An improved procedure for the purification of formiminotransferase-cyclodeaminase from pig liver. Kinetics of the transferase activity with tetrahydropteroylpolyglutamates.
Formiminotransferase-cyclodeaminase is stabilized and activated approx. 40% in the presence of low concentrations (equal or less than 0.2%) of Triton X-100, possibly because the average hydrophobicity (1.10 kcal per residue) and the frequency of large non-polar side-chains (0.34) of this protein are both somewhat higher than average. This stabilization enabled us to develop a new purification procedure for the enzyme using chromatography on Matrex Gel Orange A and heparin-Sepharose columns in the presence of Triton X-100. This procedure is easier, much more reproducible, and gives slightly higher yield than the previous method described by Drury, et al. Further investigations of the role of tetrahydropteroylpolyglutamates with formiminotransferase-cyclodeaminase reveal that the use of polyglutamylated folate substrates does not change the mechanism of the transferase reaction, but decreases the K(m) for formininoglutamate, the second substrate, more than 10-fold, bringing it closer to the expected physiological concentration.
Findlay
W A
WA
Department of Biochemistry, McGill University, Montreal, Canada.
Zarkadas
C G
CG
MacKenzie
R E
RE
D000596:Amino Acids / Q000032:analysis; D000642:Ammonia-Lyases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D002850:Chromatography, Gel; D005492:Folic Acid / Q000031:analogs & derivatives*; D005565:Formiminoglutamic Acid / Q000378:metabolism; D005971:Glutamates / Q000494:pharmacology; D018698:Glutamic Acid; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D011624:Pteroylpolyglutamic Acids / Q000378:metabolism*; D013552:Swine
D000596:Amino Acids; D005971:Glutamates; D011624:Pteroylpolyglutamic Acids; D018698:Glutamic Acid; D005565:Formiminoglutamic Acid; D005492:Folic Acid; D000642:Ammonia-Lyases; C020148:formiminotetrahydrofolate cyclodeaminase