Purification and characterization of six cytochromes P-450 from hepatic microsomes of immature female rats

Six rat hepatic cytochromes P-450, named P-450IF-1-6, were purified from hepatic microsomes of immature female rats by high-performance liquid chromatography with anion-exchange, cation-exchange, and hydroxylapatite columns. The purified forms, except for P-450IF-4, gave a single protein-staining band on SDS-polyacrylamide gel electrophoresis, with a minimum molecular weight of 50,000 for P-450IF-1, 49,000 for P-450IF-2, 47,000 for P-450IF-3, 53,500 for P-450IF-5, and 54,000 for P-450IF-6. The CO-reduced spectral maximum of these forms was at 450 nm for P-450IF-1, 448 nm for P-450IF-2, 451 nm for P-450IF-3, 449 nm for P-450IF-4, 449 nm for P-450IF-5, and 450 nm for P-450IF-6. All of these cytochromes had the low-spin state of heme in the oxidized form. P-450IF-4 had high metabolic activity for both benzphetamine and 7-ethoxycoumarin. P-450IF-5 had moderate activity toward 7-ethoxycoumarin. P-450IF-3 catalyzed the hydroxylation of testosterone at the 7 alpha-position effectively, but the other forms did not hydroxylate testosterone. Analysis of the NH2-terminal sequence showed that P-450IF-1, 2, 3, 5, and 6 differed structurally from each other. The sequences of P-450IF-1 and IF-2 were somewhat homologous, but the NH2-terminal sequences of the other forms were all different. Based on these results, we concluded that P-450IF-1 corresponded to one of the phenobarbital-inducible forms in male rat liver. P-450IF-2 was a female-specific form and its concentration was low.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation sites of bovine brain myelin basic protein phosphorylated with Ca2+-calmodulin-dependent protein kinase from rat brain

The phosphorylation sites of myelin basic protein from bovine brain were determined after phosphorylation with Ca2+-calmodulin-dependent protein kinase. Four phosphorylated peptides were selectively and rapidly separated by reversed-phase high-performance liquid chromatography. Partial sequencing of the phosphorylated peptides by automated Edman degradation revealed that Ca2+-calmodulin-dependent protein kinase phosphorylated serine-16, serine-70, and threonine-95 specifically, as well as serine-115, which is located on the experimental allergic encephalitogenic determinant of the protein. Of the four amino acid sequences determined, two sequences surrounding phosphorylated amino acids, -Lys-Tyr-Leu-Ala-Ser(P)16-Ala- and -Arg-Phe-Ser(P)115-Trp-Gly-, have both sides of each phosphoserine residue occupied by hydrophobic amino acids, and a basic amino acid, arginine or lysine, is located at the position 2 or 4 residues amino-terminal to the phosphoserine residue. In contrast, the two other sequences surrounding phosphorylated amino acids, -Tyr-Gly-Ser(P)70-Leu-Pro-Glu-Lys- and -Ile-Val-Thr(P)95-Pro-Arg-, have a basic amino acid at the position 2 or 4 residues carboxyl-terminal to the phosphoamino acid residue.     

Extraction and composition of polar lipids from the archaebacterium, Methanobacterium thermoautotrophicum: effective extraction of tetraether lipids by an acidified solvent

