE and gel filtration, respectively. It is therefore monomeric and belongs to the long-variant-type adenylate kinases. The isoelectric point of ca 4.45, as measured by IEF-PAGE and the elution profile of the Mono Q column, is characteristic for a chloroplast AK isoform. Like the chloroplast AK of maize, the activity with ATP/AMP as substrates was about two times higher than with ADP and the apparent K(m) was about 10- times higher for ATP/AMP than for ADP. In contrast to the maize enzyme and many other eukaryotic AKs, both substrate binding sites showed an exceptionally high specificity for all three adenylate substrates, together with a rather low affinity, as judged by the apparent K(m)-values. These differences at the substrate-binding sites are confirmed by a low sensitivity of the enzyme to the competitive AK inhibitor diadenosine pentaphosphate, i.e. high K(i)-values.