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J. Biol. Chem., Vol. 265, Issue 14, 8268-8274, May, 1990
SC Datta, MK Ghosh and AK Hajra
The peroxisomal acyl/alkyl dihydroxyacetone-phosphate reductase (EC
1.1.1.101) was solubilized and purified 5500-fold from guinea pig liver.
The enzyme could be solubilized by detergents only at high ionic strengths
in presence of the cosubstrate NADPH. Peroxisomes, isolated from liver by a
Nycodenz step density gradient centrifugation, were first treated with 0.2%
Triton X-100 to remove the soluble and a large fraction of the
membrane-bound proteins. The enzyme was solubilized from the resulting
residue by 0.05% Triton X-100, 1 M KCl, 0.3 mM NADPH, and 2 mM
dithiothreitol in Tris-HCl buffer (10 mM) at pH 7.5. The enzyme was further
purified after precipitating it by dialyzing out the KCl and then
resolubilized with 0.8% octyl glucoside in 1 M KCl (plus NADPH and
dithiothreitol). The second solubilized enzyme was purified to homogeneity
(370-fold from peroxisomes) by gel filtration in a Sepharose CL-6B column
followed by affinity chromatography on an NADPH-agarose gel matrix.
NADPH-agarose was prepared by reacting periodate-oxidized NADP+ to adipic
acid dihydrazide-agarose and then reducing the immobilized NADP+ with
NaBH4. On sodium dodecyl sulfate- polyacrylamide gel electrophoresis, the
purified enzyme showed a single homogeneous band with an apparent molecular
weight of 60,000. The molecular weight of the native enzyme was estimated
to be 75,000 by size exclusion chromatography. Amino acid analysis of the
purified protein showed that hydrophobic amino acid comprised 27% of the
molecule. The Km value of the purified enzyme for hexadecyldihydroxyacetone
phosphate (DHAP) was 21 microM, and the Vmax value in the presence of 0.07
mM NADPH was 67 mumol/min/mg. The turnover number (Kcat), after correcting
for the isotope effect of the cosubstrate NADP3H, was calculated to be
6,000 mol/min/mol of enzyme, assuming the enzyme has a molecular weight of
60,000. The purified enzyme also used palmitoyldihydroxyactone phosphate as
a substrate (Km = 15.4 microM, and Vmax = 75 mumol/min/mg). Palmitoyl-DHAP
competitively inhibited the reduction of hexadecyl-DHAP, indicating that
the same enzyme catalyzes the reduction of both acyl-DHAP and alkyl-DHAP.
NADH can substitute for NADPH, but the Km of the enzyme for NADH (1.7 mM)
is much higher than that for NADPH (20 microM). The purified enzyme is
competitively (against NADPH) inhibited by NADP+ and palmitoyl-CoA. The
enzyme is stable on storage at 4 degrees C in the presence of NADPH and
dithiothreitol.
Purification and properties of acyl/alkyl dihydroxyacetone-phosphate reductase from guinea pig liver peroxisomes
Department of Biological Chemistry, University of Michigan, Ann Arbor 48104-1687.
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