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Escherichia coli expression vectors containing the trc promoter and the complete DNA sequence of either the precursor or the mature form of bovine adrenocortical cholesterol side chain cleavage cytochrome P450 (P450scc) were transformed into E. coli strain JM109 and transcription induced with isopropyl-beta-D-thiogalactopyranoside (IPTG). Immunoreactive cytochrome P450scc was produced using the plasmid containing the mature P450scc sequence but not with the plasmid containing the sequence of the precursor form of P450scc, even though P450scc RNA was detectable in both cases. The mature form of P450scc was detected spectrophotometrically in a reduced CO-difference spectrum in E. coli (40-60 nmol/liter culture). Cholesterol and hydroxylated derivatives (22-hydroxycholesterol and 25-hydroxycholesterol) produce a type 1 substrate-binding spectrum in IPTG-induced, transformed E. coli. The P450scc was found to be associated with the E. coli membrane fraction and the enzymatic activity of side chain cleavage of 25-hydroxycholesterol was reconstituted using solubilized membranes, in the presence of purified bovine adrenocortical adrenodoxin and NADPH-adrenodoxin reductase (turnover number; 15.4 nmol/min/nmol P450). This bacterial expression system provides functional P450scc, in the absence of other forms of P450, which can be used for evaluation of enzymatic and spectral properties of this mitochondrial P450 by site-directed mutagenesis.
The effects of calmodulin antagonists on the capacity of hydrogen-translocating shuttles were studied in the perfused rat liver. The capacity was estimated by measuring the changes in the rate of production of glucose from sorbitol during the oxidation of ethanol [T. Sugano, T. Ohta, A. Tarui, and Y. Miyamae. Am. J. Physiol. 251 (Endocrinol. Metab. 14): E385-E392, 1986]. Thyroxine given to intact rats increased the activity of alpha-glycerophosphate dehydrogenase (alpha-GPD). Glucocorticoid replacement in adrenalectomized rats decreased the activity of the alpha-GPD to values obtained after treatment with PTU. In either thyroxine-treated or steroid-replaced rats, the capacity of hydrogen-translocating shuttles increased markedly. However, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), trifluoperazine, and chlorpromazine inhibited the increased capacity in steroid-replaced rats and had no effect on the increased capacity in thyroxine-treated rats. W-7 inhibited the stimulatory effects of norepinephrine on the capacity of the malate-aspartate shuttle without inhibition of efflux of intracellular Ca2+. The stimulatory effects of vasopressin on the malate-aspartate shuttle were also inhibited by W-7, trifluoperazine, and chlorpromazine. The results suggest that the malate-aspartate shuttle may be regulated by Ca(2+)-calmodulin.
RNA editing in the mitochondria of kinetoplastid protoza involves the insertion and/or deletion of precise numbers of uridine residues at precise locations in the numbers of uridine residues at precise locations in the transcribed RNA of certain genes. These genes are known as cryptogenes. In this paper we study computational algorithms to search for unknown cryptogenes and for the associated templates for insertion of uridines, gRNA sequences. The pairwise similarity search algorithm of Smith and Waterman (1) is modified to study this problem. The algorithm searches for unknown gRNAs given the cryptogene sequence. The method is tested on 4 known cryptogenes from L.tarentolae which are known to have 7 associated gRNAs. The statistical distribution of the longest gRNA when comparing random sequences is derived. Finally we develop an algorithm to search for cryptogenes using amino acid sequences from related proteins.
Using subtractive hybridization to identify genes that are androgen regulated in the mouse epididymis, a number of cDNAs were identified that represented mitochondrial genes including cytochrome oxidase c subunits I, II, and III, cytochrome b, NADH dehydrogenase subunit 5, a region of the displacement loop, and the 16S rRNA. Northern blot analysis of RNA from intact, castrate, or testosterone-replaced epididymides confirmed that these mitochondrial mRNAs as well as the rRNA were androgen regulated with a 2- to 5-fold reduction in expression observed after 4 weeks castration with partial to full recovery to precastrate levels upon 4 weeks of testosterone replacement. In contrast to the mitochondrial genes, the expression of the RNA component of the mitochondrial RNA-processing endoribonuclease (RNAase MRP), a nuclear factor which is thought to be involved in the regulation of mitochondrial DNA synthesis, increased in the epididymis upon castration and then returned to precastrate levels after testosterone replacement. An examination of other androgen-responsive tissues showed that mitochondrial gene expression was also regulated by androgens in the kidney. The RNAase MRP RNA levels, however, showed an increase after castration only in the reproductive tissues (epididymis, vas deferens, and seminal vesicle) and not in the kidney. No correlative increase in mitochondrial DNA levels was observed for any of the tissues. Finally, an analysis of various mouse tissues as well as the different regions of the epididymis revealed large differences in mitochondrial mRNA levels. While for most tissues the mRNA levels correlated with the mitochondrial DNA content, the levels of the RNAase MRP RNA did not. Taken together, these findings not only show the large variations in mitochondrial gene expression between tissues but also demonstrate that the expression of mitochondrial genes and ultimately mitochondrial function are androgen regulated in the epididymis and kidney.
