Cobalamin deficiency results in an abnormal increase in L-methylmalonyl-co-enzyme-A mutase expression in rat liver and COS-7 cells.
The aim of the present study was to examine the effects of cobalamin (Cbl) on the activity and expression of L-methylmalonyl-CoA mutase (MCM) in rat liver and cultured COS-7 cells. The MCM holoenzyme activity was less than 5% of the total (holoenzyme+apoenzyme) activity in the liver although rats were fed a diet containing sufficient Cbl. When weanling rats were maintained on a Cbl-deficient diet, the holo-MCM activity became almost undetectable at the age of 10 weeks. In contrast, a marked increase in the total-MCM activity occurred under the Cbl-deficient conditions, and at the age of 20 weeks it was about 3-fold higher in the deficient rats than in the controls (108 (SD 14.5) v. 35 (SD 8.5) nmol/mg protein per min (n 5); P<0.05). Western blot analysis confirmed that the MCM protein level increased significantly in the Cbl-deficient rats. However, the MCM mRNA level, determined by real-time PCR, was rather decreased. When COS-7 cells were cultured in a medium in which 10% fetal bovine serum was the sole source of Cbl, holo-MCM activity was barely detected. The supplementation of Cbl resulted in a large increase in the holo-MCM activity in the cells, but the activity did not exceed 30% of the total-MCM activity even in the presence of Cbl at 10 micromol/l. In contrast, the total-MCM activity was significantly decreased by the Cbl supplementation, indicating that Cbl deficiency results in an increase in the MCM protein level in COS-7 cells as well as in rat liver.
Nakao
Motoyuki
M
Department of Applied Biological Chemistry, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan.
Hironaka
Shoko
S
Harada
Naoki
N
Adachi
Tetsuya
T
Bito
Tomohiro
T
Yabuta
Yukinori
Y
Watanabe
Fumio
F
Miura
Takumi
T
Yamaji
Ryoichi
R
Inui
Hiroshi
H
Nakano
Yoshihisa
Y
D000818:Animals; D015153:Blotting, Western / Q000379:methods; D019556:COS Cells; D002331:Carnitine / Q000008:administration & dosage; D002522:Chlorocebus aethiops; D019587:Dietary Supplements; D015870:Gene Expression; D007668:Kidney / Q000201:enzymology*; D008099:Liver / Q000201:enzymology*; D008715:Methionine / Q000008:administration & dosage; D008765:Methylmalonyl-CoA Mutase / Q000032:analysis* / Q000235:genetics / Q000652:urine; D012333:RNA, Messenger / Q000032:analysis; D011897:Random Allocation; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014805:Vitamin B 12 / Q000008:administration & dosage; D014806:Vitamin B 12 Deficiency / Q000201:enzymology*; D014803:Vitamin B Complex / Q000008:administration & dosage; D014886:Weaning
D012333:RNA, Messenger; D014803:Vitamin B Complex; D008715:Methionine; D008765:Methylmalonyl-CoA Mutase; D014805:Vitamin B 12; D002331:Carnitine