Heavy metal detoxification in crustacean epithelial lysosomes: role of anions in the compartmentalization process

Abstract

SUMMARYCrustacean hepatopancreatic lysosomes are organelles of heavy metal sequestration and detoxification. Previous studies have shown that zinc uptake by lysosomal membrane vesicles (LMV) occurred by a vanadate- and thapsigargin-sensitive ATPase that was stimulated by a transmembrane proton gradient established by a co-localized V-ATPase associated with this organelle. In the present study, hepatopancreatic LMV from the American lobster Homarus americanus were prepared by standard centrifugation methods and 65Zn2+, 36Cl–, 35SO42– and 14C-oxalate2– were used to characterize the interactions between the metal and anions during vesicular detoxification events. Vesicles loaded with SO42– or PO43– led to a threefold greater steady-state accumulation of Zn2+ than similar vesicles loaded with mannitol,Cl– or oxalate2–. The stimulation of 65Zn2+ uptake by intravesicular sulfate was SO42– concentration dependent with a maximal enhancement at 500 μmol l–1. Zinc uptake in the presence of ATP was proton-gradient enhanced and electrogenic, exhibiting an apparent exchange stoichiometry of 1Zn+/3H+. 35SO42– and 14C-oxalate2– uptakes were both enhanced in vesicles loaded with intravesicular Cl– compared to vesicles containing mannitol, suggesting the presence of anion countertransport. 35SO42– influx was a sigmoidal function of external [SO42–] with 25 mmol l–1 internal [Cl–], or with several intravesicular pH values (e.g. 7.0, 8.0 and 9.0). In all instances Hill coefficients of approximately 2.0 were obtained, suggesting that 2 sulfate ions exchange with single Cl– or OH– ions. 36Cl– influx was a sigmoidal function of external[Cl–] with intravesicular pH of 7.0 and 9.0. A Hill coefficient of 2.0 was also obtained, suggesting the exchange of 2 Cl– for 1 OH–. 14C-oxalate influx was a hyperbolic function of external [oxalate2–] with 25 mmol l–1 internal [Cl–], suggesting a 1:1 exchange of oxalate2– for Cl–. As a group,these experiments suggest the presence of an anion exchange mechanism exchanging monovalent for polyvalent anions. Polyvalent inorganic anions(SO42– and PO43–) are known to associate with metals inside vesicles and a detoxification model is presented that suggests how these anions may contribute to concretion formation through precipitation with metals at appropriate vesicular pH.