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PubMed1991

Cytidine diphosphate diglyceride analogs of antiretroviral dideoxynucleosides: evidence for release of dideoxynucleoside-monophosphates by phospholipid biosynthetic enzymes in rat liver subcellular fractions.

We recently synthesized phospholipid analogs with antiviral nucleosides in the polar headgroup and demonstrated their antiretroviral activity in vitro in human immunodeficiency virus-infected cells (Hostetler, K.Y., Stuhmiller, L.M., Lenting, H.B.M., van den Bosch, H. and Richman, D.D. (1990) J. Biol. Chem. 265, 6112-6117). Dideoxynucleoside analogs of cytidine diphosphate diglyceride (CDP-DG) represent one class of such phospholipid prodrugs from which the antiviral active principle may be released through established pathways of cellular phospholipid metabolism. We now demonstrate that the liponucleotides of dideoxycytidine, 3'-deoxythymidine and 3'-azido-3'-deoxythymidine (AZT, Zidovudine) can substitute to varying extents for CDP-DG in the biosynthesis of phosphatidylinositol, phosphatidylglycerol or diphosphatidylglycerol by rat liver subcellular fractions. In all three biosynthetic pathways dideoxycytidine diphosphate diglyceride was the most active donor of the phosphatidyl unit. The nearly stoichiometric formation of dideoxycytidine-5'-monophosphate during phosphatidylinositol biosynthesis supports the rationale that the antiretroviral liponucleotides may provide cells with a depot form from which the antiviral drug can be released in 5'-monophosphorylated form, thus bypassing the initial phosphorylation of free dideoxynucleosides.

Animals↗

PubMed1995

Novel peroxisomal populations in subcellular fractions from rat liver. Implications for peroxisome structure and biogenesis.

According to current concepts, new peroxisomes are formed by division of pre-existing peroxisomes or by budding from a peroxisomal reticulum. Recent cytochemical and biochemical data indicate that protein content in peroxisomes are heterogenous and that import of newly synthesized proteins may be restricted to certain protein import-competent peroxisomal subcompartments (Yamamoto, K., and Fahimi, H. D. (1987) J. Cell Biol. 105, 713-722; Heinemann, P., and Just, W. W. (1992) FEBS Lett. 300, 179-182; Lüers, G., Hashimoto, T., Fahimi, H. D., and Völkl, A. (1993) J. Cell Biol. 121, 1271-1280). We have observed that substantial amounts of peroxisomal proteins are found together with "microsomes" (100,000 x g pellet) after subcellular fractionation of rat liver homogenates. In this study we have investigated the origin of these peroxisomal proteins by modified gradient centrifugation procedures in Nycodenz and by analysis of enzyme activity distributions, Western blotting, and immunoelectron microscopy. It is concluded that much of this material is confined to novel populations of "peroxisomes." Immunocytochemistry on gradient fractions showed that some vesicles were enriched in acyl-CoA oxidase and peroxisomal multifunctional enzyme ("catalase-negative") whereas others were enriched in catalase and thiolase ("acyl-CoA oxidase-negative"). Double immunolabeling experiments verified the strong heterogeneity in the protein contents of these vesicles and also identified peroxisomes varying in size from about 0.5 microns ("normal peroxisomes") to extremely small vesicles of less than 100 nm in diameter. The possibility that these vesicles may be related to different subcompartments of a larger peroxisomal structure involved in protein import and biogenesis will be discussed.

Animals↗

PubMed1992

Membrane localization of endopeptidase-24.11 and peptidyl dipeptidase A (angiotensin converting enzyme) in the pig brain: a study using subcellular fractionation and electron microscopic immunocytochemistry.

