Mitochondrial Supernatant Research Articles

Deperoxydation de l'acide hydroperoxylinoleique par la muqueuse intestinale

In the presence of glutathione, a homogenate of rat intestinal mucosa transforms linoleic acid hydroperoxide (LOOH) into the corresponding alcohol (LOH). The K m of the enzyme involved (a glutathione peroxidase) is 5.7 × 10 −6 m. The specific activity, measured as generated LOH, was found to be 0.058 μmol/mg protein/min, six times lower than that of the liver. A mitochondrial supernatant of the mucosal homogenate had 1.5 times the activity of the initial homogenate. The reduction of 1 mol LOOH requires 2 mol glutathione. Besides this enzymatic deperoxidation, 5% of the LOOH was decomposed in both the intestinal mucosa and liver by a non-enzymatic pathway, probably involving the Fe 3+ of the haemoproteins.

Effect of Vanadate, Molybdate, and Azide on Membrane-Associated ATPase and Soluble Phosphatase Activities of Corn Roots

The effects of vanadate, molybdate, and azide on ATP phosphohydrolase (ATPase) and acid phosphatase activities of plasma membrane, mitochondrial, and soluble supernatant fractions from corn (Zea mays L. WF9 x MO17) roots were investigated. Azide (0.1-10 millimolar) was a selective inhibitor of pH 9.0-ATPase activity of the mitochondrial fraction, while molybdate (0.01-1.0 millimolar) was a relatively selective inhibitor of acid phosphatase activity in the supernatant fraction. The pH 6.4-ATPase activity of the plasma membrane fraction was inhibited by vanadate (10-500 micromolar), but vanadate, at similar concentrations, also inhibited acid phosphatase activity. This result was confirmed for oat (Avena sativa L.) root and coleoptile tissues. While vanadate does not appear to be a selective inhibitor, it can be used in combination with molybdate and azide to distinguish the plasma membrane ATPase from mitochondrial ATPase or supernatant acid phosphatase.Vanadate appeared to be a noncompetitive inhibitor of the plasma membrane ATPase, and its effectiveness was increased by K(+). K(+)-stimulated ATPase activity was inhibited by 50% at about 21 micromolar vanadate. The rate of K(+) transport in excised corn root segments was inhibited by 66% by 500 micromolar vanadate.

Methodologic problems encountered in the assay of proteinases in Lewis lung carcinoma, a mouse metastasizing tumor.

The proteolytic activity in homogenates and extracts of subcellular fractions prepared from subcutaneous Lewis lung carcinoma was determined using proteins and synthetic peptides as substrates. The presence of cathepsin D, plasminogen activator, cathepsin B-, cathepsin G- and elastase-like enzymes was observed. No difference was revealed between the proteolytic activity in homogenates of Lewis lung carcinoma, at the growth stage examined, and in homogenates of normal lung. High specific activities were found in the lysosomal extract, whereas decreasing activities were found in the nuclear extract, the homogenate and the postlysosomal mitochondrial supernatant; no active or trypsin-activatable collagenase activity was detected. The presence in the tumor tissue of these enzymatic activities is in agreement with their proposed role in the process of metastasis. The lack of differences between homogenates of tumor and normal lung tissue suggests that the use of whole cells is required to selectively study tumor proteinases specifically involved in tumor malignancy.

Effects of manganese on carbohydrate metabolism and mitochondrial enzymes in rats.

The effect of daily intraperitoneal administration of Mn2+(4 mg/kg) was investigated on the metabolism of carbohydrates and certain enzymes involved in the oxidation of glucose in the rat liver and blood at the intervals of 30, 60 and 90 days after exposure. Mn2+ had no effect on the contents of blood reducing sugars and proteins, however the levels of pyruvic and lactic acids were reduced at 60 and 90 days after the metal treatment. The contents of liver glycogen and proteins remained unaffected while pyruvic acid content was decreased in Mn2+ treated rat liver throughout the experimental period. The activities of glycogen phosphorylase and lactate dehydrogenase decreased while that of phosphoglucoisomerase and glucose-6-phosphatase increased in the post mitochondrial supernatant at 60 and 90 days of Mn2+ exposure. The levels of hexokinase decreased and FDP-aldolase and fructose-1, 6-diphosphatase increased throughout the experimental period. The magnitude of alteration was found to be greater with the increase in the duration of Mn2+ treatment. Several of the mitochondrial enzymes in the liver were inhibited in the manganese exposed rats which may be responsible to inhibit the rate of dehydrogenation of Kreb cycle's intermediates along with the linked respiratory chain and eventually oxidation in the rat liver.

