In vitro synthesis of monoamine oxidase of rat liver outer mitochondrial membrane

Monoamine oxidase, an intrinsic protein of outer mitochondrial membrane, was purified from bovine liver and rabbit antibody against the enzyme was prepared. The antibody could react with the monoamine oxidase of rat liver mitochondria. When rat liver RNA was translated $\text{in}\text{vitro}$ using rabbit reticulocyte lysate and monoamine oxidase peptide in the translation products was immunoprecipitated by the antibody, the peptide was detected in the products programmed by the messenger RNA's from total and free polysomes but not that from bound polysomes. The enzyme synthesized $\text{in}\text{vitro}$ had the same apparent molecular size as the mature protein in outer mitochondrial membrane.     

Properties of rat liver mitochondria with intermembrane Cytochrome c.

When rat liver mitochondria were suspended in 0.15 m KCl, the cytochrome c appeared to be solubilized from the binding site on the outside of the inner membrane and trapped in the intermembrane space. When the outer membrane of these mitochondria was disrupted with digitonin at a digitonin concentration of 0.15 mg/mg of protein, the solubilized cytochrome c could be released from mitochondria along with adenylate kinase. When mitochondria were suspended in 0.15 m KCl instead of 0.33 m sucrose, the $\text{ADP}\text{O}$ ratio observed with succinate, β-hydroxybutyrate, malate + pyruvate or glutamate as substrates was little affected. A number of cycles of State 4-State 3-State 4 with ADP was observed. The respiratory control ratios, however, were decreased, particularly when glutamate was used as the substrate. Cytochrome c oxidase activity was also decreased to 55% when assayed using ascorbate + N,N,N′,N′-tetramethyl-p-phenylene-diamine (TMPD) as substrates. Suspension of mitochondria in 0.15 m KCl resulted in an enhancement of the very low NADH oxidation by intact mitochondria and a twofold enhancement of sulfite oxidation. Trapped cytochrome c in outer membrane vesicles prepared from untreated and trypsin-treated intact mitochondria was found to be readily reduced by NADH and suggests that some cytochrome b₅ is located on the inner surface of the outer membrane. The enhanced NADH oxidase could therefore reflect the ability of cytochrome c to mediate intermembrane electron transport. The enhanced sulfite oxidase activity was sensitive to cyanide inhibition and coupled to oxidative phosphorylation ($\text{ADP}\text{O}\text{< 1}$) unlike the activity of mitochondria in sucrose medium. These results suggest that free cytochrome c in the intermembrane space can mediate electron transfer between the sulfite oxidase and the inner membrane.     

In vitro synthesis of adrenodoxin and adrenodoxin reductase: Existence of a putative large precursor form of adrenodoxin

Cytoplasmic free and bound polysomes were isolated from bovine adrenal cortex, and used to program $\text{in}\text{vitro}$ protein synthesis in rat liver cell sap and wheat germ lysate systems. Synthesis of adrenodoxin(Ad) and adrenodoxin reductase(AdR) in the cell-free systems was determined by immunoprecipitation using monospecific antibodies, and the sizes of the $\text{in}\text{vitro}$ products were analyzed by SDS-polyacrylamide gel electrophoresis. Ad was synthesized by both free and bound polysomes as a putative large precursor having molecular weight of approximately 20,000 daltons, which was processed to mature size Ad (MW 12,000 daltons) by $\text{in}\text{vitro}$ incubation with adrenal cortex mitochondria. On the other hand, AdR was synthesized only by free polysomes apparently as the mature size product.     

Cell-free synthesis of the enzymes of peroxisomal beta-oxidation

Three enzymes of peroxisomal β-oxidation of rat liver were synthesized in a cell-free protein-synthesizing system derived from rabbit reticulocyte lysate. The $\text{in}\text{vitro}$ products of acyl-CoA oxidase and enoyl-CoA hydratase-3-hydroxyacyl-CoA dehydrogenase multifunctional protein were similar in size to or slightly larger than the subunit of the respective mature enzymes. The $\text{in}\text{vitro}$ product of peroxisomal 3-ketoacyl-CoA thiolase was about 3,000 daltons larger than the mature subunit. The hepatic levels of translatable mRNAs coding for these three enzymes were about 10 times higher in rats fed a di(2-ethylhexyl)phthalate-containing diet than in control animals.     

Cholesterol enrichment of normal mitochondria in vitro: a model system with properties of hepatoma mitochondria.

It is known that rat hepatoma mitochondria contain higher levels of endogenous cholesterol than do mitochondria from normal liver. In the experiments described here, normal liver mitochondria were enriched with cholesterol by a solid-state transfer process $\text{in}\text{vitro}$ and some of their enzymic properties were compared with literature values reported for hepatoma mitochondria. Striking parallels were seen. The data indicate that normal mitochondria, enriched with cholesterol $\text{in}\text{vitro}$, may create an interesting model system for examining some metabolic characteristics of tumor mitochondria.     

