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PubMed1989

Electrophoretic mobility of gamma-glutamyltransferase in rat liver subcellular fractions. Evidence for structure difference from the kidney enzyme.

Adult rat liver gamma-glutamyltransferase (GGT) has been poorly characterized because of its very low concentration in the tissue. In contrast with the kidney, the liver enzyme is inducible by some xenobiotics, and its relationship to hepatic ontogeny and carcinogenesis seems to be important. Liver GGT polypeptides were identified by immunoblot analysis in subcellular fractions (rough endoplasmic reticulum, smooth endoplasmic reticulum, Golgi membranes and plasma membranes). Rat liver GGT appeared as a series of polypeptides corresponding to different maturation steps. Polypeptides related to the heavy subunit of GGT were detected in rough endoplasmic reticulum at 49, 53 and 55 kDa, and in Golgi membranes at 55, 60 and 66 kDa. Two polypeptides related to the light subunit of GGT were also observed in Golgi membranes. In plasma membranes GGT was composed of 100 kDa, 66 kDa and 31 kDa polypeptides. The 66 kDa component could correspond to the heavy subunit of the rat liver enzyme, and if so has a molecular mass higher than that of the purified rat kidney form of GGT (papain-treated). These data suggest different peptide backbones for the heavy subunits of liver GGT and kidney GGT.

Animals↗

PubMedDate not supplied

[P1, P3-(5'-guanosyl-5''-[14C]adenosyl)triphosphate: preparation of the cap parent compound and its catabolic properties towards rat liver subcellular fractions (author's transl)].

We report on the chemical preparation of 14C-labeled P1, P3-(5'-guanosyl-5''-adenosyl)triphosphate (1) which is the parent compound of 5'-terminal cap structures of most eukaryotic mRNAs. Incubation experiments of cap (1)- and non cap-structured nucleotides as Ap2A (2) as well as their ribose ring opened derivatives (3, 4) with rat liver subcellular fractions show that cap-degrading nucleases being able to unblock 5' termini are present in nuclei but not in mitochondria.

Animals↗

PubMed2001

Biotransformation of flobufen enantiomers in ruminant hepatocytes and subcellular fractions.

Flobufen (F), a new antiinflammatory drug, has one chiral and one prochiral center in its structure. Reduction of rac-F, the principal biotransformation pathway, leads to the formation of four diastereoisomers of 4-dihydroflobufen (DHF). F was chosen as a model substrate for interspecies comparison of activity, stereospecificity, and stereoselectivity of biotransformation enzymes in fallow bucks, red deer stags, and roe bucks in vitro. Formation of F metabolites was examined in hepatocyte suspension and in subcellular fractions of liver homogenate. (+)-R-F, (-)-S-F and rac-F were used as substrates. After incubation of substrates, the amounts and ratios of DHF diastereoisomers and F enantiomers were assessed by HPLC, with (R,R)-ULMO and terguride-bonded columns. Considerable interspecies differences in stereoselectivity and stereospecificity of F reductases were found at the cellular and subcellular levels, although these ruminants are closely related. Chiral inversion of F enantiomers to their antipodes was detected in vitro in all ruminants tested, but individual species also differed in the direction and rate of this inversion.

Animals↗

PubMed1985

Proteolytic enzymes in tumor metastasis. II. Collagenase type IV activity in subcellular fractions of cloned tumor cell populations.

Collagenase type IV degradation activity was examined in metastatic and nonmetastatic clones of the Lewis lung carcinoma (3LL) and of the T10 sarcoma. Conditioned media prepared from cells of both tumors grown in vitro contained low degradation activities, whereas conditioned media from organ cultures of the same clones grown as solid tumors in animals exhibited higher degradation activities. Analysis of subcellular fractions of tumor cells showed that collagenase type IV activity was localized mainly in the cytoplasmic fraction. Crude homogenates or detergent lysates manifested low degradation activities. Little activity was associated with purified plasma membrane preparations and endoplasmic reticular fractions. However, addition of plasma membrane to conditioned media and to cytoplasmic fractions reduced the degradation activities of the cytoplasmic fractions. Possibly a factor that inhibits collagenase type IV exists in the cells in a vesicular form. No correlation between degradation activity and metastatic capacity was demonstrated in the models used in this study. Both metastatic and nonmetastatic clones of the same tumor similarly could degrade basement membrane components.

