A novel electrophoretic technique designed to modify the ratio of magnesium isotopes inside the creatine kinase active sites. A preliminary report.
A simple and efficient preparative electrophoretic technique has been proposed to obtain a modified creatine kinase (CK, E.C.2.7.3.2) molecule with an increased content of 25Mg in the active site. A key point of the method is the special design of a 0.9 x 12.0 cm column for ascendent electrophoresis, packed consecutively, from the bottom to the top, with layers of 30 % PAAG (polyacrylamide grade), 25Mg2+ -containing 7.5 % PAAG, enzyme-binding ADP Sepharose and 2.2 % agarose gels, based on different tris-glycine and tris-HCl separation buffer systems. The isotope substitution process was a result of simultaneous desorption of enzyme from ADP Sepharose and electrically directed extensive flow of 25Mg2+ cations through the porous gel matrix. Greater than 8-fold 25Mg enrichment, i.e. a 10.2-86.3 % increase of 25Mg contribution to total enzyme magnesium, has been reached. The modified 25Mg-rich CK samples manifest higher (2.4-fold increase) values of specific catalytic activity when compared with intact (control) ones.
Kouznetsov
Dmitri A
DA
NN Semenov Institute of Chemical Physics, Russian Academy of Sciences, Ul. Kosygina 4, 117977 Moscow. orlova@radio.chem.msu.ru
Arkhangelsky
Stanislav E
SE
Berdieva
Alana G
AG
Khasigov
Peter Z
PZ
Orlova
Marina A
MA
D001665:Binding Sites; D003402:Creatine Kinase / Q000032:analysis* / Q000378:metabolism; D004586:Electrophoresis / Q000379:methods*; D007554:Isotopes / Q000032:analysis*; D008274:Magnesium / Q000032:analysis* / Q000378:metabolism
D007554:Isotopes; D003402:Creatine Kinase; D008274:Magnesium