Hf. Zhu et al., FUNCTIONAL-STUDIES OF YEAST-EXPRESSED HUMAN HEART-MUSCLE CARNITINE PALMITOYLTRANSFERASE-I, Archives of biochemistry and biophysics, 347(1), 1997, pp. 53-61
Long-chain fatty acids are the primary source of energy production in
the heart. Carnitine palmitoyltransferase I (CPT-I) catalyzes the firs
t reaction in the transport of long-chain fatty acids hom the cytoplas
m to the mitochondrion, a rate-limiting step in beta-oxidation. In thi
s study, we report the functional expression of the human heart/skelet
al muscle isoform of CPT-I (M-CPT-I) in the yeast Pichia pastoris. Scr
eening of a human heart cDNA Library with cDNA fragments encoding the
rat heart M-CPT-I resulted in the isolation of a single full-length hu
man heart M-CPT-I cDNA clone. The clone has an open reading frame of 2
316 bp with a 5' untranslated region of 38 bp and a 256-bp 3' untransl
ated region with the poly(A)(+) addition sequence AATAAA. The predicte
d protein has 772 amino acids and a molecular mass of 88 kDa. Northern
blot analysis of mRNAs from different human tissues using the human M
-CPT-I cDNA as a probe revealed an abundant transcript of similar to 3
.1 kb that was only present in human heart and skeletal muscle tissue.
Expression of the human M-CPT-I cDNA in P. pastoris, a yeast with no
endogenous CPT activity, produced an 80-kDa protein that was located i
n the mitochondria. Isolated mitochondria from the M-CPT-I expression
strain exhibited a malonyl-coenzyme A (CoA)-sensitive CPT activity tha
t was detergent labile. The I-50 for malonyl-CoA inhibition of the yea
st-expressed M-CPT-I was 69 nM, and the K(m)s for carnitine and palmit
oyl-CoA were 666 and 42 mu M, respectively. The I-50 for malonyl-CoA i
nhibition of the heart enzyme is 30 times lower than that of the yeast
-expressed liver CPT-I, and the K-m for carnitine is more than 20 time
s higher than that of the liver CPT-I. This is the first report of the
expression of a heart CPT-I in a system devoid of endogenous CPT acti
vity and the functional characterization of a human heart M-CPT-I in t
he absence of the Liver isoform and CPT-II. (C) 1997 Academic Press.