Animals↗

PubMed1982

The subcellular fractionation of the bovine caudate nucleus.

Two synaptosomal fractions could be obtained from bovine caudate nucleus on sucrose density gradients one of which had a much greater capacity for 'high affinity' choline uptake than the other but comparable amounts of CAT and choline kinase activity. Specific binding of QNB was widely distributed among all the subcellular fractions except the mitochondrial fraction and in quantitative terms by far the greatest amount was in the microsomal fraction. Only the microsomal fraction contained measurable amounts of glycerophosphocholine phosphodiesterase.

Animals↗

PubMed1983

[3H]nimodipine specific binding to cardiac myocytes and subcellular fractions.

[3H]Nimodipine binding was studied in isolated myocytes from rat heart and in partially purified sarcolemma, sarcoplasmic reticulum and mitochondrial fractions from dog heart. In isolated myocytes, the density of [3H]nimodipine specific sites (10(6) per cell) was close to density of [3H]QNB sites (0.8 x 10(6) per cell) and higher than that of [3H]DHA sites (0.2 x 10(6) per cell). During subcellular fractionation, [3H]nimodipine binding did not copurify with plasma membrane markers. The highest densities were found in fractions enriched in sarcolemma or in sarcoplasmic reticulum. No specific binding was found in mitochondria. These results indicate that the localization of [3H]nimodipine sites is not restricted to areas of the plasma membrane rich in beta-adrenoceptors, muscarinic receptors and sodium pump sites.

Animals↗

PubMed1971

Studies on memory: distribution of peptidyl-puromycin in subcellular fractions of mouse brain.

Previous experience has led us to suggest that sustained blockage of memory in mice upon administration of puromycin is caused by the persistence of peptidyl-puromycin at synapses. The block can be removed and memory restored by intracerebral injections of water and by subcutaneous or intraperitoneal injections of several psychotropic drugs. The present experiments provide evidence that peptidyl-puromycin persists for long periods in subcellular fractions of synaptosomes (nerve endings), and that the peptide is lost from synaptosomal, but not from mitochondrial, fractions when memory is restored. These findings strengthen our interpretation of the mode of action of puromycin on memory.

Acetylcholinesterase↗

PubMedDate not supplied

Mutagenicity of some derivatives of dipyrido[1,2-a:3',2'-d]imidazoles in Salmonella typhimurium with metabolic activation by rat liver and small intestine subcellular fractions.

The mutagenic effect of 2-amino-dipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2) was compared with that of the 3-amino, 3-nitro, or 3-N-hydroxylated derivatives of the same base ring with methyl groups at positions 4 and 6 of the molecule. The compounds were tested in Salmonella typhimurium strain TA98 without metabolic activation and in the presence of different concentrations of subcellular fractions from livers or small intestines of rats pretreated with different P448/P450 inducers. The 4,6-dimethyl compounds are always more mutagenic than Glu-P-2. Pretreatment with Aroclor 1254 (ARO) is the most effective inducer in the activation of the 2- and 3-amino compounds by liver S9, whereas the same fraction decreases the mutagenicity of the 3-nitro derivative. S9 from small intestine increased the mutagenic effect of the 3-nitro and 3-N-hydroxylated compounds, but it was unable to activate the amino compounds.

Animals↗

PubMed2006

Characterization of the outer membrane protein profile from disease-related Helicobacter pylori isolates by subcellular fractionation and nano-LC FT-ICR MS analysis.

Because of the important role of membrane proteins in adhesion, invasion, and intracellular survival of pathogens in the host, membrane proteins are of potential interest in the search for drug targets or biomarkers. We have established a mass spectrometry-based method that allows characterization of the outer membrane protein (OMP) profile of clinical isolates from of the human gastric pathogen Helicobacter pylori. Subcellular fractionation and one-dimensional gel electrophoresis (1D-GE) analysis was combined with nano-liquid chromatography Fourier transform-ion cyclotron resonance mass spectrometry (nano-LC FT-ICR MS) and tandem mass spectrometry (MS/MS) analysis of fifteen H. pylori strains associated either with duodenal ulcers, gastric cancer, or isolated from asymptomatic H. pylori infected carriers. Over 60 unique membrane or membrane-associated proteins, including 30 of the 33 theoretically predicted OMPs, were identified from the strains. Several membrane proteins, including Omp11 and BabA, were found to be expressed by all strains. In the search for clinical markers we found that Omp26 was expressed by all disease-related strains but was only present in one out of five strains from asymptomatic carriers, which makes Omp26 a potential target for further investigation in the search for proteins unique to disease-related H. pylori strains. In addition, presence of Omp30 and absence of Omp6 seemed to be associated with H. pylori strains causing duodenal ulcer.

