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First published online September 14, 2007
Journal of Experimental Biology 210, 3484-3493 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.008300

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

Kenneth M. Sterling¹, Prabir K. Mandal¹, Barbara A. Roggenbeck¹, Sean E. Ahearn¹, George A. Gerencser² and Gregory A. Ahearn^1,*

¹ Department of Biology, University of North Florida, 4567 St Johns Bluff Road, S., Jacksonville, FL 32224, USA
² Department of Physiology, University of Florida, Gainesville, FL, 32610, USA

^* Author for correspondence (e-mail: gahearn{at}unf.edu)

Crustacean 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^–, ³⁵SO₄^2– and ¹⁴C-oxalate^2– were used to characterize the interactions between the metal and anions during vesicular detoxification events. Vesicles loaded with SO₄^2– or PO₄^3– led to a threefold greater steady-state accumulation of Zn²⁺ than similar vesicles loaded with mannitol, Cl^– or oxalate^2–. The stimulation of ⁶⁵Zn²⁺ uptake by intravesicular sulfate was SO₄^2– 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⁺. ³⁵SO₄^2– and ¹⁴C-oxalate^2– uptakes were both enhanced in vesicles loaded with intravesicular Cl^– compared to vesicles containing mannitol, suggesting the presence of anion countertransport. ³⁵SO₄^2– influx was a sigmoidal function of external [SO₄^2–] 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 [oxalate^2–] with 25 mmol l^–1 internal [Cl^–], suggesting a 1:1 exchange of oxalate^2– for Cl^–. As a group, these experiments suggest the presence of an anion exchange mechanism exchanging monovalent for polyvalent anions. Polyvalent inorganic anions (SO₄^2– and PO₄^3–) 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.

Key words: zinc transport, lysosomes, detoxification, sulfate, oxalate, chloride, anion exchange, heavy metals, lobster, Homarus americanus, V-ATPase, electrogenic transport