Import and processing of the precursor of cytochrome P-450(SCC) by bovine adrenal cortex mitochondria

Isolated bovine adrenal cortex mitochondria imported in vitro synthesized pre-P-450(SCC) and processed it to the mature form. Partial radio-sequencing of the processed P-450(SCC) gave a result identical with that for authentic P-450(SCC). Rat liver mitochondria also imported pre-P-450(SCC) and processed it to the mature form, whereas bovine heart mitochondria were unable to import and process pre-P-450(SCC) although both mitochondrial preparations imported and processed pre-adrenodoxin. The pre-P-450(SCC) processing activity of bovine adrenal cortex mitochondria was associated with the matrix side surface of the inner membrane. The processing protease could be solubilized by sodium cholate and partially purified by ammonium sulfate fractionation. The partially purified processing protease cleaved pre-P-450(SCC) at the correct position. It was also active in processing pre-P-450(11 beta) but inactive toward pre-adrenodoxin. Bovine heart mitochondria lacked the processing activity to pre-P-450(SCC). The localization of pre-P-450(SCC) and mature P-450(SCC) in bovine adrenal cortex mitochondria was examined. Mature P-450(SCC) processed by the mitochondria was found associated with the matrix-side surface of the inner membrane, which is the correct location of P-450(SCC) in the cell. In the presence of o-phenanthroline, pre-P-450(SCC) was imported into the organelles without being processed and remained soluble in the matrix. The incorporation of newly processed mature P-450(SCC) into the inner membrane was also observed when pre-P-450(SCC) was incubated with inner membrane vesicles. Mature P-450(SCC) generated in vitro from pre-P-450(SCC) by the partially purified processing protease was incorporated not only into the inner membrane vesicles but also into bovine adrenal cortex microsomes. These findings suggested that the processing of pre-P-450(SCC) occurred prior to the incorporation of mature-P-450(SCC) into the inner membrane.     

Synthesis and processing of mitochondrial steroid hydroxylases. In vivo maturation of the precursor forms of cytochrome P-450scc, cytochrome P-450(11)beta, and adrenodoxin

The synthesis and maturation of the precursor forms of three mitochondrial enzymes involved in steroid hormone biosynthesis have been studied in vivo. Primary cultures of bovine adrenocortical cells were radiolabeled with [35S] methionine and newly synthesized cholesterol side-chain cleavage cytochrome P-450 (P-450scc), 11 beta-hydroxylase cytochrome P-450 (P-450(11)beta), and adrenodoxin immunoisolated using specific antibodies. Both the precursor and mature forms of P-450scc and P-450(11)beta were detected during short periods of pulse labeling; however, the precursor forms were transitory in nature while their corresponding mature forms accumulated. Pulse-chase experiments showed that the precursor form of each cytochrome P-450 had an apparent half-life of 3.5 min. In contrast, the precursor form of adrenodoxin was not readily detected in pulse-labeling experiments until a substantial amount of its mature form had accumulated. When the cultured cells were treated with a chelator of divalent cations (o-phenanthroline) or a mitochondrial uncoupler (dinitrophenol), the maturation of all three precursors was inhibited. The synthesis of the P-450scc and P-450(11)beta precursors was induced in cells maintained in the presence of adrenocorticotropin, and the rates of appearance of their processed forms were also increased. The mature forms of all three proteins were immunoisolated from a trypsinized mitochondrial fraction prepared from the radiolabeled cells, demonstrating that the mature proteins were localized within the organelle. These studies establish that the maturation of the precursor forms of the mitochondrial steroidogenic enzymes are characterized by steps similar to those reported for other mitochondrial precursor proteins.     

