The ingestion of alcohol causes porphyria in man and, with it, an increase in aminolevulinic acid synthase, the initial enzyme of heme biosynthesis. Since that enzyme is synthesized in the cytosol under the control of the nuclear genome, but must move from the cytosol into the mitochondria to function in heme biosynthesis, there are obviously many steps at which ethanol might act to increase its cellular activity. Previous studies in this laboratory have shown that the digitonin fractionation of isolated hepatocytes is an unusually good method for obtaining rapid information about the subcellular distribution of macromolecules, and this project was initiated to apply that method to analyze the induction of the synthase and its movement between cytosol and mitochondria. Evidence has been presented that, in chick embryo liver, the enzyme appears in the cytosol initially as a 74,000-molecular weight precursor that is converted to a 68,000-molecular weight protein during transit into the mitochondria. In contrast, for rat liver, it has been reported that both the cytosolic and mitochondrial ALAS proteins are dimers of 51,000-molecular weight subunits. These discrepancies may really reflect species differences, or they may simply arise from technical difficulties in extracting the native protein. Another contrast between the chick embryo and rat, mouse, guinea pig, as well as the adult chicken, is that all of the other named species can acquire substantial levels of ALAS in the cytosol following treatment with inducers (see below) whereas no cytosolic ALAS occurs in the chick embryo liver. These problems are being pursued by establishing conditions for the induction of aminolevulinic acid synthase in isolated liver cells and fractionation of these cells with digitonin. A cDNA probe is being developed to permit quantitation of ALAS mRNA levels in response to nutritional, alcohol and xenobiotic treatments.
