A nucleotide-binding domain of porcine liver annexin VI. Proteolysis of annexin VI labelled with 8-azido-ATP, purification by affinity chromatography on ATP-agarose, and fluorescence studies.
Porcine liver annexin VI (AnxVI) of Mr 68.000 is an ATP-binding protein as evidenced by specific and saturable UV-dependent labelling with 8-azido-[gamma-32P]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 identify and characterize preliminary nucleotide-binding domain of the protein. AnxVI labelled with 8-azido-ATP was subjected to limited proteolysis and the proteolytic fragments of AnxVI that retained the covalently-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 Mr 34-36.000 and smaller retained the nucleotide. In a reciprocal experiment, AnxVI was digested with proteolytic enzymes and in an ATP eluate from an ATP-agarose column protein fragments of similar Mr to these labelled with 8-azido-ATP were identified. The extent of AnxVI labelling with 8-azido-ATP and the distribution of proteolytic fragments varied upon calcium concentration. These results lead to the conclusion that there is a nucleotide-binding domain within the AnxVI molecule that is functionally similar to the nucleotide-binding domains of other nucleotide-binding proteins. The nucleotide-binding domain is located close to the tryptophan residue 343 of AnxVI and in close vicinity to the Ca2+- and phospholipid-binding sites of the protein. This is confirmed by the observation that the tryptophan fluorescence intensity of AnxVI decreases in the presence of a fluorescence analog of ATP in a calcium-dependent manner, due to the quenching properties of the nucleotide and/or fluorescence energy transfer from AnxVI tryptophan to fluorophore. Both processes were modulated by the presence of phospholipid molecules.
Bandorowicz-Pikuła
J
J
Department of Cellular Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland.
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000345:Affinity Labels; D000818:Animals; D017317:Annexin A6 / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D001386:Azides; D002118:Calcium / Q000502:physiology; D002846:Chromatography, Affinity / Q000379:methods; D010450:Endopeptidases; D005456:Fluorescent Dyes; D008099:Liver / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification; D012685:Sepharose / Q000031:analogs & derivatives; D013050:Spectrometry, Fluorescence; D013552:Swine
C030548:ATP-sepharose; D000345:Affinity Labels; D017317:Annexin A6; D001386:Azides; D005456:Fluorescent Dyes; D010446:Peptide Fragments; C017040:8-azidoadenosine 5'-triphosphate; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D012685:Sepharose; D010450:Endopeptidases; D002118:Calcium