Table 3

Effects of bufuralol on testosterone 6β-hydroxylase activities of membranes containing different combinations of CYP3A4, CYP2D6, and CPR

Incubation SystemTestosterone 6β-Hydroxylase ActivityActivity
nmol/min/nmol total P-450 %
CYP3A4/pJR715.82  ± 0.59100
CYP3A4/pJR7 + bufuralol15.78  ± 0.43100
CYP3A4/pJR7 + 1′-hydroxybufuralol15.79  ± 0.57100
CYP3A4/CYP2D6/pJR78.53  ± 0.35100
CYP3A4/CYP2D6/pJR7 + bufuralol7.22  ± 0.403-a 84.8
CYP3A4/CYP2D6/pDRlacZ1.15  ± 0.12100
CYP3A4/CYP2D6/pDRlacZ + bufuralol0.79  ± 0.093-a 68.7

Membranes containing 10 pmol P-450 were incubated at 37°C for 10 min in 50 mM HEPES (pH 7.4), 30 mM MgCl2 containing 100 μM testosterone, and, when indicated, 10 μM bufuralol. Incubations were also carried out in the presence of 1′-hydroxybufuralol (10 μM) as indicated. Reaction was initiated by addition of a NADPH-regenerating system (see Experimental Procedures). Reaction was stopped by adding 100 μl ice cold methanol plus 5 μl 60% perchloric acid. Analysis of metabolites was by HPLC chromatography. Values are given as means of six experiments ±S.D.

  • 3-a Significantly different from control p≤ .01, n = 6 independent experiments each in triplicate.