The synthesis of proteolipid protein by isolated rat liver mitochondria

About 15% of the total (³H)leucine incorporated into protein by isolated rat liver mitochondria $\text{in}\text{vitro}$ could be extracted by chloroform:methanol. This incorporation was inhibited by chloramphenicol and carbomycin, both specific inhibitors of mitochondrial protein synthesis. SDS-gel electrophoresis of the mitochondrial membrane revealed 6–7 labeled bands. Label in the proteolipid fraction was present mainly in a band of 40,000 molecular weight. Several labeled bands observed in gels of the mitochondrial membrane were not removed or changed by extraction with chloroform:methanol suggesting that some, but not all, of the proteins synthesized by rat liver mitochondria are proteolipids.     

In vitro synthesis of mitochondrial cytochromes P-450(scc) and P-450(11-β) and microsomal cytochrome P-450(C-21) by both free and bound polysomes isolated from bovine adrenal cortex

$\text{In}\text{vitro}$ synthesis of mitochondrial cytochromes P-450(scc) and P-450(11-β), and microsomal cytochrome P-450(C-21) programmed by bovine adrenal cortex polysomes was carried out using rat liver cell sap and wheat germ lysate systems. Synthesis of P-450 proteins in the cell-free systems was determined by immunoprecipitation and immunoadsorption using mono-specific antibodies to each species of P-450, and the sizes of the $\text{in}\text{vitro}$ products were analyzed by SDS-polyacrylamide gel electrophoresis. Both free and bound polysomes synthesized these three species of P-450 in the cell-free systems. P-450(scc) and P-450(C-21) were synthesized apparently as the mature size products, whereas P-450(11-β) was synthesized as a putative precursor approximately 5,000 daltons larger than the mature form. Mitochondrial and microsomal P-450 proteins seem to share common sites of synthesis in the cytoplasm of adrenal cortex cells.     

Import of a putative precursor of rat liver mitochondrial glutamic oxaloacetic transaminase into mitochondria

A putative precursor of rat liver mitochondrial glutamic oxaloacetic transaminase which was about 2,000 daltons larger than the subunits of the mature enzyme synthesized in vitro was sensitive to proteases (trypsin and chymotrypsin). When this precursor was incubated with isolated mitochondria in the absence of protein synthesis, it was processed to the mature form; the mature form co-sedimented with mitochondria and was resistant to externally added proteases. Mature enzyme did not compete with this transport.     

A lysosomal factor that interconverts multiple forms of rat liver tyrosine aminotransferase.

A particulate factor of rat liver is described which interconverts three forms of rat liver cytosolic tyrosine aminotransferase $\text{in}$$\text{vitro}$ with no alteration of enzyme activity. The factor appears to be a heat- and pH-sensitive lysosomal protein. The interconversion process is stimulated $\text{in}$$\text{vitro}$ by 2.5 mM MgCl₂ and 2.5 mM ATP. Asparate aminotransferase multiple forms are also susceptible to $\text{in}$$\text{vitro}$ interconversion by the lysosomal factor. The properties of the factor explain several anomalous effects of $\text{in}$$\text{vitro}$ manipulation on the tyrosine aminotransferase forms which have been reported in the literature and implicate the form interconversion in the degradation of tyrosine aminotransferase.     

The effects of quipazine on serotonin metabolism in rat brain.

Quipazine, 2-(1-piperazinyl)-quinoline, is a drug that has been reported to stimulate serotonin receptors in brain. We therefore studied the effect of quipazine on several parameters of serotonin metabolism in rat brain. Quipazine caused a slight, dose-related elevation of serotonin levels and decrease in 5-hydroxyindoleacetic acid levels for 2–4 hrs after it was administered. The decrease in 5-hydroxyindoleacetic acid levels was probably due primarily to a depression of 5-hydroxyindole synthesis, since quipazine also decreased the rate of 5-hydroxytryptophan accumulation after NSD 1015, the rate of serotonin decline after α-propyldopacetamide, and the rate of 5-hydroxyindoleacetic acid accumulation after probenecid. The elevation of serotonin was probably due to weak inhibition of monoamine oxidase. Quipazine reversibly inhibited the oxidation of serotonin by rat brain monoamine oxidase $\text{in}$$\text{vitro}$ and protected against the irreversible inactivation of the enzyme $\text{in}$$\text{vivo}$. Quipazine also was a potent inhibitor of serotonin uptake into brain synaptosomes $\text{in}$$\text{vitro}$ and attained concentrations in brain higher than the $\text{in}$$\text{vitro}$ IC₅₀. However, quipazine did not prevent the depletion of brain serotonin by p-chloroamphetamine $\text{in}$$\text{vivo}$. In addition to stimulating serotonin receptors in brain, quipazine may inhibit monoamine oxidase and serotonin reuptake $\text{in}$$\text{vivo}$.     

m-Octopamine as a substrate for monoamine oxidase.