Isolation and characterization of subcellular membranes from canine stomach smooth muscle.

Subcellular membrane fractions were isolated from the circular muscle of the corpus of canine stomach by differential and isopycnic sucrose density gradient centrifugation. Differential centrifugation gave a mitochondrial fraction enriched (fourfold) in cytochrome c oxidase and a microsomal fraction enriched (fourfold) in 5'-nucleotidase and NADPH-cytochrome c reductase over postnuclear supernatant. On the basis of a study using continuous gradient, a discontinuous sucrose density gradient was prepared to yield F1 to F5 fractions. The F3 fraction at the interface of 18-32% (w/w) sucrose was maximally enriched (13-fold) in 5'-nucleotidase. The fraction contained very low levels of cytochrome c oxidase but did contain NADPH-cytochrome c reductase (eightfold enrichment). The F4 fraction, at the interface of 32-40% (w/w) sucrose, was maximally enriched in NADPH-cytochrome c reductase (12-fold) and cytochrome c oxidase (6-fold). The distribution of the azide-insensitive. ATP-dependent Ca2+ uptake correlated very well with that of 5'-nucleotidase but less well with NADPH-cytochrome c reductase and not at all with cytochrome c oxidase. Sodium azide and ruthenium red inhibited the ATP-dependent Ca2+ uptake by the mitochondrial fraction and postnuclear supernatant, but not by the F3 fraction. ATP-dependent Ca2+ uptake by the F3 fraction was inhibited by calcium ionophores A23187 and ionomycin, but not by the sodium ionophore, monensin. These results are consistent with the hypothesis that the plasma membrane plays a major role ih regulating intracellular Ca2+ concentration in canine corpus circular muscle.

Glutaric aciduria type II: in vitro studies on substrate oxidation, acyl-CoA dehydrogenases, and electron-transferring flavoprotein in cultured skin fibroblasts.

The previous biochemical evidence had suggested that glutaric aciduria type II (GA II) is due to deficient dehydrogenation of multiple short-chain acyl coenzyme A's (CoA's), bu the precise biochemical mechanism underlying this disease was unknown. We investigated substrate oxidation and in vitro activities of isovaleryl CoA- and butyryl CoA dehydrogenases as well as that of electron-transferring flavoprotein (ETF) in cultured skin fibroblasts from a patient with GA II. GA II cells have a markedly decreased ability to oxidize [1-14C]butyrate, [2-14C]lysine, and [2,14C]leucine (3, 9, and 9% of control values, respectively). Mitochondrial isovaleryl CoA- and butyryl CoA dehydrogenase activities in GA II cells were determined using a tritium release assay with [2,3-3H] acyl-CoA's as substrate. When an artificial electron acceptor, phenazine methosulfate (PMS) was not added in the assay media, these activities were 108 and 113% of controls, respectively. This represents the normal abilities of the dehydrogenases in GA II cells to bind the substrate and to catalyze tritium exchange between the bound substrate and solvent. When PMS was added to the assay mixture, these activities were 88 and 70% of control values, respectively, indicating that these enzymes can both dehydrogenate their substrates normally and then transfer electrons to an acceptor (PMS). ETF activity in mitochondrial sonic supernatants from GA II cells, as assessed by a newly devised method, was 159% of control values. These observations suggest that the acyl CoA dehydrogenases themselves and ETF are not defective in GA II. Therefore, the deficiency of another common gene product necessary for the function of all the affected acyl CoA dehydrogenases must be sought to explain the etiology of GA II.

