Purification and properties of lactate dehydrogenase from liver of Uromastix hardwickii.
Lactate dehydrogenase isoenzyme-1 was purified from liver of Uromastix hardwickii using colchicine-Sepharose and heat-inactivation methods. The crude enzyme showed four isoenzymes by agarose gel electrophoresis (AGE). The purified enzyme showed a single band after native AGE and SDS-PAGE corresponding to a molecular weight of 34 kDa. The enzyme did not bind with DEAE-Sepharose at pH 7.2. The optimum pH for forward reaction was 7.5, while for reverse reaction, the maximum activity was at pH 9.5. The Km values for pyruvate, NADH, lactate and NAD+ were 0.105, 0.045, 9.0 and 0.011 mM, respectively. The pyruvate showed maximum activity at about 150 microM and then starts showing inhibition at higher concentration. Pre-heating of enzyme showed that it was stable at 80 degrees C for 30 min and at 100 degrees C it became inactive immediately. Oxalate, glutamate, Cu2+, Co2+, Mn2+, and Mg2+ have shown inhibitory effects both for forward- and reverse-reactions. From these properties, we suggest that LDH-1 from Uromastix liver may be quite different from that of other vertebrates.
Javed
M U
MU
Department of Biochemistry, Aga Khan University Medical College, Karachi, Pakistan.
Yousuf
F A
FA
Hussain
A N
AN
Ishaq
M
M
Waqar
M A
MA
D000818:Animals; D002413:Cations, Divalent; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D018698:Glutamic Acid / Q000494:pharmacology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D007773:Lactates / Q000378:metabolism; D019344:Lactic Acid; D008099:Liver / Q000201:enzymology*; D008116:Lizards; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D010070:Oxalates / Q000494:pharmacology; D019815:Oxalic Acid; D011773:Pyruvates / Q000378:metabolism; D019289:Pyruvic Acid
D002413:Cations, Divalent; D007527:Isoenzymes; D007773:Lactates; D010070:Oxalates; D011773:Pyruvates; D009243:NAD; D019344:Lactic Acid; D018698:Glutamic Acid; D019289:Pyruvic Acid; D019815:Oxalic Acid; D007770:L-Lactate Dehydrogenase