Genetic regulation of malic enzyme activity in the mouse.

D.L Coleman,J.E Kuzava

doi:10.1016/s0021-9258(18)54736-0

Abstract

Cytosolic malic enzyme catalyzes the NADP(+)-dependent oxidative decarboxylation of malate to pyruvate and CO2. Additionally, this enzyme produces large amounts of reducing equivalents (NADPH) required for de novo fatty acid synthesis and provides a precursor for oxaloacetate replacement in the mitochondria. Malic enzyme is considered a key lipogenic enzyme and changes in enzyme activity parallel changes in the lipogenic rate. As would be expected, the activity of malic enzyme responds to a variety of dietary and hormonal factors acting mainly on the rate of enzyme synthesis. In the mouse, the structural locus for malic enzyme (Mod-1) is located on chromosome 9. Two alleles reflecting differences in electrophoretic mobility have been identified. This report demonstrates that the amount of hepatic malic enzyme activity is strain-dependent and is regulated by a malic enzyme regulator locus (Mod1r) located on the proximal end of chromosome 12. Two alleles have been identified: Mod1ra, conferring high enzyme activity (C57BL/6J), and Mod1rb, conferring low enzyme activity (C57BL/KsJ). Biochemical studies have demonstrated differences in the apparent Km and Vmax and in specific activity on purification and immunoprecipitation, features that suggest changes in enzyme structure even though no differences were observed by electrophoresis and isoelectric focusing. These combined data suggest that differences in both enzyme quantity and structure may be involved in the genetic regulation of malic enzyme activity in mice.

Highlights

Cytosolic malic enzyme catalyzes the NADP+-de- the adrenal androgen, dehydroepiandrosterone (DHEA)’ (6, pendent oxidative decarboxylation of malate to pyru- 7), whereas synthesis is inhibited by thyroid hormone defivate and CO
The apparent K, and V, and in specific activity on Our interest in malic enzyme started during studies on the purification and immunoprecipitation, features that rate of the lipogenesis in diabeticmice maintained on suggest changes in enzyme structure even though no two different inbred backgrounds, C57BL/6J (B6a)nd differences were observed by electrophoresis and iso- C57BL/KsJ (BKs)
Biochemical and Molecular Studies-Southern analyses with the malic enzyme probe revealed an additional unique 4.8-kb restriction fragment associated with digests from B6 but not BKs mice (Fig. 1).The DBA/2J strain, carrying the Mod-la allele lacked this 4.8-kb fragment but exhibited a different restriction fragment pattern typical of the

Summary

Genetic Regulationof Malic EnzymeActivity in the Mouse*

Cytosolic malic enzyme catalyzes the NADP+-de- the adrenal androgen, dehydroepiandrosterone (DHEA)’ (6, pendent oxidative decarboxylation of malate to pyru- 7), whereas synthesis is inhibited by thyroid hormone defivate and CO,. This report deals with the genetic, biochemical, and molecular analysis of thesestrain differences and identifies a malic enzyme regulator gene ( Modlr )located on chromosome 12 of maintained primarily by modulating its rate of synthesis Aliquots of high speed supernatant were electrophoresed on cellulose acetate, overlaid with agar solution containing the complete enzyme reaction mixture plus nitro blue tetrazolium and phenazine methosulfate to visualize enzyme bands on the gels [11].Purified enzyme from each strain was subjected to SDS-PAGE electrophoresis [18]and to isoelectric focusing according to Righetti [19], usingthe specific instructions in the LKB2117 Multiphor I1electrophoresis system laboratory manual. Blots were exposed to Kodak X-Omat AR film a t -70 "C

RESULTS

Strain or cross

Enzyme activity

DISCUSSION

MalicAcEtnivziytyme in the Mouse