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PubMed1984

Metabolic hydrolysis of isoniazid by subcellular fractions of rat liver.

The characteristics of isoniazid amidase which hydrolyzes isoniazid to isonicotinic acid and hydrazine was examined in vitro using rat liver subcellular fractions. The activity of isoniazid amidase was estimated from the amount of hydrazine produced from a substrate, isoniazid, by means of GC-MS. High activity of the amidase was observed in the microsomal and lysosomal fractions, and at pH 7.4-7.8 in the microsomal fraction. The amidase was not inhibited by acetanilide, but by procaine and bis(p-nitrophenyl)phosphate. As expected, acetylisoniazid, a main metabolite of isoniazid, also inhibited the amidase. Not only the microsomal monooxygenase but also amidase was strongly induced by pretreatment with phenobarbital, 3-methylcholanthrene and rifampicin, respectively.

Amidohydrolases↗

PubMed1982

Subcellular fractions of the adrenal medulla. Comparison by two-dimensional polyacrylamide gel electrophoresis.

Subfractions of adrenal medullary homogenates were analyzed in two-dimensional polyacrylamide gels to assess the extent of protein homology. Chromaffin granule proteins were highly acidic, with the exception of the soluble form of the enzyme dopamine beta-hydroxylase (EC 1.14.17.1). The purified granule membrane proteins were more heterogeneous, but still predominantly acidic. The soluble and membrane forms of dopamine beta-hydroxylase behaved identically in this gel system. Lactoperoxidase-catalyzed iodination of intact granules revealed that most, but not all, granule membrane proteins are accessible at the cytoplasmic face. Prominent proteins of the purified adrenal medullary mitochondria showed little if any homology with purified granule membranes. The crude microsome fraction showed significant homology with purified granule membranes despite low levels of cross-contamination between the two fractions in marker enzyme analysis. Among proteins that could be identified, dopamine beta-hydroxylase was at a low level in the microsomes, while the granule membrane protein cytochrome b-561 appeared to be in both fractions. The pattern obtained from primary cultures of adrenal chromaffin cells was very complex, but prominent proteins from the subcellular fractions were seen without difficulty. Actin and tubulin were very prominent in the whole cell pattern. Radioiodination of the whole cells resulted in a number of spots being labelled, although the majority of the label appeared to be in only two proteins of molecular weight 70000 and isoelectric point 5.7.

Adrenal Medulla↗

PubMed1978

Interactions of gonadotropins with corpus luteum membranes. I. Properties and distributions of some marker enzyme activities after subcellular fractionation of the superovulated rat ovary.

The properties of a number of enzyme activities of the superovulated rat ovary have been studied to establish optimal assay conditions and specific assay procedures for each activity. The activities were chosen on the basis of their extensive use in other tissues of the rat as marker enzymes for the major cell organelles. Homogenates of superovulated rat ovaries were subjected to fractionation by differential rate centrifugation, and sedimentation profiles were constructed for each marker enzyme activity. The various subcellular fractions were also monitored by electron microscopy. The enrichment of fractions with particular organelles by electron microscopy, and enrichment of the appropriate organelle marker enzyme activities correlated well. Sedimentation profiles of a number of plasma membrane marker enzymes demonstrated a marked discrepancy between hCG-binding activity, and 5'-nucleotidase-, alkaline phosphatase-, and Mg2+-dependent ATP-ase on the one hand, and basal, hCG-stimulated, and fluoride-stimulated adenylate cyclase activities on the other hand. Fractions enriched in hCG-binding and adenylate cyclase activities were subjected to further fractionation on discontinuous sucrose density gradients. The distributions of the various plasma membrane markers again indicated a partial dissociations between hCG-binding and adenylate cyclase activities of luteinized rat ovaries, suggesting the existence of two distinct major plasma membrane populations, with different buoyant densities, marker enzyme profiles and adenylate cyclase and hormone-binding levels.

Adenylyl Cyclases↗

PubMed2005

Distribution of conjugated linoleic acid in total and subcellular fractions from normal and cancerous parts of human testes.