Processing-independent in vitro translocation of cytochrome P-450(SCC) precursor across mitochondrial membranes

In the presence of a membrane-permeable metal chelator, bovine adrenal cortex mitochondria imported P-450(SCC) precursor without processing of the amino-terminal extension peptide. The imported precursor was bound to the matrix side surface of the inner membrane. When the inhibition due to the metal chelator was removed, the imported precursor was processed to the mature form. Unprocessed precursor was also detected in mitochondria when the import reaction was carried out at relatively low temperature. These results suggest that the translocation of P-450(SCC) precursor across mitochondrial membranes is independent of its processing to the mature form. Both membrane-bound and solubilized P-450(SCC) could be cleaved by trypsin into two fragments with molecular weights of 29 kDa and 26 kDa, respectively, suggesting a two-domain structure of the molecule. The in vitro-imported and processed P-450(SCC) was also cleaved by trypsin in the same way. This finding indicated that the in vitro-imported and processed P-450(SCC) has the same conformation as the native form.     

Contributions of cytoplasmic free and membrane-bound ribosomes to the synthesis of mitochondrial cytochrome P-450(SCC) and P-450(11 beta) and microsomal cytochrome P-450(C-21) in bovine adrenal cortex

Rabbit antibodies against cytochrome P-450 (SCC), P-450 (11 beta), and P-450 (C-21) from bovine adrenal cortex were prepared, and it was confirmed that these three cytochrome P-450 species are immunologically distinct from one another. Cytoplasmic sites of synthesis of P-450 (SCC), P-450 (11 beta), and P-450 (C-21) in bovine adrenal cortex were determined by examining the presence of their nascent peptides on isolated free and bound ribosomes. Nascent peptides were released in vitro from ribosomes by [3H]puromycin in a high salt buffer in the presence of a detergent, and the nascent peptides of P-450 (SCC), P-450 (11 beta), and P-450 (C-21) were isolated by immunoprecipitation. The nascent peptides of these three cytochrome P-450 species were found in both free and bound ribosomal fractions, suggesting that they share common sites of synthesis in the cytoplasm. However, the nascent peptides of mitochondrial P-450 (SCC) and P-450 (11 beta) were more concentrated in the free ribosomal fraction, whereas those of microsomal P-450 (C-21) were more abundant in the bound ribosomal fraction. The nascent peptides of the three cytochrome P-450 species were released from the membrane-bound ribosomes of rough microsomes into the cytoplasmic surface of microsomal vesicles by puromycin treatment.     

Discriminatory processing of the precursor forms of cytochrome P-450scc and adrenodoxin by adrenocortical and heart mitochondria

The mitochondrial proteins involved in adrenocortical steroidogenesis are synthesized as higher molecular weight precursors which require processing by the mitochondria to their mature sizes. The post-translational maturation of two of these proteins has been examined: the cholesterol side chain cleavage cytochrome P-450 (P-450scc) and the iron-sulfur protein, adrenodoxin. Total translation products synthesized in a cell-free system programmed by bovine adrenocortical poly(A+) RNA were incubated with isolated bovine adrenocortical or heart mitochondria followed by immunoisolation of radiolabeled P-450scc or adrenodoxin. In the presence of adrenocortical mitochondria, the precursor form of P-450scc was converted into a trypsin-resistant form that had the same molecular weight as mature P-450scc. Unlike adrenocortical mitochondria, heart mitochondria were unable to process the P-450scc precursor which remained unaltered and trypsin-sensitive. In addition, a matrix fraction of heart mitochondria did not cleave the P-450scc precursor. In contrast, the adrenodoxin precursor did not exhibit similar specificity as it was processed to the mature form by both adrenocortical and heart mitochondria. Also, the adrenocortical mitochondria were not restricted to processing endogenous proteins as they imported and cleaved the precursor to ornithine transcarbamylase. The results indicate that some mitochondrial precursor proteins have tertiary structures which allow them to be recognized by all mitochondria while other mitochondrial precursor proteins have structures recognizable by only specialized mitochondria.     

