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PubMed1985

In vitro and in vivo effects of vanadate on K+-dependent phosphatase activities from subcellular fractions of brain, kidney and liver.

Inhibition of K+-dependent phosphatases by sodium orthovanadate was studied in the subcellular fractions of brain, kidney and liver from male Sprague-Dawley rats. The concentration required to produce an in vitro phosphatase inhibition of 50% ranged from a low of 0.2 microM in the kidney 900 X g fraction to a peak of 400 microM in the brain soluble fraction. The concentrations required to produce 50% inhibition occurred in the micromolar range for most fractions studied. Injection of sodium vanadate (i.p., 8 mg/kg) produced phosphatase inhibition in several of the fractions, but not as pronounced as the in vitro treatment. The results indicate that vanadate is a potent inhibitor of phosphatase activity in vitro, and may have similar potential in vivo.

Animals↗

PubMedDate not supplied

[Characteristics of immunoaffinity chromatography and a new method for isolation of human thyroid peroxidase from subcellular fractions of thyroid gland].

A system for quantitative determinations of human thyroid peroxidase (TPO) in biological fluids has been obtained, based on the use of enzyme-linked immunosorbent assay. Immunochemical properties of TPO were studied under variable conditions, and a new method for isolating the protein from microsomes, mitochondria, and cytosol of thyroid glands of patients with diverse thyroid diseases was developed. The procedure involves solubilization of subcellular fractions with detergents, their sonication, two sequential runs of chromatography (on sorbents with immolbilized monoclonal antibodies against TPO and goat anti-human immunoglobulin antibodies), treatment with ribonuclease, and dialysis. Highly purified preparations of intact TPO and a product of its limited trypsinolysis are expected to be used as research tools and components of high-sensitivity immunoassays.

Antibodies, Monoclonal↗

PubMed1992

Immunocytochemical localization of capsid-related particles in subcellular fractions of poliovirus-infected cells.

The structural proteins of poliovirus can assemble into a series of different configurations (capsid-related particles, CRP). Only some seem to be true capsid precursors and the role of most CRP in morphogenesis is unclear. We used electron microscopic immunocytochemistry with monoclonal antibodies recognizing different CRP [protomers, pentamers, 65S empty capsids (EC), 74S-EC, and virions] to locate CRP in subcellular fractions containing virus-induced vesicles associated with the viral replication complex. We found pentamer antigenic CRP to be associated with the replication complex. The same pentamer antigenicity was exhibited by novel, "capsid-like" structures attached to the surface of the virus-induced vesicles. Upon solubilization of the vesicular fraction, mainly 65S-EC and only negligible amounts of pentamers were found by sucrose gradient analysis and by immunoprecipitation. We show that the pentamer antigenic particles are converted into 65S-EC when their membranous support is dissolved. We propose that the vesicular membrane prevents the assembly of 65S-EC and keeps the pentamer antigenic CRP in the appropriate concentration and configuration for association with the nascent progeny RNA.

Antibodies, Monoclonal↗

PubMed1985

Phosphatidic acid phosphatase activity in subcellular fractions of normal and dystrophic human muscle.

Biopsy samples from normal and dystrophic human muscle (Duchenne type) were fractionated by differential centrifugation and microsomes, mitochondria and cytosol were assayed for phosphatidic acid phosphatase (EC 3.1.3.4) and marker enzymes of mitochondria and cytosol. The activity of phosphatidic acid phosphatase was significantly lower in microsomes and higher in cytosol and mitochondria of dystrophic muscle than in the corresponding subcellular fractions of normal muscle. The results support an explanation of earlier findings that there is reduced G3P incorporation into diglycerides and phosphatidylcholine and a qualitative and quantitative change in the amount of phosphatidylcholine in dystrophic microsomes. The possible reasons for the reduction in the activity of only microsomal PA-P-ase were discussed.

Animals↗

PubMed1992

Contribution of dihydrodiol dehydrogenase to the metabolism of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene in fortified rat liver subcellular fractions.