The objective of the present study was to examine differences in the fatty acid composition of subcellular fractions from normal and cancerous parts of human testes. The conjugated linoleic acid (CLA) content was significantly higher in total testicular carcinoma (TC), but significantly lower in the mitochondrial fraction of TC in comparison to normal testicular tissue. The subcellular distribution pattern of CLA was similar to that of monounsaturated fatty acids, but different to that of 18:2n-6 (linoleic acid), underlining the different physiological properties of CLA and 18 : 2n-6. Because polyunsaturated fatty acids (PUFAs) have been suggested to have an effect on cancer risk and previous research has found that CLA inhibits the metabolism of 18 : 2n-6 into 20 : 4n-6, the contents of n-6 and n-3 PUFAs were determined. Significant differences were observed for 18 : 2n-6, 18 : 3n-3, 20 : 5n-3, and 22 : 6n-3, with 18 : 2n-6, 18 : 3n-3, and 20 : 5n-3 contents being higher and 22 : 6n-3 content being lower in TC than in normal testicular tissue. These results indicate a changed availability of substrates for the cyclooxygenase (COX) or lipooxygenase (LOX) pathways generating eicosanoids. Although not statistically significant, the reduced content of 20 : 4n-6 shown in this study might be due to an increased metabolism of this fatty acid into eicosanoids.

Cell Fractionation↗

PubMed1991

Big endothelin-converting enzyme activities in subcellular fractions of bovine aortic endothelial cells.

A combination of HPLC elution patterns and peptide sequencing has been used to characterize two distinct activities present in subcellular fractions of bovine aortic endothelial cells (BAECs) capable of converting human big endothelin-1 (big ET-1) to mature (ET-1). A pepstatin-inhibitable activity with an acidic pH optimum present in a lysosome-enriched fraction cleaved big ET-1 at positions 18 and 21 at similar rates. A neutral pH activity present in a postlysosomal organelles subfraction was also able to convert big ET-1, and was inhibited by EDTA, but not by 1-chloro-3-tosylamido-4-phenyl-2-butanone (TPCK), an inhibitor of chymotrypsin-like serine proteases.

Animals↗

PubMed1982

Acid:coenzyme A ligase in brain: fatty acid specificity in cellular and subcellular fractions.

We measured long-chain fatty acid: coenzyme A (CoA) ligase (EC 6.2.1.3) activity with four fatty acids in brain homogenates, and cellular and subcellular fractions to determine whether there are differences in activity that could be correlated with differences in fatty acid composition and metabolism. In rat brain homogenates, ligase activity varied appreciably with the four acids, with 18:2 greater than 18:1 greater than 16:0 greater than 22:1 (nmol acyl-CoA formed/min/mg protein: 1.46, 1.20, 0.96, and 0.57, respectively). This order was similar under all incubation conditions tested, including variable pH and fatty acid concentrations. The relative specific activities (RSA, 16:0 = 1.0) with the four substrates were similar in rat brain homogenate, mitochondria, and microsomes, with the highest specific activities in the latter fraction. The RSA were also similar in ox brain homogenates, in rabbit brain microsomes prepared from gray and white matter, in neurons isolated from rat brain, and in cultured neuroblastoma cells. Rat liver homogenates had a significantly different pattern of RSA. These results indicate that the ligase(s) has a preference for certain fatty acids, but suggest that the major control of fatty acid composition and metabolism is a function of subsequent metabolic steps.

Animals↗

PubMed1987

Chagas' disease: humoral response to subcellular fraction of Trypanosoma cruzi in symptomatic and asymptomatic patients.

The capacity of antibodies in serum from individuals with chronic Chagas' disease to react with antigens in different subcellular fractions of Trypanosoma cruzi varied according to the clinical status of the patients. Antibodies in serum of asymptomatic patients were directed mostly against antigens in the citosol of the parasite, whereas in overtly cardiopathic patients antibodies were directed mostly against antigens in the microsomal fractions.