Purification of lipoamide dehydrogenase from Ascaris muscle mitochondria and its relationship to NADH:NAD + transhydrogenase activity

Lipoamide dehydrogenase (NADH:lipoamide oxidoreductase EC 1.6.4.3) has been isolated from Ascaris suum muscle mitochondria. This activity has been purified to apparent homogeneity from both the pyruvate dehydrogenase complex and from 150,000 g mitochondrial supernatants which were devoid of pyruvate dehydrogenase complex activity. The enzymes from both sources exhibited similar kinetic, catalytic, and regulatory properties and appear to be identical as judged by polyacrylamide gel electrophoresis. The native enzyme acts as a dimer, containing 2 mol of FAD, and has a subunit molecular weight of 54,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel chromatography. The enzyme also possesses substantial NADH:NAD + transhydrogenase activity. Heat denaturation and differential solubilization experiments imply that the transhydrogenase activity previously reported is, in fact, associated with the lipoamide dehydrogenase moiety of the Ascaris pyruvate dehydrogenase complex. Whether or not this activity functions physiologically in hydride ion translocation, as previously suggested, remains to be demonstrated.

Preparation of liver microsomes with high recovery of endoplasmic reticulum and a low grade of contamination

A modified procedure for preparing the microsomal fraction from rat liver was developed with the aim of increasing the recovery without increasing the degree of contamination. 87% of the membranes of the microsomal fraction isolated from the first mitochondrial ( 10 000 × g) supernatant originates from the endoplasmic reticulum, representing a 35% yield. By gentle resuspension of the 10 000 × g pellet followed by differential centrifugation a second crop of microsomes can be prepared which, together with the first crop, gives a 55% total recovery of microsomal markers. 87% of the protein in this second crop also originates from the endoplasmic reticulum and this fraction has properties similar to those of the first crop. Contaminating membranes include Golgi membranes (0.6% of the total protein), mitochondria (2.5%), lysosomes (5%) and plasma membranes (5%). Collecting further crops increases the contamination. Subfractionation studies revealed almost identical distributions of ribosome-rich, ribosome-poor and smooth membranes in the two crops of microsomal fractions. The results obtained after treatment of the animals with phenobarbital or methylcholantrene were similar to those obtained with control animals; but in the case of methylcholantrene treatment the second crop represents a larger portion of the total membranes of the endoplasmic reticulum.

Effect of sclerin on amino acid incorporation into mitochondria isolated from rat liver.

Though sclerin (SCL) stimulated amino acid incorporation into the protein fraction of post mitochondrial supernatant of rat liver homogenate, it had no effect on the incorporation into the isolated mitochondria at pH 7.2, despite of its stimulating effect on mitochondrial oxidative phosphorylation. SCL stimulated amino acid incorporation into the mitochondria at pH 6.1, and to some extent maintained the activity on that in mitochondria during aging in hypotonic Tris-HCl buffer (pH 7.2). Since SCL prevented leakage of amino acids from the mitochondria into these buffers, it was suggested that SCL may protect a structure of mitochondrial membrane which appeared to have a significance on transport of amino acids. In liver slices, SCL stimulated amino acid incorporation only into the extra-mitochondrial fraction for the first 3 min, but gradually turned to stimulate incorporation into mitochondria within 30 min.

Membrane 32P-phospholipid labeling in early developing toad embryos

Endogenously labeled toad oocytes and early developing embryos were employed to evaluate the phospholipid metabolism in whole embryos and in subcellular fractions. The whole embryo and postmitochondrial supernatant display a linear increase in relative specific activity as development proceeds, although higher values were reached by mitochondrial and yolk platelet fractions. In all the subcellular fractions the half-lives of the acid-soluble pool were similar and ranged from 2.5 to 3.5 days. Conversely the phospholipid half-lives were only similar in mitochondrial and postmitochondrial supernatant fractions.

A sensitive [formula omitted] protein synthesizing system from ehrlich ascites mitochondria

The S-25 fraction prepared from digitonin washed mitochondria is highly active in poly(U) directed phenylanine incorporation when supplemented with t-RNA. Ribosomes prepared from the S-25 fraction contain 58S monomeric ribosomes and 40S and 29S subunits. Further, these ribosomes contain 21S and 13S rRNA. No detectable cytoplasmic specific ribosomal particles and also rRNA was detected in the mitochondrial S-25 preparation. Ribosomes from mitochondrial S-25 have specific requirement for mitochondrial specific supernatant factors for complete activity.

