The stimulation of yeast mitochondrial protein synthesis by low molecular weight cytoplasmic factors: Requirement for intact mitochondria and lack of effect by folate derivatives

• 1.1. Protein synthesis by GTP -supplemented yeast mitochondria is stimulated by a fraction of molecular weight less than 2,000 isolated from yeast high-speed supernatant (S-150).
• 2.2. The low molecular weight fraction works independently of the respiratory chain as the stimulation effect is not cyanide-sensitive.
• 3.3. Stimulation of mitochondrial protein synthesis by cytoplasmic factors is dependent upon the method of mitochondrial isolation.
• 4.4. The low molecular weight stimulatory factor(s) are not reduced folate derivatives which supply formyl groups required for initiation of mitochondrial protein synthesis.

     

The characterization of the linoleic acid desaturation and elongation system in ovine placental tissue

• 1.1. The desaturation and elongation of linoleic acid has been studied in homogenates and in subfractions of ovine placental tissue.
• 2.2. The reaction was characterized in terms of pH and temperature optima, time course and protein concentration.
• 3.3. Activity was found to be confined to the 11,000g supernatant fraction of the tissue and the results suggest that the enzymes are membrane bound.
• 4.4. The cytosolic fraction and ATP were required for full activity and the reaction was inhibited by cyanide.
• 5.5. The properties of the reaction are compared with those of other desaturation systems and their implications with regard to possible reaction mechanisms are discussed.

     

Pyrimidine degradation in tomato cell suspension cultures and in Euglena gracilis—localization of enzymes

• 1.1. Subcellular location of dihydropyrimidinase and NCβA-amidohydrolase2 was studied in a cell suspension culture of tomato (Lycopersicon esculentum cv. Lukullus) and in Euglena gracilis.
• 2.2. By differential centrifugation, crude extracts were separated into ten fractions. Activities of both enzymes were found mainly in cytosolic fractions marked by EDH (tomato) and glu-6-P-DH (E. gracilis).
• 3.3. A cytosolic location was also found by a 20–60% and a 17.5–30% sucrose density gradients.
• 4.4. Using mitochondrial marker enzymes such as fumarase, SDH, CS and MDH, a mitochondrial occurrence of both enzymes or their release from mitochondria can be excluded by sucrose gradient centrifugations. This can also be achieved using purified mitochondria prepared from tomato cells by two subsequent sucrose gradients.
• 5.5. A possible vacuolar location of dihydropyrimidinase and NCβA-amidohydrolase was excluded by comparing their activities in isolated protoplasts and purified vacuoles which were characterized by their marker enzyme α-mannosidase.
• 6.6. A nuclear location of both enzymes and/or their release from the nucleus during procedures used cannot be excluded.
• 7.7. The results are discussed in relation to subcellular location to other pyrimidine-metabolizing enzymes in plant cells.

     

Subcellular distribution of metals within the kidney of the bivalve Mercenaria mercenaria (L.)

• 1.1. The subcellular distribution of nine transition metals (plus four additional elements) was measured in the kidney tissue of the quahog, Mercenaria mercenaria.
• 2.2. Elemental analyses of the subcellular fractions indicated three main patterns of metal distribution within kidney cells.
• 3.3. Barium, iron, manganese and lead were associated primarily with kidney granules.
• 4.4. Cadmium, copper, potassium and magnesium were found mainly in the cytosolic fraction.
• 5.5. Calcium, phosphorus and zinc were found in all isolated fractions, probably reflecting the important roles that these elements play in bivalve metabolism.
• 6.6. The organelle composition of the isolated subcellular fractions was determined using marker enzyme assays and microscopic techniques.

     

Interrelationships between gluconeogenesis and uricogenesis in chicken liver

• 1.1. Experiments performed on isolated hepatocytes and perfused liver of starved chickens showed that gluconeogenesis from lactate, glycerol and fructose was inhibited by 22–100% on addition of urate precursors.
• 2.2. The inhibition was associated with an increased rate of urate formation.
• 3.3. 2,4-Dinitrophenol (40 μM), 2-bromooctanoate (2 mM) and 3-mercaptopicolinate (3MPA) (0.5 mM) were inhibitory with respect to gluconeogenesis but did not significantly affect the rate of urate formation.
• 4.4. The possible interrelationships between gluconeogenesis and uricogenesis are considered in terms of a competition for ATP and for other metabolites between the two pathways.
• 5.5. An interplay of both pathways at the level of anion transfer across the inner mitochondrial membrane is also discussed.

