Co-expression of Different Subunits of Human Phenylalanine Hydroxylase: Evidence of Negative Interallelic Complementation.
From: Unidade de Biologia Molecular e Biopatologia Experimental, Centro de Patogénese Molecular, Faculdade de Farmácia da Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal.
Biochimica et biophysica acta
- Publish Date: May 2006
- ISSN: 0006-3002
- Volume: 1762
- Issue: 5
- Pages: 544-50
- Medium: Print
- Language: English
- Citation (JAMA): Leandro João, Nascimento Cátia, de Almeida Isabel Tavares, et al. Co-expression of Different Subunits of Human Phenylalanine Hydroxylase: Evidence of Negative Interallelic Complementation.. Biochim. Biophys. Acta May 2006;1762:544-50
Abstract
To study the interaction between two different subunits of the heteromeric human phenylalanine hydroxylase (hPAH), present in hyperphenylalaninemic (HPA) compound heterozygous patients, heteroallelic hPAH enzymes were produced. A dual vector expression system was used (PRO Bacterial Expression System) in which each mutant subunit was expressed from a separate compatible vector, with different epitope tags, in a single bacterial host. Experimental conditions were selected in order that each plasmid produced equivalent levels of mutant subunits. In this study, we demonstrated that both subunits were expressed and that the purified heteroallelic enzymes, were catalytically active. As expected, the produced proteins displayed enzymatic activities levels lower than the predicted catalytic activity, calculated by averaging in vitro PAH activities from both alleles, and were strongly dependent on the proteins subunit composition. The obtained data suggest that interactions between the studied hPAH subunits, namely the I65T, R261Q, R270K and V388M, and the wild-type protein occurred. As postulated, this phenomenon could be a source of phenotypic variation in genetic diseases involving multimeric proteins.
Mesh Headings (Keywords): Alleles, Cells, Cultured, Gene Expression Regulation, Genetic Complementation Test, Humans, Mutation, Phenylalanine Hydroxylase, Protein Subunits
Check for Full Text / PubMed Unique Identifier (PMID): 16545551
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