1. A permeabilized isolated rat liver cell preparation was developed to achieve selective permeabilization of the cell membrane to metabolites and to allow the assay of mitochondrial overt
carnitine palmitoyltransferase (
CPT I) activity in situ. By performing the
digitonin-induced permeabilization in the presence of
fluoride and bivalent-
metal-
cation sequestrants, it was possible to demonstrate that the activity of other
enzymes, which are regulated by reversible phosphorylation, was preserved during the procedure and subsequent washing of cells before assay. 2.
CPT activity at a sub-optimal
palmitoyl-CoA concentration was almost totally (approximately 90%) inhibited by
malonyl-CoA, indicating that mitochondrial
CPT I was largely measured in this preparation. 3. The
palmitoyl-CoA-saturation and
malonyl-CoA-inhibition curves for
CPT activity in permeabilized cells were very similar to those obtained previously for the
enzyme in isolated liver mitochondria. Moreover,
starvation and diabetes had the same effects on
enzyme activity, affinity for
palmitoyl-CoA and
malonyl-CoA sensitivity of
CPT I in isolated cells as found in isolated mitochondria. These physiologically induced changes persisted through the cell preparation and incubation period. 4. Neither incubation of cells with
glucagon or
insulin nor incubation with
pyruvate and
lactate before permeabilization resulted in alterations of these parameters of
CPT I in isolated cells. 5. The results are discussed in relation to the temporal relationships of changes in the activity and properties of
CPT I in vivo in relation to the effects of
insulin and
glucagon on
fatty acid metabolism in vivo.