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Two methods for determining the activity of δ-aminolevulinate synthetase within intact liver cells in culture

Analytical Biochemistry, ISSN: 0003-2697, Vol: 79, Issue: 1, Page: 380-393
1977
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Two methods are described by which δ-aminolevulinate synthetase activity is assayed within cultures of intact chick embryo liver cells. The methods depend on blocking further metabolism of δ-aminolevulinate with levulinate (25 m m ). Under these conditions, the δ-aminolevulinate accumulates, much of it leaking out into the medium. The δ-aminolevulinate is measured colorimetrically in a 10-cm light-path cuvette. As little as 0.5 mg of cell protein/assay is required, and the lower limit of the spectrophotometric assay is 600 pmol/2 ml of culture medium. With the first method, the accumulation of δ-aminolevulinate can be followed during the induction, by chemicals, of δ-aminolevulinate synthetase within the intact cell. The second method serves as a quantitative assay for the amount of enzyme present at any one time point. Method 1 is sufficiently sensitive to measure the low activity of the uninduced enzyme as the accumulation of δ-aminolevulinate over a period of 16 hr. With cells in which δ-aminolevulinate synthetase has been chemically induced, method 1 yields a rate of δ-aminolevulinate production by these intact cells, 50–60% of the rate of δ-aminolevulinate produced by homogenates of the same cells incubated in an optimal assay medium. Glycine added to the culture medium at 5 m m increased by three-fold δ-aminolevulinate production by the induced intact cells. With method 2, the enzyme activity of intact cells which have been induced for a defined time, can be measured merely by replacing the culture medium with an “assay medium” containing levulinate (30 m m ), glycine (50 m m ), citrate (5 m m ) and buffer and then incubating the cells for 5 hr. During this time, δ-aminolevulinate accumulates at a linear rate. The rate is equivalent to that of the homogenates of the same cells. The advantage of method 2 compared with the assay of homogenized cells is that the enzyme can be assayed in small amounts of cells without harvesting. One 3.5-cm dish containing 0.7 mg of cell protein is sufficient for one determination.

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