Identification of mammalian aminotransferases utilizing glyoxylate or pyruvate as amino acceptor. Peroxisomal and mitochondrial asparagine aminotransferase

The subcellular distribution of asparagine:oxo-acid aminotransferase (EC 2.6.1.14) in rat liver was examined by centrifugation in a sucrose density gradient. About 30% of the homogenate activity after the removal of the nuclear fraction was recovered in the peroxisomes, about 56% in the mitochondria, and the remainder in the soluble fraction from broken peroxisomes. The mitochondrial asparagine aminotransferase had identical immunological properties with the peroxisomal one. Glucagon injection to rats resulted in the increase of its activity in the mitochondria but not in the peroxisomes. Immunological evidence was obtained that the enzyme was identical with alanine:glyoxylate aminotransferase 1 (EC 2.6.1.44) which had been reported to be identical with serine:pyruvate aminotransferase (EC 2.6.1.51) (Noguchi, T. (1987) in Peroxisomes in Biology and Medicine (Fahimi, H. D., and Sies, H., eds) pp. 234-243, Springer-Verlag, Heidelberg). The same results as described above were obtained with mouse liver. All of alanine:glyoxylate aminotransferase 1 in livers of mammals other than rodents, which cross-react with the antibody against rat liver alanine:glyoxylate aminotransferase 1, had no asparagine aminotransferase activity.     

Structure and mapping of the fosB gene. FosB downregulates the activity of the fosB promoter.

We have determined the genomic structure of the fosB gene and shown that it consists of 4 exons and 3 introns at positions also found in the c-fos gene. By deletion analysis we have characterized a region upstream of the TATA box which is the promoter region of the gene. Several consensus sequences have been identified, including an SRE and AP-1 binding site whose relative positions are identical to those in the 5' upstream region of the c-fos gene. We have also shown that FosB and c-Fos can downregulate the activity of the fosB promoter to a similar extent. The fosB gene is located in the [A1-B1] region of mouse chromosome 7.     

Detection of tetrodotoxin by thin-layer chromatography/fast atom bombardment mass spectrometry

A new method for detection of tetrodotoxin (TTX) by thin-layer chromatography/fast atom bombardment (FAB) mass spectrometry was developed. TTX and/or related substances were separated by TLC on LHP-K high-performance precoated plates, with a solvent system of pyridine:ethyl acetate:acetic acid:water (15:5:3:4). The plates were subjected to positive FAB mass spectrometry, under scanning within a mass range from m/z 100 to 500. TTX was identified by selected ion-monitored chromatograms at m/z 320 (M + H)+ and 302 (M + H - H2O)+, along with full scan positive ion FAB mass spectrometry. The limit of detection for TTX was about 0.1 micrograms. TTX was also detected by cellulose acetate membrane electrophoresis/FAB mass spectrometry.     

Sensitive sandwich enzyme immunoassay of human growth hormone (hGH) in serum and urine using monoclonal antibody-coated polystyrene balls

A sensitive sandwich enzyme immunoassay for human growth hormone (hGH) using monoclonal antibody is described. A monoclonal anti-hGH IgG-coated polystyrene ball was incubated with hGH and subsequently with affinity-purified rabbit anti-hGH Fab'-horseradish peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was assayed by fluorimetry using 3-(4-hydroxyphenyl) propionic acid as a substrate. The detection limits of hGH in serum and urine were 1.5 ng/l using 20 microliters of serum and 0.2 ng/l using 0.15 ml of urine, respectively. The specificity and assay precision were satisfactory. hGH levels in serum and urine determined by the present sandwich enzyme immunoassay using monoclonal anti-hGH IgG-coated polystyrene balls were well correlated to those determined by the previous sandwich enzyme immunoassay using rabbit anti-hGH IgG-coated polystyrene balls. Levels of hGH in urine collected as first morning voids from healthy subjects aged 19-28 yr were 6.4 +/- 3.2 (SD) ng/g creatinine. However, the present assay gave lower hGH levels than the previous assay. This was at least partly explained by the fact that hGH in urine was less efficiently bound to monoclonal anti-hGH IgG-polystyrene balls than standard hGH, while the binding of hGH in urine and standard hGH to rabbit anti-hGH IgG-coated polystyrene balls was equally efficient. In addition, gel filtration showed that 22K hGH, a major component, in urine was less efficiently bound to monoclonal anti-hGH IgG-coated polystyrene balls than standard 22K hGH. The nature of hGH in serum and urine remains to be investigated.     

A single amino acid substitution converts cytochrome P450(14DM) to an inactive form, cytochrome P450SG1: complete primary structures deduced from cloned DNAS

Genes for lanosterol 14-demethylase, cytochrome P450(14DM), and a mutated inactive cytochrome P450SG1 were cloned from S. cerevisiae strains D587 and SG1, respectively. A single nucleotide change resulting in substitution of Asp for Gly-310 of cytochrome P450(14DM) was found to have occurred in cytochrome P450SG1. In this protein the 6th ligand to heme iron is a histidine residue instead of a water molecule, which may be the ligand for the active cytochrome P450(14DM). Molecular models of the active sites of the cytochrome P450(14DM) and cytochrome P450SG1 were built by computer modeling on the basis of the known structure of that of cytochrome P450CAM whose crystallographic data are available. The mechanisms which may cause a histidine residue to gain access to the heme iron are discussed.     

