DNA biosynthesis in rat liver mitochondria: Inhibition by sulfhydryl compounds and stimulation by cytoplasmic proteins

The sulfhydryl compounds, 2-mercaptoethanol, dithiothreitol, cysteine. and glutathione inhibit the incorporation of [³H]dTTP or [³H]dATP into mitochondrial DNA by rat liver mitochondria in vitro. The lack of inhibition by non-SH-containing analogs indicates that the SH group is responsible for the inhibition.The inhibition does not result from an effect of the sulfhydryl compounds on precursor permeability, ATP formation, or respiration, or the action of the thiol on the outer mitochondrial membrane. An intact inner membrane is not required for the action of the inhibitor. Furthermore, SH compounds do not appear to exert their effect by activation of a mitochondrial nuclease, chemical breakdown of high molecular-weight mitochondrial DNA or dissociation of membrane-bound DNA from the inner mitochondrial membrane. Incorporation of labeled precursor into DNA by mitochondrial DNA polymerase, when removed from the inner mitochondrial membrane, is not inhibited by SH compounds.Cytoplasmic extracts prepared from rat and mouse tumors and 22-h regenerating rat liver contain a protein(s) not detectable in normal rat liver which can reverse the inhibition by SH compounds of the synthesis of mitochondrial DNA in rat liver mitochondria in vitro.More importantly, when the stimulatory protein(s) is partially purified by affinity chromatography on DNA-cellulose, it is possible to demonstrate that this protein(s) also stimulates the synthesis of mitochondrial DNA by normal rat liver mitochondria in vitro in the absence of the sulfhydryl inhibitor.     

Resolution and reconstitution of soluble components of rat liver microsomal vitamin D3-25-hydroxylase

Solubilized components of the vitamin D₃-25-hydroxylase, isolated from intact rat liver microsomes known to catalyze the C-25 oxidation of vitamin D₃in vitro, have been separated into two submicrosomal fractions enriched in detergent-solubilized NADPH-cytochrome c reductase and cytochrome P-450 or P-448. The P-450 hemoprotein-containing fraction was obtained by solubilization with cholic acid followed by treatment with the nonionic detergent, Emulgen 911, yielding a final preparation with a specific content of 7.25 nmol/mg microsomal protein. The reduced triphosphopyridine nucleotide-dependent cytochrome P-450 reductase activity, as detected by its ability to reduce the artificial electron acceptor, cytochrome c, was isolated free of cytochromes b₅ or P-450 by solubilization with deoxycholate and chromatography on DEAE-cellulose. The reductase component was found to exhibit kinetic properties with Michaelis constants: K_m(NADPH) = 3.14 μM, K_m(NADH) = 31.25 μM, and K_{m(cyt c)} = 12.34 μM. The NADPH-cytochrome c reductase activity was sensitive to NADPH-reversible inhibition by NADP, but not rotenone or cyanide. When the isolated components were incubated in the presence of an NADPH-generating system and carbon monoxide under anaerobic conditions, enzymatic reduction of the P-450 hemoprotein was measured by the appearance of characteristic absorbances at 420 and 450 nm of the reduced carbon monoxide vs. reduced difference spectrum. Furthermore, when the soluble submicrosomal components were reconstituted with excess reduced triphosphopyridine nucleotide, ³H-labeled vitamin D₃, and soluble cytosolic supernatant, full vitamin D₃-25-hydroxylase activity was restored at rates of up to 7.68 pmol/h/mg protein, with an apparent turnover number of cytochrome P-450 of 1.16 to 1.20 under conditions where the concentrations of the hemoprotein were rate limiting for net product formation. These results strongly support the hypothesis that the rat liver microsomal mixed-function oxidase, vitamin D₃-25-hydroxylase, consists of at least two membrane-bound protein components, NADPH-cytochrome c reductase and a cytochrome P-450 terminal oxidase, for the catalytic conversion of vitamin D₃ to 25-hydroxyvitamin D₃.     

