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.     

Purification of Protein Body-I of Rice Seed and its Polypeptide Composition

Protein body type one (PB-I) was isolated and purified fromdeveloping rice grain by a combination of sucrose density gradientcentrifugation and treatment with pepsin. SDS-PAGE analysisshowed that isolated PB-I contains several polypeptide groups,the largest having an apparent molecular size of 13 kDa andtwo smaller ones of 10 kDa and 16 kDa. The 13-kDa group wasfound to be composed of two polypeptides of slightly differentmolecular sizes, 13a (larger component) and 13b (smaller component).Most of the 13a and 13b polypeptides were shown to be largelyprolamins, although there were also some salt- and alcohol-insolublepolypeptides with an apparent molecular size of 13 kDa. It wasconcluded that PB-I is the accumulation site of rice prolamin.It was further estimated that the protein amount in PB-I accountedfor about 20% of the total protein of rice endosperm. (Received March 20, 1987; Accepted September 8, 1987)     

A New Method for Preservation of Actin Filaments in Higher Plant Cells

Treatment with tropomyosin before fixation of tobacco BY-2 cellswith aldehydes improved the preservation of actin filamentsin the cells and enabled us to visualize fine networks of bothcortical actin filaments and cortical microtubules in the sameinterphase cells by the double staining technique using rhodaminephalloidin and antitubulin antibodies. (Received June 25, 1987; Accepted August 31, 1987)     

Structural analysis and specific expression of microsomal cytochrome P-450(M-1) mRNA in male rat livers

cDNA clones for the P-450(M-1) mRNA, which exhibits a male-specific expression in rat livers, were isolated by using synthetic oligonucleotides as the probes. Sequence analysis of the cDNAs showed that P-450(M-1) mRNA contains 1,853 nucleotides in addition to a poly(A) chain, and a single open reading frame of 1,500 nucleotides encodes a polypeptide of 500 amino acids with a Mr = 57,187. The predicted NH2-terminal sequence of 30 amino acids agrees well with that of the purified protein determined by Edman degradation, and the predicted primary structure included all the partial sequences of six internal peptides of P-450(M-1) obtained by the proteolytic digestion and a conserved amino acid sequence containing a putative heme-binding cysteine, proximate to the COOH terminus of the molecules. P-450(M-1) showed relatively high sequence similarity with P-450b (Fujii-Kuriyama, Y., Mizukami, Y., Kawajiri, K., Sogawa, K., and Muramatsu, M. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2793-2797) (52% similarity), P-450-3b (Ozols, J., Heinemann, F. S., and Johnson, E. F. (1985) J. Biol. Chem. 260, 5427-5434) (64%), P-450-1 (Tukey, R. H., Okino, S., Barnes, H., Griffin, K. J., and Johnson, E. F. (1985) J. Biol. Chem. 260, 13347-13354) (74%), P-450PBc1 (Leighton, J. K., DeBrunner-Vossbrinck, B. A., and Kemper, B. (1984) Biochemistry 23, 4598-4603) (71%), while its sequence similarity with 3-methylcholanthrene-inducible P-450c and P-450d is rather low. Consequently, P-450(M-1) could be structurally classified into the phenobarbital-inducible type of P-450 gene family. RNA blot analysis using a synthetic oligonucleotide specific for P-450(M-1) revealed that P-450(M-1) mRNA was expressed exclusively in the livers of mature male rats in a sex-specific manner, but not in other tissues so far examined.     

Regional distribution of DNA and RNA in rat brain: A sensitive determination using high-performance liquid chromatography with electrochemical detection

DNA and RNA contents in 20 brain regions or nuclei of the rat were determined by a highly sensitive method using high-performance liquid chromatography with electrochemical detection. The high DNA and RNA contents were found in the hypothalamic nuclei, especially the median eminence-arcuate nucleus. These results may be available for the preparation of nucleic acids as the regional control.     

Ultrastructure of the kidney of a South American caecilian,Typhlonectes compressicaudus (Amphibia,Gymnophiona)

Summary The ultrastructure of the distal nephron, the collecting duct and the Wolffian duct was studied in a South American caecilian, Typhlonectes compressicaudus (Amphibia, Gymnophiona) by transmission and scanning electron microscopy (TEM, SEM). The distal tubule (DT) is made up of one type of cell that has a well-developed membrane labyrinth established both by interdigitating processes and by interlocking ramifications. The processes contain large mitochondria, the ramifications do not. The tight junction is shallow and elongated by a meandering course. The connecting tubule (CNT) is composed of CNT cells proper and intercalated cells, both of which are cuboidal in shape. The CNT cells are characterized by many lateral interlocking folds. The intercalated cells have a dark cytoplasm densely filled with mitochondria. Their apical cell membrane is typically amplified by microplicae beneath which a layer of globular particles (studs) is found. The collecting duct (CD) is composed of principal cells and intercalated cells, again both cuboidal in shape. The CD epithelium is characterized by dilated intercellular spaces, which are often filled with lateral microfolds projecting from adjacent principal cells. The apical membrane is covered by a prominent glycocalyx. The intercalated cells in the CD are similar to those in the CNT. The Wolffian duct (WD) has a tall pseudostratified epithelium established by WD cells proper, intercalated cells and basal cells. The WD cells contain irregular-shaped dense granules located beneath the apical cell membrane. The intercalated cells of the WD have a dark cytoplasm with many mitochondria; their nuclei display a dense chromatin pattern.Research fellow of the Alexander von Humboldt Foundation