Studies on aldehyde dehydrogenases in mouse brain: Electrophoretic analysis with agarose gel isoelectric focusing.

Abstract

It has been known that mice of C57BL/6J strain prefer a 10% ethanol solution to tap water and DBA/2J strain mice avoid alcohol. The F1 hybrid (C57BL/6J X DBA/2J) has been shown to have intermediate preference. From our previous studies on the activity of aldehyde dehydrogenase(ALDH) in the brain subcellular fractions of various regions, the possibility that the ALDH activity in the mitochondrial fraction(MT-fr) of midbrain-diencephalon was associated with ethanol preference was assumed. In the present study, we investigated the electrophoretic properties of ALDH in the MT-fr of midbrain-diencephalon from these strains. Mouse brain MT-fr was separated by the method of Gray and Whittaker and solubilized with 0.7% Triton X-100 and sonicated. The supernatant after the centrifugation at 15,000 rpm for 10 min was subjected to agarose gel isoelectric focusing (AG-IEF). AG-IEF was performed for 3.5 hr according to the instructions by Pharmacia Fine Chemicals using 1% agarose gel containing Pharmalyte mixture of pH 3-10 and pH 5-8. The enzymes were stained for 2 hr in the dark at 37°C in 50 ml of 75 mM sodium pyrophosphate buffer, pH 9.0, that contained 51 mg NAD, 9 mg MTT, 35 mg pyruvate and substrate aldehydes. In the conclusion, there was no difference in the isozyme patterns of ALDH localized in MT-fr of midbrain-diencephalon from mice of inbred strains with different alcohol preference when acetaldehyde was used as substrate with AG-IEF.