Amino Acid Sequence↗

PubMed1985

[Binding by subcellular fractions of rat liver homogenate and intracellular distribution of antibiotics in acute radiation injury].

In vitro binding of tetracycline, gentamicin, erythromycin and rifampicin by the liver organoids and intracellular distribution of the antibiotics in the hepatocytes of rats with acute irradiation disease were studied. Total gamma-irradiation of the animals with 60Co in a dose of 750 R resulted in the development of a typical radiation disease and induced pronounced changes in binding of tetracycline, gentamicin and erythromycin by isolated subcellular fractions. Simultaneous impairment of the antibiotic intracellular distribution was observed. Rifampicin interaction with the cell structures was unchanged. It is suggested that there is a relation between the level of the impairment of the antibiotic cell pharmacokinetics in acute radiation disease and the mechanism of the drug intracellular binding in health.

Animals↗

PubMed1984

Alterations in the fatty acid composition of rat brain cells (neurons, astrocytes, and oligodendrocytes) and of subcellular fractions (myelin and synaptosomes) induced by a diet devoid of n-3 fatty acids.

Rats were fed through four generations with a semisynthetic diet containing 1.0% sunflower oil (6.7 mg/g n-6 fatty acids, 0.04 mg/g n-3 fatty acids). Ten days before mating, half of the animals received a diet in which sunflower was replaced by soya oil (6.6 mg/g n-6 fatty acids, 0.8 mg/g n-3 fatty acids) and analyses were performed on their pups. Fatty acid analysis in isolated cellular and subcellular material from sunflower-fed animals showed that the total amount of unsaturated fatty acids was not reduced in any cellular or subcellular fraction (except in 60-day-old rat neurons). All material from animals fed with sunflower oil showed an important reduction in the docosahexaenoic acid content, compensated (except in 60-day-old rat neurons) by an increase in the n-6 fatty acids (mainly C22:5 n-6). When comparing 60-day-old animals fed with soya oil or sunflower oil, the n-3/n-6 fatty acid ratio was reduced 16-fold in oligodendrocytes, 12-fold in myelin, twofold in neurons, sixfold in synaptosomes, and threefold in astrocytes. No trienes were detected. Saturated and monounsaturated fatty acids were hardly affected. This study provides data on the fatty acid composition of isolated brain cells.

Animals↗

PubMed1975

Biosynthesis and hydrolysis of cholesteryl esters by rat skin subcellular fractions. Regulation by prostaglandins.

The properties and subcellular distribution of the enzymes involved with the synthesis and hydrolysis of cholesteryl esters were investigated in skin of normal and essential fatty acid-deficient rats. Most of the activity of the cholesterol-esterifying enzyme(s) is associated with the 12000g and 105000g particulate fractions. The dependence of the enzyme reaction on ATP and CoA suggests that the esterification of cholesterol by rat skin is mediated by a fatty acyl-CoA-cholesterol acyltransferase (EC 2.3.1.-). On the other hand, most of the activity of the cholesteryl ester hydrolase (EC 3.1.1.13) is localized in the 105000g supernatant fraction. Although the activity of the cholesterol-esterifying enzyme(s) was elevated in skin preparations from essential fatty acid-deficient rats, the activity of the hydrolase was significantly decreased. These observations may explain in part the elevated concentrations of sterol esters in the skin of these animals. Prostaglandin E(2) at low concentrations exerted marked inhibitory effect on the activity of the cholesterol-esterifying enzyme(s), whereas no effect was observed on the activity of the hydrolase at similar concentrations. However, at high concentrations prostaglandin E(2) exerted moderate stimulatory effect on the activity of the hydrolase. These results suggest a possible physiological role of this substance in regulating the production of sterol esters in this tissue.

Adenosine Triphosphate↗

PubMed2001

Cardiac creatine kinase metabolite compartments revealed by NMR magnetization transfer spectroscopy and subcellular fractionation.

