Genistein inhibits glucose and sulphate transport in isolated rat liver lysosomes

Genistein and daidzein are known to have both beneficial and adverse effects on human health due to their many biological actions at the cellular level. Both isoflavones have been shown to inhibit GLUT-mediated glucose transport across the plasma membrane of mammalian cells. Since lysosomal membrane transport is essential for maintaining cellular homeostasis, the present study examined the effects of genistein and daidzein on glucose and sulphate transport in isolated rat liver lysosomes. Both genistein and daidzein significantly inhibited lysosomal glucose uptake. Genistein was a more potent glucose transport inhibitor than daidzein, with a half-maximum inhibitory concentration (IC50) of 45 μmol/l compared with 71 μmol/l for daidzein. Uptake kinetics of d-glucose showed a significant decrease in Vmax (control:genistein treat = 1489 (sem 91):507 (sem 76) pmol/unit of β-hexosaminidase per 15 s) without a change in Km. The presence of 50 μm-genistein in the medium also reduced glucose efflux from lysosomes preloaded with 100 mm-d-glucose. Genistein also inhibited lysosomal sulphate transport. Similar to its effects on glucose uptake kinetics, genistein treatment caused a significant decrease in sulphate uptake Vmax (control:genistein treat = 87 (sem 4):59 (sem 5) pmol/unit of β-hexosaminidase per 30 s), while the Km was not affected. The evidence provided by the present study suggests that the most likely mechanism of lysosomal glucose transport inhibition by genistein is via direct interaction between genistein and the transporter, rather than mediation by tyrosine kinase inactivation. Genistein likely has a similar mechanism of directly inhibiting sulphate transporter.

Intracellular localization of p40, a protein identified in a preparation of lysosomal membranes

Unlike lysosomal soluble proteins, few lysosomal membrane proteins have been identified. Rat liver lysosomes were purified by centrifugation on a Nycodenz density gradient. The most hydrophobic proteins were extracted from the lysosome membrane preparation and were identified by MS. We focused our attention on a protein of approx. 40 kDa, p40, which contains seven to ten putative transmembrane domains and four lysosomal consensus sorting motifs in its sequence. Knowing that preparations of lysosomes obtained by centrifugation always contain contaminant membranes, we combined biochemical and morphological methods to analyse the subcellular localization of p40. The results of subcellular fractionation of mouse liver homogenates validate the lysosomal residence of p40. In particular, a density shift of lysosomes induced by Triton WR-1339 similarly affected the distributions of p40 and β-galactosidase, a lysosomal marker protein. We confirmed by fluorescence microscopy on eukaryotic cells transfected with p40 or p40–GFP (green fluorescent protein) constructs that p40 is localized in lysosomes. A first molecular characterization of p40 in transfected Cos-7 cells revealed that it is an unglycosylated protein tightly associated with membranes. Taken together, our results strongly support the hypothesis that p40 is an authentic lysosomal membrane protein.