Brains from piglets were dissected and a block of tissue including the substantia nigra, globus pallidus, and entopeduncular nucleus was homogenized and then fractionated on discontinuous Percoll gradients. Ligand-binding assays using (-)-[3H]nicotine and [3H]quinuclidinyl benzilate served to delineate fractions containing nicotinic and muscarinic acetylcholine receptors. In this system endopeptidase-24.11 exhibited a biphasic distribution, consistent with its presence on both pre- and postsynaptic membranes. Peptidyl dipeptidase A (angiotensin converting enzyme; ACE) was associated with membrane fractions containing muscarinic receptors. An immunoblot of these fractions with an affinity-purified polyclonal antibody to ACE revealed only the neuronal form of ACE (Mr 170,000), the endothelial form (Mr 180,000) being undetectable. Electron microscopic immunoperoxidase staining of the substantia nigra, with an affinity-purified antibody to endopeptidase-24.11 at the preembedding stage, showed this antigen to be confined to the plasma membranes of boutons, axons, and some dendrites. Both pre- and postsynaptic membranes were stained, and occasionally other regions of the dendritic membrane were positive. No staining of synaptic vesicles within the boutons was observed. Thus, two independent approaches indicate that endopeptidase-24.11 is present on both pre- and postsynaptic membranes in the pig substantia nigra. The subcellular fractionation suggests that neuronal ACE is confined to dendritic membranes.

Animals↗

PubMed1990

Soluble and membrane-bound leucyl- and arginyl-aminopeptidase activities in subcellular fractions of young and adult rat brains.

The subcellular distribution of soluble and membrane-bound leucyl- and arginyl-aminopeptidase activities were analyzed in one and five month old rat brains using Leu- and Arg-2-naphthylamide as substrates. Both soluble leucyl- and arginyl-aminopeptidase activities showed the highest levels in the synaptosomal fraction in the two groups of rats. The highest levels of membrane-bound leucyl- and arginyl-aminopeptidase activities were found in the microsomal fraction in the two ages studied. There were no differences between the two ages in soluble leucyl- and arginyl-aminopeptidase activities. However, a significant decrease in both membrane-bound enzymatic activities was evidenced in the synaptosomal fraction of older rats. Developmental changes of these aminopeptidase activities in a determined subcellular localization, may reflect modifications in their effect on neuropeptides susceptible to be hydrolyzed in this particular location.

Aminopeptidases↗

PubMed1993

Structural and functional heterogeneity among peroxidase-negative granules in human neutrophils: identification of a distinct gelatinase-containing granule subset by combined immunocytochemistry and subcellular fractionation.

The existence of separate gelatinase granules in human neutrophils has been a matter of debate in recent years. We have demonstrated that the 135-kD form of neutrophil gelatinase is a complex of 92-kD gelatinase and a novel 25-kD protein termed neutrophil gelatinase-associated lipocalin (NGAL) that, in addition to being complexed with part of the gelatinase, is localized in free form in peroxidase-negative specific granules. Because this association was not appreciated in earlier studies, we decided to reassess the ultrastructural localization of gelatinase using specific antibodies without immunoreactivity towards NGAL. Double-labeling immunogold electron microscopy was performed on frozen thin sections of human neutrophils. Twenty-four percent of all peroxidase-negative granules were labeled with antigelatinase antibody, but not with antilactoferrin antibody. These granules are defined as gelatinase granules. Sixteen percent reacted with antilactoferrin antibody but not with antigelatinase antibody. The rest (60%) reacted with both antibodies. All granules labeling for lactoferrin are defined as specific granules. Gelatinase granules were observed as round and oval forms of considerably smaller size than specific granules, and were less electron dense. Isolated granules obtained by subcellular fractionation were also examined by immunoelectron microscopy. This demonstrated that peroxidase-negative granules comprise a continuum from the most dense granules that contain lactoferrin but no gelatinase to the lightest that contain gelatinase but no lactoferrin. Thus, gelatinase granules do exist as a subpopulation of peroxidase-negative granules and may allow for exocytosis of gelatinase during neutrophil diapedesis without substantial mobilization of other peroxidase-negative granules, ie, specific granules.