Adult↗

PubMed1984

Localization of proteoglycan core protein in subcellular fractions isolated from rat chondrosarcoma chondrocytes.

Chondrocytes from the Swarm rat chondrosarcoma were pulse-labeled with [3H]serine for 30 min and chased, in the presence of cycloheximide, for times up to 300 min. The movement of newly synthesized core protein precursor of the proteoglycan through elements of the endoplasmic reticulum and Golgi complex was examined. Rough and smooth microsome fractions were obtained by centrifuging postmitochondrial supernatants from cell homogenates on discontinuous sucrose gradients. The core protein precursor was identified in subcellular fractions by (a) immunoprecipitation with an antiserum directed against the hyaluronate binding region of the core protein and the link protein and (b) its size on polyacrylamide gels. Labeled core protein precursor decreased from the microsomes with a t1/2 of 60 +/- 8 min, nearly the same as for the appearance of label in completed proteoglycan monomer (t1/2 = 58 +/- 13 min), consistent with a precursor-product relationship. After correcting for incomplete recovery of the core protein precursor in the microsomal fractions and for cross-contamination of the smooth microsomes by elements of rough endoplasmic reticulum, the redistribution of core protein precursor and completed proteoglycan in the intracellular compartments and of labeled extracellular proteoglycan were fit to a three-compartment model. A t1/2 of 98 +/- 7 min for the loss of core protein precursor from the rough microsomes and a t1/2 = 10 +/- 4 min for the completed proteoglycan in the intracellular compartment (Golgi and secretory vesicles) was obtained. The data indicate that at least 70% of the intracellular transit time for the core protein precursor is spent in the rough endoplasmic reticulum. The addition of glycosaminoglycan chains followed by secretion from the cell occurs relatively rapidly, occupying less than 30% of the total intracellular dwell time.

Animals↗

PubMed1983

Cellular immunity in Chagas' disease patients. Lymphoproliferative response to subcellular fractions of Trypanosoma cruzi.

The ability of peripheral mononuclear cells from Chagas' disease patients (CDP) to generate specific proliferative responses in vitro to whole homogenate (WH) and subcellular fractions of T. cruzi was investigated. Flagellar (F), microsomal (Mc) and cell sap (CS) fractions were isolated from a WH obtained by disruption by pressure-depressure. Parasite-free human peripheral mononuclear cells were isolated by centrifugation of leukocyte-rich plasma over discontinuous gradients of Ficoll-Hypaque and cultured for 6 days in the presence of the different fractions of T. cruzi. Eighty-six percent (54/63) of the CDP showed good capacity to respond to at least one of the fractions assayed while only 18% (4/22) of the controls reacted. The Mc fraction showed the best specificity as well as the maximal stimulating effect in CDP (37/55 vs 0/18 in controls) while WH (62% vs 10%), F (54% vs 16%) and CS (52% vs 6%) showed a lesser antigenic capacity. These results suggest that recognition mechanisms and proliferative responses are functional in CDP. No correlation was found between the stimulating capacity of fractions in vitro and their immunogenic capacity previously observed in vivo.

Cell Count↗

PubMed1989

Intracellular distribution of copper in the liver of copper-loaded sheep--a subcellular fractionation study.

Eighteen ewes in two groups were dosed orally with CuSO4 to induce chronic Cu toxicity. Copper dosing was stopped at the first rise of acid AP activity in the serum in group 1 sheep and on the first day of haemolysis in group 2 sheep. Liver samples were obtained 1 week prior to the start of Cu dosing, at the first rise of acid phosphatase (AP) activity in serum and on the first day of haemolysis. These liver samples were homogenized and were separated into nuclear (N), heavy mitochondrial (MH), light mitochondrial (ML), microsomal (MI) and cytosolic (CY) fractions by centrifugation. The Cu concentration and specific activities of AP were determined in the liver, LH and subcellular fractions. The composition of the fractions was studied by light and electron microscopy. In the predosing biopsies, the concentration and percentage of Cu and the total specific activity of AP were highest in the ML fractions. With increasing Cu loading, the concentration of Cu in all fractions increased; the percentage of Cu increased in the N and MH fractions, decreased in the ML and MI fractions and was maintained at a constant level in the CY fractions. The total specific activities of AP in LH, N, MH, MI and CY fractions were increased and the activity was highest in the MH fraction. The results indicate that the increase in the concentration of Cu in liver cells was predominantly in lysosomes and cytosol. Furthermore, it is suggested that the necrosis of isolated hepatocytes observed in chronic Cu-poisoned sheep may be due to a saturation of the uptake of Cu into the lysosomal system of the cell, leading to the accumulation of toxic levels of Cu in the cytosol.