     

Some properties of the NADP-malic enzyme from mango fruit,Mangifera indica

• 1.1. Malic enzyme l-malate-NADP-oxidoreductase (oxaloacetate-decarboxylating) (EC 1.1.1.40) was located in the cytosolic fraction of ripening mango fruit.
• 2.2. The purified enzyme has an isoelectric point of 6.86 and an activation energy of 11.9kcal/mol.
• 3.3. The amino acid composition of the enzyme was determined and revealed a low cysteine and tryptophan content.
• 4.4. The enzyme has an ultraviolet absorption maximum at 266 nm with maxima for fluorescence excitation and emission at 285 and 328 nm.
• 5.5. The enzyme shows positive cooperativity between the malate binding sites and the effect of allosteric regulators and structural analogues on the activity were investigated.

     

Properties of enzymes hydrating epoxides in human epidermis and liver

• 1.1. Cytosolic and microsomal epoxide hydrolyzing enzymes of human skin and liver were compared and found to be different.
• 2.2. Epidermal and hepatic cytosolic epoxide hydrolases were different in terms of substrate selectivity, pI, inhibitor sensitivity and affinity Chromatographic properties.
• 3.3. Microsomal epoxide hydrolases had the same pIs but different substrate selectivities.
• 4.4. Cytosolic epoxide hydrolase from adults had higher specific activity than that from neonates or cultured epidermis, but lower activity than adult hepatic enzymes.
• 5.5. The sizes of cytosolic epoxide hydrolase from epidermis and liver were similar and lower than that from cultured fibroblasts.
• 6.6. Cytosolic epoxide hydrolase from all sources shared similar antigenic determinants.

     

Malate dehydrogenase isoforms from ribbed mussel (Modiolus demissus) gill tissue

• 1.1. The malate dehydrogenase (MHD) activity from the ribbed mussel gill is polymorphic with two distinct mitochondrial forms (M1 and M2) and five forms that could be resolved from cytosolic extracts (C1 to C5) by DEAE-cellulose chromatography and starch gel electrophoresis.
• 2.2. Two of the cytosolic forms (C3 and C4) may represent interchangeable conformational states.
• 3.3. With kinetic analysis there appear to be three distinct cytosolic forms (C1, C2 and C3–C4), with C2 possibly behaving as a heterodimer.
• 4.4. The identity of C5 is uncertain.
• 5.5. The forms isolated from the mitochondria (M1 and M2) exhibited lower apparent K_ms for oxaloacetate (OAA) than the cytosolic forms.
• 6.6. For all isozymic forms, the apparent K_ms for OAA increased as the pH increased between pH 6 and 9
• 7.7. Increasing the salt concentration raised the K_m for OAA for all forms.
• 8.8. The mMDHs were more sensitive to inhibition by NaCl than the cMDHs.
• 9.9. Representative cMDH (C1) and mMDH (M2) isozymes exhibited substrate inhibition by high concentrations of OAA with the mMDH possessing lower K_is for substrate inhibition than the cMDH at each pH tested.
• 10.10. Differences and similarities in K_m app. for OAA at the different pHs and salt concentrations indicated that C1, C2 and C3–C4 and C5 were distinct forms, that M1 and M2 were distinct but very similar to each other, and that C1, C2, C3–C4 and C5 were distinct from M1 and M2.

     

Non-transferrin-bound-iron in serum and low-molecular-weight-iron in the liver of dietary iron-loaded rats

• 1.1. The feeding of 0.5% (3,5,5-trimethylhexanoyl)ferrocene (TMH-ferrocene) in rats resulted in a severe and progressive liver siderosis (total liver iron, 30 mg/g liver wet weight, after 30 weeks).
• 2.2. High concentrations of an iron-rich ferritin (up to 250 mg/l) were detected in serum of heavily iron-loaded rats forming a large fraction of non-transferrin-bound-iron (5000 μg/dl in maximum).
• 3.3. Ferritin and not haemosiderin was the major iron storage protein in the liver.
• 4.4. The total liver iron concentration (from 0.4 to > 30 mg Fe/g wet wt) but not the cytosolic low-molecular-weight-iron fraction (from 0.5 to 2.5 μM) was extremely increased during iron-loading.

     

A Combined N-terminomics and Shotgun Proteomics Approach to Investigate the Responses of Human Cells to Rapamycin and Zinc at the Mitochondrial Level

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Highlights

• •Rapamycin and zinc induce moderate but significant mitochondrial proteome changes.
• •The mitochondrial proteins processing system is robust under subtoxic conditions.
• •Rapamycin and zinc perturb the mitochondrial proteins processing system.
• •Rapamycin and zinc perturb the mitochondrial proteins homeostasis.