Cytoplasmic Granules↗

PubMed2000

Role of redox-active iron ions in the decomposition of S-nitrosocysteine in subcellular fractions of porcine aorta.

We recently reported that degradation of S-nitrosocysteine in homogenates of porcine aorta increased severalfold in the presence of Mg2+ ions [Kostka, P., Xu, B. & Skiles, E.H. (1999) J. Cardiovasc. Pharmacol. 33, 665-670]. The objective of the present study was to examine this in greater detail. The rate of S-nitrosocysteine degradation by aortic homogenates in the presence of Mg2+ ions exhibited differential sensitivity to chelators of iron ions. Terpyridine and diethylenetriamine penta-acetic acid (5-500 microM) caused a concentration-dependent inhibition of S-nitrosocysteine decay, whereas deferoxamine (100 microM) was ineffective. o-Phenanthroline (250 microM), a selective chelator of Fe2+ ions, potentiated the reaction at low initial concentrations of S-nitrosocysteine (< or = 15 microM) and inhibited the reaction at higher concentrations. The inhibitory effects of o-phenanthroline were related to suppression of S-nitrosocysteine decay by cysteine-mediated reduction of Fe3+. In the presence of o-phenanthroline, S-nitrosocysteine decomposition followed saturable kinetics with K0.5 = 3.8 +/- 0.3 microM and h = 1.8 +/- 0.1 (mean +/- SE, n = 4). Comparison of the rates of S-nitrosocysteine decay in different subcellular fractions showed selective association with the cytosolic fraction, as documented by copurification with lactate dehydrogenase activity. At non-limiting concentrations of S-nitrosocysteine, the rate of degradation in the cytosolic fraction was 4.1 +/- 0.3 nmol.min-1.(mg protein)-1 (n = 4). It is concluded that the cytosolic fraction of porcine aorta contains a protein factor, presumably an enzyme, capable of catalyzing heterolytic decomposition of the S-NO bond of S-nitrosocysteine in a process involving redox cycling of iron ions.

Animals↗

PubMed1965

FURTHER OBSERVATIONS ON THE PRODUCTION OF AMINO ACIDS BY RAT-LIVER MITOCHONDRIA AND OTHER SUBCELLULAR FRACTIONS.

1. Rat-liver mitochondria showed a decrease in amino acid production after preparation in 0.25m-sucrose containing EDTA (1mm), but an increase in water content. When EDTA was replaced by Mn(2+) (1mm) or succinate (1mm), both amino acid production and water content were lowered, whereas preparation in 0.9% potassium chloride caused an increase in both. 2. Amino acid production by rat-liver homogenates prepared in 0.9% potassium chloride or 0.25m-sucrose was similar (q(amino acid) 0.047 and 0.042 respectively aerobically). After freezing-and-thawing q(amino acid) values were approximately doubled, and approached that of a homogenate prepared in water. 3. All cations tested inhibited amino acid production by mitochondria, Hg(2+) and Zn(2+) being the most effective in tris-hydrochloric acid buffer. In phosphate buffer Mg(2+) and Mn(2+) had no effect. Of the anions tested only pyrophosphate and arsenate had any inhibitory effect at final concn. 1mm. 4. Iodosobenzoate (1mm) and p-chloromercuribenzenesulphonate (1mm) inhibited mitochondrial amino acid production by 70-80%, whereas soya-bean trypsin inhibitor, EDTA and di-isopropyl phosphorofluoridate inhibited by a maximum of 30%. Respiratory inhibitors had no effect. 5. Rat-liver homogenate and subcellular fractions each showed an individual pattern of inhibition when a series of inhibitors was tested. 6. Amino acid production by mitochondria was decreased by up to 50% in the presence of oxidizable substrate, apart from alpha-glycerophosphate and palmitate, which had no effect. CoA stimulated amino acid production in tris-hydrochloric acid but not in phosphate buffer, alpha-oxoglutarate abolishing the stimulation. 7. Cysteine and glutathione stimulated amino acid production by whole mitochondria by 30%, but only reduced glutathione stimulated production in broken mitochondria. 8. Adrenocorticotrophic hormone and growth hormone stimulated mitochondrial amino acid production by 21-24%, whereas insulin inhibited production by 25%. 9. Coupled oxidative phosphorylation increased amino acid production by up to 154% at 25 degrees and 40 degrees . The increase was abolished by 2,4-dinitrophenol. 10. Amino acid incorporation in mitochondria was accompanied by an increase in amino acid production, both being decreased by chloramphenicol. 11. Mitochondrial production of ninhydrin-positive material was increased in the presence of albumin. The biggest increase was noted for the soluble fraction of broken mitochondria. No increase was found in the presence of (14)C-labelled algal protein or denatured mitochondrial protein.

