Faculty Bibliography

The mitochondrial fraction of adult rat lung contains choline phosphotransferase (EC 2.7.8.2) activity which can not be explained by microsomal contamination estimated on the basis of marker enzyme distribution. Mitochondrial (14C)glycerol-3-phosphate incorporation into PC (phosphatidylcholine) can be distinguished from the microsomal incorporation by different sensitivity to N-ethylmaleimide inhibition. The data indicate that rat lung mitochondria have the intrinsic capability to synthesize PC. Both synthesis of PC and PG (phosphatidylglycerol) are susceptible to isotonic tryptic attack against the cytoplasmic face of isolated rat lung mitochondria, suggesting the outer membrane location of crucial activities involved in the formation of these phospholipids. Rat liver mitochondria are different from rat lung mitochondria with respect to their capability to synthesize PC, their rate of (14C)glycerol-3-phosphate incorporation into PG as well as the submitochondrial site of PG formation

It is not yet completely understood how a cell is able to export specific phospholipids, like dipalmitoylphosphatidylcholine (dipalmitoyl-PC), which is secreted by pneumocytes type II, into pulmonary surfactant. The acyl species composition of [3H]PC which was synthesized in type II cells in the presence of [2-3H]glycerol resembled the species composition of PC localized in intracellular pneumocyte membranes. This species pattern was different from the pattern of PC of lamellar bodies, i.e., intracellularly stored surfactant, by a higher proportion of dipalmitoyl-PC mainly at expense of 1-palmitoyl-2-oleoyl-PC. Lamellar body PC in turn showed the same species distribution as surfactant PC. The data suggest that subcellular compartmentation and/or intracellular transfer of PC destined to storage in lamellar bodies, but not secretion of lamellar bodies, involves an enrichment of dipalmitoyl-PC and a depletion of 1-palmitoyl-2-oleoyl-PC. In contrast, the acyl species pattern of phosphatidylglycerol does not seem to undergo gross changes on the path from synthesis to secretion

The acyl species of mitochondrial phosphatidylcholine from rat liver and lung were analysed by HPLC separation of the 1,2-diacyl-3-naphthylurethane derivatives. Comparison of phosphatidylcholine species patterns in microsomal, mitochondrial and submitochondrial fractions revealed only minor differences, whereas mitochondria from liver and lung differed markedly in the molecular composition of their respective phosphatidylcholine species

To investigate whether or not the mitochondrial intermembrane space together with the extramitochondrial space form a homogeneous pool for adenine nucleotides, rat-heart mitochondria were studied in reconstituted systems with pyruvate kinase and ADP-producing enzymes with varied localization. In the hexokinase system, ADP is produced extramitochondrially by added yeast hexokinase, whereas in the creatine kinase system mitochondrial creatine kinase is responsible for ADP regeneration in the intermembrane space. The dependence of mitochondrial respiration on the extramitochondrial [ATP]/[ADP] ratio in both systems was investigated experimentally and by means of computer simulation. Near the resting state, higher [ATP]/[ADP] ratios were found in the creatine kinase system than in the hexokinase system at the same rate of respiration. This and the maintaining of a substantial creatine kinase-stimulated respiration in the presence of pyruvate kinase in excess is explained by a two-compartment model considering diffusion limitations of adenine nucleotides. A diffusion rate constant of (8.7 +/- 4.7) 10(4) microliters X mg-1 X min-1 for ADP and ATP was estimated, resulting in rate-dependent concentration differences up to 13.7 microM AdN between the extramitochondrial space and the AdN-translocator at the maximum rate of oxidative phosphorylation of rat-heart mitochondria. The results support the assumption that ADP diffusion towards the AdN-translocator is limited if its extramitochondrial concentration is low, resulting in a dynamic compartmentation of adenine nucleotides in the mitochondrial intermembrane space

The subcellular site of phosphatidylglycerol (PG) formation for lung surfactant has not been convincingly clarified. To approach this problem we analysed the acyl species pattern of lung PG in mitochondria, microsomes and surfactant by h.p.l.c. separation of its 1,2-diacyl-3-naphthylurethane derivatives. Both mitochondrial and microsomal PG proved identical with surfactant PG, containing the major species 1-palmitoyl-2-oleoyl-PG and 1,2-dipalmitoyl-PG. The fatty acid composition of mitochondrial PG differs markedly from that of diphosphatidylglycerol. This may be taken as an indication that mitochondrial PG is synthesized on purpose to form surfactant, rather than being only the precursor of diphosphatidylglycerol. In vitro, sn-[U-14C]glycerol 3-phosphate incorporation into PG of mitochondria or microsomes occurs in the presence of CTP, ATP and CoA but independently of the supply of exogenous lipoidic precursors. Although the rate in vitro of autonomous PG synthesis, and the endogenous PG content, are higher in mitochondria than in microsomes, it is assumed that both subcellular fractions are involved in PG formation for surfactant

Cleavage of mitochondrial phosphatidylethanolamine (PE), phosphatidylcholine (PC) and cardiolipin (CL) by phospholipase A2 but not selective degradation of PE and PC by phospholipase C dissociates creatine kinase from rat heart mitochondria. Creatine kinase exhibits a high resistance against Triton X-100 solubilization up to concentrations of 0.05-0.1%. Scatchard plot of rebinding experiments using mitoplasts revealed the presence of both low and high affinity binding sites; the latter may account for the originally bound creatine kinase activity. It is suggested that creatine kinase is specifically bound to a CL containing domain of the inner mitochondrial membrane

The kinetic properties of heart mitochondrial creatine kinase were measured with and without inhibitors of the adenine nucleotide translocator. No significant differences were observed suggesting that mitochondrial creatine kinase is not acting together with the adenine nucleotide translocator as a functional multienzyme complex. Adenine nucleotides from the bulk phase are able to enter the active center of creatine kinase without the necessity of transport via the adenine nucleotide translocator

The submitochondrial distribution of hexokinase was studied by repeated specific solubilizations and by tryptic digestion of isolated rabbit reticulocyte mitochondria. Whereas most of the enzyme is dissociably bound to the outer side of outer mitochondrial membrane, a small tightly bound portion is localized more internally. Electrophoretic separations did not reveal a specific isoenzyme pattern of the internal mitochondrial enzyme. Relationships between mitochondrial hexokinases and the intramitochondrial ATP pool, generated by oxidative phosphorylation, were studied by measuring 32P-fluxes following gamma-32P-ATP pulses on phosphorylating and non-phosphorylating mitochondria. Under both conditions, the specific activities in deoxyglucose-6-phosphate correspond closely to that of total gamma-ATP, thus not supporting a preferential use of intramitochondrial generated ATP by part of the mitochondrial hexokinases