Purification and cloning of the GTP cyclohydrolase I feedback regulatory protein, GFRP.
The activity of GTP cyclohydrolase I, the initial enzyme of the de novo pathway for biosynthesis of tetrahydrobiopterin, the cofactor required for aromatic amino acid hydroxylations and nitric oxide synthesis, is sensitive to end-product feedback inhibition by tetrahydrobiopterin. This inhibition by tetrahydrobiopterin is mediated by the GTP cyclohydrolase I feedback regulatory protein GFRP, previously named p35 (Harada, T., Kagamiyama, H., and Hatakeyama, K. (1993) Science 260, 1507-1510), and -phenylalanine specifically reverses the tetrahydrobiopterin-dependent inhibition. As a first step in the investigation of the physiological role of this unique mechanism of regulation, a convenient procedure has been developed to co-purify to homogeneity both GTP cyclohydrolase I and GFRP from rat liver. GTP cyclohydrolase I and GFRP exist in a complex which can be bound to a GTP-affinity column from which GTP cyclohydrolase I and GFRP are separately and selectively eluted. GFRP is dissociated from the GTP agarose-bound complex with 0.2 NaCl, a concentration of salt which also effectively blocks the tetrahydrobiopterin-dependent inhibitory activity of GFRP. GTP cyclohydrolase I is then eluted from the GTP-agarose column with GTP. Both GFRP and GTP cyclohydrolase I were then purified separately to near homogeneity by sequential high performance anion exchange and gel filtration chromatography. GFRP was found to have a native molecular mass of 20 kDa and consist of a homodimer of 9.5-kDa subunits. Based on peptide sequences obtained from purified GFRP, oligonucleotides were synthesized and used to clone a cDNA from a rat liver cDNA library by polymerase chain reaction-based methods. The cDNA contained an open reading frame that encoded a novel protein of 84 amino acids (calculated molecular mass 9665 daltons). This protein when expressed in Escherichia coli as a thioredoxin fusion protein had tetrahydrobiopterin-dependent GTP cyclohydrolase I inhibitory activity. Northern blot analysis indicated the presence of an 0.8-kilobase GFRP mRNA in most rat tissues, the amounts generally correlating with levels of GTP cyclohydrolase I and tetrahydrobiopterin. Thus, mRNA levels were relatively high in liver and kidney and somewhat lower in testis, heart, brain, and lung. These results suggest that GFRP is widely expressed and may play a role in regulating not only phenylalanine metabolism in the liver, but also the production of biogenic amine neurotransmitters as well as nitric oxide synthesis.
Milstien
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Laboratory of Cell Biology, National Institute of Mental Health.
Jaffe
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Kowlessur
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Bonner
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001708:Biopterins / Q000031:analogs & derivatives* / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D005246:Feedback; D006136:GTP Cyclohydrolase / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D047908:Intracellular Signaling Peptides and Proteins; D008099:Liver / Q000201:enzymology; D008297:Male; D008969:Molecular Sequence Data; D010649:Phenylalanine / Q000494:pharmacology; D011506:Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley
D017931:DNA Primers; C491065:Gchfr protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; D012333:RNA, Messenger; D001708:Biopterins; D010649:Phenylalanine; D006136:GTP Cyclohydrolase; C003402:sapropterin