A NUCLEOTIDE-BINDING DOMAIN OF PORCINE LIVER ANNEXIN VI - PROTEOLYSISOF ANNEXIN-VI LABELED WITH 8-AZIDO-ATP, PURIFICATION BY AFFINITY-CHROMATOGRAPHY ON ATP-AGAROSE, AND FLUORESCENCE STUDIES

Porcine liver annexin VI (AnxVI) of M-r 68.000 is an ATP-binding prote in as evidenced by specific and saturable UV-dependent labelling with 8-azido-[gamma-P-32]ATP or the fluorescent analog of ATP, 2'-(or 3')-O -(2,4,6-trinitrophenyl)adenosine triphosphate and by binding of AnxVI to ATP-agarose. These characteristics of purified AnxVI were used to i dentify and characterize preliminary nucleotide-binding domain of the protein. AnxVI labelled with 8-azido-ATP was subjected to limited prot eolysis and the proteolytic fragments of AnxVI that retained the coval ently-bound nucleotide were separated by means of gel electrophoresis and visualized by exposure of the gel to a phosphor storage screen. It was found that the AnxVI proteolytic fragments of M-r 34-36.000 and s maller retained the nucleotide. In a reciprocal experiment, AnxVI was digested with proteolytic enzymes and in an ATP eluate from an ATP-aga rose column protein fragments of similar M to these labelled with 8-az ido-ATP were identified. The extent of AnxVI labelling with 8-azido-AT P and the distribution of proteolytic fragments varied upon calcium co ncentration. These results lead to the conclusion that there is a nucl eotide-binding domain within the AnxVI molecule that is functionally s imilar to the nucleotide-binding domains of other nucleotide-binding p roteins. The nucleotide-binding domain is located dose to the tryptoph an residue 343 of AnxVI and in close vicinity to the Ca2+ - and phosph olipid-binding sites of the protein. This is confirmed by the observat ion that the tryptophan fluorescence intensity of AnxVI decreases in t he presence of a fluorescence analog of ATP in a calcium-dependent man ner, due to the quenching properties of the nucleotide and/or fluoresc ence energy transfer from AnxVI tryptophan to fluorophore. Both proces ses were modulated by the presence of phospholipid molecules.