Presence of a prepiece at the NH2-terminal end of pre-cytochrome P-450(SCC) peptide

Mild acid treatment of in vitro translated cytochrome P-450(SCC) (pre-P-450(SCC] peptide cleaved the peptide into two fragments. Comparison of the sizes and the NH2-terminal amino acids of the fragments with those of the corresponding fragments from mature P-450(SCC) suggested that the prepiece of pre-P-450(SCC) was present at the NH2-terminal end of the peptide. This conclusion was confirmed by radio-sequencing of the NH2-terminal portion of pre-P-450(SCC).     

Synthetic partial extension peptides of P-450(SCC) and adrenodoxin precursors: effects on the import of mitochondrial enzyme precursors

Various portions of the extension peptides of P-450(SCC) precursor were chemically synthesized. The effects of these peptides on the import of enzyme precursors into mitochondria were examined. Peptides SEP1-15 and SEP1-20, corresponding to the amino terminal portion of the extension peptides, strongly inhibited the import of P-450(SCC) precursor into mitochondria. These peptides were effective at concentrations above 30 microM, and complete inhibition was observed at 100 microM. SEP1-11, which is shorter than SEP1-15 and SEP1-20, showed very weak inhibition. SEP25-39, which corresponds to the carboxy terminal portion of the extension peptide, did not affect the import of the precursor. The import of P-450(11 beta) and adrenodoxin precursors were also inhibited by SEP1-15. Another peptide, AEP1-14, which corresponds to the amino terminal portion of the extension peptide of adrenodoxin precursor, was also synthesized. The peptide inhibited the import of both adrenodoxin and P-450(SCC) precursors into mitochondria. The import of malate dehydrogenase was also inhibited by SEP1-15 and AEP1-14. The rate of the internalization of the precursor into mitochondria was decreased by the peptides. The amount of the precursor bound to the surface of mitochondria and the processing of adrenodoxin precursor were not affected. The respiratory activities of isolated mitochondria were not influenced by SEP1-15 even at 100 microM. We conclude that the inhibitory activities of the synthetic partial extension peptides on the import of enzyme precursors into mitochondria require the presence of about fifteen amino acid residues in the amino terminal portion of the extension peptides, and the inhibition of the import by the peptides was dependent on the blockage of the internalization of the precursors into mitochondria.     

Cytochrome P-450 of bovine adrenal mitochondria. Ligand binding to two forms resolved by EPR spectroscopy.

The binding of cholest-5-ene-3beta,20alpha-diol (20alpha-hydroxycholesterol), 11-deoxycorticosterone, and aminoglutethimide to cytochrome P-450 in bovine adrenal mitochondria was measured by changes in optical spectra at room temperature and by EPR spectra at 14 K. The two methods provided nearly identical quantitation of these interactions with cytochrome P-450. Two distinct high spin forms of cytochrome P-450 were revealed by EPR spectra. The predominant high spin species (g = 8.2) was decreased by addition of 20alpha-hydroxycholesterol and elevated pH but was increased by addition of cholesterol. The minor high spin species (g = 8.1) was incrreased by addition of deoxycorticosterone but decreased by low concentrations of metyrapone. The two forms were evidently not in equilibrium and have been assigned to distinct forms of cytochrome P-450 involved in, respectively, cholesterol side chain cleavage (P-450scc) and steroid 11beta hydroxylation (P-450(11)beta). The high spin states are derived from complexes of these P-450 cytochromes with endogenous substrates, which are, respectively, cholesterol and deoxycorticoids. A high to low spin transition was observed when these complexes were turned over by initiating hydroxylation with malate. The contributions of cytochromes P-450(11)beta and P-450scc to the low spin spectrum were also resolved by similar means. At least 20% of P-450scc is in the low spin state while about 90% of P-450(11)beta is low spin in isolated beef adrenal mitochondria. Low spin complexes of cytochrome P-450scc with 20alpha-hydroxycholesterol and 3beta-hydroxypregn-5-ene-20-one (pregnenolone) gave distinct EPR spectra. Aminoglutethimide interacted with the total cytochrome P-450 content of the bovine adrenal mitochondria forming low spin complexes. Both optical and EPR data indicated binding to two forms of cytochrome P-450. These results suggest a detailed correlation between the spin state and absorbance changes seen at room temperature, illustrate that EPR allows the distinction of two principal forms of P-450, and suggest that there is no appreciable change in the spin state of either cytochrome between 14 K and 300 K.     

