Lysosomal sulphate transport is dependent upon sulphydryl groups Available to Purchase

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 K_m to 867 μM versus a control value of 126 μM and a modest decrease in V_max 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 K_m to 448 μM and a V_max 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.