A method for determining δ-aminolevulinic acid synthase activity in homogenized cells and tissues
| Autor: | John D. Phillips, Michael R. Franklin, James P. Kushner, Hector A. Bergonia |
|---|---|
| Rok vydání: | 2015 |
| Předmět: |
Clinical Biochemistry
Reaction intermediate Mitochondrion Article chemistry.chemical_compound Mice Cell Line Tumor Cytotoxic T cell Animals Humans Succinyl-CoA Heme Fluorescent Dyes chemistry.chemical_classification ATP synthase biology Heme biosynthesis General Medicine Mice Inbred C57BL Enzyme chemistry Biochemistry Liver biology.protein Leukemia Erythroblastic Acute 5-Aminolevulinate Synthetase Chromatography Liquid |
| Zdroj: | Clinical biochemistry. 48(12) |
| ISSN: | 1873-2933 |
| Popis: | In mammalian cells the rate-limiting step in heme biosynthesis is the formation of δ-aminolevulinic acid (ALA). The reaction intermediates, porphyrins and iron and the final product, heme can be highly cytotoxic if allowed to accumulate. The importance of maintaining the levels of metabolic intermediates and heme within a narrow range is apparent based on the complex homeostatic system(s) that have developed. Ultimately, determining the enzymatic activity of ALA synthase (ALAS) present in the mitochondria is highly beneficial to confirm the effects of the transcriptional, translational and post-translational events. The aim of this study was to develop a highly sensitive assay for ALAS that could be used on whole tissue or cellular homogenates.A systematic approach was used to optimize steps in formation of ALA by ALAS. Reducing the signal to noise ratio for the assay was achieved by derivatizing the ALA formed into a fluorescent product that could be efficiently separated by ultra performance liquid chromatography (UPLC) from other derivatized primary amines. The stability of ALAS activity in whole tissue homogenate and cellular homogenate was determined after extended storage at -80 °C.A method for assaying ALAS has been developed that can be used with tissue homogenates or cellular lysates. There is no need to purify mitochondria and radiolabeled substrates are not needed for this assay. General laboratory reagents can be used to prepare the samples. Standard UPLC chromatography will resolve the derivatized ALA peak. Samples of tissue homogenate can be stored for approximately one year without significant loss of enzymatic activity. |
| Databáze: | OpenAIRE |
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