Multiple molecular forms of cytochrome P-450SCC purified from bovine corpus luteum mitochondria

Cytochrome P-450 related to side-chain cleavage of cholesterol (P-450SCC) was isolated from bovine corpus luteum mitochondria in the form of its stable cholesterol complex. The isolation procedure included ammonium sulfate fractionation and chromatography on omega-aminohexyl-Sepharose (AH-Sepharose). Corpus luteum P-450SCC was resolved into one minor (AH-I) and two major (AH-II and AH-III) fractions by the chromatography. Results of re-chromatography suggested the possibility that AH-III Fraction was originally complexed with lipidic material. The two major fractions purified by the re-chromatography (AH-IIR and AH-IIIR Fractions) showed essentially a single band on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and their absorption spectra were indistinguishable from each other. Both fractions were further resolved into two major and some minor bands of P-450SCC by isoelectric focusing on polyacrylamide gel in the presence of a non-ionic detergent, as detected by protein staining, heme staining and immunoblot analysis with anti-bovine P-450SCC monoclonal antibody. Both AH-IIR and AH-IIIR Fractions were further resolved by high-performance liquid chromatography (HPLC) on SP-TSK gel column into two fractions, SP-I and SP-II. These fractions had the same N-terminal amino acid sequence, showed similar catalytic activity and resolved into one major and a few minor bands on isoelectric focusing on polyacrylamide gel. Much more heterogeneity was observed in purified P-450SCC preparations from bovine adrenal cortex mitochondria. These results indicated the presence of multiple molecular forms of corpus luteum P-450SCC as well as adrenal cortex P-450SCC. Computer simulation studies were carried out in order to analyze the mechanism of formation of multiple bands on isoelectric focusing. The multiple bands of corpus luteum P-450SCC could be explained by postulating the presence of two isozymes (or molecular forms) having a pair of sites each with or without a charged group.     

Characterization of a mitochondrial matrix protease catalyzing the processing of adrenodoxin precursor

Adrenodoxin (Ad) is synthesized as a larger precursor (preAd) by cytoplasmic polysomes and then transported into mitochondria concomitant with its proteolytic processing to the mature form. The protease in bovine adrenal cortex mitochondria, which converts preAd to the mature form, is a metalloprotease in the matrix (Sagara, Y., Ito, A. & Omura, T. (1984) J. Biochem. 96, 1743-1752). In this study, the protease was purified about 100-fold from the matrix fraction of bovine adrenal cortex mitochondria. The partially purified protease converted not only preAd, but also the precursors of malate dehydrogenase (MDH) and 27 kDa protein (P-27) to the corresponding mature forms. However, it was inactive toward the precursors of P-450(SCC) and of P-450(11 beta). Since isolated rat liver mitochondria can import and process preAd as efficiently as bovine adrenal cortex mitochondria, we partially purified a preAd-processing protease from rat liver mitochondria and compared its properties with those of the bovine adrenal cortex enzyme. The properties of the rat liver protease were indistinguishable from those of the bovine adrenal cortex enzyme in molecular weight determined from Sephadex G-150 gel filtration, metal requirement and ability to process preMDH and preP-27. The rat liver enzyme was also inactive toward the precursors of P-450(SCC) and P-450(11 beta). These results indicate the presence in both adrenal cortex and liver mitochondria of the same type of processing protease, which processes preAd and also the precursors of some other mitochondrial proteins.     

