Mediated calcium transport by isolated human fibroblast lysosomes.
Lysosomes purified by Percoll gradient from normal human fibroblasts (GM0010A) show uptake of Ca2+ in a mediated manner. The uptake is linear over the first 1.5 min and approaches a steady state by 10 min. Uptake is saturable, displaying a Vmax of about 10 pmol/min/unit hexosaminidase at 20 mM Ca2+ (7 nmol/min/mg protein), and a Km of 5.7 mM. Ca2+ uptake increases with increasing extralysosomal pH from 5.0 to 8.5. The Q10 is 1.6, and Ea 8.7 kcal/mol. Uptake of 0.1 mM Ca2+ was inhibited to the extent indicated by 1.0 mM of the following: Cd2+, 100%; Hg2+, 100%; Zn2+, 89%; Mg2+, 77%; Ba2+, 60%; Sr2+, 37%; Fe2+, 20%; Cu2+, 0%. Mono- and trivalent cations had no effect. ATP (1.0 mM) inhibited uptake by 80%, and chloroquine (0.1 mM) inhibited by 60%, as did 1.0 mM L-cystine. Cysteamine, N-ethylmaleimide, and the anions Cl-, SO(2-)4, and acetate had no effect. The calcium ionophore A23187 augmented uptake by 10-fold at 10 microM. Surprisingly, Pb2+ greatly augmented lysosomal Ca2+ uptake in a concentration-dependent manner. Pb2+, however, adversely affected lysosomal latency. Lysosomal calcium uptake was not affected by inositol 1,4,5-triphosphate, and calcium-induced calcium release from lysosomes was not observed. A role for lysosomes in cellular calcium homeostasis has not been previously suggested. This work shows that Ca2+ can be transported into and out of lysosomes and could assist in lysosomal proteolysis. The extent of further lysosomal participation in cellular calcium regulation is unclear.
Lemons
R M
RM
Department of Pediatrics, University of Michigan, Ann Arbor 48109-2029.
Thoene
J G
JG
D000255:Adenosine Triphosphate / Q000378:metabolism; D001692:Biological Transport; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D003543:Cysteamine / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D007854:Lead / Q000494:pharmacology; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D013696:Temperature; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
D007854:Lead; D000001:Calcimycin; D003543:Cysteamine; D000255:Adenosine Triphosphate; D012964:Sodium; D001619:beta-N-Acetylhexosaminidases; D005033:Ethylmaleimide; D002118:Calcium