Purification and characterization of animal porphobilinogen synthases--II. Dog liver porphobilinogen synthase.
1. A method is described for the purification of porphobilinogen synthase (PBG-S) from dog liver (acetone dry powder; ammonium sulfate fractionation; controlled heat denaturation; ion exchange chromatography; gel filtration). The 911-fold purified enzyme with a specific activity of 372 mU/mg and 6.2 nkat/mg enzyme protein, respectively, appears as a single band in disc electrophoresis. 2. The optimum pH of the enzyme is 6.8; the Michaelis constant Km = 1.77 x 10(-4) M with 5-amino-levulinic acid as substrate. 3. The determination of the molecular weight of the native multisubunit enzyme (268,000 dalton) and of the subunit (33,500 dalton) are in favour of an octameric structure. 4. The acyl group blocking the N-terminus has been identified as an acetyl group (hydrazinolysis; dansylation; TLC; HPLC). 5. The amino acid composition of the PBG-S subunit is as follows CH3CO-NH-(Asx22Thr9-10Ser18Glx28-29Pro20-21Gly22Ala35-37Val23-25Met5 Ile8-9Leu32-33Tyr10Phe12Lys12Cys3His7Arg22Trp3)-Ala-Leu-COOH. 6. In the presence of ganidinium chloride only one free sulfhydryl group per subunit (3 cysteine residues) could be detected. 7. The purified enzyme contains 0.56 gram atoms of zinc per octamer (neutron activating analysis).
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D004285:Dogs; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D000623:Porphobilinogen Synthase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D011487:Protein Conformation; D013438:Sulfhydryl Compounds; D015032:Zinc / Q000032:analysis
D000596:Amino Acids; D008670:Metals; D013438:Sulfhydryl Compounds; D000623:Porphobilinogen Synthase; D015032:Zinc