Chapter 2 Preparation of RNA from Animal Cells

Publisher Summary This chapter presents standard procedures for isolation of undegraded RNA from various tissues and cell fractions. In animal cells, ribosomal particles contain RNA species with sedimentation coefficients of 28 S, 18 S, 7 S, and 5 S. The large ribosomal subunit contains one molecule of 28 S, 7 S, and 5 S RNA. 7 S RNA is known to be hydrogen-bonded with 28 S RNA, whereas 5 S RNA appears to exist as a mini subunit in association with a protein. The small ribosomal subunit contains one 18 S RNA molecule. Isolation of these RNAs can be accomplished either from whole cells, post mitochondrial supernatant, ribosomes, or ribosomal subunits. Theoretically, the purest ribosomal RNA should be obtainable from ribosomal subunits. High temperature not only increases the rate of RNA extraction, but also suppresses the release of DNA into the aqueous phase, resulting in a lower contamination of DNA. At least 55°C is required for efficient extraction of nuclear RNA. Higher temperatures result in a higher rate of extraction of tightly bound nuclear RNA.

The enzymic incorporation of arachidonic acid into ether-containing choline and ethanolam1ne phosphoglycerides by deacylation-acylat1on reactions

Microsomal preparations and mitochondrial supernatant fractions from rat testes incorporated [ 3H]arachidonic acid into the 2-position of [ 14C]alkylacylglycero-phosphorylcholine and endogenous alk-1-enyl-acylglycerophosphorylethanolamine. The reaction required ATP and CoA and was stimulated by Mg 2+ when microsomes washed with EDTA were used. Under the same conditions, only insignificant quantities of [ 3H]palmitic acid were incorporated into [ 14C]alkylacylglycerophosphorylcholine or the alk-I-enylacylglycerophosphorylethanolamine. However, both the [ 3H]palmitic and the [ 3H]arachidonic acids were incorporated into diacylglycero-phosphorylcholine and diacylglycerophosphorylethanolamine; the palmitate was found predominately in the 1-position and the arachidonate in the 2-position. When α-glycerophosphate was added to the system, incorporation of the 3H-labeled acids into phosphatidic acid showed little positional selectivity. These findings indicated that the labeled acids were incorporated into the choline- and ethanolamine-containing phospholipids by deacylation-acylation reactions.

An alternate enzymic route for the synthesis of the alkyl analog of phosphatidic acid involving alkylglycerol

A key intermediate in the biosynthesis of complex ether lipids is 1-alkyl-2-acyl- sn -glycero-3-phosphate. This lipid is known to be formed by enzymic reduction of alkyldihydroxyacetone phosphate and subsequent acylation. We have now obtained evidence for an alternate pathway for its formation from hexadecylglycerol. The reaction is catalyzed by a mitochondrial supernatant fraction prepared from mouse preputial gland tumors; CoA, ATP, and Mg ++ are required as cofactors.

The biosynthesis of ethanolamine plasmalogens by a postmitochondrial fraction from rat brain

Ethanolamine plasmalogens were synthesized from 1- 14C-alkyl- sn-glycero-3-phosphorylethanolamine by mitochondrial supernatants of rat brain. The data indicate that the desaturation of the O-alkyl moiety to the O-alk-l-enyl moiety occurs on intact 1-alkyl-2-acyl- sn-glycero-3-phosphorylethanolamine.

Hepatic acetic thiokinase: Possible regulatory step in lipogenesis

Acetic thiokinase activity was measured in rat liver mitochondrial supernatants in conditions known to affect lipogenesis. The enzyme's activity was reduced in livers of starved or alloxandiabetic rats. Insulin treatment reversed the effect of diabetes. Adding oleate in vitro, to mitochondria-free liver homogenates of normal rats inhibited acetic thiokinase activity. To compare the relative sensitivity to inhibition of this enzyme with that of the enzymes converting acetyl CoA to fatty acid, rat liver microsomal supernatants were incubated with acetate-2- 14C and acetyl CoA- 3H and the ratio 14C 3H determined in biosynthesized fatty acid. The results demonstrate that acetic thiokinase was inhibited to a greater extent by the addition of exogenous oleate or by fasting than were the enzymes involved in the conversion of acetyl CoA to fatty acid. It is proposed that, in addition to acetyl CoA carboxylase and fatty acid synthetase, hepatic lipogenesis may be controlled by the activity of acetic thiokinase.