Dihydrodiol dehydrogenase (DD; EC 1.3.1.20) purified to homogeneity from rat liver cytosol will catalyze the NAD(P)(+)-dependent oxidation of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-diol) to yield benzo[a]pyrene-7,8-dione (BPQ). To verify that BPQ is a metabolite of B[a]P-diol in rat liver, an S100 fraction was supplemented with NAD+ and NADP+, and the formation of BPQ was followed by reverse-phase HPLC. The identity of BPQ was established by co-chromatography with an authentic standard (under different solvent conditions) and by RP-HPLC using a diode-array detector which established that the metabolite shared spectral identity with BPQ. The formation of BPQ in the S100 fraction was blocked by either a competitive inhibitor (indomethacin) or a suicide substrate [1-(4-nitrophenyl)-propen-1-ol] for DD, indicating that BPQ was being formed by this enzyme. To assess the contribution of DD to the metabolism of [3H]B[a]P-diol, subcellular fractions obtained from uninduced rat liver were fortified with co-factors to optimize the activity of enzymes that would compete for this proximate carcinogen. Under these conditions, S100 fractions fortified with NAD+ and NADP+ metabolized 25% of the B[a]P-diol, producing 731 +/- 154 pmol of BPQ. In contrast, rat liver microsomes fortified with an NADPH generating system metabolize 75% of the B[a]P-diol producing 2614 +/- 379 pmoles of benzo[a]pyrene-tetrahydrotetrols. Rat liver homogenates (S10) fortified with either uridine diphosphoglucuronic acid or phosphoadenosine phosphosulfate produced 180 +/- 56 and 95 +/- 31 pmoles of conjugates respectively, which were recovered as B[a]P-diol after treatment of the aqueous phase with either beta-glucuronidase or aryl sulfatase. Of the metabolites analyzed BPQ was formed in the second largest amount. These studies show that in uninduced rat liver DD may play a significant role in the metabolism of B[a]P-diol. The metabolic fate of BPQ remains to be determined.

Alcohol Oxidoreductases↗

PubMed1978

Analytical subcellular fractionation studies on different cell types isolated from normal rat liver.

1. Parenchymal, Kupffer and biliary tract cells were isolated from normal rat liver by perfusion with collagenase solution. 2. The specific activities (munits of enzyme activity/mg of protein) of marker enzymes for the principal subcellular organelles were determined in the isolated cell homogenates and compared with whole liver homogenates. 3. The cells were disrupted and the extracts subjected to analytical subcellular fractionation by sucrose-density-gradient centrifugation. Lysosomal integrity was determined by assaying latent beta-N-acetylglucosaminidase in the extracts. 4. Similar subcellular distributions were found for lysosomal, endoplasmic reticulum and plasma membrane marker enzymes in the whole liver and in parenchymal and biliary tract cells. In Kupffer cells, the proportion of these enzymes in the cytosol was significantly increased compared with the other fractions. In addition the equilibrium densities of the various organelles in these cells were lower than those from parenchymal cells.

Animals↗

PubMed1997

Generation of proteoliposomes from subcellular fractions.

Intracellular membranes are highly dynamic, yet they retain their identity and functional characteristics. Integral membrane proteins, which must confer this specific membrane identity, remain poorly characterized at the biochemical level, largely because detergent-mediated solubilization is required for purification and analysis, and several properties of integral membrane proteins can only be investigated when the molecule is properly embedded in a lipid bilayer. We present a method for the efficient reconstitution into proteoliposomes of integral membrane proteins from subcellular fractions. Integral membrane proteins were identified on high-resolution two-dimensional gels after selective extraction of soluble and peripheral membrane proteins; they accounted for 8% of the number of resolved polypeptides. A reconstitution procedure based on membrane solubilization with dodecyl-octaoxyethylene (C12E8) and subsequent detergent removal with BioBeads SM-2 resulted in the efficient reconstitution of several membrane proteins into proteoliposomes of uniform density. The generated proteoliposomes strongly resemble the starting membrane fraction in protein composition. This reconstitution allows the functional characterization of integral membrane proteins after enrichment and/or specific (immuno)depletion.

Animals↗

PubMed1984

Effect of inositol-1,4,5-trisphosphate on isolated subcellular fractions of rat pancreas.

