Purification and characterization of 6-pyruvoyl tetrahydropterin synthase from salmon liver.
Salmon liver was chosen for the isolation of 6-pyruvoyl tetrahydropterin synthase, one of the enzymes involved in tetrahydrobiopterin biosynthesis. A 9500-fold purification was obtained and the purified enzyme showed two single bands of 16 and 17 kDa on SDS/PAGE. The native enzyme (68 kDa) consists of four subunits and needs free thiol groups for enzymatic activity as was shown by reacting the enzyme with the fluorescent thiol reagent N-(7-dimethylamino-4-methylcoumarinyl)-maleimide. The enzyme is heat-stable up to 80 degrees C, has an isoelectric point of 6.0-6.3, and a pH optimum at 7.5. The enzyme is Mg2+ -dependent and has a Michaelis constant for its substrate dihydroneopterin triphosphate of 2.2 microM. The turnover number of the purified salmon liver enzyme is about 50 times as high as that of the enzyme purified from human liver. It does not bind to the lectin concanavalin A, indicating that it is free of mannose and glucose residues. Polyclonal antibodies raised against the purified enzyme in Balb/c mice were able to immunoprecipitate enzyme activity. The same polyclonal serum was not able to immunoprecipitate enzyme activity of human liver 6-pyruvoyl tetrahydropterin synthase, nor was any cross-reaction in ELISA tests seen.
Hasler
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Department of Pediatrics, University of Zürich, Switzerland.
Curtius
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HC
D000429:Alcohol Oxidoreductases / Q000276:immunology / Q000302:isolation & purification*; D000818:Animals; D000917:Antibody Formation; D001708:Biopterins / Q000031:analogs & derivatives / Q000096:biosynthesis; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability; D004797:Enzyme-Linked Immunosorbent Assay; D006160:Guanosine Triphosphate / Q000378:metabolism; D006358:Hot Temperature; D008099:Liver / Q000201:enzymology* / Q000378:metabolism; D008301:Maleimides / Q000494:pharmacology; D010449:Peptide Mapping; D019761:Phosphorus-Oxygen Lyases; D012474:Salmon; D013438:Sulfhydryl Compounds / Q000494:pharmacology
D008301:Maleimides; D013438:Sulfhydryl Compounds; D001708:Biopterins; C019347:N-(7-dimethylamino-4-methylcoumarinyl)maleimide; D006160:Guanosine Triphosphate; D000429:Alcohol Oxidoreductases; D019761:Phosphorus-Oxygen Lyases; C043497:6-pyruvoyltetrahydropterin synthase; C003402:sapropterin