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PubMed1989

[Subcellular kinetics of In-111 monoclonal antibody in the liver].

To evaluate the mechanism for prolonged retention of In-111 monoclonal antibody (MoAb) by the liver, we investigated the subcellular kinetics of the In-111 225.28S, an antimelanoma IgG2a. Forty microCi/10 micrograms of the In-111 MoAb was injected IV into groups of SD rats. The liver was excised at 3, 8, 17, 24, 48 and 72 hr and homogenized with 0.25 M sucrose containing 0.01 M Tris-HCl buffer (pH 7.6). Subcellular fractionation was carried out by 3-step (ultra-)centrifugation. The radioactivity of each fraction including nuclear, mitochondrial (lysosomal), microsomal and supernatant fraction was measured and expressed as a percentage of the total radioactivity of all the fractions. Moreover, the supernatant fraction was assessed by size exclusion HPLC. The supernatant was a predominant fraction of the In-111 radioactivity, 61% at 3 hr, and was decreased with time, 21% at 72 hr (p less than 0.01), while the activity in the mitochondrial fraction continued to increase from 11% to 44%. As compared to these two fractions, the nuclear and microsomal radioactivities were relatively constant, 10-25%, throughout the study. The HPLC analysis revealed that the supernatant In-111 activity at early time points was mainly eluted with an intact IgG and thereafter this major peak activity was reduced with associated activity peaks found in smaller moiety fractions. To compare the In-111 MoAb with the iodine labeled MoAb, we also investigated the subcellular kinetics of the I-125 MoAb in the same manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMedDate not supplied

[Subcellular localization of glutamine synthetase activity in carp muscle tissue and liver].

Subcellular distribution of the glutaminesynthetase activity (EC 6.3.1.2) is studied in muscle tissue and liver of carp. It is established that this activity is distributed by subcellular fractions analogously to the glutamatedehydrogenase activity--enzyme-marker activity of mitochondria. An additional evidence for the association of the two given enzymes is obtained during equilibrium density-gradient centrifugation of mitochondria on 30-60% gradient of sorbitol. Comparison of the obtained results with data from literature testifies to a coincidence of the subcellular localization of the processes of ammonia formation and binding into glutamine, thus confirming a view of the leading role of glutaminesynthetase in ammonia detoxication in carps.

Ammonia↗

PubMed1985

Subcellular distribution of dihydropyridine isothiocyanate binding in guinea-pig ileal smooth muscle.

The subcellular distribution of the 3H--2,6-dimethyl-3,5-dicarbomethoxy-4(2-isothiocyano) phenyl-1,4-dihydropyridine (DPSCN) binding to guinea-pig ileal smooth muscle was studied by subcellular fractionation. Initial experiments on subcellular fractionation of 3H-DPSCN-labelled tissues by differential centrifugation showed that there was an excellent correlation between the levels of the label present in a fraction and the plasma membrane marker phosphodiesterase I (r = 0.98) but not between the label and the putative endoplasmic reticulum marker NADPH: cytochrome-c-reductase (r = 0.56) or the inner mitochondrial marker cytochrome-c-oxidase (r = 0.36). Centrifugation of the microsomes on a continuous sucrose density gradient showed an excellent correlation of the migration of the label with phosphodiesterase I activity (r = 0.93) but not with the activities of NADPH: cytochrome-c-reductase (r = 0.66) or cytochrome-c-oxidase (r = 0.44). Treatment of microsomes with digitonin (1 mg/ml) followed by centrifugation on continuous sucrose density gradients increased the weighted mean densities of the phosphodiesterase activity (plasma membrane marker) and the labelling by similar magnitudes (0.04 to 0.06 g/ml). The weighted mean densities of NADPH: cytochrome-c-reductase and the cytochrome-c-oxidase were not altered significantly. It is concluded that in the guinea-pig ileal smooth muscle, DPSCN labels the plasma membrane specifically.

Animals↗

PubMed1987

Subcellular localization of retinoids, retinoid-binding proteins, and acyl-CoA:retinol acyltransferase in rat liver.

