Membrane microenvironment regulation of carnitine palmitoyltranferases I and II

CPT (carnitine palmitoyltransferase) 1 and CPT2 regulate fatty acid oxidation. Recombinant rat CPT2 was isolated from the soluble fractions of bacterial extracts and expressed in Escherichia coli. The acyl-CoA chain-length-specificity of the recombinant CPT2 was identical with that of the purified enzyme from rat liver mitochondrial inner membranes. The Km for carnitine for both the mitochondrial preparation and the recombinant enzyme was identical. In isolated mitochondrial outer membranes, cardiolipin (diphosphatidylglycerol) increased CPT1 activity 4-fold and the Km for carnitine 6-fold. It decreased the Ki for malonyl-CoA inhibition 60-fold, but had no effect on the apparent Km for myristoyl-CoA. Cardiolipin also activated recombinant CPT2 almost 4-fold, whereas phosphatidylglycerol, phosphatidylserine and phosphatidylcholine activated the enzyme 3-, 2- and 2-fold respectively. Most of the recombinant CPT2 was found to have substantial interaction with cardiolipin. A model is proposed whereby cardiolipin may hold the fatty-acid-oxidizing enzymes in the active functional conformation between the mitochondrial inner and outer membranes in conjunction with the translocase and the acyl-CoA synthetase, thus combining all four enzymes into a functional unit.

Oxygenation of 18-hydroxycorticosterone as the final reaction for aldosterone biosynthesis

Incubation of 18-hydroxycorticosterone with the sonicated mitochondrial preparation of bovine adrenal glomerulosa tissue leads to the production of aldosterone, as measured by radioimmunoassay. The in vitro production of aldosterone from 18-hydroxycorticosterone requires both molecular oxygen and NADPH, and is inhibited by carbon monoxide. Cytochrome P-450 inhibitors such as metyrapone, SU 8000. SU 10603, SKF 525A, amphenone B and spironolactone decrease the biosynthesis of aldosterone from 18-hydroxycorticosterone. These results support the conclusion that the final reaction in aldosterone synthesis from 18-hydroxycorticosterone is catalyzed by an oxygenase, but not by 18-hydroxysteroid dehydrogenase. By the same preparation, the production of [3H]aldosterone but not [3H]18-hydroxycorticosterone from [1,2-3H ]corticosterone is decreased in a dose-dependent manner by addition of non-radioactive 18-hydroxycorticosterone.

Preparation of rat enterocyte mitochondria

Rat enterocyte mitochondria were prepared with respiratory control ratios of 4 or 5 and occasionally 6. When EGTA was excluded from the mitochondrial incubation medium the calculated P/O ratios were high, especially those based on the first addition of ADP. These ratios were lowered by increasing the EGTA concentration from 1 mM to 2 mM in the mitochondrial preparation medium and including 1 mM-EGTA in the incubation medium. The use of EDTA in the enterocyte isolation medium led to the mitochondria requiring added cytochrome c. Substituting EGTA for EDTA abolished this requirement. The mitochondrial fraction consisted of two components, an upper cream-coloured layer rich in DNA and a lower brown-coloured layer poor in DNA. Both components were capable of oxidative phosphorylation with succinate or the glutamate/malate couple as substrates. The mitochondrial yield was assessed by assaying succinate dehydrogenase activity, and the contamination of the mitochondrial fraction by other cell organelles was assessed by assays for appropriate marker enzymes.

Immunochemical characterization of monoamine oxidase from human liver, placenta, platelets and brain cortex

1. Antiserum raised to purified human liver monoamine oxidase was used to characterize the monoamine oxidase from human liver, brain cortex, placenta and platelets. 2. Antibodies to monoamine oxidase were purified by adsorption with a mitochondrial preparation. 3. Monoamine oxidase was present in liver particle-free supernatant as measured by enzyme activity and immunodiffusion. 4. Multiple precipitin lines were obtained on immunodiffusion analysis against the purified liver enzyme. It is proposed that this is due to either aggregation or to differential lipid binding. 5. The results suggest that the functionally different enzymes found in liver, brain cortex, platelets and placenta are immunochemically related and may be identical.

Acetoacetate metabolism in rat brain. Development of acetoacetyl-coenzyme A deacylase and 3-hydroxy-3-methylglutaryl-coenzyme A synthase

1. Data are provided that indicate that the rat brain acetoacetyl-CoA deacylase is almost exclusively mitochondrial. Developmental studies show that this enzyme more than doubles its activity during suckling (0–21 days) and then maintains this activity in adults (approx. 1.1 units/g wet wt.). 2. Kinetic studies (on the acetoacetyl-CoA deacylase) in a purified brain mitochondrial preparation give a Vmax. of 47 nmol/min per mg of protein, and a Km for acetoacetyl-CoA of 5.2 micron and are compatible with substrate inhibition by acetoacetyl-CoA above concentrations of 47 micron. 3. The total brain 3-hydroxy-3-methyl-glutaryl-CoA synthase remains constant in the developing and adult rat brain (approx. 1.2 units/g wet wt.). This enzyme is located in both the mitochondrial and cytosolic fractions. During suckling (0–21 days) the mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase represents approx. one-third of the total, but this increases markedly to about 60% of the total in the adult. The cytosolic enzyme correspondingly falls to approx. 40% of the total. 4. The role of the acetoacetyl-CoA deacylase in providing cytosolic acetoacetate for biosynthetic activities in the developing brain is discussed.