Inhibition of cathepsin L-induced degradation of epidermal growth factor receptors by c-Ha-ras gene products

The inhibitory activities of c-Ha-ras gene products (p21s) toward several cysteine proteinases have been investigated. The activity of cathepsin L was inhibited by p21s most effectively while those of cathepsin B and papain were slightly inhibited by p21s. p21s did not show any inhibitory activity toward cathepsin H. In order to connect the protease-inhibitor activity of p21s with cell growth, the degradation of epidermal growth factor receptors (EGF-receptors) was investigated. EGF-receptors were preferentially cleaved by cathepsin L but not by cathepsin B or H. The cleavage of EGF-receptors by cathepsin L was inhibited by p21s dose-dependently. These results raise the possibility that p21s can suppress the degradation of growth-related proteins such as EGF-receptors and thereby affect cell growth.     

Cytochrome P-450C-M/F, a new constitutive form of microsomal cytochrome P-450 in male and female rat liver with estrogen 2- and 16 alpha-hydroxylase activity

A new cytochrome P-450 isozyme, P-450C-M/F, has been purified from untreated rat liver microsomes. The purified preparation was electrophoretically homogeneous and contained 12-15 nmol of P450/mg of protein and had a minimum molecular weight of 48,500. The NH2-terminal amino acid sequence of P-450C-M/F was different from that of other P-450's. Immunoblot analysis of microsomes demonstrated that P-450C-M/F was present in the liver of untreated male as well as female rats. Treatment of rats with phenobarbital, 3-methylcholanthrene, or beta-naphthoflavone did not induce P-450C-M/F. Cytochrome P-450C-M/F exhibited little activities of 7-ethoxycoumarin and 7-ethoxyresorufin O-deethylation or hydroxylation of arylhydrocarbon, testosterone, androstenedione, and progesterone. In contrast, it was highly active in N-demethylation of ethylmorphine and benzphetamine and in 2- and 16 alpha-hydroxylation of estrogens, particularly that of estradiol. These studies establish that cytochrome P-450C-M/F is constitutively present in both male and female rats and suggest that it may be involved in the oxidative metabolism of estradiol, particularly in the formation of estriol, the uterotropic metabolite of estradiol.     

Formation of 5 alpha-products as major C19-steroids in estrogen-responsive mouse Leydig cell tumor lines

Homogenates of estrogen-responsive mouse Leydig cell tumors (T 124958-R and T 22137) or 28- and 120-day-old mouse testes were incubated with [3H]progesterone or [14C]4-androstene-3,17-dione in the presence of NADPH, and progesterone metabolism and enzyme activities were estimated. The growth of T 124958-R tumor transplanted in BALB/c mice was markedly stimulated by estrogenization of host mice, but the growth of T 22137 tumor was evidently suppressed by the estrogenization. The major C21-17-OH-steroids and C19-steroids formed from progesterone by both tumors and the testes of immature mice were 5 alpha-steroids, such as 3 alpha,17-dihydroxy-5 alpha-pregnan-20-one, 5 alpha-androstane-3,17-dione, androsterone, 3 beta-hydroxy-5 alpha-androstan-17-one and 5 alpha-androstane-3 alpha,17 beta-diol. In contrast, the major steroids formed by the testes of adult mice were testosterone and 4-androstene-3,17-dione, and no or little 5 alpha-steroids were produced. 5 alpha-Reductase activities in both tumor cells (40-50 nmol/l X 10(8) cells per h) were also found to be approx. 5-6 times higher than that in Leydig cells of adult mouse testes (8 nmol/l X 10(8) Leydig cells per h), though 17-hydroxylase activity was much higher in the Leydig cells of adult testes (730 nmol/l X 10(8) Leydig cells per h) than in both tumor cells (1-7 nmol/l X 10(8) cells per h). Furthermore, the presence of significant amounts of endogenous androsterone and/or 5 alpha-androstane-3 alpha,17 beta-diol was demonstrated in both tumors by radioimmunoassay. The present results demonstrate for the first time that C19-5 alpha-steroids are major C19-steroid products (immature type of testicular androgen production) in Leydig cell tumor lines.     

Phototropism in Hypocotyls of Radish : I. Isolation and Identification of Growth Inhibitors, cis- and trans-Raphanusanins and Raphanusamide, Involved in Phototropism of Radish Hypocotyls

Three growth inhibitors which might be involved in phototropism of Sakurajima radish (Raphanus sativus var. hortensis f. gigantissimus Makino) hypocotyls, were isolated as crystalline forms from light-exposed radish seedlings and identified as cis- and trans-raphanusanins and 6-methoxy-2,3,4,5-tetrahydro-1,3-oxazepin-2-one (designated raphanusamide). The cis- and trans-raphanusanins inhibited growth of etiolated radish hypocotyls at concentrations higher than 1.5 micromolar, raphanusamide at concentrations higher than 20 micromolar.