The usual Bligh and Dyer method could extract only a small part of the lipids of Methanobacterium thermoautotrophicum. When the water in the solvent was replaced by 5% trichloroacetic acid, the lipid recovery reached the maximum level, which was 6 times higher than that by the former method. The use of HCl (2 M) or disruption of cells was also effective but prolonged extraction with the HCl-containing solvent caused degradation of some phosphoglycolipids. Twenty-three spots of polar lipids were detected on a thin-layer chromatogram of the total lipid. These were 10 phospholipids (18%), 6 aminophospholipids (17%), 3 aminophosphoglycolipids (15%), 2 phosphoglycolipids (31%), and 2 glycolipids (19%). The predominant polar lipids were a highly polar phosphoglycolipid (PGL1, 30%) and a glycolipid (GL1a, 16%). The other major lipids included an aminophospholipid (PNL1a, 9%), and an aminophosphoglycolipid (PNGL1, 7%). The complete structure determination of PNL1a, GL1a, and PNGL1 is described in the accompanying paper. Acetolysis of the total lipids followed by acid methanolysis was required for the complete cleavage of polar head groups, releasing core residues of diphytanyl glycerol diether (C20 diether) and dibiphytanyl diglycerol tetraether (C40 tetraether). A densitometric assay of a thin-layer chromatogram showed that the ratio of C20 diether and C40 tetraether was 1:14. GLC analysis of alkyl chlorides prepared from the total lipid by BCl3 treatment showed that phytanyl (C20), biphytanyl (C40), and unidentified alkyl chains accounted for 10, 83, and 7 mol% of the total alkyl chains, respectively. Strong acid hydrolysis of the macromolecular residue obtained after lipid extraction gave a significant amount of C40 tetraether, which had probably been bound covalently to other substances in the cells.     

Calcium influx in a single rat basophilic leukemia cell as revealed with a digital imaging fluorescence microscope

Using a digital imaging fluorescence microscope, we have detected a rapid transient increase in the free cytosolic calcium concentration in a single rat basophilic leukemia cell (RBL-2H3) after antigen stimulation. Calcium ions were transported very rapidly (within 1 s) after a lag time (about 10 s at 37 degrees C) from the external environment into the cytoplasm. On the basis of the present experimental results we conclude that the gradual changes in the overall fluorescence intensity observed for a cell suspension are due to the distribution of different lag times shown by different cells as to the calcium influx through membrane calcium channels.     

Coupling between phosphoinositide breakdown and early mitogenic events in fibroblasts. Studies with fluoroaluminate, vanadate, and pertussis toxin

In the preceding paper (Paris, S., and Pouysségur J. (1987) J. Biol. Chem. 262, 1970-1976), AlF4- and vanadate have been shown to induce inositol phosphate formation in resting hamster fibroblasts (CCL39). In this study, we show that these two phosphate analogs are good tools to explore the causal relationship between phosphoinositide breakdown and early mitogenic events. AlF4- can activate, very similarly to the mitogen alpha-thrombin: the amiloride-sensitive Na+/H+ antiport, the bumetanide-sensitive Na+/K+/Cl- co-transport, and the expression of c-myc mRNA. The link between phospholipase C activation and these early events of the mitogenic response is demonstrated by the similarity of all dose-response curves for NaF and AlCl3 and by the common sensitivity of the four events to pertussis toxin. Vanadate likewise stimulates the Na+/H+ antiport through a pertussis toxin-sensitive pathway. On longer incubations, both fluoride and vanadate were found to be toxic and failed to induce DNA synthesis. Therefore, we have used pertussis toxin to investigate the link between phospholipase C activation and commitment to DNA synthesis. We show that pertussis toxin strikingly inhibits thrombin-induced reinitiation of DNA synthesis but does not affect the stimulation by the epidermal or fibroblast growth factors, two mitogens that do not stimulate phosphoinositide breakdown in CCL39 cells. In conclusion, these studies demonstrate that activation of phospholipase C, if not an obligatory step in the action of all growth factors, plays a crucial role in the mitogenic signaling pathway of alpha-thrombin.     

cDNA cloning and amino acid sequence of bovine brain 2',3'-cyclic-nucleotide 3'-phosphodiesterase