     

Intracellular localization and characterization of 3-hydroxykynureninase in human liver

• 1.1. 3-hydroxykynureninase in human liver was present in cytosol and mitoehondria.
• 2.2. The cytosolic enzyme and mitochondrial enzyme had the same physiological and enzymic properties.
• 3.3. The enzyme had a mol. wt of 130,000 by gel filtration and isoelectric point of pH 5.9.
• 4.4. The enzyme was active for 3-hydroxykynurenine and kynurenine, and its activity ratio was 15:1. The apparent K_m values of the enzyme were 7.7 × 10⁻⁵M for 3-hydroxykynurenine, 1.0×10⁻³M for kynurenine and 2.5 × 10⁻⁶M for pyridoxal 5'-phosphate with 3-hydroxykynurenine.
• 5.5. Some other properties of purified enzymes are described.

     

Initial characterization of human thymocyte sialidase activity: Evidence that this enzymatic system is not altered during the course of T-cell maturation

• 1.1. The sialidase activity of human thymocyte was examined by a fluorogenic assay.
• 2.2. These studies revealed that human thymocyte sialidase activity is essentially acid-active and membrane-bound since 59.6% and 33% of the total activity was recovered in the lysosome-enriched and microsomal fractions, respectively.
• 3.3. A weak activity was also detected in the cytosolic fraction.
• 4.4. However, the acidic optimum pH of this soluble sialidase was at variance with the general concept of mammalian soluble sialidases which are known to be optimally active at more neutral pH.
• 5.5. This acidic soluble sialidase seems to be a general characteristic of the human T-cell lineage since examination of mature circulating T-cells revealed that they contain a soluble sialidase activity similar to that observed in thymocytes.
• 6.6. Analysis of mature and immature thymocyte subpopulation obtained by differential PNA agglutination indicated that this enzymatic system was not altered during the course of thymic maturation.
• 7.7. These results suggest that unlike in T-cell activation where changes in the level of sialidase activity were shown to influence the extent of cell surface sialylation and thereby the cell physiology, this enzymatic system seems not to be involved in the fluctuation of cell surface sialic acid content observed during thymic maturation.

     

Relative bioavailability and DNA adduct formation of benzo[a]pyrene and metabolites in the diet of the winter flounder

• 1.1. Radiolabeled metabolites of the carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) were shown to be absorbed through the diet of the winter flounder, Pseudo pleuronectes americanus.
• 2.2. Oral bioavailability of a mixture of naturally produced metabolites was significantly less than that of the parent BaP.
• 3.3. Oral bioavailability of a pure metabolite, BaP-7,8-dihydrodiol (7,8-D) was found to be similar to that of BaP.
• 4.4. Both metabolites and BaP formed DNA adducts in liver.

     

Proline synthesis from glutamate in mitochondria from the blowfly Aldrichina grahami

• 1.1. Glutamyl kinase in a homogenate of blowfly abdomens was not inhibited by proline and its analogues, and was found only in the mitochondrial fraction.
• 2.2. Proline was biosynthesized from glutamate only in a cell-free system prepared from blowfly abdomen.

     

Biomines-stimulated phosphorylation and (Na+, K+-ATPase in the gills of the chinese crab,Eriocheir sinensis

• 1.1. Subcellular distribution of (NA⁺, K⁺-ATPase and ouabain-insensitive ATPase (Mg²⁺-ATPase) are compared in branchial tissues of the euryhaline crab, Eriocheir sinensis, acclimated to fresh water.
• 2.2. Both the anterior and posterior gills contain cAMP-dependent protein kinase and endogenous protein substrate for phosphorylation.
• 3.3. Phosphorylation occurs in both “particulate” and “soluble” subcellular fractions but its stimulation by cAMP is restricted to the “soluble” fraction.
• 4.4. serotonin (5-HT) and dopamine receptors are present only in the “light particulate” fraction isolated from the posterior gills.

• 1.(a) Serotonin and dopamine have no effect on the phosphorylation observed in a subcellular fraction alone.
• 2.(b) Activation of the phosphorylation by serotonin and dopamine is found when the soluble fraction (source of cAMP-dependent protein kinase) is added to the fraction P₃ from the posterior gills.
• 3.(c) No activation occurs with the fractions P₃ as well as P₁ or P₂ (not shown) from anterior gills of fresh water crab.
• 4.(d) Cyproheptadine, a serotonin receptor antagonist, inhibits the 5-HT dependent increase in phosphorylation.
• 5.(e) The dopamine receptor antagonist, chlorpromazine, inhibits dopamine-stimulated phosphorylation.
• 6.5. Ouabain mimics the effect of cyproheptadine on the serotonin-stimulated phosphorylation found in the posterior gills.

