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 CuCl 2 , the alkylating agent, N -ethylmaleimide (NEM), and NADH all inhibited lysosomal sulphate transport. The inhibitory effects of NEM and Cu 2+ were not additive, suggesting that they both act upon the same critical sulphydryl group(s). Unlike the case for NEM, the inhibitory effects of Cu 2+ were reversed by the reducing agent, dithiothreitol. Exposure to NEM resulted in a seven-fold increase in K m to 867 μ M versus a control value of 126 μ M 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 Cu 2+ with an increase of K m to 448 μ M 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.