Pyrenemethyl laurate (
PMLes), a
fluorogenic substrate for determining in vitro
lipase activity [Nègre, Salvayre,
Dagan and Gatt (1989) Biochim. Biophys. Acta 1006, 84-88], has been administered to cultured lymphoblastoid cells from normal subjects and from a patient affected with
Wolman disease, which is characterized by a deficiency of
lysosomal acid lipase. The intracellular degradation of
PMLes was dependent on the mode of administration of the substrate into the cells, and occurred by two separate pathways involving lysosomal and extra-lysosomal
hydrolases.
PMLes incorporated into
LDL was taken up by normal lymphoblastoid cells through the
apolipoprotein-B/E-receptor-mediated pathway and degraded in the lysosomal compartment, as suggested by the degradation block in Wolman cells. In contrast, when
PMLes dissolved in 2%
dimethyl sulphoxide was added directly to the culture medium, its hydrolysis was similar in lymphoblastoid cells from controls and from patients affected with
Wolman disease, neutral
lipid storage disease or familial hypercholesterolaemia. This suggested that the administered
PMLes was degraded by a non-lysosomal
enzyme which is not deficient in Wolman cells. This
enzyme also differs from the neutral
lipase system which is deficient in lymphoblastoid cells from patients with neutral
lipid storage disease. When
pyrenemethanol was administered directly to the cell culture, it was only poorly acylated and was rapidly released into the culture medium. These results and the fluorescence properties of
PMLes ('monomeric' emission in a hydrophobic environment and 'excimeric' emission in a hydrophilic environment) and
pyrenemethanol ('monomeric' emission in a hydrophilic environment) allowed us to design a 'direct reading' procedure by monitoring (without any
lipid extraction) the fluorescence of intact living cells and that of the culture medium during pulse-chase experiments. This method allowed the direct evaluation of the time course of in situ degradation of
PMLes. In pulse-chase experiments with
LDL-
PMLes, the fluorescence of normal cells decreased relatively rapidly with time whereas the fluorescence of the culture medium increased concomitantly. With Wolman cells, the cellular fluorescence decreased only very slightly, whereas that of the culture medium remained at the basal level; this demonstrates the catabolic block in intact living cells from patients with
Wolman disease. In vitro degradation of
PMLes indicated the existence of two
PMLes-degrading
enzymes in lymphoblastoid cell homogenates: one is the
acid lipase which is involved in
PMLes degradation in the lysosomal compartment (and is deficient in Wolman cells), while the second is a cytoplasmic
enzyme (not deficient in Wolman cells).