We have previously shown that inositol-1,4,5-trisphosphate (IP3) releases Ca2+ from an intracellular calcium store in permeabilized acinar cells of rat pancreas (H. Streb et al., 1983, Nature (London) 306:67-69). This observation suggests that IP3 might provide the missing link between activation of the muscarinic receptor and Ca2+ release from intracellular stores during stimulation. In order to localize the intracellular IP3-sensitive calcium pool, IP3-induced Ca2+ release was measured in isolated subcellular fractions. A total homogenate was prepared from acinar cells which had been isolated by a collagenase digestion method. Endoplasmic reticulum was separated from mitochondria, zymogen granules and nuclei by differential centrifugation. Plasma membranes and endoplasmic reticulum were separated by centrifugation on a sucrose step gradient or by precipitation with high concentrations of MgCl2. IP3-induced Ca2+ release per mg protein in the total homogenate was the same as in leaky cells and was sufficiently stable to make short separation procedures possible. In fractions obtained by either differential centrifugation at 7000 X g, sucrose-density centrifugation, or MgCl2 precipitation there was a close correlation of Ip3-induced Ca2+ release with the endoplasmic reticulum markers ribonucleic acid (r = 0.96, 1.00, 0.91, respectively) and NADPH cytochrome c reductase (r = 0.63, 0.98, 0.90, respectively). In contrast, there was a clear negative correlation with the mitochondrial markers cytochrome c oxidase (r = -0.64) and glutamate dehydrogenase (r = -0.75) and with the plasma membrane markers (Na+ + K+)-ATPase (r = -0.81) and alkaline phosphatase (r = -0.77) in all fractions analyzed. IP3-induced Ca2+ release was distributed independently of zymogen granule or nuclei content of the fractions as assessed by electron microscopy. The data suggest that inositol-1,4,5-trisphosphate releases Ca2+ from endoplasmic reticulum in pancreatic acinar cells.

Animals↗

PubMedDate not supplied

[Content of nucleic acid in subcellular fractions of cow milk].

The article deals with the content of nucleic acids in mitochondrial and cell-nuclear fractions isolated from fresh milk as well as with changes in the content of phosphoric compounds and activity of alkaline phosphatase in milk in different periods between milkings. It is shown that prolongation of the intervals between milkings decreases the intensity of the phosphoric compounds secretion and the content of nucleic acids in the subcellular fractions and simultaneously rises activity of alkaline phosphatase, that evidences for a decrease in the intensity of the secretory processes in the mammary gland.

Alkaline Phosphatase↗

PubMed1989

Calmodulin-binding proteins in subcellular fractions of zones of the adrenal cortex.

The guinea pig adrenal cortex consists of a steroidogenic ACTH-responsive outer zone and an ACTH-unresponsive inner zone. It has been suggested that calmodulin plays an important role in ACTH-stimulated steroidogenesis. Thus, in an effort to examine the calmodulin 'system' in the guinea pig adrenal cortex model, Ca2+-dependent binding of calmodulin to proteins in subcellular fractions of the outer and inner zones was examined by the [125I]iodocalmodulin overlay technique and compared to similar studies utilizing pancreas, brain and liver tissue. Although the general pattern of calmodulin-binding proteins was similar for the two adrenocortical zones, quantitatively there was a striking difference with greater binding in the outer zone; this was particularly noteworthy for the mitochondrial fraction. The two most prominent calmodulin-binding proteins isolated from cytosol by calmodulin-Sepharose column chromatography had Mr of 60,000 and 47,000. The size of these two proteins suggested the presence of Ca2+/calmodulin-dependent protein kinase II. Western blot analysis, however, failed to demonstrate calmodulin kinase II in either zone, although it was clearly detectable in brain cytosol. The 60 K calmodulin-binding protein in the adrenal cortex also suggested the presence of the calmodulin-binding A subunit of the Ca2+/calmodulin-stimulated protein phosphatase, calcineurin. Western blot analysis did reveal the presence of calcineurin in the outer adrenocortical zone; it was not detectable, however, in the inner adrenocortical zone. The relation between the striking zonal differential for calmodulin-binding proteins and the zonal differential in ACTH-stimulated steroidogenesis in the guinea pig adrenal cortex will require further investigation.

Adrenal Cortex↗

PubMed2001

Comparison of the metabolism of L-erythro- and L-threo-sphinganines and ceramides in cultured cells and in subcellular fractions.

Ceramide (Cer) is a key intermediate in the synthetic and degradative pathways of sphingolipid metabolism, and is also an important second messenger. Natural Cer exists in the D-erythro configuration. Three additional, non-natural stereoisomers exist, but conflicting reports have appeared concerning their metabolism. We now compare the stereospecificity of three enzymes in the sphingolipid biosynthetic pathway, namely dihydroceramide (dihydroCer), sphingomyelin (SM) and glucosylceramide synthases, in subcellular fractions and in cultured cells. The L-erythro enantiomers of sphinganine, dihydroCer and Cer do not act as substrates for any of the three enzymes. In contrast, the diastereoisomer, L-threo-sphinganine, is acylated by dihydroCer synthase, and L-threo-dihydroCer and L-threo-Cer are both metabolized to dihydroSM and SM, respectively, but not to dihydroglucosylceramide and glucosylceramide. No significant difference was detected in the ability of SM synthase to metabolize Cer containing a short (hexanoyl) versus long acyl chain (palmitoyl), demonstrating that short-acyl chain Cers mimic their natural counterparts, at least in the sphingolipid biosynthetic pathway.

