Respiratory and calcium transport properties of spiny lobster hepatopancreas mitochondria

Abstract

Mitochondria were isolated from the hepatopancreas of the Florida spiny lobster Panulirus argus using a high osmolarity medium containing 600 m m mannitol, 83 m m sucrose, 5 m m 4-morpholinepropanesulfonic acid, pH 7.6, 0.5% bovine serum albumin (BSA), and 1 m m EDTA. O 2 uptake and Ca 2+ transport were measured by electrode methods in similar media (plus 4 m m K P i , 3.3 m m MgCl 2, and 0.67 mg/ml BSA, with 80 m m KCl replacing a portion of the osmotic support). Substrate-supported respiration was observed to be coupled to phosphorylation of ADP or uptake of Ca 2+ ions. State 3 rates (nanogram atoms O × minute −1 × milligram protein −1 ± SEM ( N)) were: 49.2 ± 3.9 (19), succinate; 30.9 ± 3.9 (6), dl-palmitoyl carnitine; 29.0 ± 2.7 (9), l-malate; 40.0 ± 2.3 (3), l-glutamate; 27.7 ± 2.2 (5), d-3-hydroxybutyrate; and 26.4 ± 2.4 (18), l-proline ± pyruvate. α-Glycerol phosphate was not oxidized. Ca 2+ uptake driven by succinate oxidation proceeded with Ca:O ratios of 4.0 ± 0.2 (SEM). Hepatopancreas mitochondria were not uncoupled by Ca 2+ uptake in excess of 1100 ng atoms × mg protein −1. Ca 2+ efflux could be induced by ruthenium red, indicating the presence of an active Ca 2+ cycle. These mitochondria may provide a favorable model system in which to study regulation of the Ca 2+ cycle.