Synthesis of nuclear and cytoplasmic proteins in the early development of fishes and echinoderms

ABSTRACT Synthesis of nuclear and cytoplasmic proteins was studied biochemically and autoradio-graphically in early loach (Misgurnus fossilis) and sea-urchin (Strongylocentrotus nudus) embryos. After incubation with [14C]amino acids for 5–120 min the ratio of the specific activities of nuclear, mitochondrial and 12000g supernatant proteins was shown to be equal approxi-mately to 6:1:2 in loach embryos and to 8:4:3 in sea-urchin embryos independently of the duration of labelling. After incubation with [3H]amino acids the number of silver grains per unit section was on the average 2·4 times higher for nuclei than it was for cytoplasm at mid-blastula and mid-gastrula stages. At the mid-gastrula the vegeto-animal gradient of protein synthesis was found. A higher level of the synthesis of nuclear proteins as compared with that of cytoplasmic proteins appears to be related to an increase in the nuclear volume and the nucleo-cytoplasmic ratio during the early development of the loach and sea-urchin embryos.

Zonal Centrifuge Applied to the Purification of Herpesvirus in the Lucké Frog Kidney Tumor

A series of "winter" and "summer" Lucké kidney tumors of the frog (Rana pipiens) were homogenized and fractionated by differential centrifugation into nuclear, mitochondrial, and mitochondrial supernatant fractions. Winter tumors often contained high concentrations of herpesvirus, whereas no virus was observed in any of the summer tumors. The crude tumor fractions were further purified by rate-zonal sucrose gradient centrifugation in a B-XV zonal rotor. Gradient fractions rich in an enveloped, nucleated form of the herpesvirus from certain winter tumors have induced renal tumors when injected into developing frog embryos. Zonal centrifugation was followed by isopycnic banding of the virus zones for further purification of the different morphological forms of the virus.

Zonal centrifuge applied to the purification of Herpesvirus in the Lucké frog kidney tumor.

A series of "winter" and "summer" Lucké kidney tumors of the frog (Rana pipiens) were homogenized and fractionated by differential centrifugation into nuclear, mitochondrial, and mitochondrial supernatant fractions. Winter tumors often contained high concentrations of herpesvirus, whereas no virus was observed in any of the summer tumors. The crude tumor fractions were further purified by rate-zonal sucrose gradient centrifugation in a B-XV zonal rotor. Gradient fractions rich in an enveloped, nucleated form of the herpesvirus from certain winter tumors have induced renal tumors when injected into developing frog embryos. Zonal centrifugation was followed by isopycnic banding of the virus zones for further purification of the different morphological forms of the virus.

Activation in vitro of rat liver polyribosomes.

The increase in the incorporation of amino acids into protein in vitro by preparations obtained from protein-fed rats as compared with preparations obtained from carbohydrate-fed rats has been described previously. After molecular sieving through Sephadex G-25 of cell-free preparations, the difference in incorporating activity between the two types of rats was diminished in systems containing ATP, phosphoenolpyruvate, pyruvate kinase, GTP, and a mixture of amino acids. When, after molecular sieving, a mitochondrial (15,000 g) supernatant was incubated for 4 min at 35 degrees C the polysomal pattern of the preparations was unchanged. In the presence of ATP, phosphoenolpyruvate, and pyruvate kinase the polysomal incorporating activity was low and the polysomal pattern was only slightly changed. Addition of GTP increased the activity markedly, and a more pronounced activity was observed when a mixture of amino acids was added as well. As the amino acid incorporation ability increased, monosomes were formed from the polyribosomes. The activity of the polyribosomes was severalfold higher than that of non-Sephadex-treated preparations, indicating an activation of polysomal aggregates which under the usually applied conditions of incubation and prior to molecular sieving show little or insignificant activity. It was possible to activate polyribosomes from carbohydrate-fed and protein-fed rats to almost the same extent.