The relationship between NADPH-dependent lipid peroxidation and degradation of cytochrome P-450 in adrenal cortex mitochondria

The relationship between NADPH-dependent lipid peroxidation and the degradation of cytochrome P-450 has been studied in bovine adrenal cortex mitochondria. Malondialdehyde formation is accompanied by a corresponding decrease in total cytochrome P-450 content. Inhibitors of lipid peroxidation also prevent the loss of cytochrome P-450, further demonstrating a direct relationship between NADPH-dependent lipid peroxidation and degradation of P-450. To differentiate between cytochrome P-450(11)beta and P-450scc, steroid-induced difference spectra were used to evaluate P-450 degradation. These measurements provide the first evidence that both P-450's are degraded during NADPH-dependent lipid peroxidation with P-450(11)beta being much more susceptible to this process.     

Gene structure of human cytochrome P-450(SCC), cholesterol desmolase

Four independent clones containing a part of the P-450(SCC), cholesterol desmolase, gene were isolated from human genomic libraries using bovine P-450(SCC) cDNA as a probe. These clones covered the entire P-450(SCC) gene except for a part of the 1st intron. The gene is at least 20 kb long and is split into 9 exons by 8 introns. The sequence analysis revealed that the nine separated exons code for a primary structure consisting of 521 amino acids which shows 72% homology with that of bovine P-450(SCC). A CATT sequence and a TATAAT sequence, which are possibly a "CAT" box, and a "TATA" box, respectively, are present 129 and 91 bp upstream from the initiation codon. An unusual exon/intron junctional sequence that begins with GC was found in the 6th intron of the gene. A putative extension peptide consisting of 39 amino acids was found in the sequence of human P-450(SCC) by comparison with that of the bovine counterpart. Two conserved regions were found in the extension peptide of these two forms of P-450(SCC), suggesting a functional role of the portions in the mitochondrial localization and processing of P-450(SCC) precursor. The mature form of human P-450(SCC) has only one cysteine residue, which was located in the center of the HR2 region (Gotoh et al. (1983) J. Biochem. 97, 807-817). This observation established beyond doubt that the sole cysteine residue in the HR2 region is the 5th ligand to the heme.     

Cloning and characterization of bovine cytochrome P-450(11 beta) genes

We isolated 4 different clones of the P-450(11 beta) gene from a bovine genomic library. These genomic clones were highly homologous with each other. Two of the isolated clones were pseudogenes. Determination of its nucleotide sequences indicated that the bovine P-450(11 beta) gene is divided into 9 exons by 8 introns and that it is about 8.5 kb in total length. The number of exons and the locations of intron insertion into the P-450(11 beta) gene are identical with those in the case of P-450(SCC), but different from those of other microsomal P-450s.     

Isolation of two distinct cytochromes P-45011 beta with aldosterone synthase activity from bovine adrenocortical mitochondria

Two distinct forms of cytochrome P-45011 beta, with apparent molecular weights of 48,500 (48.5K) and 49,500 (49.5K), have been isolated from bovine adrenocortical mitochondria. Their amino acid sequences up to the 19th position from the N-terminus were only different at the 6th position (Val and Ala for the 48.5K and 49.5K enzymes, respectively). Each sequence was assignable to a distinct cDNA clone for cytochrome P-450(11) beta (Kirita, S., et al. [1988] J. Biochem. 104, 683-686), indicating that the two proteins originate from different genes in bovine adrenocortical cells. Both forms of cytochrome P-450(11) beta were capable of catalyzing aldosterone synthesis as well as the 11 beta- and 18-hydroxylation of 11-deoxycorticosterone. Thus, at least two distinct cytochrome P-450(11) beta species exist in the adrenal cortex and participate in steroidogenesis.     

Expression and transport into mitochondria of bovine cytochrome P-450(SCC) in insect cells using the baculovirus expression system.

Bovine cytochrome P-450(SCC) introduced with the baculovirus host vector system was found to be expressed in Spodoptera frugiperda cells. Cell fractionation analysis indicated that the P-450(SCC) expressed as the precursor form was transported into mitochondria and converted to a mature form. However, this form did not exhibit definite activity for cholesterol side chain cleavage. These findings suggest that most of the P-450(SCC) expressed by this system is an inactive protein within mitochondria that is not folded to the conformation of the active enzyme and/or does not incorporate heme appropriately.     

