Understanding cerebral L-lysine metabolism: the role of L-pipecolate metabolism in Gcdh-deficient mice as a model for glutaric aciduria type I.

Inherited deficiencies of the L-lysine catabolic pathway cause glutaric aciduria type I and pyridoxine-dependent epilepsy. Dietary modulation of cerebral L-lysine metabolism is thought to be an important therapeutic intervention for these diseases. To better understand cerebral L-lysine degradation, we studied in mice the two known catabolic routes -- pipecolate and saccharopine pathways -- using labeled stable L-lysine and brain peroxisomes purified according to a newly established protocol. Experiments with labeled stable L-lysine show that cerebral L-pipecolate is generated along two pathways: i) a minor proportion retrograde after ε-deamination of L-lysine along the saccharopine pathway, and ii) a major proportion anterograde after α-deamination of L-lysine along the pipecolate pathway. In line with these findings, we observed only little production of saccharopine in the murine brain. L-pipecolate oxidation was only detectable in brain peroxisomes, but L-pipecolate oxidase activity was low (7 ± 2µU/mg protein). In conclusion, L-pipecolate is a major degradation product from L-lysine in murine brain generated by α-deamination of this amino acid.

Isolation of peroxisomal subpopulations from mouse liver by immune free-flow electrophoresis.

Peroxisomes (PO) are a heterogeneous population of cell organelles which in mammals are most abundant in liver and kidney. Commonly, differential and density gradient centrifugation are used for their isolation which, however, give only rise to the so-called "heavy" PO with a buoyant density of 1.22-1.24 g/cm(3). Subpopulations other than the heavy PO which are also present in both of these tissues have escaped adequate purification because of their sedimentation characteristics which are close to those of other major organelles, in particular microsomes. Since the purification of these subpopulations has become an essential task in view of the putative importance of peroxisomal subpopulations in the biogenesis of this organelle, alternatives to density gradient centrifugation are required. Recently, we have introduced such a novel approach, named immune free flow electrophoresis (IFFE). IFFE combines the advantages of eletrophoretic separation with the high selectivity of an immune reaction. It makes use of the fact that the electrophoretic mobility of a subcellular particle, complexed with an antibody directed against the cytoplasmic domain of one of its integral membrane proteins is greatly diminished, provided the pH of the electrophoresis buffer is adjusted to pH approximately 8.0, the pI of immunoglobulin G (IgG) molecules. pH-values other than 8.0 proved to be less efficient, probably because IgG molecules only focus at pH 8.0 but are scattered at any other. Applying IFFE to heavy and light mitochondrial as well as microsomal fractions of rat liver not only regular PO (rho = 1.22-1.24 g/cm(3)) but also other subpopulations could be isolated. To substantiate the validity of this approach, we now have subfractionated mouse liver homogenates accordingly. Of the PO subpopulations collected, mainly that obtained from the heavy mitochondrial fraction differed in its composition of matrix and membrane proteins as revealed by immunoblotting. This is in line with the data reported on rat liver thus confirming the potential of IFFE in the isolation of distinct subpopulations of hepatic PO.