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.

Proline residues in two tightly coupled helices of the sulphate transporter, SHST1, are important for sulphate transport

The sulphate transporter SHST1, from Stylosanthes hamata, features three tightly coupled transmembrane helices which include proline residues that are conserved in most related transporters. We used site-directed mutagenesis and expression of the mutant transporters in yeast to test whether these proline residues are important for function. Four proline residues were replaced by both alanine and leucine. Only one of these proline residues, Pro-144, was essential for sulphate transport. However, mutation of either Pro-133 or Pro-160 resulted in a severe decrease in sulphate transport activity; this was due more to a decrease in transport activity than to a decrease in the amount of mutant SHST1 in the plasma membrane. These results suggest that all three proline residues are important for transport, and that the conformation of the three tightly coupled helices may play a critical role in sulphate transport. We also show that SHST1 undergoes a post-translational modification that is required for trafficking to the plasma membrane.

Lysosomal sulphate transport is dependent upon sulphydryl groups

Using thiol blocking agents, we examined the role of sulphydryl groups for function of the lysosomal sulphate transport system. Monothiol binding reagents, p-hydroxymercuribenzoic acid (p-HMB) and p-chloromercuribenzene sulphonic acid (p-CMBS), dithiol binding reagents such as CuCl2, the alkylating agent, N-ethylmaleimide (NEM), and NADH all inhibited lysosomal sulphate transport. The inhibitory effects of NEM and Cu2+ were not additive, suggesting that they both act upon the same critical sulphydryl group(s). Unlike the case for NEM, the inhibitory effects of Cu2+ were reversed by the reducing agent, dithiothreitol. Exposure to NEM resulted in a seven-fold increase in Km to 867 μM versus a control value of 126 μM and a modest decrease in Vmax to 99 pmol per unit β-hexosaminidase per 30 s versus a control value of 129 pmol per unit β-hexosaminidase per 30 s. Similar although somewhat less dramatic results were obtained using Cu2+ with an increase of Km to 448 μM and a Vmax of 77 pmol per unit β-hexosaminidase per 30 s. The sulphate transport activity of detergent solubilized lysosomal membranes could be bound to a p-chloromercuribenzoic acid (p-CMB)-Sepharose sulphydryl affinity resin and eluted with mercaptoethanol. Sulphydryl groups thus appear to play a role in sulphate transport through effects on substrate affinity. Sulphydryl-binding appears to be a strategy that may be useful for purification of the transporter.

ATP stimulates lysosomal sulphate transport at neutral pH: evidence for phosphorylation of the lysosomal sulphate carrier

ATP markedly stimulated sulphate uptake by rat liver lysosomes that had been treated with N-ethylmaleimide to block the effects of the lysosomal proton-translocating ATPase (H+-ATPase). Maximal stimulation required millimolar concentrations of ATP and neutral buffer pH. ATP-stimulated transport exhibited saturation kinetics with a Km of 175 μM, identical with the Km for lysosomal sulphate uptake at pH 5.0, a process that does not require ATP. The requirement for ATP was specific: other nucleotides such as AMP, ADP, CTP, GTP, ITP and UTP failed to stimulate transport. Adenosine 5ʹ-[β,γ-imido]triphosphate, the non-hydrolysable analogue of ATP, also failed to stimulate sulphate uptake, suggesting a requirement for ATP hydrolysis. Lysosomal pH, membrane potential and glucose transport were unchanged by the presence of ATP under the experimental conditions, consistent with a direct effect of ATP on the sulphate transporter. Exposure of lysosomes to protein kinase A and protein kinase C inhibitors did not alter the stimulation of sulphate transport by ATP. The lysosomal sulphate transport protein might be subject to regulation by a phosphorylation pathway that is not dependent on protein kinase A or protein kinase C.