Myelin proteolipid protein is not esterified at the site of synthesis

Brain slices prepared from 20-day old rats were incubated with [³H]palmitic acid to study its incorporation into myelin proteins. After separation by SDS-PAGE, most of the label was found to be associated with the major proteolipid protein (PLP) and with the intermediate protein (I). The radioactivity measured in PLP at short incubation times was shown to be due to palmitic acid bound to the protein by ester linkages. Time-course incorporation of [³H]palmitic acid into PLP of fraction SN₄ (a myelin like membrane) and of purified myelin showed that the former was poorly labeled and no relationship of the type ‘precursor-product’ between these fractions could be detected. Incorporation of the fatty acid into PLP was not affected by inhibition of the synthesis or transport of myelin PLP with cycloheximide or colchicine, indicating that the pool of PLP that can be acylated must be larger than the extramyelin pool. Addition of unlabeled palmitic acid to the incubation medium, 30 min after the addition of [³H]palmitate, stopped the appearance of label in myelin PLP almost immediately, indicating that there is no significant extramyelin pool of PLP destined for transport into myelin. The results presented in this paper strongly suggest that esterification of PLP takes place in the myelin membrane or at a site very close to it.     

Influence of concanavalin A on protein synthesis and protein release in BHK 21 cells

Addition of concanavalin A to baby-hamster kidney cells (strain BHK 21/C13) grown in Eagle's minimal essential medium devoid of serum but supplemented with insulin as growth-promoting factor, caused a marked reduction of the total protein content of the cells: as early as 1 h after treatment, the amount of protein decreased to about 60–70% of the values found in untreated cultures.Pulse-labelling experiments performed with [³H]leucine demonstrated that the uptake and incorporation of the labelled amino acid was not affected by the lectin up to 6 h after treatment. Pulse-chase experiments gave no evidence for an enhanced degradation of proteins.Examination of the supernatants of concanavalin A-treated cultures as well as their controls, pre-labelled with [³H]fucose and [¹⁴C]leucine revealed that the amount of membrane-derived glycoproteins which were shed into the culture medium was considerably higher in concanavalin A-treated cultures.However, the bulk of protein which accounts for the difference between lectin-treated and untreated cultures consists of intracellular material which was released during the cell harvest procedure. The loss of protein was prevented by α-methyl-d-mannoside (10^?2 M).Scanning electron microscopy of concanavalin A-treated cells showed a change from the smooth surface of the fibroblastic cells to a retracted one as early as 30 min after addition of the lectin. The surface of the altered cells was characterized by the presence of numerous bleb-like protuberances.The viability of the cells was not affected by concanavalin A-treatment during the course of the experiments.Experiments performed with variable concentrations of insulin excluded the possibility that the observed effects might be due to a competition between the lectin and the hormone.     

Vitamin E dependent reduced glutathione inhibition of rat liver microsomal lipid peroxidation

Effects of reduced glutathione (GSH) were investigated on $\text{in}$$\text{vitro}$ lipid peroxidation of hepatic microsomes obtained from Long-Evans Hooded rats fed chemically defined, purified diets containing adequate or documented deficiencies of vitamin E (E), selenium (Se) or both. Glutathione inhibited lipid peroxidation mediated by both NADPH-dependent enzymatic and ascorbate-dependent non-enzymatic systems. The inhibitory effect of GSH was observed in microsomes obtained from E supplemented groups whereas it had no effect on microsomes from E deficient animals. Selenium status had no effect on GSH inhibition. Glutathione was found to be specific for the E dependent inhibition of lipid peroxidation and could not be substituted by other sulfhydryl compounds tested. Also, GSH did not inhibit non-enzymatic lipid peroxidation of heat-denatured microsomes from either E-supplemented groups or any of the other dietary regimens.     

Lipid peroxidation increases the molecular order of microsomal membranes

The effect of enzymatic lipid peroxidation on the molecular order of microsomal membranes was evaluated by ESR spectroscopy using the spin probes 5-, 12-, and 16-doxyl-stearic acid. Rat liver microsomal membranes were peroxidized by the NADPH-dependent reaction in the presence of the chelate ADP-Fe3+. Peroxidation resulted in a preferential depletion of polyenoic fatty acids and an increase in the percentage composition of shorter fatty acyl chains. There was no change in the cholesterol/phospholipid ratio of the peroxidized microsomes. The molecular order of both control and peroxidized membranes decreased toward the central region of the bilayer, and the order parameter (S) of each probe was temperature dependent. Peroxidation of the microsomal membrane lipids resulted in an increase in the order parameter determined with the three stearic acid spin probes. Of the three probes, 12-doxylstearic acid was the most sensitive to the changes in membrane organization caused by peroxidation. These data indicate that ESR spectroscopy is a sensitive method of detecting changes in membrane order accompanying peroxidation of membrane lipids.     