Interaction of cytochrome P-450scc with cytochrome b5

Spectrophotometric, affinity chromatography and cross-linking experiments provided evidence that cytochrome P-450scc from bovine adrenocortical mitochondria forms a tight complex with cytochrome b5 from rabbit liver microsomes. In the reconstituted system cholesterol side chain activity of cytochrome P-450scc was enhanced by the addition of cytochrome b5.     

Protein rotation study of cytochrome P-450 in submitochondrial particles: effect of KCl and intermolecular interactions with redox partners

The rotational diffusion of cytochrome P-450 in submitochondrial particles (SMP) of bovine adrenocortical mitochondria was measured by detecting the decay of absorption anisotropy, r(t), after photolysis of the heme.CO complex by a vertically polarized laser flash. Analysis of r(t) was based on a "rotation-about-membrane normal" model. The measurements were used to investigate the effect of KCl on intermolecular interactions involving cytochrome P-450 and to investigate the interactions of cytochrome P-450 with other redox partners. The rotational diffusion of cytochrome P-450 was significantly dependent on KCl concentration. When the KCl concentration was increased from 0 to 1,000 mM, the mobile population of cytochrome P-450 was increased from 33 to 82%. After removing the KCl, the mobile population of cytochrome P-450 returned to the original 33%. These results suggest that nonspecific protein aggregates are dissociated by the presence of KCl, possibly due to the change in electrostatic interactions, resulting in mobilization of cytochrome P-450. SMP were observed to be nearly free from adrenodoxin and adrenodoxin reductase. The addition of adrenodoxin to SMP increased the mobile population of cytochrome P-450 from 35 to 54%. Further addition of adrenodoxin reductase to SMP containing adrenodoxin immobilized cytochrome P-450 by 6%. The addition of only adrenodoxin reductase to SMP, however, did not immobilize cytochrome P-450. The present results are consistent with our previous observations [Ohta, Y., Mitani, F., Ishimura, Y., Yanagibashi, K., Kawamura, M., & Kawato, S. (1990) J. Biochem. 107, 97-104] that cholesterol-bearing P-450SCC forms a transient ternary association with adrenodoxin and adrenodoxin reductase.     

Active site of bovine adrenocortical cytochrome P-450(11) beta studied by resonance Raman and electron paramagnetic resonance spectroscopies: distinction from cytochrome P-450scc

Cytochrome P-45011 beta was purified as the 11-deoxycorticosterone-bound form from bovine adrenocortical mitochondria and its active site was investigated by resonance Raman and EPR spectroscopies. Resonance Raman spectra of the purified sample revealed that the heme iron adopts the pure pentacoordinated ferric high-spin state on the basis of the nu 10 (1629cm-1) and nu 3 (1490 cm-1) mode frequencies, which are higher than those of the hexacoordinated ferric high-spin cytochrome P-450scc-substrate complexes. In the ferrous-CO state, a Fe2(+)-CO stretching mode was identified at 481.5 cm-1 on the basis of an isotopic substitution technique; this frequency is very close to that of cytochrome P-450scc in the cholesterol-complexed state (483 cm-1). The EPR spectra of the purified sample at 4.2 K showed ferric high-spin signals (at g = 7.98, 3.65, and 1.71) that were clearly distinct from the cytochrome P-450scc ferric high-spin signals (g = 8.06, 3.55, and 1.68) and confirmed previous assignments of ferric high-spin signals in adrenocortical mitochondria. The EPR spectra of the nitric oxide (NO) complex of ferrous cytochrome P-45011 beta showed EPR signals with rhombic symmetry (gx = 2.068, gz = 2.001, and gy = 1.961) very similar to those of the ferrous cytochrome P-450scc-NO complex in the presence of 22(S)-hydroxycholesterol and 20(R),22-(R)-dihydroxycholesterol at 77 K.(ABSTRACT TRUNCATED AT 250 WORDS)