2',3'-Cyclic-nucleotide 3'-phosphodiesterase (EC 3.1.4.37) has been widely used as a marker for myelin-oligodendrocytes in the central nervous system. Evidence has been provided that the enzyme is identical with one of the Wolfgram proteins of central nervous system myelin. The amino acid sequence of bovine 2',3'-cyclic-nucleotide 3'-phosphodiesterase was determined by both protein and cDNA sequence analyses. Protein sequence analysis was done on bovine elastase 2',3'-cyclic-nucleotide 3'-phosphodiesterase, a low molecular weight enzyme obtained by solubilization with pancreatic elastase (EC 3.4.21.36) (Nishizawa, Y., Kurihara, T., and Takahashi, Y. (1980) Biochem. J. 191, 71-82; Kurihara, T., Nishizawa, Y., Takahashi, Y., and Odani, S. (1981) Biochem. J. 195, 153-157). Based on the carboxyl-terminal sequence of bovine elastase 2',3'-cyclic-nucleotide 3'-phosphodiesterase, synthetic oligodeoxyribonucleotides were prepared and used as probes for screening a cDNA library of bovine brain. A cDNA of 2305 base pairs was obtained and sequenced, and the complete amino acid sequence of bovine 2',3'-cyclic-nucleotide 3'-phosphodiesterase was deduced. Bovine 2',3'-cyclic-nucleotide 3'-phosphodiesterase deduced contains 400 amino acids including initiation methionine and has a molecular weight of 44,850. Bovine elastase 2',3'-cyclic-nucleotide 3'-phosphodiesterase corresponds to the 236 amino acids of bovine 2',3'-cyclic-nucleotide 3'-phosphodiesterase. RNA blot analysis revealed a single-species mRNA of about 2600 bases.     

Effect of forskolin on collagen production in clonal osteoblastic MC3T3-E1 cells

The effect of forskolin on collagen production in osteoblasts was investigated by using clonal osteoblastic MC3T3-E1 cells cultured in a-minimum essential medium containing 0.1% bovine serum albumin. Forskolin increased the adenylate cyclase activity in membranes pelleted from homogenates of the cell line in a dose-dependent manner. The drug caused a 13-fold stimulation at 10(-4) M, indicating that the compound directly acts on adenylate cyclase, leading to an increase in the intracellular cAMP content of the cells. Collagen accumulation in the cultures was elevated by one-day treatment with 5 X 10(-5) M forskolin to about twice that in the controls. The stimulation was mainly due to an elevation in collagen synthesis but not to an inhibition of intracellular collagen degradation because forskolin dose-dependently increased collagen synthesis; it also significantly increased the amount of low-molecular-weight hydroxyproline found in the cultures. Cells treated with forskolin produced mainly type I collagen, as found in bone matrix in situ, with only small amounts of other types of collagen. Furthermore, forskolin time-dependently inhibited DNA synthesis in the cells, indicating that the increase in type I collagen synthesis by forskolin was not due to stimulated cell proliferation. These results suggest that cAMP is closely linked to the differentiation of osteoblasts in vitro.     

Identification of I:A mismatch base-pairing structure in DNA

Deoxyoligonucleotides containing deoxyinosine residues at positions corresponding to ambiguous nucleotides derived from an amino acid sequence have been successfully used as hybridization probes. It is assumed that the hypoxanthine residue can make base pairs with multiple bases. In order to obtain direct evidence for I:A base-pairing, a self-complementary deoxyoligonucleotide, d(G-G-I-A-C-C), was synthesized and its properties were examined by NMR spectroscopy. Three hydrogen-bonded imino proton resonances are observed at low temperatures in H2O suggesting the formation of a self-duplex with complete base pairing. Nuclear Overhauser effect (NOE) experiments showed that a signal at 15.1 ppm originated from the imino proton (H1) of the dI residue (I3) which is hydrogen-bonded to the dA residue (A4). Both the I3 and A4 residues were assumed to have taken an anti glycosidic conformation since irradiating the H1 of I3 gave NOEs both to its own H2 and to that of A4, an NOE also being observed between the H2 protons of I3 and A4. Comparison of the 31P NMR spectra of d(G-G-I-A-C-C) and d(G-G-I-C-C-C) showed the backbone structure of d(G-G-I-A-C-C) to have been disturbed by the presence of purine:purine base pairs in the middle of the hexamer duplex.     

Cloning and sequence analysis of adrenodoxin reductase cDNA from bovine adrenal cortex

cDNA clones for bovine adrenodoxin reductase were isolated, and the primary structure of the enzyme precursor was deduced from their nucleotide sequences. The precursor consists of 492 amino acids including an extrapeptide of 32 amino acids at the amino terminus. The extrapeptide is hydrophilic [corrected] and rich in arginine. The amino terminal sequence of the precursor is homologous with that of the adrenodoxin precursor. A possible FAD- or NADPH-binding site is present near the amino terminus of the mature enzyme.