Three
biotin-dependent
enzymes,
pyruvate carboxylase (PC),
propionyl CoA carboxylase (PCC), and
beta-methylcrotonyl CoA carboxylase (beta MCC), were biochemically characterized in fibroblasts from two patients with neonatal
multiple carboxylase deficiency. Genetic complementation analyses indicated that both cell lines, designated lines 1 and 2, were deficient in the various carboxylase activities and belonged to the bio complementation group. The activities of the three carboxylases became normal when line 2 cells were incubated in medium supplemented with
biotin (1 mg/l) for 24 hrs, whereas 4-6 days were required to achieve maximum activities of PC, PCC, and beta MCC (57%, 46%, and 29% of mean normal
enzyme activity, respectively) in line 1 cells incubated in medium containing up to 10 mg/1
biotin. Furthermore, PC activity in line 2 continued to increase under apparent gluconeogenic conditions in culture, but not in line 1. Thermostability studies suggested that
biotin stabilizes PC and beta MCC in both cell lines. PC in line 1 cells incubated with or without
biotin was less stable than that in normal or line 2 cells, and the less than normal increase of
enzyme activities in line 1, especially that of PC, may represent incomplete biotination. These results indicate that there is biochemical heterogeneity within the bio complementation group. Immunotitration with
antibodies prepared against purified pig heart PCC demonstrated normal quantities of cross-reacting material in both lines and no differences in the amount of this material after incubation with supplemental
biotin, despite the seven- to 20-fold increase in PCC activity. Thus, the increase in carboxylase activity in both bio lines appears to represent activation of rpe-existing apocarboxylase rather than de novo
enzyme synthesis. The primary defect in this form of
multiple carboxylase deficiency may be in a common holocarboxylase
synthetase or in
biotin transport. If the defect is in the
synthetase, the differences noted between the two bio lines could be explained by a difference in the
enzyme's Km for
biotin.