Determination of malic Acid using a malate dehydrogenase reactor after purification and immobilization in non-denaturing conditions and staining with ponceau S.
Mouse liver cytosolic malate dehydrogenase was separated by non-denaturing two-dimensional electrophoresis and identified. Furthermore, the activity of the enzyme was preserved even after separation, electroblotting onto a membrane and staining with Ponceau S in acidic buffer solution (pH 5.1). Using the membrane-immobilized enzyme, the malic acid content was estimated by measuring absorbance changes due to the conversion of nicotinamide adenine dinucleotide (NAD) to NADH. These results indicate that enzyme reactors can be systematically produced after purification, immobilization and staining with Ponceau S.
Shimazaki
Youji
Y
Graduate School of Science and Engineering (Science Section) and Venture Business Laboratory, Ehime University, Matsuyama, Japan. yoji@dpc.ehime-u.ac.jp
Sakikawa
Takahiro
T
D000595:Amino Acid Sequence; D000818:Animals; D001391:Azo Compounds / Q000737:chemistry; D003600:Cytosol / Q000201:enzymology; D004800:Enzymes, Immobilized / Q000378:metabolism*; D008099:Liver / Q000201:enzymology; D008291:Malate Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008293:Malates / Q000032:analysis* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D013379:Substrate Specificity
D001391:Azo Compounds; D004800:Enzymes, Immobilized; D008293:Malates; C030298:malic acid; D008291:Malate Dehydrogenase; C032756:ponceau S