Animals↗

PubMed1984

Effects of papaverine on the relative distribution of 45Ca ions in subcellular fractions of hog biliary muscles.

The mechanisms by which papaverine (Pap) relaxes smooth muscles of hog bile ducts were investigated with respect to the effects of Pap on the distribution of Ca ions in intracellular organelles. Muscles loaded with 45Ca ions in 139 mM KCl-Tyrode's solution were incubated with Pap, [3H]Pap or theophylline (ThP) in Tyrode's solution. The Pap and Ca contents of various subcellular fractions were measured. Pap (40 microM) had no effect on Ca influx into the muscle in normal and 139 mM KCl-Tyrode's solutions. Pap was distributed to a greater extent in the cytosol fraction (F4) than in microsome and mitochondria-enriched fractions (F3) and in the nuclei-enriched fraction (F2). Pap selectively accumulated in the plasma membrane-enriched fraction (PM-F) and was negligible in the sarcoplasmic reticulum-enriched fraction (SR-F) and the mitochondria-enriched fraction (Mit-F). The Ca release of PM-F was greater and more rapid than the release from SR-F or Mit-F. The increase in the content of Ca in PM-F induced by Pap was enhanced by oxalate, suggesting that the increase is based on the increase in Ca uptake, not on the reduced efflux. Pap increased dose-dependently the Ca content of PM-F and appeared to decrease the Ca contents of SR-F and Mit-F. ThP and cyclic AMP (4 microM) had no effect on the Ca uptake of PM-F and no influence on the effects of Pap. These results suggest that Pap relaxation of hog biliary smooth muscle results from the interaction of the drug with the plasma membrane causing cyclic AMP-independent sequestration of Ca.

Animals↗

PubMed1990

Effect of methionine sulfoximine on pyruvate dehydrogenase, citric acid cycle enzymes and aminotransferases in the subcellular fractions isolated from rat cerebral cortex.

The effect of acute and subacute doses of L-methionine-DL-sulfoximine (MSI) were studied on the activities of pyruvate dehydrogenase, enzymes of citric acid cycle and aspartate and alanine aminotransferases in the mitochondria, synaptosomes and cytosol of rat brain. In general, the activities of pyruvate dehydrogenase and of the citric acid cycle enzymes, except malate dehydrogenase (malate----oxaloacetate), were elevated in all 3 subcellular fractions. Malate dehydrogenase activity (malate----oxaloacetate) was suppressed in the mitochondria while the activity of this enzyme in the reverse direction was enhanced in the cytosol. Activities of aspartate and alanine aminotransferases were suppressed under these conditions. As the effects of MSI on these enzymes were similar to those observed upon the administration of ammonium salts, it is suggested that the hyperammonemic state induced by MSI might derange the operation of the malate-aspartate shuttle. Increased activities of citric acid cycle enzymes in the cytosol suggested the existence of a small population of mitochondria which was highly vulnerable either to ammonia or to MSI.

Ammonia↗

PubMed1980

Analytical subcellular fractionation of rabbit alveolar macrophages with particular reference to the subcellular localisation of pyridine nucleotide-dependent superoxide-generating systems and superoxide dismutase.

Normal and Bacillus Calmette-Guerin (BCG) vaccine-induced rabbit alveolar macrophage homogenates were fractionated by isopycnic density gradient centrifugation. Superoxide dismutase-inhibitable NAD(P)H-dependent nitro-blue tetrazolium reductase was found localised to endoplasmic reticulum and mitochondria. The normal macrophages tended to contain more of this activity than the BCG-induced macrophages. Two superoxide dismutases were found: cyanide-sensitive superoxide dismutase was predominantly present in the cytosol, with a small proportion in mitochondria; cyanide-resistant superoxide dismutase was found confined to mitochondria. Neither differed in specific activity betw-en the normal and BCG-induced macrophages.

Animals↗

PubMed1973

Metabolism of palmitic acid in the subcellular fractions of mouse brain.