In the perfused rat heart NMR inversion transfer revealed the existence of a compartment of ATP not exchanging through creatine kinase (CK), as demonstrated by an apparent discrepancy between the forward (F(f)) and reverse (F(r)) CK flux if this compartment was neglected in the analysis [Joubert et al. (2000) Biophys. J. 79, 1-13]. To localize this compartment, CK fluxes were measured by inversion of PCr (inv-PCr) or gamma ATP (inv-ATP), and the distribution of metabolites between mitochondria and cytosol was studied by subcellular fractionation. Physiological conditions were designed to modify the concentration and distribution of CK metabolites (control, adenylate depletion, inhibition of respiration, KCl arrest). Depending on cardiac activity, mitochondrial ATP (mito-ATP) assessed by fractionation varied from 11% to 30% of total ATP. In addition, the apparent flux discrepancy increased together with mito-ATP (F(f)/F(r) ranged from 0.85 to 0.50 in inv-PCr and from 1.13 to 1.88 in inv-ATP). Under conditions masking the influence of the ATP-P(i) exchange on CK flux, the ATP compartment could be directly quantified by the apparent flux discrepancy; its size was similar to that of mito-ATP measured by fractionation. Thus NMR inversion technique is a potential tool to assess metabolite compartmentation in the whole organ.

Adenosine Triphosphate↗

PubMed1976

Abnormal membrane phospholipid content in subcellular fractions from the Morris 7777 hepatoma.

1. Mitochondrial and microsomal fractions were prepared from normal rat liver and the Morris 7777 hepatoma and characterized by the use of the marker enzymes, succinate dehydrogenase and rotenone-insensitive NADPH-cytochrome c reductase. 2. The phospholipid content per mg membrane protein of Morris 7777 hepatoma mitochondria was increased by 75% as compared with mitochondria from normal rat liver. Microsomes from this poorly-differentiated tumor were found to have a 45% decrease in the content of phospholipid. These abnormalities were independent of tumor size or age. 3. The percent phospholipid content of the subcellular fractions was determined, and revealed an increase in the percent sphingomyelin in both the microsomal and mitochondrial fractions of the tumor. Decreases in the percent phosphatidylcholine and phosphatidylethanolamine were noted in tumor microsomes as compared with normal liver. Diphosphatidylglycerol was not found in significant quantities in the microsomal fraction of this hepatoma line. 4. The content of the various phospholipid classes per mg protein in the respective mitochondrial and microsomal fractions was determined. Large increases in nearly all the major phospholipid classes were found in tumor mitochondria; tumor microsomes were characterized by an increased content of sphingomyelin but the content of nearly all other phospholipids was significantly decreased. These findings suggest the presence of disturbances in the regulation of phospholipid metabolism in subcellular organelle membranes of the Morris 7777 hepatoma.

Animals↗

PubMed1975

Labelling kinetics of RNA containg poly(A) in liver subcellular fractions.

Kinetics of incorporation of (3H) uridine into cytoplasmic RNA fractions of rat liver is investigated. The fractions include free and membrane bound polysomes, rough membranes sedimenting with mitochondria and free cytoplasmic RNA particles. (1) Poly(A) containing RNA, isolated by oligo-dT cellulose, amounts to 0.4% of the total RNA in the homogenate, 0.5% in bound polysomes, 3.4% in free polysomes and 16% in free cytoplasmic RNA particles. (2) The rate of (3H) uridine incorporation into RNA lacking poly(A) proceeds uniformly in all subcellular fractions except for free cytoplasmic RNA particles, which accumulate negligible amounts of radioactivity. (3) The initial labelling of RNA containing poly(A) is most active in free cytoplasmic RNA particles supporting their identity as mRNA en route to polysomes. The initial specific radioactivities decrease in the following order: homogenate, bound polysomes, rough membranes sedimenting with mitochondria, free polysomes. The data suggest that mRNA is supplied to free and membrane-bound polysomes via different routes. The kinetic analysis indicates that free cytoplasmic RNA particles may be a precursor of mRNA of free polysomes rather than that of bound polysomes. (4) The kinetic differences of free and membrane bound polysomes are also demonstrated by comparing the radioactivity of RNA containing poly(A) to the total radioactivity at various incorporation times. In bound polysomes this decreases from 31% at 1 h to 10% at 25 h, whereas in free polysomes the corresponding ratio increases from 10 to 13%. RNA containing poly(A) of free cytoplasmic RNA particles represents 64% of the total radioactivity throughout the experiment.

