Analysis of mutant NADH-cytochrome b5 reductase: apparent "type III"
methemoglobinemia can be explained as type I with an unstable reductase
T Nagai, K Shirabe, T Yubisui and M Takeshita
Department of Biochemistry, Oita Medical University, Japan.
A patient in Kurobe, Japan, was previously reported to have a new class of
hereditary methemoglobinemia, type III. In this patient, NADH cytochrome b5
reductase deficiency was observed in lymphocytes and platelets as well as
in erythrocytes, but this was not associated with mental retardation. A
base change was identified in the gene for NADH cytochrome b5 reductase,
causing an amino acid substitution from Leu- 148 to Pro. In the present
study, the mutant enzyme (Leu-148-->Pro) was expressed in Escherichia
coli, purified, and characterized. The mutant enzyme retained about 60% of
the catalytic activity of the wild type, but was remarkably heat unstable.
By incubating the mutant enzyme at 42 degrees C for 10 minutes, 80% of the
enzyme activity was lost, whereas the wild-type enzyme lost < 20%
activity after incubation at 50 degrees C for 30 minutes. Another mutant in
which Leu-148 was replaced by Ala was prepared to establish the role of the
residue. This mutant was apparently less heat stable than the wild type,
implying a structural role for Leu-148. Reinvestigation of the enzyme
activity in the blood cells and fibroblasts of the type III Kurobe patient,
revealed that about 40% of the normal activity was detected in these cells,
in contrast to the previous report. Thus, this patient reported previously
as having hereditary meth-hemoglobinemia type III was shown to have type I.
Volume 81,
Issue 3,
pp. 808-814,
02/01/1993
Copyright © 1993 by The American Society of Hematology
