Effect of plasma biotinyl-peptides on biotinidase activity.
Purified biotinidase (enriched 24,000-fold) from fresh human plasma exhibited reduced catalytic activity when incubated with heat-inactivated dialyzed plasma. The polypeptide fractions separated from the heat-inactivated dialyzed plasma using streptavidin-Sepharose resin showed the same effect on purified biotinidase. These inhibitory effects on biotinidase were partial (25-45%) rather than complete. The polypeptide fraction from streptavidin-Sepharose resin was analyzed by SDS-PAGE in the Laemmli system and by various types of HPLC. Analyses by ion-exchange and reversed-phase HPLC revealed the existence of three relatively small mol. wt polypeptides. Each of these peak fractions exhibited similar inhibitory effects on biotinidase activity. SDS-PAGE analysis indicated that the streptavidin affinity resin fraction was composed of four major polypeptides whose mol. wts were 120,000, 76,000, 53,000 and 27,000. The two bands of 120,000 and 76,000 corresponded to the mol. wts of the biotinyl subunit of pyruvate carboxylase, beta-methyl-crotonyl-CoA and/or propionyl-CoA carboxylase respectively. However, the polypeptides of mol. wts 53,000 and 27,000 were found to be two unique biotinyl-peptides present in human plasma. These bands on the gels were transblotted and exhibited a fluorescent activity after incubated with a FITC-avidin. These findings strongly suggest the existence of circulating plasma biotinyl-polypeptides as inhibitory factor(s) on human plasma biotinidase.
Oizumi
J
J
National Children's Medical Research Center, Division of Metabolism, Tokyo, Japan.
Hayakawa
K
K
D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000097:blood* / Q000302:isolation & purification; D001710:Biotin / Q000097:blood*; D043523:Biotinidase; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007525:Isoelectric Focusing; D008970:Molecular Weight; D010455:Peptides / Q000097:blood* / Q000302:isolation & purification
D010455:Peptides; D001710:Biotin; D000581:Amidohydrolases; D043523:Biotinidase