RESUMO
Porcine kidney betaine aldehyde dehydrogenase (EC 1.2.1.8) kinetic properties were determined at low substrate concentrations. The double-reciprocal plots of initial velocity versus substrate concentration are linear and intersect at the left of the 1/v axis and showed substrate inhibition with betaine aldehyde. Studies of inhibition by NADH and dead-end analogs showed that NADH is a mixed inhibitor against NAD(+) and betaine aldehyde. AMP is competitive with respect to NAD(+) and mixed with betaine aldehyde. Choline is competitive against betaine aldehyde and uncompetitive with respect to NAD(+). The kinetic behavior is consistent with an Iso-Ordered Bi-Bi Steady-State mechanism.
Assuntos
Aldeído Oxirredutases/metabolismo , Rim/enzimologia , Aldeído Oxirredutases/antagonistas & inibidores , Animais , Betaína-Aldeído Desidrogenase , Cinética , NAD/metabolismo , Especificidade por Substrato , SuínosRESUMO
Significant betaine aldehyde dehydrogenase activity was found in porcine kidney. The enzyme was purified 320-fold with an overall recovery of 11%. It had a specific activity of 115.8 nkats/mg protein and proved to be homogeneous by SDS-PAGE with a subunit molecular mass of 52 kDa. IEF studies showed three bands with pI values of 5.74, 5.68 and 5.58, respectively. The enzyme was stable in a pH range between 5.0 and 10.0 and the optimum pH was 9.5. The reaction is highly specific for NAD+ and betaine aldehyde, although acetaldehyde, butyraldehyde and glyceraldehyde can be used. Estimated values of Km at pH 8.0 and 25 degrees C were 127 microM for betaine aldehyde and 40 microM for NAD+. The reaction could not be reversed even at high glycine betaine concentrations. The enzyme was not activated by salts at high concentrations but it was salt tolerant-retaining 50% of maximal activity at 1.0 M K+ and Na+. It is inferred that salt tolerance is an essential property for an enzyme participating in the cellular synthesis of an osmoprotectant. Proline, glycerol, sucrose and mannitol had a little effect on the enzyme activity while glycine betaine had an inhibitory effect.