Studies were conducted to define the subcellular localization of endogenous retinoids (vitamin A), retinoid-binding proteins, and acyl-CoA:retinol acyltransferase (ARAT) in liver and to determine whether their distributions were affected by hepatic vitamin A content. Quantitative subcellular fractionation techniques were used. Rats were fed purified diets either containing or lacking vitamin A to obtain animals with total retinoid stores ranging from 0.5 to 172 micrograms of retinol equivalent per gram of liver. Liver homogenates were fractionated by differential centrifugation to yield nuclear (N), mitochondrial-lysosomal (ML), microsomal (P), and high-speed supernatant (S) fractions. N, ML, and P were washed two more times by resuspension and centrifugation to remove constituents bound nonspecifically. S was further resolved into "floating lipid" and underlying "cytosol" by prolonged ultracentrifugation. The distributions of marker constituents were not affected by vitamin A status. Most of the retinyl ester in the liver was recovered in the S fraction where it was entirely (greater than 95%) associated with floating lipid. About half of the total free retinol was also recovered in the S fraction, but it was mostly (2/3) associated with cytosol per se. A substantial portion (30%) of the free retinol was recovered in the 3 X -washed microsomal (P) fraction. Sufficient binding capacity for retinol was present in both P (as retinol-binding protein) and S (as cellular retinol-binding protein) to quantitatively account for the amounts of free retinol present in the two fractions. ARAT activity in the liver was distributed among the subcellular fractions in a manner identical with an endoplasmic reticulum marker enzyme (NADPH-cytochrome C reductase).(ABSTRACT TRUNCATED AT 250 WORDS)

Acyltransferases↗

PubMed1985

Subcellular localization of the phospholipases A of rat heart: evidence for a cytosolic phospholipase A1.

During myocardial ischemia increased levels of lysoglycerophospholipids have been reported which may be deleterious to myocardial function. Phospholipases are presumed to be important in the regulation of this process. To further quantify and characterize the activity of heart phospholipases, we carried out a systematic analysis of phospholipase A activity in rat heart subcellular fractions isolated by the method of Palmer et al. (J. Biol. Chem. 1972. 262: 8731-8739). Neutral phospholipase A was recovered predominately in the cytosolic (soluble) fraction which represented 46% of recovered activity, while the microsomal and subsarcolemmal mitochondrial fractions represented 15% and 12% of the total recovered activity, respectively. Cytosolic phospholipase A differed from the two principal membrane-bound phospholipases A in its pH dependence and apparent Km for substrate. The cytosolic enzyme had a Km (apparent) for dioleoylphosphatidylcholine of 0.07 mM versus 0.28-0.33 mM for the membrane-associated phospholipases A. Acid phospholipase A activity had a subcellular distribution consistent with a lysosomal localization. Lysophospholipase was found principally in the cytosolic, microsomal, and the subsarcolemmal and interfibrillar mitochondrial fractions where it represented 46, 17, 6.3, and 6.9% of the recovered activity, respectively. The positional specificity of the respective phospholipases was assessed. This analysis was complicated by the fact that in heart, lysophospholipase has an observed Vmax 3.6- to 4.5-fold greater than that of phospholipase A in the various subcellular fractions. Equations were derived to obtain corrected values for the activity of phospholipases A1 and A2. Using this method we found that the cytosolic and lysosomal fractions contained phospholipase A1, while the mitochondrial fractions contained primarily phospholipase A2. In heart microsomes, the positional specificity of phospholipase A could not be determined because lysophospholipase activity was very high and lysophosphatidylcholine did not accumulate.

Animals↗

PubMed1974

Phospholipids and acyl groups of subcellular membrane fractions from human intracranial tumors.