After an intracerebral injection of [(14)C]palmitic acid to C57BL/10J mice, the radioactivity in the brains decreased rapidly with time. The incorporated radioactivity was primarily in the 16:0 acyl groups of the diacyl phosphoglycerides at 1 and 3 days after injection. At longer times, increasing proportions of the radioactivity were found in cerebrosides, alkenyl groups, and other acyl groups. The specific radioactivities of the phosphoglycerides were highest in the microsomal fraction at 1 day after injection. The exchange of the diacyl glycerophosphorylcholines and diacyl glycerophosphorylethanolamines between the microsomes and the myelin required 8-14 days. When calculated on the basis of the radioactivity in the 16:0 acyl groups, the half-lives for both of these phosphoglycerides were 6-8 days in all subcellular fractions during the period from 14 to 30 days after injection. The radioactivity in the total lipids from the purified myelin fraction did not decline until more than 14 days after injection because of the reutilization of labeled 16:0 acyl groups for lipid biosynthesis. Recycling of the acyl groups explains the long half-lives reported for myelin phosphoglycerides after injection of [(14)C]acetic acid. Lipids with a relatively high specific radioactivity were lost from the myelin fraction during the purification procedure. The most likely source of these lipids is the most recently formed myelin that is not consolidated into the myelin sheath.

Acylation↗

PubMed1985

Intracellular fate of liposome-encapsulated DNA in mouse liver. Analysis using electron microscope autoradiography and subcellular fractionation.

Transient expression of liposome-encapsulated DNA in liver after intravenous injection to rats and mice has raised questions concerning the intracellular fate of this DNA. Electron microscope autoradiography shows that at 10 min after injection the highest concentration of liposomal DNA which is taken up by the liver is associated with lysosomes and vesicles. The proportion of DNA associated with the mitochondria steadily increases for 1 h after injection, up to 48% of the exogenous DNA found in the tissue. Part of this DNA follows the subcellular fractionation profile of the mitochondrial matrix marker, malate dehydrogenase. In contrast, 14% of the liposomal DNA taken up by the liver is found in the nuclei at 3 min after injection, and this percentage decreases over a period of 1 h. These results permit us to establish the distribution of liposome-encapsulated DNA among subcellular organelles in liver at different times after injection.

Animals↗

PubMedDate not supplied

Subcellular fractionation of bone marrow-derived macrophages: localization of phospholipase A1 and A2 and acyl-CoA:1-acylglycero-3-phosphorylcholine-0-acyltransferase.

Analysis of the subcellular distribution of lipid-metabolizing enzymes was carried out in bone marrow-derived macrophages with special respect to a comparison of the subcellular localisation of phospholipase A1 and A2 and to acyl-CoA:1-1-acylglycero-3-phosphorylcholine-0-acetyltransferase. After cell disruption differential centrifugation was followed by additional sucrose gradient purification of three main fractions. Satisfactory enrichment factors were obtained by this method for the following marker enzymes. The plasma-membrane enzyme alkaline phosphodiesterase I was enriched up to 25-fold and the acyl-CoA:1-acylglycero-3-phosphorylcholine-0-acyltransferase was enriched up to 30-fold. The marker enzyme for the endoplasmic reticulum, NADPH-cytochrome c reductase showed a similar enrichment and distribution as the acyltransferase. Therefore it was concluded that the acyl-CoA:1-acylglycero-3-phosphorylcholine-0-acyltransferase of bone marrow-derived macrophages is mainly located in the endoplasmic reticulum. Phospholipase A1 and A2 occurred in a high proportion together with the lysosomal marker enzyme N-acetyl-beta-glucosaminidase in the soluble supernatant and in the gradient fractions. In the endoplasmic reticulum phospholipase A2 occurred only in trace activities whereas phospholipase A1 was maximally enriched in this subcellular fraction. No subcellular fraction could be obtained where phospholipase A2 was enriched exclusively. However, it can be concluded that the two enzymes which are responsible for the balance of fatty acid liberation and re-acylation are located in two different cellular compartments. Furthermore it can be claimed that in the cell there has to exist an exchange of substrates and products between these compartments to achieve a complete metabolic cycle of the de- and re-acylation reaction of phospholipids in bone marrow-derived macrophages.

Acyltransferases↗

PubMed1989

Radioactive metal complexes with affinity for tumors. II. Biodistribution of radioactivity in cellular and subcellular fractions of tumor tissues.

The 99mTc and 57Co complexes of ethylenediamine-N,N-diacetic acid (EDDA) are accumulated in tumor tissue. The complexes and related radioactive compounds were administered to experimental animals bearing Ehrlich tumor, and the blood, tumor, abscess, and other tissues were separated, fractionated and analyzed. In blood, the EDDA complexes of 99mTc and 57Co were in dialyzable forms, whereas other tumor-nonlocalizing compounds were in undialyzable or protein-bound forms. The tumor/blood and tumor/muscle ratios of the radioactivity showed that the complexes had the high affinity for tumor tissues. Density gradient centrifugation analysis of the ascites tumor tissues showed that a significant amount of the radioactivity of the complexes was present in tumor cells. Subcellular fractionation of solid tumor tissue showed that the radioactivity was present in nuclear fraction.

Animals↗