Characterization of porphobilinogen deaminase from rat liver.
Porphobilinogen deaminase (porphobilinogen ammonia-lyase, EC 4.3.1.8) was isolated from rat liver. The final preparation was homogeneous according to polyacrylamide gel electrophoresis and immunodiffusion criteria. Electrophoresis of the native enzyme revealed a single band of activity which was distributed into three bands after incubation with porphobilinogen. When electrophoresed under denaturing condition it displayed a single polypeptide band with a molecular weight of 42,000 confirmed by exclusion chromatography and by sucrose density gradient centrifugation. The enzyme showed a pH optimum of 7.5 both in 0.1 M sodium phosphate and 0.05 M Tris-HCl buffer, when assayed at 37 degrees C. An isoelectric point of 4.9 for the native purified protein was found. Hepatic porphobilinogen deaminase was remarkably heat-stable showing maximum activity at 55-60 degrees C with one break in the Arrhenius plot. The kinetic behaviour of the purified enzyme followed the typical Michaelis-Menten kinetics with values of Km = 17 microM and Vmax = 29.4 units power mg in 0.1 M phosphate buffer at 37 degrees C. The amino acid composition was determined, showing that the enzyme had a low content of sulphur-containing amino acids and a considerable number of acidic residues per mol of polypeptide chain. Reagents known to interact with sulphydryl groups have small effect on rat liver enzyme activity.
Mazzetti
M B
MB
Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina.
Tomio
J M
JM
D000596:Amino Acids / Q000032:analysis; D000642:Ammonia-Lyases / Q000378:metabolism*; D000818:Animals; D006863:Hydrogen-Ion Concentration; D011163:Hydroxymethylbilane Synthase / Q000302:isolation & purification / Q000378:metabolism*; D007526:Isoelectric Point; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008970:Molecular Weight; D011489:Protein Denaturation; D051381:Rats; D013057:Spectrum Analysis; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013696:Temperature
D000596:Amino Acids; D046911:Macromolecular Substances; D013439:Sulfhydryl Reagents; D011163:Hydroxymethylbilane Synthase; D000642:Ammonia-Lyases