The phospholipids from subcellular fractions of human intracranial tumors were examined. For comparison, microsomes were isolated from a fetal human brain and from the gray matter of adult human brains. The subcellular membranes of tumors had a higher protein-to-phospholipid ratio than the normal brain membranes. The microsomes from tumors had a lower proportion of diacylglycerophosphorylethanolamine and higher proportions of alkenylacylglycerophosphorylcholine and sphingomyelin (plus diacylglycerophosphorylinositol) than microsomes from the gray matter. Also, the ratios of alkenylacylglycerophosphorylethanolamine to diacylglycerophosphorylethanolamine were higher in the tumors than in the normal controls. The acyl groups of ethanolamine phosphoglycerides in tumor microsomes had relatively more 18:1, 18:2, and 20:4(n - 6) and less 18:0, 22:4(n - 6), and 22:6(n - 3) than the adult brain gray matter. Except for the increase in 18:2, acyl group changes in choline phosphoglycerides between tumors and controls were not as extensive as in the ethanolamine phosphoglycerides. The characteristic features of phospholipids and their constituent acyl groups of tumors were often present in all the subcellular fractions. Although the acyl group profiles of the tumor phosphoglycerides were in closer resemblance to the fetal brain than to the adult brain, other differences were observed. Results indicate that neoplastic brain cells are unique in their cellular composition, and consequently they deviate from the normal neurons and glials in metabolism and functions.

Adult↗

PubMed1972

Cholesterol metabolism in myelin and other subcellular fractions of rat brain.

For many years the bulk of myelin in adult brain was believed to be metabolically stable, although some metabolic activity of a small myelin fraction, especially in the gray matter of the brain, was recognized. We have attempted to compare the composition of myelin fractions isolated from two different areas of the brain. No differences in chemical composition were observed. We have also investigated the metabolism of cholesterol in myelin and other subcellular fractions from the two areas. Both young (16-day-old) and adult rats were used. Results show an uptake of radioactive cholesterol by all subcellular fractions of the brain, including myelin, in both young and adult animals, with ultimate uniform distribution of the radioactive sterol and its persistence in all uniformly labeled subcellular fractions of the brain. On the basis of these results we suggest that there is a pool of cholesterol in the brain from which all metabolizing structures, including myelin, draw their cholesterol supplies. There is continuous exchange of cholesterol between the brain pool and the blood. The rate of this exchange may be related to the rate of blood flow through the tissue.

Acetates↗

PubMed1984

Subcellular localization of enzymes involved in leukotriene formation within human polymorphonuclear granulocytes.

The subcellular localization of enzymes involved in leukotriene formation was analysed according to biological (chemotaxis, spasmogenic properties) and analytical methods. By subcellular fractionation the major activity for 5--lipoxygenase and L-gamma-glutamyltranspeptidase coeluted with the 200,000 g precipitate while glutathione-S-transferase activity was mainly present in the 200,000 g supernatant. Our data were supported by results indicating that the 200,000 g precipitate and supernatant fractions proved to be most active in generating 5-HETE and leukotriene C4 (LTC4) respectively. The 200,000 g pellet was the most active fraction in transforming synthetic LTC4 into LTD4 and LTE4. When synthetic LTD4 was incubated with the various subcellular fractions and the appearance of LTE4 was analysed the 20,000 and 200,000 g pellets were the most potent fractions. Several discrepancies observed using biological, biochemical and analytical (synthetic substrates) methods may be in part due to the formation of leukotriene isomers which interfere with the biological assays.

Chemotactic Factors↗

PubMed1984

Heterogeneity of human whole blood platelet subpopulations. III. Density-dependent differences in subcellular constituents.

Structurally intact platelet cohorts of differing densities can be isolated from normal subjects by the use of isosmolar arabinogalactan density gradients. Using platelets separated in this fashion, we have studied the density-dependent distribution of four subcellular organelles: mitochondria, lysosomes, dense bodies, and alpha granules. Mitochondria, which are not secreted during platelet release, demonstrate a slow decline in monoamine oxidase activity within the gradient. Lysosomal beta-glucuronidase does not vary significantly with platelet density. In contrast, dense body number and endogenous serotonin content decrease significantly with decreasing platelet density, primarily as the result of differences in the number of storage organelles. Platelet factor 4 content declines rapidly in comparison to lysosomal activities (P less than .001 from bottom to top of the gradient); but beta-thromboglobulin, also an alpha granule component, exhibits considerably less change than platelet factor 4 (P less than .001). Thus, specific platelet subcellular constituents have different density distributions. We postulate that these density differences may be due to differential in vivo loss of selective biochemical constituents from unique subcellular compartments.