Acid Phosphatase↗

PubMed1990

Trace element imbalances in isolated subcellular fractions of Alzheimer's disease brains.

Concentrations of 13 trace elements (Ag, Br, Co, Cr, Cs, Fe, Hg, K, Na, Rb, Sc, Se, Zn) in isolated subcellular fractions (whole brain, nuclei, mitochondria, microsomes) of temporal lobe from autopsied Alzheimer's disease (AD) patients and normal controls were determined utilizing instrumental neutron activation analysis. Comparison of AD and controls revealed elevated Br (whole brain) and Hg (microsomes) and diminished Rb (whole brain, nuclear and microsomes), Se (microsomes) and Zn (nuclear) in AD. The elevated Br and Hg and diminished Rb are consistent with our previous studies in AD bulk brain specimens. Comparison of element ratios revealed increased Hg/Se, Hg/Zn and Zn/Se mass ratios in AD. Se and Zn play a protective role against Hg toxicity and our data suggest that they are utilized to detoxify Hg in the AD brain. Overall our studies suggest that Hg could be an important toxic element in AD. Whether Hg deposition in AD is a primary or secondary event remains to be determined.

Aged↗

PubMed1987

Protein turnover in subcellular fractions of brain from the ethanol-fed rat.

The effect of ethanol consumption (27% of energy intake) for 21 days, on protein synthesis and degradation in subcellular fractions of rat brain was investigated using the Na2(14)CO3 labelling procedure. Ethanol administration elicited an increase in the rate of protein synthesis in all fractions which was accompanied by an increase of similar magnitude in the rate of breakdown. These results imply that whilst ethanol increases the rate of protein turnover it is without effect on the rate of protein accretion.

Animals↗

PubMed1989

Detection of low molecular mass GTP-binding proteins in chromaffin granules and other subcellular fractions of chromaffin cells.

A homogenate of purified chromaffin cells was fractionated, after removal of the nuclear fraction, by sucrose density gradient ultracentrifugation. The presence and subcellular localization of low molecular mass GTP-binding proteins was explored by incubation of blots of proteins from different subcellular fractions with [alpha-32P]GTP in the presence of Mg2+. The fractions enriched in intact chromaffin granule markers, i.e. catecholamines, chromogranin A, chromogranin B and cytochrome b-561 were also enriched in labelled GTP-binding proteins. Two major labelled components of 23 and 29 kDa were rapidly detected by autoradiography. Traces of 26 and 27 kDa components were also present. These components were detectable in both plasma and granule membranes. In addition to these components, the cytosolic fraction contained another GTP-binding protein of about 20 kDa. Binding of [alpha-32P]GTP was specific and dependent on Mg2+. By analogy to the findings reported in non-mammalian systems, the observations described here suggest the involvement of low molecular mass GTP-binding proteins in the chromaffin cell secretory process.

Animals↗

PubMed1982

[Organ and species factor in the phenomenon of tetracycline interaction with animal subcellular fractions].

Binding of the tetracyclines by the cell components of various organs of rats was studied comparatively. It was shown that binding of the tetracyclines by the subcellular fractions was decreasing as follows: the liver, the heart, the kidneys. The mitochondria were the fraction most actively binding the antibiotics. For determination of the features of this process the characteristics of tetracycline binding by the liver organoids of the rats and rabbits were compared. The quantitative indices of binding of the tetracyclines by the cell components of the rats and rabbits were rather similar.

