Table 4

Determination of the CPR orientation on plasma membrane

Preimmune antibodies	Anti-CPR antibodies	Dextromethorphan conversion per unit of CPR^4-a	CPR activity
			nmol/min/μg of ergosterol
		11 ± 2	118 ± 15
External^4-b		11 ± 1	115 ± 10
	External^4-b	10 ± 1	111 ± 12
External^4-b + internal^4-c		10 ± 2	113 ± 11
External^4-b	Internal^4-c	1.0 ± 0.2	11 ± 1
	External^4-b + internal^4-c	0.8 ± 0.2	0
Internal^4-c	External^4-b	10 ± 1	95 ± 9

To determine CPR orientation by immuno-inhibition, spheroplasts were incubated for 1 hr on ice with anti-CPR or preimmune antibodies (diluted 1:50 in 1.2 m sorbitol, PBS), that led to the selective immunoinhibition of the externally oriented enzymes. After three washes, incubated spheroplasts were mixed with silica microbeads and treated to prepare PM. As controls, these PM were incubated a second time with anti-CPR or preimmune antibodies to inhibit the inside oriented enzymes that were not inhibited during the first incubation. NADPH-supported CYP2D6 and detergent solubilized CPR activities were measured as described under Materials and Methods. The values shown represent the mean ± standard deviation of results on four independent cultures.

↵4-a Arbitrary unit.
↵4-b Spheroplasts were incubated with sera and washed before disruption.
↵4-c Isolated PM were incubated with sera before the activity assays.