Amino Acids↗

PubMed1977

Adenylate cyclase activity in lymphocyte subcellular fractions. Characterization of a nuclear adenylate cyclase.

Nuclei from purified human peripheral lymphocytes were prepared by incubations with Triton X-100 to disrupt the cells, followed by sucrose-density gradient centrifugation. The nuclei were pure as judged by phase-contrast microscopy and had low contents of non-nuclear marker enzymes. In addition, nuclei prepared from lymphocytes surface-labelled with 125I had only 2-7% of the radioactivity bound to intact lymphocytes. At 3.3 mM-Ca2+ and 100 micronM-ATP a fluoride-sensitive adenylate cyclase was demonstrated in nuclei prepared in 0.2% Triton X-100 or 0.33% Triton X-100. There was linear accumulation of cyclic AMP for 10 min in both preparations. The apparent Km for ATP was 90 micronM. Adenylate cyclase activity was augmented by 1.0 mM-Mn2+ and inhibited at higher concentrations. Ca2+ showed two peaks of stimulation, at 1.0-2.5 mM- and above 10 mM-Ca2+. Mg2+ was inhibitory at all concentrations. EDTA OR EGTA only slightly decreased adenylate cyclase activity, suggesting that another metal ion may be necessary for activity. Adenylate cyclase activity was stimulated by 10mM-isoproterenol and 10 micronM-adrenaline in the presence of a phosphodiesterase inhibitor. Phytohaemagglutinin and prostaglandin E1 alone or in combination with isoproterenol had no effect on nuclear adenylate cyclase activity in either nuclei preparation. These results indicate that human lymphocyte nuclei contain one or several adenylate cyclases which differ from adenylate cyclases found in other subcellular fractions of these cells with regard to their bivalentcation requirements and responsiveness to pharmacological agents.

Adenylate Kinase↗

PubMed1985

Effect of netilmicin on the phospholipid composition of subcellular fractions of rat renal cortex.

The purpose of this study was to determine the subcellular site(s) of the renal cortical phospholipidosis induced by aminoglycosides. For this purpose we injected male Sprague-Dawley rats s.c. with netilmicin, containing tracer quantities of [3H]netilmicin, at 100 mg/kg/day for 2 days; control rats were injected with saline. Twenty-four hours after the second injection of drug the rats were sacrificed and the renal cortex was fractionated by differential ultracentrifugation and Percoll gradient density techniques to obtain purified lysosomes, mitochondria, microsomes, brush border membranes and basolateral membranes. The total phospholipid content of the renal cortex was 300 +/- 5 nmol/mg of protein in control rats and 340 +/- 5 nmol/mg of protein in netilmicin-injected rats. The total phospholipid content of the lysosomal fraction of netilmicin rats, which was enriched in myeloid bodies and [3H]netilmicin, was 91% greater than that of control rats and reflected significant increases of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol. This pattern is identical to that reported previously for the rat renal cortical phospholipidosis induced by aminoglycosides. The total phospholipid contents of the mitochondrial, microsomal, brush border membrane and basolateral membrane fractions of netilmicin-injected rats were higher by approximately 10% than the respective fractions of control rats and each fraction exhibited a significant increase of one or more of the four phospholipids elevated in the renal cortical homogenate and in the lysosomal fraction. The data indicate that the myeloid body is the primary source of the lysosomal phospholipidosis induced by netilmicin which provides support for the hypothesis that the lysosomal phospholipidosis is secondary to aminoglycoside-induced inhibition of phospholipid degradation. In addition the findings of increased phospholipid content and altered phospholipid composition of the other subcellular fractions raise the possibility that aminoglycoside antibiotics cause a more generalized disturbance of phospholipid metabolism characterized by altered synthesis as well as degradation in renal proximal tubular cells.