Blood Platelets↗

PubMed1984

Breakdown of proteins from mouse liver subcellular fractions. Effect of nutritional changes.

The rapid restoration of liver protein mass observed in protein-depleted mice when they are fed with an adequate diet is quantitatively explained by a large decrease in the average rate of breakdown of total liver proteins. This study was performed in order to know whether this inhibition of breakdown affects in the same way all the protein constituents of the tissue, or only affects a group of these proteins belonging to a particular subcellular fraction. Subcellular fractions were obtained by differential centrifugation. The relative rates of breakdown of their proteins were estimated by the conservation of radioactivity in these proteins previously labelled by the administration of NaH14CO3 to mice. The results obtained indicated: 1) a general decrease in the rate of breakdown of proteins of subcellular fractions from re-fed livers compared with livers of protein-depleted mice; 2) a decrease of breakdown of proteins from cytosol in re-fed mice which is higher as lower is the molecular weight of the proteins subunits.

Animals↗

PubMed1981

Subcellular fractionation studies on the post-translational processing of pro-adrenocorticotropic hormone/endorphin in rat intermediate pituitary.

The subcellular localization of the post-translational processing steps which occur in the conversion of pro-adrenocorticotropic hormone (ACTH)/endorphin into beta-endorphin-sized molecules in rat intermediate pituitary has been studied. Primary cell cultures were incubated in radioactively labeled amino acids, and a subcellular fraction containing secretory granules was separated from a subcellular fraction containing rough endoplasmic reticulum and Golgi apparatus by centrifugation of homogenates on gradients on Percoll (Pharmacia Fine Chemicals). The radiolabeled beta-endorphin-related material in the granule and rough endoplasmic reticulum/Golgi apparatus fractions was quantitated by immunoprecipitation and sodium dodecyl sulfate polyacrylamide gel electrophoresis. A pulse-chase labeling experiment demonstrated that newly synthesized beta-endorphin-related material first appeared in the rough endoplasmic reticulum/Golgi apparatus fraction and after longer incubations (chase) appeared in the secretory granule fraction. After 2 h of chase incubation, about 85% of the beta-endorphin-related material synthesized during the 30-min pulse incubation had been transferred from the rough endoplasmic reticulum/Golgi apparatus to the secretory granule fraction. The conversion of most of the newly synthesized pro-ACTH/endorphin into beta-lipotropin occurred in the rough endoplasmic reticulum/Golgi apparatus fraction, whereas the conversion of most of the beta-lipotropin into beta-endorphin-sized molecules occurred in the secretory granule fraction.

Adrenocorticotropic Hormone↗

PubMed1983

Internalization and subcellular localization of transferrin and transferrin receptors in HeLa cells.

The subcellular location of radiolabeled transferrin (125I-Tf), internalized during cellular iron uptake, and the cellular distribution of transferrin (Tf) receptors were studied in cultured HeLa cells. Cells were incubated at 37 degrees C with 125I-Tf(Fe)2. Forty per cent of the labeled ligand was associated with cell surface receptors. The remaining 60% was internalized as shown by the inability to dissociate 125I-Tf from cells by competition with excess Tf(Fe)2 or treatment of cells with 0.2 M acetic acid containing 0.5 M NaCl. Subcellular fractionation studies using sucrose density gradients indicated that internalized Tf was localized in a membranous vesicle distinct from lysosomes, Golgi apparatus, endoplasmic reticulum, or plasma membranes. The subcellular distribution of Tf receptors was studied using an assay for detergent solubilized receptors. Even without preincubation with ligand, the majority of cellular Tf receptors were localized intracellularly in a vesicle with the same buoyant density as the vesicle containing internalized 125I-Tf. Using an assay for occupied receptors, we demonstrated that the same vesicle contained both internal receptors and internalized ligand. A portion (20%) of the intracellular receptor pool was insensitive to trypsin treatment of whole cells at 37 degrees C suggesting that during the experimental time period (20-30 min) this portion did not recycle to the cell surface. We propose that during cellular iron uptake, Tf receptor-ligand complexes are internalized and directed to a nonlysosomal compartment where iron is released, followed by recycling to the cell surface of an intact Tf receptor-apo-Tf complex.

