Role of polyvalent anions in lysosomal heavy metal detoxification

Abstract

Crustacean hepatopancreatic lysosomes are organelles of heavy metal sequestration and detoxification. In this study hepatopancreatic lysosomal membrane vesicles (LMV) from the American lobster (Homarus americanus) were prepared by standard centrifugation methods and 65Zn2+, 36Cl−, and 35SO42− were used to characterize the transport processes responsible for vesicular accumulation of these ions. 65Zn2+ influx into LMV was a hyperbolic function of external [Zn] and followed Michaelis-Menten kinetics (Km = 32 ±10 μM; Jmax = 21 ± 3 pmol/mg protein x sec). This transport was stimulated by 1 mM ATP and abolished by the simultaneous addition of 1 mM ATP + 250 μM vanadate. Thapsigargin (10 μM) was also a significant inhibitor of zinc influx, but not as effective as vanadate. Cadmium and copper were competitive inhibitors of 65Zn2+ influx (Cd2+ Ki = 68 ± 3 μM; Cu2+ Ki = 33 ± 2 μM). An outwardly-directed proton gradient, in the absence of ATP, stimulated 65Zn2+ influx, while an inwardly-directed proton gradient inhibited metal uptake. Vesicles loaded with SO42− or PO43− led to a 3-fold greater steady-state accumulation of Zn2+ than similar vesicles loaded with mannitol, Cl−, or oxalate2−. 35SO42− influx was a hyperbolic function of internal [Cl] and 36Cl− influx appeared to occur by diffusion in the absence of an antiport substrate. These results suggest that 65Zn2+ influx occurred by a vanadate- and thapsigargin-sensitive ATPase transporter that was stimulated by an outwardly-directed proton gradient. Polyvalent inorganic anions (SO42− and PO43−) associate with the metal inside the vesicles and may result in concretion formation through precipitation at appropriate vesicular pH. Supported by NSF grant IBN04-21986.