$\text{m}$-Octopamine was characterized as substrate for monoamine oxidase (MAO) in rat brain and liver mitochondria. The $\text{K}$m and $\text{V}$max values of the brain enzyme were 735 μM and 32.5 nmoles/mg protein/30 min, and those of the liver enzyme 351 μM and 125 nmoles/mg protein/30 min, respectively. The inhibition experiments with clorgyline and deprenyl showed that $\text{m}$-octopamine was a common substrate for type A and type B MAO, though a major part of the activity was due to type A enzyme.     

A new assay for protoporphyrinogen oxidase — Evidence for a total deficiency in that activity in a heme-less mutant of Saccharomyces cerevisiae

A new spectrophotometric assay for protoporphyrinogen oxidase activity has been developed, involving enzymatic generation of protoporphyrinogen in the incubation medium. This assay, more sensitive and reliable than those previously described, can be used to measure this activity in yeast mitochondrial membranes, rat liver mitochondria and $\text{E. coli}$ membranes. By measuring protoporphyrinogen oxidase activity in different wild type and heme-mutant yeast strains, it was shown that 1) one heme-mutant was totally lacking this activity, 2) different factors might control its level in yeast.     

Protein synthesis in mitochondrial and microsomal fractions from rat brain and liver after acute or chronic ethanol administration

Incorporation of C¹⁴ Leucine was determined $\text{in vitro}$ or $\text{in vivo}$ in isolated mitochondria and microsomes of rat brain and liver after acute or chronic ethanol administration $\text{in vivo}$.The protein synthesis in mitochondrial and microsomal preparation was inhibited respectively by chloramphenicol and cycloeximide, specific inhibitors for the two systems tested. The experimental data demonstrate that the $\text{in vitro}$ protein synthesis in both systems, mitochondrial and microsomal, is strongly affected only after chronic treatment which produces significant activation at the mitochondrial and microsomal level in the liver and an inhibition on the same systems of the brain.The data for $\text{in vivo}$ protein synthesis instead show strong inhibition after acute administration, except for brain mitochondria, which are practically unaffected, while after chronic treatment no significant alterations are observed.     

Precursor of a mitochondrial enzyme accumulates in the cytoplasm of preen glands for a specialized function

Malonyl-CoA decarboxylase in the mitochondria of the liver of goose is immunologically identical with the decarboxylase in the cytoplasm of the uropygial gland (Buckner et al. (1978) $\text{Arch. Biochem. Biophys.}$ 186, 152–163). Messenger RNA was isolated from the liver and the uropygial gland and translated in a rabbit reticulocyte system. Specific immunoprecipitation of the translation products with anti malonyl-CoA decarboxylase showed that in both cases the primary translation product was a 50 K dalton peptide identical in size to the cytoplasmic enzyme in the gland. Specific immunoprecipitation of malonyl-CoA decarboxylase from liver slices which had been incubated with [³⁵S]methionine showed that the mature mitochondrial enzyme was a 47 K dalton peptide, 3 K daltons smaller than the primary translation product and the isolated cytoplasmic enzyme. These results suggest that the decarboxylase is proteolytically processed during transport into the mitochondria and that the large amount of the cytoplasmic decarboxylase found in the gland represents accumulation of the unprocessed precursor form of the normally mitochondrial enzyme.     

Danazol inhibits human adrenal 21-and 11β-hydroxylation invitro

The effects of danazol on steroidogenesis $\text{in}$$\text{vitro}$ in the 16–20 week old human fetal adrenal were examined by studying: 1) danazol binding to adrenal microsomal and mitochondrial cytochrome P-450, and 2) enzyme kinetics of danazol inhibition of the adrenal microsomal 21-hydroxylase and the mitochondrial llβ-hydroxylase. The addition of danazol to preparations of adrenal microsomes or mitochondria elicited a type I cytochrome P-450 binding spectrum. Danazol bound to microsomal cytochrome P-450 with a high affinity apparent spectral dissociation constant (Kg) of 1 μM and with a lower affinity K's of 10 μM. Danazol bound to mitochondrial cytochrome P-450 with a Kg of 5 μM. In addition, danazol competitively inhibited the microsomal 21-hydroxylase (apparent enzymatic inhibition constant K_I = 0.8 μM) and the mitochondrial 11β-hydroxylase (K_I = 3 μM). These findings demonstrate that low concentrations of danazol directly inhibit steroidogenesis in the human fetal adrenal $\text{in}$$\text{vitro}$.     

