Abstract
The cleavage of reductively alkylated rat liver dihydropteridine reductase with cyanogen bromide afforded a mixture of peptides, six of which (CB-1 to CB-6) were isolated and purified by C8 reverse-phase high performance liquid chromatography. Portions of peptides CB-1, CB-4, and CB-6 were sequenced by automated Edman degradation and high performance liquid chromatography and the carboxyl-terminal region by conventional procedures. Further proteolytic digestion of CB-6 and isolation of the products afforded a seven-amino acid peptide. A low degeneracy probe comprising 20 nucleotides was synthesized from the sequence of this peptide and was used to screen a rat liver cDNA expression library constructed in the vector lambda gt 10. Positive clones were isolated, and detailed examination of five of these by restriction endonucleases and dideoxy sequence analyses allowed identification of the entire coding region for dihydropteridine reductase. The gene was found to code for a protein of 240 amino acids (excluding the methionine initiator) of Mr = 25,420. Each of the sequences corresponding to the peptides CB-1, CB-4, CB-6, and the carboxyl terminus were identified in the deduced protein sequence. The rat enzyme is highly homologous to the human dihydropteridine reductase; the two proteins differ in only 10 amino acids, and all are conservative substitutions. In contrast, the sequence shows little homology with that of mammalian dihydrofolate reductase: reduced pyridine nucleotide-requiring enzymes with superficial mechanistic similarities.
Highlights
The cleavage of reductively alkylated rat liver di- result from an absence of a functional phenylanine hydroxhydropteridine reductase with cyanogen bromide af- ylase ordihydropteridine reductase andin somecases by forded a mixture of peptides, six of which
The purified samples of DNA were the sample in 70% formic acid (2 ml) containing a 100-fold molar precipitated with ethanol, dried, and resuspended in T E buffer excess of cyanogen bromide, and the (10 mM Tris-C1, 1mM EDTA, pH 8) prior to digestion with EcoRI
After cooling,an aliquot (25pl) was removedto provide a zero time reading, and carboxypeptidase Y was added and the mixture incubated quencing, further cleavage of this peptide was carried out using a protease from S. aureus strainV8 [28], and theproduct was subjected toHPLC on the Cs reverse-phase column
Summary
Dihydropteridine Reductase Sequence procedure [14]. Rat liver (600 g) was homogenized in 0.01 M acetic and samples were lyophilized. Isolation of Rat Dihydropterine Reductase cDNAClones-The to the procedure of Crestfield et al [15], enzyme (10 mg in 2 ml of XgtlO library was screened (250,000 plaques) for dihydropteridine standard buffer) was dialyzed against 0.1 M Tris-HC1 containing 0.5 reductase inserts using the 32P-labeledoligonucleotideprobe. The purified samples of DNA were the sample in 70% formic acid (2 ml) containing a 100-fold molar precipitated with ethanol, dried, and resuspended in T E buffer excess of cyanogen bromide (relative to methionine content), and the (10 mM Tris-C1-, 1mM EDTA, pH 8) prior to digestion with EcoRI stirred solution was incubated in the dark a t 4 "C for 16h. 12.5% gelscontaining 1.25%bisacrylamide and 6 M urea, which were run at a constant current of 3 mA/gel, fixed in 10% trichloroacetic
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