The reversal of inhibition of protein synthesis by double-stranded RNA in lysed rabbit reticulocytes with fructose 6-phosphate.

Fructose 6-phosphate (1.4 mM – 3.0 mM) effectively prevents the inhibition of protein synthesis in unfractionated rabbit reticulocyte lysates by the presence of double-stranded RNA (poly rI:poly rC, 1 μg/ml). Glucose 6-phosphate, but not fructose 1,6-diphosphate, is equally as effective as fructose 6-phosphate. The data suggest that fructose 6-phosphate prevents the formation of a protein synthesis inhibitor induced by double-stranded RNA.     

The synthesis of α-isomalto-oligosaccharide derivatives and their protein conjugates

2-[4-(p-Toluenesulfonamido)phenyl]ethyl 2,3,4-tri-O-benzyl-α-D-glucopyranoside was condensed with 2,3,4-tri-O-benzyl-6-O-(N-phenylcarbamoyl)-1-O-tosyl-D-glucopyranose to give 2-[4-(p-toluenesulfonamido)phenyl]ethyl 2,3,4,2′,3′,4′-hexa-O-benzyl-6′-O-(N-phenylcarbamoyl)α-isomaltoside. The disaccharide was decarbanilated in ethanol with sodium ethoxide. The sequence of coupling with the 1-O-tosyl-glucose derivative followed by decarbanilation was repeated to form the tri- and tetra-saccharide derivatives. The di-, tri-, and tetra-oligo-saccharides, were deblocked with sodium in liquid ammonia to give the 2-(4-aminophenyl)ethyl α-isomalto-oligosaccharides, which were diazotized with sodium nitrite in acid, and then coupled to bovine serum albumin and edestin to give the protein conjugates.     

DNA Replication by a membrane-DNA complex from rat liver mitochondria

The results presented here indicate that mitochondrial DNA (mtDNA) synthesis occurs on the inner mitochondrial membrane and that a membrane-DNA complex, enriched in newly synthesized DNA, can be isolated. The complex is able to synthesize DNA in vitro. Enrichment studies demonstrated that mtDNA synthesis occurs on an intact membrane-DNA complex in vitro and that pulse-labeled mtDNA could be chased from the membrane-DNA complex to the top fraction of the discontinuous sucrose gradient. The membrane-DNA complex was also shown to carry out replicative synthesis of mtDNA in vitro. Replication was shown to be asynchronous with heavy-strand synthesis preceding light-strand synthesis. The progression of mtDNA replication by the membrane-DNA complex was shown to be from small fragments (<13 S) to larger fragments (14–24 S) liberated from closed circular molecules, to a heat-stable 27 S molecule, and finally to a 38 S heat-stable molecule. The time estimated to progress from small fragments to the 38 S molecule is 120 min.     

Influence of free fatty acid anion on the binding of warfarin to cytoplasmic proteins from rat liver

In vitro binding studies have shown that warfarin binds strongly to both ligandins (Y) and Z protein obtained from rat liver cytosol with dissociation constants of 11.7 and 10.1 μM respectively. Increasing concentrations of oleate ion significantly increased the dissociation constant of warfarin with either protein, whereas laurate ion showed the same behavior only with Z protein. On the other hand, the binding of warfarin to liver cytoplasmic proteins in vivo was decreased in 72-h-pre-fasted rats, although such fasting failed to produce any increase in the in vivo levels of the cytoplasmic free fatty acids (FFA). However, based on the results of the in vitro binding study, it is suggested that changes in the composition of hepatic cytoplasmic free fatty acids as a result of fasting could reduce the in vivo binding of warfarin to Y and Z proteins and hence could lead to an increase of unbound warfarin in liver cytosol.     

Purification of S-2-hydroxyacylglutathione hydrolase (glyoxalase II) from rat erythrocytes.

Glyoxalase II, a specific glutathione thiolesterase, has been purified 9100-fold from rat erythrocytes using a purification scheme which employs Affi-Gel blue as a hydrophobic affinity column and also employs a glutathione-affinity column prepared by coupling S-(p-chlorophenacyl)glutathione to Affi-Gel 202. This procedure offers a convenient method for the preparation of highly purified glyoxalase II. Also described is a convenient method for the preparation of S-lactoyl-glutathione, a substrate for glyoxalase II.     

