title: Method for the simultaneous assay of initial velocities of lactate dehydrogenase isoenzymes following gel electrophoresis.
abstract: A method was developed for determining initial isoenzyme velocity in a model based on lactate dehydrogenase (LD, EC. 1.1.1.27). First, purified LD was fractionated by polyacrylamide disc gel electrophoresis. Following electrophoresis, the gel was cut into slices, each containing an isoenzyme, and the isoenzymes were subsequently extracted by slice homogenization into a buffer and their activity was measured by the reverse (pyruvate to lactate) LD reaction. The volume of LD solution positively correlated with the activity of all five isoenzymes. Isoenzyme recovery in all cases exceeded 80%, compared to isoenzyme activity before electrophoresis. The lower limit of detection was 6.5 mU/microliter for LD 1, as measured using this method. The kinetic parameters of isoenzymes following recovery were similar to literature values. The results indicate that the method described here represents an improvement in the measurement of initial LD isoenzyme velocity.
authors.affiliation: Department of Biochemistry, Tokyo Women's Medical College, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Yoshida
authors.forename: Y
authors.initials: Y
authors.lastname: Takakuwa
mesh_terms: D020106:Acrylamide; D000178:Acrylamides; D000818:Animals; D002021:Buffers; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D012680:Sensitivity and Specificity
chemical_list: D000178:Acrylamides; D002021:Buffers; D007527:Isoenzymes; D020106:Acrylamide; D007770:L-Lactate Dehydrogenase