A 96-kDa glycyrrhizin-binding protein (gp96) from soybeans acts as a substrate for casein kinase II, and is highly related to lipoxygenase 3.
A 96-kDa glycyrrhizin (GL)-binding protein (gp96) was purified to apparent homogeneity from an aqueous extract of soybeans by means of successive DEAE-cellulose column chromatography, gel filtration on Superdex 200pg, GL-affinity column chromatography, and ion-exchange chromatography on a Mono S column (HPLC). The protein was identified as a GL-binding protein since it specifically binds to [3H]GA. Moreover, it is a lipoxygenase (an enzyme that catalyzes the oxygenation of unsaturated fatty acids) since (i) it displays lipoxygenase (LOX) activity at pH 6.5; (ii) it is recognized on Western blot analysis by antibodies against LOX-1 and LOX-2; and (iii) the sequence of the N-terminal 21 amino acid residues (SNDVYLPRDEAFGHLKSSDFL) of a 42-kDa fragment (p42) proteolytically generated from gp96 is identical to a sequence of soybean LOX-3. In addition, GL, glycyrrhetinic acid (GA) and soyasaponin beta g slightly inhibited LOX activity of the purified gp96 fraction, whereas oGA (a GA derivative) greatly inhibited its activity. Furthermore, CK-II catalyzed phosphorylation of gp96 was stimulated significantly by GL at doses between 1 and 10 microM, but this phosphorylation was inhibited completely by 50 microM GL. All these results taken together suggest that (i) gp96 purified from soybeans as a GL-binding protein belongs to the LOX family; and (ii) triterpenoid saponins, including GL, are involved in the regulation of the activities of CK-II and LOXs in plants, such as soybeans and roots of liquorice, which contain large quantities of saponins.
Ohtsuki
K
K
Laboratory of Genetical Biochemistry, Kitasato University School of Allied Health Sciences.
Nakamura
S
S
Shimoyama
Y
Y
Shibata
D
D
Munakata
H
H
Yoshiki
Y
Y
Okubo
K
K
D000595:Amino Acid Sequence; D002240:Carbohydrate Sequence; D002352:Carrier Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D047390:Casein Kinase II; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003636:DEAE-Cellulose; D006034:Glycyrrhetinic Acid / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D019695:Glycyrrhizic Acid; D007700:Kinetics; D008084:Lipoxygenase / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D030262:Soybean Proteins; D013025:Glycine max / Q000378:metabolism*; D013379:Substrate Specificity
D002352:Carrier Proteins; D030262:Soybean Proteins; C086010:glycyrrhizin-binding proteins; D019695:Glycyrrhizic Acid; D003636:DEAE-Cellulose; C490096:glycyrrhizin-binding lipoxygenase protein, Glycine max; C095864:lipoxygenase 3; D008084:Lipoxygenase; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D006034:Glycyrrhetinic Acid