A novel electrophoretic technique designed to modify the ratio of magnesium isotopes inside the creatine kinase active sites. A preliminary report

Abstract

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.

Reexamination of magnetic isotope and field effects on adenosine triphosphate production by creatine kinase

“…Furthermore, the magnesium isotope effect reported by BK occurs in a number of different kinases including pyruvate kinase, phosphoglycerate kinase, and ATP synthase, so it would be surprising if the effect occurred in one species variant of CK but not in either variant of MM CK that we tested. The CK isozyme used by BK is extracted from the venom of a snake Vipera Xanthia (sic) (15,20), but details regarding the isozyme and type are not reported. The purified protein is an active monomer (20) not a dimer like CK MM, MB, and BB (30)(31)(32).…”

“…These enzymes are mitochondrial ATP synthase (8,11,16), phosphoglycerate kinase (9, 13), pyruvate kinase (12), and creatine kinase (9,12,15,20). The effects increased linearly with the amount of 25 Mg in the isotopic mixture (13).…”

“…In a series of papers by Buchachenko and Kuznetsov (BK) together with various co-workers (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22), a radical pair mechanism has been proposed for enzymatic phosphorylation of adenosine diphosphate (ADP) to adenosine triphosphate (ATP). This mechanism is influenced by the presence of a magnesium isotope with a nuclear spin.…”

Spin Biochemistry: Magnetic Isotope Effect in the Reaction of Creatine Kinase with CH3HgCl

“…A salt-free preparation of the lyophylized electrophoretically homogeneous 40 kDa CK monomer has been employed. The V. xanthia enzyme was isolated and purified followed by chromatography on hydrophobic (Phenyl Sepharose 6B-CL) anion exchange (Mono Q) and affinity gel (ADP-Sepharose 4B-CL) sorbents, as we have described earlier . All sorbents listed were purchased from Pharmacia Fine Chemicals AB.…”

Magnetic isotope effect of magnesium in phosphoglycerate kinase phosphorylation

“…To isolate the ATP fraction from postincubation and preincubation mixtures, our HPLC technique (ODS-S5CN reversed-phase separation column, 10-60% pyridine linear gradient) was used (17). All incubation mixtures used were balanced for catalysis-optimal conditions according to ref.…”