Animals↗

PubMed1978

The evidence for the existence of chymotrypsin-like esterase activity in the plasma membranes of rabbit neutrophils and the specific chemotactic peptide binding activity of the subcellular fractions.

1. Light- and heavy-plasma membrane fractions have been isolated from rabbit neutrophils and a chymotrypsin-like esterase has been shown to be present in these fractions. 2. The molecular weight of the chymotrypsin-like esterase of rabbit neutrophil plasma membrane was estimated to be about 200 000. 3. About 93% of the chymotrypsin-like esterase of the plasma membranes is esterase 1 and the susceptibility to potential inhibitors was similar in light- and heavy-plasma membrane. 4. Chemotactic peptide, [3H]formyl-norleucyl-leucyl-phenylalanine [3H]formyl-Nle-Leu-Phe) binding by subcellular fractions shows that the highest specific binding was observed in the light-plasma membrane was about 2-fold higher than the heavy-plasma membrane, about 37-fold higher than the nuclear fraction, about 3-fold higher than lysosomal fraction and about 10-fold higher than the microsomal fraction.

Animals↗

PubMed1972

Lysosomes of the arterial wall. I. Isolation and subcellular fractionation of cells from normal rabbit aorta.

Smooth muscle cells were dissociated from normal rabbit aorta by incubating the tissue in Hanks' solution containing elastase, collagenase, and hyaluronidase. The isolated cells contained significant amounts of the following acid hydrolases: N-acetyl-beta-glucosaminidase, N-acetyl-beta-galactosaminidase, beta-galactosidase, beta-glucuronidase, alpha-mannosidase, beta-glucosidase, acid phosphatase, and cathepsins C and D. The cells were disrupted and fractionated by isopycnic centrifugation on sucrose density gradients in the Beaufay automatic zonal rotor. Lysosomes with a modal density of 1.16 were identified by the distribution of these acid hydrolases and by the latency of N-acetyl-beta-glucosaminidase and beta-galactosidase. Other particulate enzymes studied in these sucrose gradients included cytochrome oxidase and monoamine oxidase (mitochondria), 5'-nucleotidase and leucyl-beta-naphthylamidase (plasma membrane), and catalase (? peroxisome). This microanalytical subcellular fractionation technique is applicable to the study of milligram quantities of many other tissues, both normal and pathological.

Acetyltransferases↗

PubMed1986

Subcellular localization of fucose incorporation into mouse thyrotropin and free alpha-subunits: studies employing subcellular fractionation and inhibitors of the intracellular translocation of proteins.

To determine the subcellular sites of fucose incorporation into TSH subunits, pituitaries from hypothyroid mice were incubated with [3H]fucose and fractionated by sucrose gradient centrifugation. To assess potential molecular cross-contamination between subcellular fractions enriched in rough endoplasmic reticulum (RER) or Golgi elements, trace amounts of exogenous [35S]methionine-labeled proteins or [125I]rat TSH were added before tissue homogenization. Particulate contamination of fractions was monitored by electron microscopy. TSH subunits were immunoprecipitated from fractions and analyzed by gel electrophoresis. After both a 2-h pulse incubation and a 2-h pulse, 3-h chase incubation, about half (range, 44-71%) of the [3H]fucose-labeled TSH subunit precursors present in microsomes were in the RER (amounts in excess of estimated contamination by nonspecific readsorption of molecules to vesicles or the presence of Golgi vesicles in the RER fractions); [3H]fucose-labeled free alpha-subunits were also detected in RER as well as in Golgi fractions. During chase incubations, both monensin and carboxyl cyanide m-chlorophenylhydrazone inhibited the appearance of [35S]methionine- or [3H]fucose-labeled TSH subunits in medium in a dose-dependent manner, suggesting that [3H] fucose was added to subunits, in part, early in the secretory pathway. Free alpha-subunits were more fucosylated than was TSH; in TSH heterodimers, beta-subunits were richer in fucose than were alpha-subunits. Thus, the fucosylation of TSH and free alpha-subunits in pituitaries of hypothyroid mice appears to begin at an unusually early stage of intracellular transport and may represent an adaptation to special posttranslational processing requirements.

Acetylglucosaminidase↗