Liver Enzyme Analyses
Immunoblot analyses of liver homogenates with anti-MCAD antisera demonstrated that the 42 kDa MCAD monomer was present in MCAD+/+ mice, but not in MCAD−/− mice (Figure 3). As a control analysis, anti–short-chain acyl-CoA dehydrogenase (SCAD) antisera revealed no differences in levels of expression of SCAD protein between MCAD+/+ and MCAD−/− mice (Figure 3).
Figure 3  Immunoblots of Liver Homogenates from MCAD+/+ and MCAD−/− Mice
These were probed with anti-MCAD antibody or anti-SCAD antibody. Homozygous MCAD−/− mice had no detectable MCAD protein. MCAD protein is only detectable under the MCAD protein–spiked (positive control) lane. As a control analysis, liver homogenates probed with anti-SCAD antibody revealed that SCAD protein was present in both MCAD+/+ and MCAD−/− mice. No MCAD positive-control protein is detected by anti-SCAD antibodies (MCAD lane). mw, molecular weight standards. Enzyme activity was analyzed in mouse liver homogenates using the electron transport flavoprotein (ETF) reduction assay with octanoyl-CoA (C8:0) and palmitoyl-CoA (C16:0) as substrates. MCAD−/− mice had a significant reduction in ability to dehydrogenate octanoyl-CoA and a modest reduction in activity toward palmitoyl-CoA compared to MCAD+/+ mice (Table 1). Specifically, the dehydrogenation of octanoyl-CoA and palmitoyl-CoA substrates were reduced by 75% and by 30%, respectively, in MCAD−/− mice as compared to MCAD+/+ controls.
Table 1  Characteristics of MCAD-Deficient Mice
Values given are mean ± SD.
aMCAD+/+ n = 5 and MCAD−/− n = 5.
bMCAD+/+ n = 8 litters and MCAD−/− n = 10 litters.
cMCAD+/+ n = 5 and MCAD−/− n = 6.
dMCAD+/+ n = 4 and MCAD−/− n = 5.
eExpressed as a ratio relative to the internal standard.