title: Cleavage of hexokinase II to two domains by trypsin without significant change in catalytic activity.
abstract: Hexokinase II prepared from Ehrlich-Lettre hyperdiploid tumor cells (ELD cells) was subjected to a limited digestion by trypsin. After 60 min digestion, hexokinase II (100 kDa) was completely cleaved to two fragments with the molecular weight of about 60 kDa and 40 kDa as manifested in SDS-PAGE. It was noteworthy that the enzyme activity was observed even at the time when the native enzyme molecule was no more detectable. These fragments were separated by SDS-PAGE irrespective of the presence of a reducing agent, but neither by native PAGE nor by cellulose acetate membrane electrophoresis under the nondenaturing conditions. Neither kinetic parameters such as Km values for ATP and glucose nor an ability of binding to mitochondria were changed significantly by the tryptic digestion. These results indicate that an essential conformation of hexokinase II can be restored by the self-association of two fragments produced as a result of the cleavage by trypsin at the middle of the molecule. Affinity labeling with 2',3'-dialdehyde ATP followed by the trypsin digestion showed that ATP binding site resided in the 40 kDa fragment. Furthermore, the mode of the response in the incorporation of this ATP analog to hexose phosphate, moreover, was similar to that in the catalytic activity.
authors: Department of Physiological Chemistry, Hiroshima University School of Medicine, Japan.
authors: H
authors: H
authors: Okazaki
authors: Y
authors: Y
authors: Takebayashi
authors: M
authors: M
authors: Ando
authors: S
authors: S
authors: Date
authors: H
authors: H
authors: Tokuda
authors: S
authors: S
authors: Ishibashi
mesh_terms: D000818:Animals; D002384:Catalysis; D006593:Hexokinase / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification*; D011485:Protein Binding; D011869:Radioligand Assay; D014357:Trypsin
chemical_list: D010446:Peptide Fragments; D006593:Hexokinase; D014357:Trypsin