The effect of experimental folacin deficiency on the uptake and distribution of radioactive folic acid in the rat was investigated. Less radioactivity was taken up by livers of deficient rats than controls 24 hours after intraperitoneal injection of [3H]-folic acid, although more radioactivity was incorporated by the brain and kidneys of deficient rats. The distribution of radioactivity among the three folacin-binding proteins of rat liver cytosol and the binding protein of mitochondria was also studied. In deficiency, very little radioactivity was incorporated into cytosol binding proteins I and II, while more radioactivity was incorporated into cytosol binding protein II and the mitochondrial binding protein. A decrease in the endogenous folacin associated with all protein-bound and free forms was seen in deficiency with the major decrease coming at the expense of unbound folacin, and cytosol binding protein I. This latter protein may have a primary storage role in the liver.
Patients with a metabolic block in the conversion of THCA into cholic acid develop cirrhosis and hemolysis, and die of hepatic failure. In these patients, THCA is largely conjugated to taurine (tauro-THCA) and excreted instead of being converted into cholic acid. In the present study, the effects of tauro-THCA on hemolysis, bile flow, and hepatic morphology were evaluated in bile fistula rats. All rats infused with tauro-THCA at rates of 0.25, 0.50 or 0.75 micronmol/min developed hemolysis with hemoglobinuria. A direct toxic effect of tauro-THCA on washed human red blood cell membranes was demonstrated at a concentration of 8 X 10(-4) M. Liver biopsy sections from rats infused for a 2 hr period with tauro-THCA were examined by electron microscopy and showed dilation of the rough endoplasmic reticulum and distortion of mitochondrial membranes. Cholestasis was not induced, since tauro-THCA actually caused a greater choleretic response for a given rate of bile salt excretion than did taurocholate. This study raises the possibility that the clinical liver disease seen in patients with a metabolic block in the conversion of THCA into cholic acid may be caused by tauro-THCA.
Intraperitoneal injection of tritiated folic acid (PteGlu) into rats has revealed the presence of three separate protein fractions in the cytosol fraction of the liver and one in the mitochondria which bind folate derivatives. The proteins in the cytosol (cytosol I, II and III) have approximate molecular weights of 350,000, 150,000, and 25,000 and the protein in the mitochondria has an approximate molecular weight of 90,000 as estimated by gel filtration. The bound folate derivatives are primarily polyglutamate forms while cytosol II contains primarily bound 5-methyltetrahydrofolate polyglutamate derivatives. Little binding of radioactively labeled folic acid or 5-methyltetrahydrofolate to these fractions was observed when binding was carried out in vitro. Significant binding in vitro was observed, however, when a mixture of biosynthetically labeled natural folate derivatives was used. These proteins have not been purified, but cytosol III partially consists of the enzyme, tetrahydrofolate dehydrogenase (EC 22.214.171.124). Studies on the time course of folic acid incorporation into the liver showed that soon after injection nonmetabolized folic acid was bound to the plasma membrane fraction of the liver cell. It is suggested that at least one of the binding proteins in the cytosol may be involved in storage of the vitamin while the binding of nonmetabolized folic acid to the plasma membrane may reflect the existence of a carrier for folic acid transport into the cell.
An assay has been developed to study the steady-state kinetics of the reduction of cytochrome c by purified beef heart mitochondrial cytochrome c reductase (cytochrome bc(1) complex, complex III). An analogue of coenzyme Q(2) (2,3-dimethoxy-5-methyl-6-decylhydroquinone) was employed as an antimycin-sensitive reductant. The kinetics of reaction of ten different mono(4-carboxy-2,6-dinitrophenyl) derivatives of horse cytochrome c were determined. The modified proteins showed higher apparent K(m) values than the native protein and greater sensitivity to ionic strength, defining an interaction domain on cytochrome c for purified cytochrome c reductase. This interaction site is located on the front surface of the molecule (which contains the exposed heme edge) and surrounds the point at which the positive end of the dipole axis crosses the surface of the protein. The site is similar to that previously determined for mitochondrial cytochrome c oxidase and yeast cytochrome c peroxidase, suggesting that the primary interaction with redox partners is directed by the dipolar charge distribution on cytochrome c. The extensive overlapping of the interaction domains for the mitochondrial cytochrome c oxidase and reductase indicates that cytochrome c must be mobile in order to transfer electrons between them, depending on their relative positions in the membrane. Whether such mobility is necessary in intact mitochondria depends on whether the interactions with the complete membrane-bound system are the same as with the purified components.