Studies on Hepatic δ-Aminolevulinic Acid Synthetase

Abstract

Abstract Optimal conditions for the measurement of δ-aminolevulinic acid synthetase activity were determined by means of a radiochemical assay. When the results obtained with this assay were compared with the commonly used colorimetric methods for determining δ-aminolevulinic acid, it was demonstrated that under identical conditions, the two assays yielded identical results. δ-Aminolevulinic acid synthetase activity in the mitochondria was increased 6- to 10-fold upon disruption of the mitochondria by either sonication or freezing and thawing. The addition of exogenous coenzyme A and a succinyl-CoA-generating system was necessary to assay the enzyme in the disrupted mitochondria. Activity in the sonicated mitochondria was linear with time and with enzyme concentration provided that less than 200 µg of protein was added to the incubation mixture. Using the activities measured with sonicated mitochondria as a source of enzyme, it was determined by assays of microsomes, sonicated mitochondria, and supernatant fractions that δ-aminolevulinic acid synthetase is localized exclusively in the mitochondria. The δ-aminolevulinic acid synthetase activity present in either the microsomal or the supernatant fractions is due to mitochondrial contamination as determined by the presence of the mitochondrial markers glutamic and succinic dehydrogenases in these fractions. δ-Aminolevulinic acid synthetase appears to be partitioned between the inner mitochondrial membrane and the matrix, i.e. it is less tightly bound to the inner membrane than is succinic dehydrogenase, an inner membrane marker, and more tightly bound than isocitric or glutamic dehydrogenases, matrix markers. No increase in δ-aminolevulinic acid synthetase activity was observed during porphyria when the enzyme was assayed under optimal conditions with dilute suspensions of sonicated mitochondria as a source of enzyme and [14C]succinate as substrate. However, δ-aminolevulinic acid synthetase activity was elevated during porphyria when intact mitochondria were used as the source of enzyme with [14C]succinate as substrate. Alternately, when either α-keto[14C]glutarate or [14C]citrate were used as substrates, an increase in δ-aminolevulinic acid synthetase activity was observed in both intact and sonicated mitochondria. These results suggest that the increased rate of heme synthesis observed during experimental porphyria may result from an increased activity of δ-aminolevulinic acid synthetase in the intact mitochondria rather than from an actual increase in the amount of enzyme present.