Two methods for determining the activity of δ-aminolevulinate synthetase within intact liver cells in culture
✍ Scribed by Peter Sinclair; S. Granick
- Book ID
- 102984019
- Publisher
- Elsevier Science
- Year
- 1977
- Tongue
- English
- Weight
- 935 KB
- Volume
- 79
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
Two methods are described by which b-aminolevulinate synthetase activity is assayed within cultures of intact chick embryo liver cells. The methods depend on blocking further metabolism of &aminolevulinate with levulinate (25 mM). Under these conditions, the S-aminolevulinate accumulates, much of it leaking out into the medium. The Gaminolevulinate is measured calorimetrically in a IO-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 pmoV2 ml of culture medium.
With the first method, the accumulation of B-aminolevulinate can be followed during the induction, by chemicals, of 8-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 O-aminolevulinate over a period of 16 hr. With cells in which 8-aminolevulinate synthetase has been chemically induced, method 1 yields a rate of B-aminolevulinate production by these intact cells, 50-60% of the rate of O-aminolevulinate produced by homogenates of the same cells incubated in an optimal assay medium. Glycine added to the culture medium at 5 mM increased by three-fold &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 mM), glycine (50 mM), citrate (5 mM) and buffer and then incubating the cells for 5 hr. During this time, S-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 35cm dish containing 0.7 mg of cell protein is sufficient for one determination.