Evidence for participation of the inner membrane in the import of sulfite oxidase into the intermembrane space of liver mitochondria

Sulfite oxidase, a soluble enzyme in mitochondrial intermembrane space, was synthesized as a precursor protein larger than the authentic enzyme when rat liver RNA was translated $\text{in}\text{vitro}$ using reticulocyte lysate. When the $\text{in}\text{vitro}$ translation products were incubated with isolated rat liver mitochondria, the precursor of sulfite oxidase was converted to the size of the mature enzyme. The $\text{in}\text{vitro}$ processed mature enzyme was no longer susceptible to externally added proteases and was extractable by a hypotonic treatment of the mitochondria, suggesting its location in the intermembrane space. When mitochondria were subfractionated, most of the processing activity was recovered in the mitoplast fraction. The import-processing activity of mitochondria was inhibited by CCCP, oligomycin, or atractyloside in the presence of KCN. These results suggest that the import of sulfite oxidase into mitochondrial intermembrane space requires the participation of 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.     

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}$.     

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.     

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.     

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.     

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.     

Cell-free synthesis of pigeon liver fatty acid synthetase.

Pigeon liver fatty acid synthetase proteins (apo- and holo-forms) have been synthesized in a cell-free system reconstituted from polysomes and a soluble enzyme fraction. Identification of the cell-free synthesized products as fatty acid synthetase was achieved by affinity chromatography, by immuno-precipitation and by the simultaneous conversion of both the authentic carrier protein and the $\text{in vitro}$ synthesized products from the holo- to the apo-form of the synthetase. The reverse conversion was also effected.     

Evidence for the regulation of pyridoxal 5′-phosphate formation in liver by pyridoxamine (pyridoxine) 5′-phosphate oxidase

Pyridoxamine (pyridoxine) 5′-phosphate oxidase (EC 1.4.3.5) purified from rabbit liver is competitively inhibited by the reaction product, pyridoxal 5′-phosphate. The K_i, 3 μM, is considerably lower than the K_m for either natural substrate (18 and 24 μM for pyridoxamine 5′-phosphate and 25 and 16 μM for pyridoxine 5′-phosphate in 0.2 M potassium phosphate at pH 8 and 7, respectively). The K_i determined using a 10% rabbit liver homogenate is the same as that for the pure enzyme; hence, product inhibition $\text{in}\text{vivo}$ is probably not diminished significantly by other cellular components. Similar determinations for a 10% rat liver homogenate also show strong inhibition by pyridoxal 5′-phosphate. Since the reported liver content of free or loosely bound pyridoxal 5′-phosphate is greater than K_i, the oxidase in liver is probably associated with pyridoxal 5′-phosphate. These results also suggest that product inhibition of pyridoxamine-P oxidase may regulate the $\text{in}\text{vivo}$ rate of pyridoxal 5′-phosphate formation.     

A new type of enzyme electrode: The ascorbic acid eliminator electrode

A new type of enzyme electrode has been developed for $\text{in}$$\text{vivo}$ electrochemical measurements which allows discrimination between ascorbic acid and catecholamines and their metabolites. The electrode employs the enzyme ascorbic acid oxidase held between the voltammetric electrode and solution by a dialysis membrane or immobilized on the outer surface of serous membrane from rat small intestine. The electrode gives linear calibration curves for all catecholamines and metabolites independent of any ascorbic acid concentrations significant in physiological measurements. The electrode has been tested in brain slice measurements and shown to respond to releases of catecholamines initiated by potassium ion stimulation.     

Synthesis of liver cytochrome P-450b in a cell-free protein synthesizing system

Live ppolysomes isolated from rats that had been treated with phenobarbital (PB) are able to incorporate [³H]leucine into total protein $\text{in}\text{vitro}$ at a rate almost five times that of polysomes prepared from control animals. Specific immunoprecipitation of translational products has shown that polysomes from induced animals synthesize cytochrome P-450b at a rate almost seven times greater than polysomes from control animals. The increased protein and cytochrome P-450b synthesis can be detected as early as 6 h following phenobarbital administration and reaches a maximum at 12–18 h. The results suggest that PB administration effects an increase in mRNA for cytochrome P-450b.     

The effect of beta-naphthoflavone on rabbit liver protein synthesis, and on the induction of cytochrome P-450 LM4 mRNA

A single injection of β-naphthoflavone dispersed in corn oil causes significant changes in rabbit liver polysome and polysomal poly(A+)mRNA driven $\text{in vitro}$ protein synthesis. The changes occur between 6–18 hr and 30–36 hr after the injection. Our data indicate that the first effect is due to the β-naphthoflavone and the second effect is due to the oil vehicle. $\text{In vitro}$ translation of rabbit liver polysomes obtained from treated rabbits followed by specific immunoprecipition and gel electrophoresis, showed that maximal levels of translatable cytochrome P-450 LM₄ occurred 18–24 hr after β-naphthoflavone treatment.     

Lactate dehydrogenase-C mRNA: Its isolation and invitro translation

Lactate dehydrogenase-C (LDH-C) mRNA was purified from $\text{DBA}\text{2}$ mouse testes and translated $\text{in}\text{vitro}$. First, the LDH-C synthesizing polysomes were isolated by double immunoprecipitation using specific anti-LDH-C and anti-horse immunoglobulin antibodies. Extraction of mRNA was made from the isolated polysomes using hot sodium dodecyl sulfate-phenol method at alkaline pH. In a wheat germ cell-free translation system, the mRNA coded for a polypeptide chain that could be immunoprecipitated with specific anti LDH-C antibody and comigrated with authentic LDH-C in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Oxygenation of mitochondrial membranes by the reticulocyte lipoxygenase. Action on monoamine oxidase activities A and B

Incubation of isolated rat liver mitochondria with the pure rabbit reticulocyte lipoxygenase caused a time-dependent inactivation of the monoamine oxidase activities A and B. Furthermore, a conversion of the monoamine oxidase into a diamine oxidase was observed. The inactivation kinetics for both monoamine oxidase activities A and B showed a biphasic behaviour; a reversible short-term inhibition during the first 5 min of incubation was followed by an irreversible inactivation of the enzyme. The kinetic studies suggest that the slow irreversible inactivation of the monoamine oxidase activities is due to secondary reactions subsequent to the initial attack of the lipoxygenase on the mitochondrial outer membrane. During the interaction of the lipoxygenase with the mitochondria, only about 1.5% of the polyenoic fatty acids present in the mitochondrial membranes were oxygenated. The predominant products formed during the interaction of the lipoxygenase with the mitochondrial membranes are (13S)-hydro(pero)xy-9Z,11E-octadecadienoic acid and (15S)-hydro(pero)xy-5,8,11,13(Z,Z,Z,E)-eicosatetraenoic acid.     

Purification of rat liver monoamine oxidase by octyl glucoside extraction and reconstitution

Catalytically active isoenzymes of rat liver monoamine oxidase have been copurified from the outer mitochondrial membrane by a novel method involving repetitive solubilization with octyl-β-d-glucopyranoside followed by reconstitution into lipid vesicles. As analyzed using sodium dodecyl sulfate-gel electrophoresis, the purified enzyme migrates as a single band of protein of molecular weight 60,000. The preparation is capable of metabolizing 576 nmol serotonin and 777 nmol β-phenylethylamine/min/mg protein. Apparent K_m values and sensitivity to the inhibitor clorgyline are very similar for the purified and outer mitochondrial membrane-bound enzyme when determined with the substrates β-phenylethylamine, serotonin, and tyramine.     

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.