The isolation of uroporphyrinogen III synthases from recombinant strains of Escherichia coli and human erythrocytes and their properties
The isolation of uroporphyrinogen III synthases from recombinant strains of Escherichia coli and human erythrocytes and their properties
The development of uroporphyrinogen III synthase in a strain of Escherichia coli, ST1046, containing the hemD gene was investigated. Other plasmids containing the hemD gene locus were studied. One of these recombinant strains TB1/pBM2, in the presence of ImM IPTG, gave synthase activity three times that of the hitherto used ST1046. Maximum production of the synthase was attained after 15 hours, a marked improvement over the 36 hours needed for peak synthase in ST1046. The availability of the uroporphyrinogen III synthase overproducing E. coli strains, permitted the purification of the enzyme to homogeneity. For enzyme purification and characterization, a sensitive coupled assay was used to generate the hydroxymethylbilane substrate preuroporphyrinogen. The purified enzyme had a specific activity of 1,500μmoles/hr/mg protein. Determination of the M_r of the enzyme by SDS gel electrophoresis (27,000 Daltons) and gel filtration (26,000 Daltons) was consistent with the enzyme being a monomer and agreed with the gene derived molecular weight of 27,887. The enzyme had a K_m of 5.5μM, an isoelectric point of 5.2 and an N-terminal sequence NH2-SILVTRPSPAG and was thermolabile (t1/2 at 60oC 1 minute). The pH optimum was 7.8. The enzyme was inhibited by idoacetic acid, p-chloromercuribenzoic and diethylpyrocarbonate. The enzyme contained four sulphydryl groups as shown by its reaction with the thiophilic reagent, DTNB. The study on interactions between the enzymes porphobilinogen deaminase and uroporphyrinogen III synthase from different sources suggested that the two enzymes do not form a complex during the biosynthesis of uroporphyrinogen III. Uroporphyrinogen III synthase was also purified to homogeneity from human erythrocytes in excess of 6% yield. The purified enzyme had a specific activity of 252.2μmoles//hr/mg protein. Determination of the M_r of the human enzyme by SDS gel electrophoresis (29,000 Daltons) showed it to be a similar size to the E. coli enzyme. The first report of the purification of uroporphyrinogen III synthase to homogeneity from R. sphaeroides was also presented. The enzyme had a specific activity of 150μmoles/hr/mg protein. An estimated Mr of 31,000 Daltons was made by SDS-gel electrophoresis.
University of Southampton
1989
Alwan, Asam Fadel
(1989)
The isolation of uroporphyrinogen III synthases from recombinant strains of Escherichia coli and human erythrocytes and their properties.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
The development of uroporphyrinogen III synthase in a strain of Escherichia coli, ST1046, containing the hemD gene was investigated. Other plasmids containing the hemD gene locus were studied. One of these recombinant strains TB1/pBM2, in the presence of ImM IPTG, gave synthase activity three times that of the hitherto used ST1046. Maximum production of the synthase was attained after 15 hours, a marked improvement over the 36 hours needed for peak synthase in ST1046. The availability of the uroporphyrinogen III synthase overproducing E. coli strains, permitted the purification of the enzyme to homogeneity. For enzyme purification and characterization, a sensitive coupled assay was used to generate the hydroxymethylbilane substrate preuroporphyrinogen. The purified enzyme had a specific activity of 1,500μmoles/hr/mg protein. Determination of the M_r of the enzyme by SDS gel electrophoresis (27,000 Daltons) and gel filtration (26,000 Daltons) was consistent with the enzyme being a monomer and agreed with the gene derived molecular weight of 27,887. The enzyme had a K_m of 5.5μM, an isoelectric point of 5.2 and an N-terminal sequence NH2-SILVTRPSPAG and was thermolabile (t1/2 at 60oC 1 minute). The pH optimum was 7.8. The enzyme was inhibited by idoacetic acid, p-chloromercuribenzoic and diethylpyrocarbonate. The enzyme contained four sulphydryl groups as shown by its reaction with the thiophilic reagent, DTNB. The study on interactions between the enzymes porphobilinogen deaminase and uroporphyrinogen III synthase from different sources suggested that the two enzymes do not form a complex during the biosynthesis of uroporphyrinogen III. Uroporphyrinogen III synthase was also purified to homogeneity from human erythrocytes in excess of 6% yield. The purified enzyme had a specific activity of 252.2μmoles//hr/mg protein. Determination of the M_r of the human enzyme by SDS gel electrophoresis (29,000 Daltons) showed it to be a similar size to the E. coli enzyme. The first report of the purification of uroporphyrinogen III synthase to homogeneity from R. sphaeroides was also presented. The enzyme had a specific activity of 150μmoles/hr/mg protein. An estimated Mr of 31,000 Daltons was made by SDS-gel electrophoresis.
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Published date: 1989
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Local EPrints ID: 460498
URI: http://eprints.soton.ac.uk/id/eprint/460498
PURE UUID: 80110244-e574-483c-8258-20c28ad71625
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Date deposited: 04 Jul 2022 18:23
Last modified: 04 Jul 2022 18:23
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Author:
Asam Fadel Alwan
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