Oligomeric structure and autophosphorylation of nucleoside diphosphate kinase from rat mucosal mast cells.
Nucleoside diphosphate (NDP) kinases have been found to be involved in a wide range of fundamental biological processes ranging from developmental control to signal transduction and metastasis. We have recently cloned and sequenced a cDNA encoding an NDP-kinase of the rat mucosal mast cell line RBL-2H3 [Hemmerich, S., Yarden, Y., & Pecht, I. (1992) Biochemistry (preceding paper in this issue)]. The enzyme itself has been isolated by means of its affinity to the bischromone cromoglycate. Here we report several of its biochemical characteristics: A structural model for the native protein is proposed in which two disulfide-linked pairs of similar 18-kDa subunits (p18) associate to form a 72-kDa tetramer (p72). This is based on the migration properties of the purified enzyme on gel filtration columns, sodium dodecylsulfate gel electrophoresis, and two-dimensional electrophoresis, together with peptide mapping data. In the absence of NDP, both intact p72 and the dissociated 18-kDa subunits (p18) were shown to undergo Mg(2+)-dependent stoichiometric autophosphorylation utilizing adenosine and guanosine triphosphate or gamma-thiotriphosphate as phosphate donor. This autophosphorylation activity was found to be retained by the 18-kDa subunits even following fractionation by SDS-PAGE and electrophoretic transfer to nitrocellulose. The Michaelis constant of this autophosphorylation reaction with either ATP, ATP gamma S, GTP, or GTP gamma S was determined to be 6.5 +/- 1 microM, and maximally 2 mol of phosphate were found to be incorporated per p72 molecule, thus indicating that phosphorylation occurs at a single site on only two of the four 18-kDa subunits of the holoenzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
Hemmerich
S
S
Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel.
Pecht
I
I
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D006801:Humans; D015471:Leukemia, Basophilic, Acute / Q000201:enzymology; D008407:Mast Cells / Q000201:enzymology*; D051379:Mice; D009092:Mucous Membrane / Q000201:enzymology; D009701:Nucleoside-Diphosphate Kinase / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D011817:Rabbits; D051381:Rats; D013045:Species Specificity; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014018:Tissue Distribution
D009701:Nucleoside-Diphosphate Kinase