Purification and partial characterization of the catalytic subunit of cAMP-dependent protein kinases from reticulocytes.

The catalytic subunit of cyclic AMP-dependent protein kinases from rabbit reticulocytes has been purified to near homogeneity. It has a molecular weight of 43,000 as judged from gel filtration and by polyacrylamide gel electrophoresis in the presence of sodium dodecyi sulfate and appears to be similar in physical properties and substrate specificity to the comparable enzyme isolated from muscle or liver. The enzyme phosphorylates histones, a protein of 40 S ribosomal subunits from reticulocytes and from Artemia salina, and the low molecular weight heat-stable phosphatase inhibitor (G. A. Nimmo and P. Cohen, 1978, Eur. J, Biochem.87, 341–351). No evidence has been obtained for a direct or indirect role of this enzyme in the regulation of protein synthesis.     

Occurrence of NI and NII type protein kinases in the nuclei from various tissues of the rat

Cyclic nucleotide-independent protein kinases that preferentially phosphorylated casein and phosvitin as substrate were surveyed in various tissue nuclei of the rat. Enzymes were extracted from the isolated nuclei of liver, kidney, spleen, brain, heart, or testis tissue with a buffer solution containing 0.4 m NaCl, and analyzed by DEAE-Sephadex, phosphocellulose, and Bio-Gel A-1.5m column chromatographies. The chromatographic study together with characterization of the enzymes demonstrated that all the tissues contained in their cell nuclei commonly two protein kinases, the NI and NII types, and that these were exclusively found as main nuclear casein kinases. NII enzyme activity was stimulated by polyamines and strongly inhibited by heparin. By contrast, the NI enzymes were little influenced by these compounds. We interpret the present results as suggesting that NI and NII type protein kinases may be found in the cell nuclei from many tissues of rat, and have distinct functions in the cell nuclei.     

Structural studies of a mitochondria-free plasma membrane fraction from rat liver

Liver plasma membranes virtually free of contaminating mitochondria have been prepared. Sodium dodecylsulfate-polyacrylamide gel electrophoresis reveals a membrane protein resistant to papain digestion in the intact membranes but readily hydrolyzed in membranes disrupted by detergent or sonication.Electron microscopy of mechanically deformed membranes reveals fibrils within the membrane which appear to be protein in nature but which also persist in papain digested membranes.     

A biochemical study of flagellar dynein from starfish spermatozoa: protein components of the arm structure.

Half of the adenosine triphosphatase (dynein) activity of starfish sperm tail axonemes was extracted with 0.6 m KCl-10 mm Tris · HCl (pH 7.8)-0.1 mm EDTA-0.5 mm dithiothreitol (KCl-EDTA), while with 1 mm Tris · HCl (pH 7.8)-0.1 mm EDTA-0.5 mm dithiothreitol (Tris-EDTA) around 90% of the activity was extracted. The main adenosine triphosphatase (ATPase) in the KCl-EDTA extract had a sedimentation coefficient of 20S and that in the Tris-EDTA extract had a sedimentation coefficient of 12S. The effects of divalent cations, pH, and an SH-blocking reagent and the K_m for ATP were different for the activities of the two forms of dynein ATPase. These two forms of dynein can interconvert to some extent when the ionic strength of the medium is changed. In a medium suitable for recombination of dynein to outer doublet microtubules (recombination buffer, 20 mm Tris-HCl (pH 7.6)-2 mm MgCl₂-0.5 mm dithiothreitol), the 20S ATPase converted to a 24S ATPase. Recombination of the ATPase activity from the KCl-EDTA extract was almost complete while that from the Tris-EDTA extract was around 50%. Outer arms disappeared preferentially by the treatment with KCl-EDTA, and the extracted arms could be reconstituted in the recombination buffer. In the case of the Tris-EDTA extraction, both the outer and inner arms disappeared and the reconstitution of the arms could not be confirmed. From the above results it can be considered that the 20 or 24S dynein represented the arm structure. The 20 or 24S ATPase fraction contained two large polypeptide chains as main components having electrophoretic mobilities in the presence of sodium dodecylsulfate similar to those of Tetrahymena ciliary dyneins and of sea urchin sperm flagellar dyneins. The relationship between these chains and dynein subunits is discussed.