Cyclic AMP-dependent phosphorylation of rat liver 6-phosphofructo 2-kinase, fructose 2,6-bisphosphatase

Incorporation of ³²P from [γ-³²P]ATP into a homogeneous preparation of rat hepatic 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase was catalyzed by a homogeneous preparation of the catalytic subunit of the cyclic AMP dependent protein kinase from rat liver. Approximately 2 mol of phosphate were incorporated per mol of the dimeric enzyme and this was associated with inhibition of the phosphotransferase activity and activation of the phosphohydrolase activity. Acid hydrolysis of the enzyme that was phosphorylated $\text{in}$,$\text{vitro}$ revealed that only seryl residues were labeled. Fructose 2,6-bisphosphate inhibited the initial rate of phosphorylation of the enzyme. It is concluded that both activities of this bifunctional enzyme are regulated in a reciprocal manner by cyclic AMP-dependent phosphorylation and that this phosphorylation can be modulated by fructose 2,6-bisphosphate.     

Studies on the action of porphyrinogenic trace metals on the activity of hepatic uroporphyrinogen decarboxylase

Trace metals which produce experimental uroporphyrinuria in animals during prolonged exposure inhibit uroporphyrinogen (uro) decarboxylase in rat liver extracts $\text{in}\text{,}\text{vitro}$. Inhibitory effects are prevented by sulfhydryl reagents, suggesting metal binding to sulfhydryl groups of the enzyme as the likely mode of action. Mercury, the most potent of the metals tested with respect to sulfur binding kinetics, produces the greatest inhibition of enzyme activity. In contrast, iron, which is considered to play a role in the etiology of porphyria cutanea tarda (PCT) via inhibition of uro decarboxylase, did not inhibit the enzyme in the present test system, suggesting an indirect mode of action $\text{in}\text{,}\text{vivo}$. These results suggest that direct inhibition of uro decarboxylase underlies uroporphyrinuria produced by prolonged trace metal exposure. Experimental inhibition of uro decarboxylase by trace metals may serve as a model for studying metal-induced uroporphyrinuria and PCT in humans.     

Influence of progesterone on the initial rate of cholesterol esterification in rat plasma

The effect of progesterone on the initial rate of cholesterol esterification in rat plasma was measured after daily injections of the hormone or following addition of progesterone $\text{in}$$\text{vitro}$. The administration of progesterone did not modify the lecithin:cholesterol acyltransferase (LCAT) activity nor the progress of the enzyme reaction with time. When increasing concentrations of progesterone were added to the medium the percentage of cholesterol esterified per minute decreased progressively. The addition of progesterone also decreased the slope of the time-course reaction. It is suggested that the inhibition of the LCAT activity due to the presence of the hormone would be masked by an increased hepatic production of the enzyme and/or by the alterations that the hormonal treatments produced in the plama lipid levels.     

Catabolite repression of the formation of precursors of electron transfer complexes III and IV in adapting bakers' yeast: dependence upon a mitochondrially translated repressor.

When bakers' yeast cells which had been grown anaerobically in galactose were aerated in the presence of 10% glucose, they showed a 40% decrease in $\text{in}\text{vivo}$ [¹⁴C]-leucine incorporation into a washed mitochondrial membrane fraction compared with cells which had been aerated in a low glucose medium. The observed catabolite repression of membrane protein synthesis was primarily due to a decrease in cytoplasmic translational activity, but this repression was entirely dependent upon concomitant mitochondrial translation. The inductions of reduced coenzyme Q cytochrome $\text{c}$ reductase (complex III) and of cytochrome $\text{c}$ oxidase (complex IV) activities were repressed 30 and 60%, respectively, by aeration of the cells for 8 hours in 10% glucose. The catabolite repression of the formation of these two inner membrane complexes was again shown to be dependent upon concomitant mitochondrial translation. Both the amino acid incorporation and enzyme induction data suggest that catabolite repression of both cytoplasmically and mitochondrially translated mitochondrial membrane proteins is mediated through a mitochondrially translated repressor.     

NADPH-dependent reductase solubilized from microsomes by peroxidation and its activity

Isolated rat liver microsomes were subjected to enzymatic or non-enzymatic lipid peroxidation $\text{in vitro}$. NADPH-dependent cytochrome $\text{c}$ reductase activity was released from the microsomes into the media during peroxidation. This activity could be recovered from the media by DEAE-cellulose chromatography. The recovered enzyme retained high activity for the reduction of cytochrome $\text{c}$ and a lower level of activity for the reduction of cytochrome P-450. The active fractions were capable of enzymatically supporting the peroxidation of isolated mitochondria in the presence of organically complexed Fe⁺³ and NADPH, and in this respect the specific activity was found to be about ten times higher than in microsomes.