The induction and properties of the enzyme delta-aminolaevulinic acid synthetase in mammalian and avian liver

Crozier, Malcolm James (1973) The induction and properties of the enzyme delta-aminolaevulinic acid synthetase in mammalian and avian liver. PhD thesis, University of Glasgow.

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1. A new sensitive radiochemical assay method has been described for the measurement of pmole quantities of 6-aminolaevulinic acid (ALA) in minute amounts of normal tissues, for which the existing conventional colourimetric assay method is inadequate. 2. This isotopic procedure, based on the incorporation of [2-14C.] glycine into ALA, utilized crude homogenate as the source of enzyme. 3. Electrophoresis of the trichloroacetic acid supernatant of the incubation mixture on silica gel thin-layer sheet separates the reaction generated [14C] ALA from glycine and aminoacetone liaximal[14.4C] ALA production in liver homogenate was obtained in the presence of glycine, citrate, Tris, and EDTA (pH 7.2-7.4). 5. A comparison of this radiochemical assay with conventional colourimetric assay was made. Several advantages of my radiochemical assay method when compared to the colourimetric assay procedure are as follows :- (i) Only 70 to 100 mg protein were required in order to measure ALA synthesis in pmoles per min. In contrast, at least 2 mg protein is required for the colourimetric assay in order to obtain an optical density difference of 0.1. When both assay methods were performed on the same porphyric liver homogenate, 104 dpm were obtained in ALA by the 14C method per mg of protein while the colourimetric method gave an observed optical density difference of 0.053 for the same amount of protein. 100 (ii) As many as 100 assays may be performed in one day whereas more than 30 assays using the colourimetric technique proved cumbersome and laborious. (iii) Once the trichloroacetic acid deproteinised supernatant of the reaction mixture was spotted and allowed to dry on the silica gel polyester thin-layer sheets, [.1401 ALA that was generated in the reaction did not appear to degrade with time. 6. Certain chemical agents, allylisopropylacetamide (AIA), and 3,5-dicarbethoxy-1,4-dihydrocollidine (DDC) are known to increase the activity of b-aminolaevulinic acid synthetase (ALA-S), the rate-limiting enzyme in porphyrin biosynthesis, in rat and chick embryo livers and cultured chick embryo liver cells. There is conflicting evidence on the mode of action of these drugs. It has been suggested that AIA acts at the transcriptional level. Initial investigations described in this thesis involved an attempt to isolate mRNA for ALA-S from porphyric rat livers and test its biological activity in cultured chick embryo liver cells. Although results from the colourimetric assay indicated some evidence for induction of ALA-S by RNA, the actual optical differences for RNA-treated cultured cells was extremely low (i.e. maximum = 0.05) and thus colourimetric assay was found to be inadequate. 7. Attempts were made to purify the mRNA for ALA-S from poly-ribosomal RNA and total hepatic cellular RNA, obtained from livers of AIA-treated rats, by-such methods as sucrose density centrifugation, adsorption to nitrocellulose filters, or oligodeoxythymidylate-cellulose. However, the RNA fractions isolated by these methods did not increase 101 ALA-S activity, as measured by the radiochemical assay, in cultured chick embryo liver cells above control levels. Even the addition of either diethylaminoethyl-dextran or dextran sulphate to porphyric poly-ribosomal RNA prior to incubation with cells did not enhance ALA-S activity, as measured by the radiochemical assay method. 8. The failure by the radiochemical assay to detect any RNA mediated increase in ALA-S activity, contrary to previous reports by Hickman et al., (1967, 1968) and Sheaetpa., (1970) which indicated RNA induced ALA-S activity (measured by the colourimetric assay) in cultured liver cells, suggested two feasible explanations (i) The cultured chick embryo liver cell system is not sensitive enough to detect mRNA for ALA. (ii) The porphyrogenic drug, AIA, does not increase or stimulate synthesis of the mRNA for ALA-S. 9. The AIA- and DDC-induced ALA-S of rat and chick embryo livers and cultured chick embryo liver cells have been compared physic-chemically in vitro. (i) ALA-S in crude homogenates from AIA- and DDC-induced chick embryo liver appeared to require similar Na+ cation concentrations. In contrast, addition of Mg-1-1- to the incubation mixtures decreased the enzyme activity for both drug induced enzymes but between 0.02 M and 0.1 M Me+ there was a significant increase of the DLC-induced ALA-S but not for the AIA-S induced enzyme. 102 (ii) The AIA- and DDC induced ALA-S in rat liver had identical pH optima (pH 6.7). In contrast, the DDC-induced ALA-S in chick embryo liver had a higher pH optimum (pH 7.7). Moreover, the AIA-induced ALA-S in chick embryo liver showed two distinct pH optima, one which coincided with the AIA-induced enzyme in rat liver (pH 6.7), and the other was identical to the DDC-induced ALA-S pH optimum (pH 7.7), (iii) Heat stability curves for the induced enzyme were examined. The results suggested that the enzyme having ALA-S activity in rat and chick embryo liver may differ from the enzyme in cultured chick embryo liver cells.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: W S Stevely
Keywords: Analytical chemistry
Date of Award: 1973
Depositing User: Enlighten Team
Unique ID: glathesis:1973-72840
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 11 Jun 2019 11:06
Last Modified: 11 Jun 2019 11:06

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