Animals↗

PubMed1982

Rapid purification of proteolipids from rat brain subcellular fractions by chromatography on a lipophilic dextran gel.

Proteolipids from adult rat brain subcellular fractions were purified by a one-step procedure involving chromatography through Sephadex LH-60 eluted with an acidified chloroform-methanol mixture. The protein peak was eluted with the void volume and was free of adventitious lipids. The degree of purification was similar to that attained with the neutral-acidified chloroform-methanol dialysis method with the advantage that this new procedure can be carried out in only 3 h, with a recovery of proteins of 95-100%. Samples containing different lipid/protein ratios passed through the gel gave similar elution profiles. When labeled amino acids or palmitic acid were added to myelin total lipid extracts, no radioactivity was eluted with the protein, indicating that the proteolipid apoproteins purified by this method do not adsorb hydrophobic low-molecular-weight compounds.

Animals↗

PubMed1977

Thyrotropin binding and long acting thyroid stimulator absorbing activities in subcellular fractions from isolated thyroid cells and thyroid homogenates.

The ability of various thyroid subcellular fractions to bind [125I]iodo TSH and to absorb long-acting thyroid stimulator (LATS) and thyroid stimulating immunoglobulins (TSI) activities was examined. Membranes purified from thyroid homogenates or isolated thyroid cells absorbed LATS/TSI activities and specifically bound [125I]iodo TSH. Purified thyroglobulin, nuclei, mitochondria and ribosomes did not bind [125I]iodo TSH nor did they absorb LATS/TSI activities. Cell sap obtained by gentle lysis of isolated thyroid cells failed to absorb LATS/TSI activities and to bind labeled hormone. However, freeze-thawing of the cells fragmented the membranes, releasing [125I]iodo TSH binding as well as LATS/TSI absorbing activities into the soluble (cell sap) fraction. The results suggest that the LATS/TSI antigen is of cell surface origin, includes the TSH receptor or larger membrane fragments containing the receptor, and that its release into the soluble fraction is due to the fragmentation of the thyroid membrane during homogenization and preparative procedures.

Animals↗

PubMed1989

Distribution of activity converting 4-aminobutyraldehyde to gamma-aminobutyric acid in subcellular fractions of mouse brain.

The subcellular distribution of activity of 4-aminobutyraldehyde dehydrogenase (ABAL-DH) was studied in mouse brain. ABAL-DH was localized mainly in the crude mitochondrial fraction; most of the activity in this fraction was found in the subfraction containing synaptosomes, and the remainder was in the mitochondrial fraction. After osmotic disruption of synaptosomes, most of the activity was located in the synaptic cytosol, and the remainder was in the synaptic mitochondria. Sucrose density subfractionation of synaptosomes revealed that gamma-aminobutyric acid, glutamic acid decarboxylase, and ABAL-DH localized in a denser region of gradient fraction than the region containing acetylcholinesterase and choline acetyltransferase.

Acetylcholinesterase↗

PubMed1978

Decorporation of copper from liver subcellular fractions after alimentary loading of rats.

Subcellular copper distribution was studied after single alimentary loading of rats with copper sulfate and after 2- and 4-day spontaneous decorporation of the metal. Copper content in mitochondria and in the soluble fraction was found to rise as early as the 1st day. Then it decreased reaching the normal values after 2-day and 4-day autodecorporation for cytosol and mitochondria, respectively. The activities of rotenone-sensitive NADH-cytochrome c reductase, succinate-cytochrome c reductase and cytochrome oxidase were inhibited by copper treatment but after a 4-day decorporation they became normal. On the contrary, rotenone-insensitive NADH-cytochrome c reductase was activated. Single alimentary copper treatment induced changes in electron transport and oxidative phosphorylation with succinate and glutamate as substrates. The changes established were in accordance with the decreased enzyme activities. After chronic copper loading (7-8 weeks) the interruption of copper-enriched diet for 5 days led to restoration of the copper content in the subcellular fractions. Treatment with unitiol did not change the spontaneous decorporation.

Animals↗