L-Aminocarnitine [p,M]

Figure 1. Inhibition of the activities of total CPT (squares), CPT I (circles) and CPT II (triangles) by L-aminocarnitine Activity of the total CPT was measured radiochemical^ as described in Methods using palmitoyl-CoA and [l4C]-L-carnitine (forward assay). CPT II-activity was assayed in the additional presence of 0.4 mM malonyl-CoA. CPT I activity was calculated by subtracting CPT II from the sum of CPT I and CPT II activities. L-aminocarnitine was varied between 0-30 mM as indicated. Values are means of three typical experiments ± S.D. Activities are expressed as percentage of the activity seen without the addition of amino-carnitine and malonyl-CoA. The error bars indicate the standard deviation.

incubated with 1% Tween 20. After incubation time of l0min the homogenate was cen-trifugated to sediment the cell structures including the particulate CPT I-activity which remaines under these conditions. The supernatant should contain the solubilized CPT II. As shown in Fig. 2, in the pellet fraction the restidual activity in the presence of malonyl-CoA was reduced to 20% indicating that part of CPT II activity was released from the mitochondria but a substantial part remained in this fraction. This remaining activity was sensitively inhibited by L-AC (I50 approximately 20 (iM).

In comparison to the pellet fraction, the CPT in the supernatant fraction was much less sensitive to inhibition by 0.4 mM malonyl-CoA as it can be seen from the residual activity of about 88% but the sensitivity to L-AC was increased. At 100|iM L-AC the residual activity of CPT was about 20% indicating that a part of CPT I remained still in the supernatant probably due to incomplete sedimentation of mitochondrial membranes during the centrifugation.

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