Abstract
4-Hydroxyphenylpyruvic acid dioxygenase is an important enzyme in tyrosine catabolism in most organisms. From porcine and human liver cDNA libraries we isolated complementary DNA inserts for the enzyme. Protein sequence analysis of the porcine enzyme revealed a block of the amino terminus of the mature enzyme. Comparison of the amino acid sequence determined by Edman degradation of peptides derived from porcine liver 4-hydroxyphenylpyruvic acid dioxygenase with the nucleotide sequences revealed the primary structure of the porcine and human enzymes. The mature human and porcine enzymes have an 89% amino acid sequence identity in amino acid residues and are composed of 392 amino acid residues. A computer-assisted homology search revealed that the enzyme is 88% identical in amino acid sequence to rat liver-specific alloantigen F. A monoclonal antibody (mob 51), which can immunoprecipitate both the human and porcine enzymes, was developed. Cultured BMT-10 cells transfected with the cDNA insert of the human enzyme, using the expression vector pCAGGSneodE, produced a polypeptide with an M(r) of 43,000, which was immunoprecipitated with mob 51. Enzymic activity of the enzyme was detected in the transfected cells but not in the mock transfected cells. These findings suggest that the human 4-hydroxyphenylpyruvic acid dioxygenase is a homodimer of two identical subunits with an M(r) of 43,000. Liver-specific alloantigen F seems to be closely related to the enzyme or possibly to the subunit of the enzyme itself. Elucidation of the complete amino acid sequence of the enzyme is expected to reveal structure-function relationships of this metabolically important enzyme and to shed light on inherited disorders related to tyrosine metabolism, especially tyrosinemia types 1 and 3.
Highlights
Tide (M8-K2) generatebdy Achromobacter protease I indicated that 6 amino acidresidues at the carboxyl terminus were missing in the purified enzyme
Activity, whereas the mock transfected cells showed little activity (Fig. 6). These results show that thecDNA insert carried all of the sequence information necessary for full expression of the enzyme protein and activity
Microheterogeneity of purified enzymes from various sources revealed three major forms of avian enzyme that are enzymatically active with isoelectric points of I = 6.0, I1 = 6.2, and I11 = 6.4 [6]
Summary
Society for Inborn Errorsof Metabolism, Takamatsu, Japan, November, 1990. The costs of publication of this article were defrayed in part by the payment of page charges. Antiserum and specific IgG directed to the enzyme protein were preparedas described [13]. Forpreparation of mousemonoclonal antibody directed against theenzyme protein, mice were immunized a t 2-week intervalswith 25-50pgof the purified protein, using Freund’s adjuvant, and hybrid cells were isolated, as described [14]. These hybrid cells were monitored for production of IgG that crossreacted with the porcineliver enzyme. Immunoblot analyses each, Tosoh, Tokyo, Japan) in M guanidine hydrochloride contain- with anti-HPD rabbitIgG were carried outby the method of Towbin ing 10mM sodium phosphate (pH6.0), and S-pyridylethy(lPE)- et al [35] as described [13].
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