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Characterization of the N314D allele of human galactose-1-phosphate uridylyltransferase using a yeast expression system.
Biochem Mol Med. 1995 Dec; 56(2):121-30.BM

Abstract

Transferase-deficiency galactosemia is an inborn error of metabolism resulting from impairment of the enzyme galactose-1-phosphate uridylyltransferase (GALT), which normally catalyzes the second step of the Leloir pathway of galactose metabolism. Several recent studies have linked a previously reported substitution, N314D (asn to asp at position 314), with both the Duarte and Los Angeles (LA) variant alleles of GALT. While both variants demonstrate similar mobility shifts relative to the normal enzyme on isoelectric focusing (IEF) gels, one (Duarte) is associated with diminished activity, while the other (LA) is associated with greater than normal activity. Therefore, although the concordance rates between N314D and both of these phenotypes are compelling, the question remains as to whether N314D alone is sufficient to cause either or both variants. To address the question of precisely what properties of variant GALT can be attributed to the N314D substitution alone, we have modeled both the wildtype and N314D-GALT alleles in a previously defined yeast expression system, and characterized each with respect to activity, abundance, subunit interaction, and mobility on isoelectric focusing gels. Our results indicate that the N314D subunit dimerizes well both with wildtype GALT and with itself and that the N314D substitution is sufficient to confer the expected shift of IEF banding pattern associated with both the Duarte and LA variant proteins isolated from human cells. However, our results also suggest that N314D-GALT retains full specific activity, thereby calling into question the suggestion that N314D encodes the Duarte variant of GALT.

Authors+Show Affiliations

Department of Genetics and Molecular Medicine, Emory University School of Medicine, Atlanta, Georgia 30322, USA.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

8825075

Citation

Fridovich-Keil, J L., et al. "Characterization of the N314D Allele of Human Galactose-1-phosphate Uridylyltransferase Using a Yeast Expression System." Biochemical and Molecular Medicine, vol. 56, no. 2, 1995, pp. 121-30.
Fridovich-Keil JL, Quimby BB, Wells L, et al. Characterization of the N314D allele of human galactose-1-phosphate uridylyltransferase using a yeast expression system. Biochem Mol Med. 1995;56(2):121-30.
Fridovich-Keil, J. L., Quimby, B. B., Wells, L., Mazur, L. A., & Elsevier, J. P. (1995). Characterization of the N314D allele of human galactose-1-phosphate uridylyltransferase using a yeast expression system. Biochemical and Molecular Medicine, 56(2), 121-30.
Fridovich-Keil JL, et al. Characterization of the N314D Allele of Human Galactose-1-phosphate Uridylyltransferase Using a Yeast Expression System. Biochem Mol Med. 1995;56(2):121-30. PubMed PMID: 8825075.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Characterization of the N314D allele of human galactose-1-phosphate uridylyltransferase using a yeast expression system. AU - Fridovich-Keil,J L, AU - Quimby,B B, AU - Wells,L, AU - Mazur,L A, AU - Elsevier,J P, PY - 1995/12/1/pubmed PY - 1995/12/1/medline PY - 1995/12/1/entrez SP - 121 EP - 30 JF - Biochemical and molecular medicine JO - Biochem Mol Med VL - 56 IS - 2 N2 - Transferase-deficiency galactosemia is an inborn error of metabolism resulting from impairment of the enzyme galactose-1-phosphate uridylyltransferase (GALT), which normally catalyzes the second step of the Leloir pathway of galactose metabolism. Several recent studies have linked a previously reported substitution, N314D (asn to asp at position 314), with both the Duarte and Los Angeles (LA) variant alleles of GALT. While both variants demonstrate similar mobility shifts relative to the normal enzyme on isoelectric focusing (IEF) gels, one (Duarte) is associated with diminished activity, while the other (LA) is associated with greater than normal activity. Therefore, although the concordance rates between N314D and both of these phenotypes are compelling, the question remains as to whether N314D alone is sufficient to cause either or both variants. To address the question of precisely what properties of variant GALT can be attributed to the N314D substitution alone, we have modeled both the wildtype and N314D-GALT alleles in a previously defined yeast expression system, and characterized each with respect to activity, abundance, subunit interaction, and mobility on isoelectric focusing gels. Our results indicate that the N314D subunit dimerizes well both with wildtype GALT and with itself and that the N314D substitution is sufficient to confer the expected shift of IEF banding pattern associated with both the Duarte and LA variant proteins isolated from human cells. However, our results also suggest that N314D-GALT retains full specific activity, thereby calling into question the suggestion that N314D encodes the Duarte variant of GALT. SN - 1077-3150 UR - https://www.unboundmedicine.com/medline/citation/8825075/Characterization_of_the_N314D_allele_of_human_galactose_1_phosphate_uridylyltransferase_using_a_yeast_expression_system_ DB - PRIME DP - Unbound Medicine ER -