Cytochrome P450 4A, peroxisomal enzymes and nicotinamide cofactors in koala liver
Ngo, S, Kong, S, Kirlich, A, McKinnon, RA & Stupans, I 2000, Comparative Biochemistry and Physiology Part C, vol. 127, no. 1, pp. 327-334.
Microsomal lauric acid hydroxylase activity and cyanide-insensitive palmitoyl coenzyme A oxidative (CIPCO) activity were examined in koala, tammar wallaby and rat liver. The microsomal lauric acid hydroxylation was higher in the koala than either the wallaby or rat, whereas CIPCO activity was completely absent in the koala. Hepatic nicotinamide cofactors were observed for all three species, with nicotinamide-adenine dinucleotide (NAD) levels and the ratio of NAD to NAD phosphate (NADP) higher in koalas than either of the other species. A cloned cDNA for CYP4A from the koala was found to be ~70% similar to human CYP4A11, prompting its naming as CYP4A15.
The assays for microsomal lauric acid hydroxylase activity and CIPCO activity in koalas found higher lauric acid hydroxylase activity (8.52 nmol/mg protein per minute for koalas, compared to 2.44 and 1.93 nmol/mg protein per minute for rat and wallaby respectively) and no CIPCO activity. Both activity levels for rat liver microsomes were comparable with other studies, demonstrating that the effect in koalas is significant. The NAD concentration and NAD/NADP ratio in koala hepatic tissue is significantly higher than either rat or wallaby, with the koalas having an average 924 nmol/g tissue NAD, compared to 519 nmol/g for wallabies and 296 nmol/g for rats, and a ratio of 4.6 compared to wallabies at 2.5 and rats at 1.4. Real time polymerase chain reaction (PCR) was used to create a partial CYP4A cDNA clone of a length of 1188 bp, which was sequenced and compared to other CYP4A sequences previously reported. The deduced amino acid sequence was named CYP4A15, which is described in the paper.
The experiment reproduced a high CYP content in koala hepatic tissue, supporting previous research. Other studies have found that all CYP4A family members have fatty acid ω-hydroxylating activity, so the high lauric acid hydroxylase activity in koala liver is consistent. The lack of CIPCO activity is unusual, however, given previous studies have found several enzymes capable of this activity, which are likely not expressed in koala liver tissue to produce this result. The NAD concentration and NAD/NADP ratio of the rats in this study was consistent with previously determined values, although there is a considerable range of these values in literature. Peroxisome proliferators have been found to cause significant increases in hepatic NAD levels, suggesting that the koala’s levels are high due to exposure to Eucalyptus terpenes, which are a type of peroxisome proliferator. The koala CYP4A15 sequence contains the signature CYP sequence that ligates to haem iron, as well as several other conserved motifs.
This study increases our understanding of enzymatic activity in koala hepatic tissue, furthering knowledge into the function of the koala liver and how the consumption of Eucalyptus leaves impacts its activity.
Summarised by Laura Wait
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