Acetylglucosaminidase↗

PubMed1983

Effects of corticotropin-(1-24)-tetracosapeptide on polyphosphoinositide metabolism and protein phosphorylation in rabbit iris subcellular fractions.

Effects of the neuropeptide corticotropin-(1-24)-tetracosapeptide (ACTH) on the endogenous and exogenous phosphorylation of lipids and endogenous phosphorylation of proteins were investigated in microsomes and a 110,000 X g supernatant fraction [30-50% (NH4)2SO4 precipitate; ASP 30-50] obtained from rabbit iris smooth muscle. Subcellular distribution studies revealed that both of these fractions are enriched in diphosphoinositide (DPI) kinase. The 32P labeling of lipids and proteins was measured by incubation of the subcellular fractions with [gamma-32P]ATP. The labeled lipids, which consisted of triphosphoinositide (TPI), DPI, and phosphatidic acid (PA) were isolated by TLC. The microsomal and ASP 30-50 fractions were resolved into six and nine labeled phosphoprotein bands, respectively, by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The basal labeling of both lipids and proteins was rapid (30-60 s), and it was dependent on the presence of Mg2+ in the incubation medium; in general it was inhibited by high concentrations (greater than 0.2 mM) of Ca2+. ACTH stimulated the labeling of TPI and inhibited that of PA in a dose-dependent manner, with maximal effect observed at 50-100 microM of the peptide. ACTH appears to increase TPI labeling by stimulating the DPI kinase. Under the same experimental conditions ACTH (100 microM) inhibited significantly the endogenous phosphorylation of six microsomal phosphoproteins (100K, 84K, 65K, 53K, 48K, and 17K). In the ASP 30-50 fraction, ACTH inhibited the phosphorylation of three phosphoproteins (53K, 48K, and 17K) and stimulated the labeling of six phosphoprotein bands (117K, 100K, 84K, 65K, 42K, and 35K).(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenocorticotropic Hormone↗

PubMed1991

Distinct localization of renin and angiotensins in separate subcellular fractions of the rat adrenal cortex.

The subcellular localization of renin and immunoreactive angiotensins I and II was studied in rat adrenal cortical tissues. The identity of the immunoreactive angiotensins was confirmed as angiotensin I and angiotensin II by radioimmunoassay and high-performance liquid chromatography, respectively, with reference to standard compounds. By differential centrifugation of tissue homogenate in 0.25 M sucrose/30 mM Tris-HCl/l mM EDTA, pH 7.4, specific immunoreactive renin was found to be localized principally (60%) in the mitochondrial fraction (P2), whereas about 40% of both angiotensins I and II was contained in the soluble fraction; only 18-20% of both peptides was contained in the P2 fraction. On Percoll density gradient centrifugation of P2, renin was fractionated mostly in a denser band whereas angiotensins I and II were contained in a lighter density area closely corresponding to mitochondrial and lysosomal marker enzymes. These results suggest that renin and angiotensins in the cells of the rat adrenal gland reside in different subcellular compartments and argue against intracellular formation of angiotensins by renin in renin granules.

Adrenal Cortex↗

PubMed1979