title: Biochemical characterization of an effective substrate and potent activators of CK2 copurified with Bowman-Birk-type proteinase inhibitor from soybean seeds in vitro.
abstract: By means of Mono P column chromatography, an effective phosphate acceptor (EPA) of casein kinase 2 (CK2) was purified from the Bowman-Birk-type proteinase inhibitor (BBI) fraction of soybean seeds. The most acidic EPA (aEPA, pI=approx. 3.7) was heavily phosphorylated when incubated with CK2 and 5 microM [gamma-(32)P]ATP in the presence of poly-Arg (a CK2 activator) in vitro. However, aEPA was slightly phosphorylated by casein kinase 1 (CK1) as effective as C-kinase and not at all by A-kinase in vitro. The 13 N-terminal amino acid residues (SDHSSSDDESSKP) of aEPA were 100% homologous to the corresponding sequence of soybean BBI-type proteinase inhibitor CII (SBI CII). Polyamine at 3 mM stimulated 4.6-fold the CK2-mediated phosphorylation of aEPA, and this phosphorylation was sensitive to quercetin (ID(50)=approx. 0.1 microM) in vitro. Furthermore, two basic proteins [Mr=29,000 (p29) and 17,000 (p17)] copurified with BBI were identified as proteolytic cleavage products of basic 7S globulin and functioned as potent CK2 activators in vitro. aEPA fully phosphorylated by CK2 in the presence of poly-Arg or basic proteins formed a complex with trypsin, whereas unphosphorylated aEPA was digested by trypsin in vitro. These results suggest that (i) aEPA (a BBI isoform) may coexist with two basic proteins (p29 and p17) generated from basic 7S globulin; and (ii) the physiological interaction between aEPA and its binding trypsin-like proteinases may be regulated through specific phosphorylation of aEPA by CK2 activated with the two basic proteins in legume seeds.
authors.affiliation: Genetical Biochemistry and Signal Biology, Graduate School of Medical Sciences, Kitasato University, 1-15-1 Kitasato, Sagamihara 228-8555, Japan.
authors.forename: Tayo
authors.initials: T
authors.lastname: Katano
authors.forename: Yayoi
authors.initials: Y
authors.lastname: Kamata
authors.forename: Takashi
authors.initials: T
authors.lastname: Ueno
authors.forename: Teisuke
authors.initials: T
authors.lastname: Furuya
authors.forename: Takeshi
authors.initials: T
authors.lastname: Nakamura
authors.forename: Kenzo
authors.initials: K
authors.lastname: Ohtsuki
mesh_terms: D000595:Amino Acid Sequence; D047390:Casein Kinase II / Q000302:isolation & purification / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010766:Phosphorylation; D011794:Quercetin / Q000378:metabolism; D012639:Seeds / Q000737:chemistry*; D013025:Glycine max / Q000737:chemistry*; D013096:Spermine / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D014358:Trypsin Inhibitor, Bowman-Birk Soybean / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D014358:Trypsin Inhibitor, Bowman-Birk Soybean; D013096:Spermine; D011794:Quercetin; D047390:Casein Kinase II; D014357:Trypsin