Respiratory control in hyperpermeable adult heart muscle cells. Effects of calcium.

Spontaneously beating myocardial fragments prepared by mechanical disaggregation have hyperpermeable sarcolemmae. Such preparations were used to study mitochondrial function in situ. The myocardial fragments suspended in a phosphate-buffered salt solution containing 1-3 mM MgCl2 showed a low rate of oxygen uptake. Addition of succinate, pyruvate plus malate or glutamate was followed by an increase in the rate of O2 uptake. Addition of ADP to fragments engaged in State 4 respiration was followed by initiation of more rapid State 3 respiration, with respiratory control ratios routinely greater than 3 for succinate and glutamate. If the fragments were suspended in the same medium containing 3 mM ATP (a medium in which contractile activity occurs), State 3 was initiated upon addition of substrate. The suspension medium used in these experiments contained about 8 muM calcium as contamination. Addition of calcium chloride to give a final concentration of 0.14 to 0.57 mM stimulated State 4 respiration of the myocardial fragments. In contrast similar additions made during State 3 inhibited respiration. The maximum degree of inhibition brought respiration close to the State 4 rate. If calcium was added prior to ADP, respiratory stimulation by the nucleotide was diminished. Respiratory function of myocardial fragments and of mitochondria isolated from them was similar in terms of response to substrate, ADP, and calcium addition in State 4. Response to calcium in State 3 was different in that inhibition was long-lived only at low [Pi] in the case of mitochondria, but at low or high [Pi] in the case of the fragments.

Isolation and characterization of the mitochondrial DNA from the florida spiny lobster, Panulirus argus.

1. Mitochondria and mitochondrial DNA were isolated from the digestive gland of Panulirus argus. 2. The mitochondrial DNA has an average contour length of 5.13 microns which corresponds to a molecular weight of 10.10 X 10(6) daltons. 3. The molecular weight based on agarose gel electrophoresis of restricted individual DNA samples ranges from 10.04 to 10.4 X 10(6) daltons. 4. Restriction endonuclease analysis with Bam H1 and Eco R1 demonstrate variation in nucleotide sequence between individual lobsters.

Respiratory and calcium transport properties of spiny lobster hepatopancreas mitochondria.

Mitochondria were isolated from the hepatopancreas of the Florida spiny lobster Panulirus argus using a high osmolarity medium containing 600 mM mannitol, 83 mM sucrose, 5 mM 4-morpholinepropanesulfonic acid, pH 7.6, 0.5% bovine serum albumin (BSA), and 1 mM EDTA. O2 uptake and Ca2+ transport were measured by electrode methods in similar media (plus 4 mM KPi, 3.3 mM MgCl2, and 0.67 mg/ml BSA, with 80 mM KCl replacing a portion of the osmotic support). Substrate-supported respiration was observed to be coupled to phosphorylation of ADP or uptake of Ca2+ ions. State 3 rates (nanogram atoms O X minute-1 X 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. alpha-Glycerol phosphate was not oxidized. Ca2+ uptake driven by succinate oxidation proceeded with Ca:O ratios of 4.0 +/- 0.2 (SEM). Hepatopancreas mitochondria were not uncoupled by Ca2+ uptake in excess of 1100 ng atoms X mg protein-1. Ca2+ efflux could be induced by ruthenium red, indicating the presence of an active Ca2+ cycle. These mitochondria may provide a favorable model system in which to study regulation of the Ca2+ cycle.