     

microRNA-222 Attenuates Mitochondrial Dysfunction During Transmissible Gastroenteritis Virus Infection

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Highlights

• •microRNA-222 attenuates TGEV-induced mitochondrial dysfunction.
• •microRNA-222 downregulates THBS1 and CD47.
• •THBS1 is the target of microRNA-222 during TGEV infection.
• •THBS1 and CD47 increase mitochondrial Ca²⁺ level and reduced mitochondrial membrane potential (MMP).

     

Domain structure of the 90-kDa stress protein: Heparin- and antibody-binding domain

• 1.1. To understand the physiological roles of the 90-kDa stress protein (HSP90), we investigated the heparin- and antibody-binding domains of the protein.
• 2.2. For heparin-binding sites, HSP90 was digested completely with trypsin, and the digests were applied to a heparin-Sepharose column and eluted with 1.0 M NaCl, followed by 8.0 M urea.
• 3.3. Each elutant was purified by a reverse-phase C₁₈ column.
• 4.4. Two peptides from the NaCl-eluted fraction and no peptide from the urea-eluted fraction were purified.
• 5.5. The purified peptides were sequenced by an automated peptide sequencer.
• 6.6. One of the heparin-binding sites was present between Leu-362 and Arg-365; another was present between Leu-645 and Lys-648.
• 7.7. These two peptides were basic and considerably hydrophilic.
• 8.8. For antibody-binding sites, HSP90 was mildly digested with trypsin, electrophoresed on SDS-polyacrylamide gels and transferred to PVDF membranes.
• 9.9. The four bound of the trypsin fragments could be sequenced with a peptide sequencer.
• 10.10. There was only one antibody-binding peptide, 38 kDa, starting from Pro-2. The others showed no cross-reactivity with the antibody and started from Leu-283.
• 11.11. Therefore, the epitopes of HSP90 are present between Pro-2 and Leu-282.
• 12.12. The heparin-binding sites are present from the middle region of the HSP90 molecule, and the antigen sites are at the N-terminal domain.

     

A peptide inhibitor for S-adenosyl-l-methionine-dependent transmethylation reactions: Purification and characterization

• 1.1. A proteinaceous inhibitor for S-adenosyl-l-methionine (AdoMet)-dependent transmethylation reactions has been purified to apparent homogeneity from rat liver cytosolic fraction.
• 2.2. The peptide was made up of 29 amino acid residues with a molecular weight of 2,584. Glycine accounted for 52% of the total amino acids.
• 3.3. Employing AdoMet: protein-carboxyl O-methyltransferase (Protein methylase II) and bovine serum γ-globulin as in vitro substrate, the mode of inhibition was found to be non-competitive with K_i value of 1.9 × 10⁻⁸ M.
• 4.4. When the inhibitor was present in the reaction mixture together with S-adenosyl-l-homocysteine (AdoHcy), which is a competitive inhibitor for AdoMet, the extent of inhibition exceeded that exerted by each individual inhibitor alone, suggesting that the sites of the inhibitors on the enzyme molecule are different.
• 5.5. Almost a stoichiometric relationship exists between the enzyme and the inhibitor molecule, the ratio being approx one.

     

In vitro-studies on the anaerobic formation of ethanol by the larvae of Chironomus thummi thummi (Diptera)

• 1.1. The anaerobic formation of ethanol by the larvae of Chironomus thummi thummi was investigated in homogenates and isolated mitochondria.
• 2.2. It was found that homogenates transform fructose-1,6-bisphosphate into ethanol and acetate. The accumulation of ethanol decreases substantially and the formation of acetate almost ceases when arsenite, an inhibitor of pyruvate dehydrogenase, is present in the incubation medium.
• 3.3. The cytosolic fraction of the homogenate was shown to degrade fructose-1,6-bisphosphate to pyruvate only. No ethanol could be detected, although there is high activity of alcohol dehydrogenase in the cytosol.
• 4.4. Isolated mitochondria produce large quantities of ethanol from pyruvate during anoxia. This ethanol production was shown to depend on the presence of NADH. It is deduced that this cosubstrate originates from intramitochondrial formation of acetate from pyruvate, which was always found to accumulate alongside ethanol.