Skate liver Cytochrome

Table II. Comparison of the spectral properties of partially purified little skate liver cytochrome P-l+50 with partially purified rat liver cytochrome P-l+50 and highly purified rat liver cytochro-me P-l+50 and P-1+1+8. ...

Partially purified Little Skate liver cytochrome P-l+50. The data is from (25.) and is as accurate as can be read from published... 

Recently Bend et al. have succeeded to solubilize and partially purify little skate liver microsomal cytochrome P-l+50 and have thus been able to record the absolute spectra of fish cytochrome P-l+50 (25.). The spectra are very similar to those of rat liver microsomal cytochrome P-l+50 and purified rat liver cytochrome P-1+50 and P-1+1+8 (Table II). 

An obvious difference was also noted between control and induced skate hepaticdnicrosomal AHH activity in the presence of a-naphthoflavone (10 M). This compound, when added in vitro at this or higher concentrations, caused significant stimulation of AHH activity in control animals (about 3-fold) but inhibition (80%) was found in DBA-pretreated skates. Similar results were earlier reported for control and 3-methylcholanthrene-treated rats (23), where it appears that the response is due to differential effects of a-naphthoflavone on hepatic microsomal cytochrome P-450 (stimulated) and cytochrome P-448 (inhibited) (24). Our data suggests that there may be a novel form of cytochrome P-450 synthesized in skate liver in response to polycyclic hydrocarbon administration, even though there was no hypsochromic shift in the carbon monoxide difference spectrum of dithionite reduced hepatic microsomes from DBA-treated skates (relative to hepatic microsomes from control fish). 

Very similar results to those described in Fig. 3-6 were obtained when sodium cholate solubilized hepatic microsomes from DBA-treated female little skates were subjected to chromatography on DEAE-cellulose as described above (data not shown). Also not shown are the results obtained with hepatic microsomes from untreated male and female little skates. With untreated animals, 80-90% of the cytochrome P-450 eluted from the DEAE-cellulose column only at higher ionic strength (i.e., with the KC1 gradient). However, in all preparations studied, an appreciable amount of cytochrome P-450 (10-20%), having its absorption maximum in the carbon monoxide-ligated and reduced state at 450 nm, was eluted from the column with buffer II, as was observed with cytochrome P-448 of hepatic microsomes from DBA-treated skates. The further purification of the various forms of cytochrome P-450 from control and DBA-pretreated little skate livers is currently in progress in our laboratory. 

Collectively, these experiments have demonstrated that substantial amounts of cytochrome P-448 are indeed present in liver of DBA-pretreated male and female skates but not in untreated skates. Sufficient cytochrome P-448 is present to account for the increased AHH activities observed in hepatic microsomes of DBA-treated skates. 

Solubilization and Partial Purification of Cytochrome P-450 from Hepatic Microsomes of Male, DBA-Pretreated Little Skates. Washed hepatic microsomes (3) from the livers of 10 skates were suspended in 0.25 M sucrose and frozen under nitrogen (-5 to -10°) at the Maine laboratory. They were then packed in dry ice and transported to NIEHS, Research Triangle Park, NC, within 14 days of preparation and were stored at -62°C until use. Microsomes... 

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