Biological Transport↗

PubMed1978

Regional and subcellular distribution and some factors in the regulation of reduced pterins in rat brain.

We have studied the regional and subcellular distribution, functional role, and pharmacology of quinoid dihydropterin reductase (QDPR) and endogenous reduced pterins (PH4) subserving tyrosin hydroxylase (TOH) and tryptophan hydroxylase in the rat brain. There is a significant correlation between the regional distribution of PH4 and TOH but not between PH4 and tryptophan hydroxylase or between either TOH or tryptophan hydroxylase and QDPR. This suggests that a major portion of PH4 is associated with the biosynthetic activity of brain catecholaminergic systems. The regional and subcellular distribution of QDPR was inconsistent with a regulatory function for QDPR in monoamine synthesis. In vitro measures of PH4, TOH, and synaptosomal dopamine (DA) and serotonin synthesis in the striate cortex of untreated animals and animals subjected to neurotoxin or electrolytic lesions of the dorsal raphe or substantia nigra exhibit significant covariation of PH4 with synaptosomal DA but not serotonin synthesis and a significant partial correlation of PH4 with DA synthesis. The subcellular distribution of PH4 in the striatum demonstrates an association of PH4 with the biosynthetic function of dopaminergic nerve terminals. Reserpine and d-amphetamine in vivo elicited an increase and decrease, respectively, in striatal PH4 paralleling induced changes in synaptosomal DA synthesis. Other drugs altering central catecholaminergic function did not alter striatal PH4 levels significantly. The data suggest that 1) a major portion of total PH4 (as much as 90% in the striatum) is related to the function of catecholaminergic rather than serotonergic systems, 2) PH4 levels is a determinant of the velocity of DA synthesis and 3) PH4 levels are altered by some psychoactive drugs in association with changes in synaptosomal catecholamine biosynthetic rates.

Animals↗

PubMed1984

Subcellular distribution and regulation of hepatic bilirubin UDP-glucuronyltransferase.

We have investigated the subcellular location and regulation of hepatic bilirubin UDP-glucuronyltransferase, which has been presumed to be located largely in the smooth endoplasmic reticulum. Purity of subcellular membrane fractions isolated from rat liver was assessed by electron microscopy and marker enzymes. Bilirubin UDP-glucuronyltransferase activity was measured by radiochemical assay using a physiologic concentration of [14C]bilirubin, and formation rates of bilirubin diglucuronide and monoglucuronides (C-8 and C-12 isomers) were determined. Activity of the enzyme was widely distributed in subcellular membranes, the majority being found in smooth and rough endoplasmic reticulum, with small amounts in nuclear envelope and Golgi membranes. No measurable activity was found in plasma membranes or in cytosol. Synthesis of bilirubin diglucuronide as a percentage of total conjugates and the ratio of C-8/C-12 bilirubin monoglucuronide isomers formed were comparable in all membranes, suggesting that the same enzyme is present in all locations. However, the regulation of bilirubin UDP-glucuronyltransferase activity differed among intracellular membranes; enzyme activity measured in the presence of the allosteric effector uridine 5'-diphospho-N-acetylglucosamine exhibited latency in smooth endoplasmic reticulum and Golgi membranes, but not in rough endoplasmic reticulum and nuclear envelope. Since rough membranes comprise 60% of hepatocyte endoplasmic reticulum and bilirubin UDP-glucuronyltransferase activity in vitro is maximal in this membrane fraction under presumed physiologic conditions, it is likely that the rough endoplasmic reticulum represents the major site of bilirubin glucuronidation in hepatocytes.

