In a search for the pathophysiologic mechanisms, we estimated isoprenoid synthesis and concentration, cellular growth, and the activity of the LDL receptor pathway in fibroblasts from patients with mevalonate kinase deficiency (MKD), a severe multisystemic disorder of cholesterol and non-sterol isoprenoid biosynthesis. In response to different concentrations of LDL and non-lipoprotein-bound cholesterol, MKD cells partially counteracted their enzyme defect by increased activities of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase (results from earlier studies) and the LDL receptor pathway, responses similar to the pharmacologic effects seen upon administration of HMG-CoA reductase inhibitors. Rates of N-linked protein glycosylation, estimated as the amount of [14C]galactose-labeled macromolecules secreted into cell culture medium, were significantly decreased in MKD fibroblasts in comparison with control cells which may indicate alterations in the dolichol or dolichol phosphate pool. In response to exogenous cholesterol, the major feedback inhibitor of isoprenoid biosynthesis, growth velocities of MKD fibroblasts declined in comparison with control cells, further suggesting an impairment of non-sterol isoprenoid biosynthesis in MKD. Our results suggest an imbalance in the multilevel regulation of the biosynthesis of cholesterol and non-sterol isoprenoids in MKD, representing an additional causative factor responsible for the pre- and postnatal pathology of MKD.
Hyperammonemia interferes with normal brain function. The effect of ammonia
on free and membrane-bound lysosomal enzymes and on mucopolysaccharide metabolism
was studied in cultured rat brain cells (ROC-1, hybridoma between C(6)-astrocytoma and
oligodendrocytes). Intralysosomal ammoniagenesis was achieved from urea by endocytosed
Jackbean urease followed by incubation of the cultures with urea. The intralysosomal location
of urease was evidenced by the protective effects of leupeptin and urea on the stability of
intracellular urease. Ammonia formed from urea resulted in an increased secretion of lysosomal
arylsulfatase-A (AS-A), but not of the membrane-bound lysosomal β-glucosidase into
the culture medium, thus intralysosomal AS-A activity decreased. Lysosomal, membranebound
β-glucosidase activity increased, presumably due to intralysosomal proteolytic protection
following an increased lysosomal pH. Intralysosomal ammoniagenesis temporarily
impaired ^35S0(4)-glycosaminoglycan degradation of prelabeled cells. The results support the
hypothesis that hyperammonemic states may interfere with lysosomal functions in vivo as
well in cultured cells.
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