Animals↗

PubMedDate not supplied

[Subcellular distribution of the Ca++-dependent protein regulator in the gray matter of the rat brain in normal conditions and on exposure to roentgen radiation].

It was shown that calmodulin is nonuniformly distributed in subcellular fractions of rat brain gray substance. The largest calmodulin content is found in the synaptosomal-mitochondrial fraction and the lowest, in the microsomal fraction. Different subcellular fractions of rat brain gray substance are characterized by diverse specific activities of calmodulin. Lethal X-radiation doses elicit substantial, oppositely directed, changes in the content and specific activity of calmodulin in different subcellular fractions of rat brain gray substance.

Animals↗

PubMed1981

Subcellular localization of indium-111 in indium-111-labeled platelets.

Human platelets labeled with [111In]oxine have been suggested for use in platelet kinetic studies, thrombus localization, detection of atherosclerotic lesions, and other scintigraphic techniques. To demonstrate the subcellular localization of indium-111 in labeled platelets, we did subcellular fractionation and measured the amount of indium-111 localized in major platelet components. Human platelets were labeled with [111In]oxine and disrupted by nitrogen cavitation. The cellular material was fractionated on sucrose density gradients. When the subcellular fractions were analyzed, more than 70% of the indium-111 was located in the cytoplasmic pool of the platelets. Chromatography on Sephadex G-100 of aliquots of the labeled cytosol material showed that most of the indium-111 was associated with components of approximately 46,000 daltons. This demonstrated that indium-111 in human platelets labeled with [111In]oxine is located predominantly in platelet cytosol and is associated with cytoplasmic components.

Blood Platelets↗

PubMed1981

Subcellular distribution of human asialotransferrin type 3 in the rat liver.

A small quantity of 125I-labeled human asialotransferrin type 3 (2 to 4 microgram/100 g) was injected in intact rats and the distribution of the hepatic radioactivity analyzed by fractionation of liver homogenates on continuous sucrose density gradient. The ligand rapidly partitioned between plasma membrane and the interior of the cell at an approximate ratio of 1 to 4. The ratio remained constant between 3 min and 1 h. Intracellular 125I was encapsulated in a particle that was of a median equilibrium density of 1.11 (1.109 to 1.114) g/cm3 at 20 degrees C. The ligand recovered from the particles showed no sign of proteolytic digestion and was bound by the immobilized asialoglycoprotein-binding lectin from rabbit liver. The electron microscopic appearance of the subfractions containing of the entrapped ligand closely resembled that of an intermediate Golgi preparation. Various attempts were made to separate the ligand-containing particles from sialyltransferase and phosphodiesterase I activities, but complete separation could not be accomplished. 125I-Asialoorosomucoid studied in the same quantities and under the same conditions as asialotransferrin, yielded a subcellular distribution which was distinct from that of asialotransferrin type 3. Increasing the dose of asialotransferrin, to a level at which rapid catabolism of this asialoprotein occurs, profoundly changed the subcellular distribution of radioactivity. The subcellular distribution thus obtained was comparable with that found for asialoorosomucoid. These findings suggest that asialotransferrin type 3 is associated with different intracellular vehicles (different endosomes?) depending on whether the protein is simply diacytosed or is en route to lysosomes.

Animals↗

PubMed1980

[Autoantibodies to subcellular structures in chronic liver diseases].

The frequency of detecting autoantibodies to different subcellular structures in chronic liver diseases was studied and compared with the activity of the pathological process. The activity of antibodies to subcellular structures of the cytoplasm and nucleus was related in the first turn to IgG and also to IgM. Detection of autoantibodies to different subcellular structures in chronic liver diseases has a definite diagnostic implication and serves as reflection of the destructive process activity in various tissues and cells.

Autoantibodies↗