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PubMed1987

Biogenesis of the rat hepatocyte plasma membrane in vivo: comparison of the pathways taken by apical and basolateral proteins using subcellular fractionation.

We have used pulse-chase metabolic radiolabeling with L-[35S]methionine in conjunction with subcellular fractionation and specific protein immunoprecipitation techniques to compare the posttranslational transport pathways taken by endogenous domain-specific integral proteins of the rat hepatocyte plasma membrane in vivo. Our results suggest that both apical (HA 4, dipeptidylpeptidase IV, and aminopeptidase N) and basolateral (CE 9 and the asialoglycoprotein receptor [ASGP-R]) proteins reach the hepatocyte plasma membrane with similar kinetics. The mature molecular mass form of each of these proteins reaches its maximum specific radioactivity in a purified hepatocyte plasma membrane fraction after only 45 min of chase. However, at this time, the mature radiolabeled apical proteins are not associated with vesicles derived from the apical domain of the hepatocyte plasma membrane, but instead are associated with vesicles which, by several criteria, appear to be basolateral plasma membrane. These vesicles: (a) fractionate like basolateral plasma membrane in sucrose density gradients and in free-flow electrophoresis; (b) can be separated from the bulk of the likely organellar contaminants, including membranes derived from the late Golgi cisternae, transtubular network, and endosomes; (c) contain the proven basolateral constituents CE 9 and the ASGP-R, as judged by vesicle immunoadsorption using fixed Staphylococcus aureus cells and anti-ASGP-R antibodies; and (d) are oriented with their ectoplasmic surfaces facing outward, based on the results of vesicle immunoadsorption experiments using antibodies specific for the ectoplasmic domain of the ASGP-R. Only at times of chase greater than 45 min do significant amounts of the mature radiolabeled apical proteins arrive at the apical domain, and they do so at different rates. Approximate half-times for arrival are in the range of 90-120 min for aminopeptidase N and dipeptidylpeptidase IV whereas only 15-20% of the mature radiolabeled HA 4 associated with the hepatocyte plasma membrane fraction has become apical even after 150 min of chase. Our results suggest a mechanism for hepatocyte plasma membrane biogenesis in vivo in which all integral plasma membrane proteins are shipped first to the basolateral domain, followed by the specific retrieval and transport of apical proteins to the apical domain at distinct rates.

Animals↗
PubMed1999

GH kinase activity in bovine anterior pituitary subcellular fractions.

Growth hormone (GH) and prolactin (PRL) share significant structural homology. We have previously characterized the phosphorylation of bovine PRL and wish to determine whether a similar kinase activity phosphorylates bovine GH. Phosphorylation of bovine GH was performed using [alpha-32P]ATP labeling of subcellular fractions. Bovine GH phosphorylation was dependent on Zn2+ or Cu2+ with apparent Km's of 0.9 and 1.0 mM, respectively, and a pH maxima of 7.0. The apparent Km's of bovine GH kinase activity for exogenous bovine GH and ATP were 30 microM and 376 microM, respectively. Exogenous bovine PRL served as a competitive substrate, increasing the apparent Km for bovine GH by threefold compared to the Km determined without exogenous bovine PRL. We conclude: 1) in vitro phosphorylation of bovine GH occurs under conditions that are consistent with those found in anterior pituitary cells, and 2) a similar kinase activity phosphorylates both bovine PRL and GH.

Adenosine Triphosphate↗
PubMed1983

Evaluation of preformed Percoll and reorientating sucrose density gradient centrifugation for the analytical subcellular fractionation of dog liver.

A low-speed supernatant from dog liver was prepared and subjected to analytical subcellular fractionation using either preformed Percoll or reorientating sucrose density gradient centrifugation. In Percoll the following organelles, with marker enzymes and modal densities between brackets, were characterised: plasma membrane (alkaline phosphatase, 5' nucleotidase, gamma-glutamyl transferase, 1.039); endoplasmic reticulum (neutral alpha-glucosidase, 1.039); lysosomes (N-acetyl-beta-glucosaminidase, 1.087; alpha-mannosidase, 1.081) peroxisomes (catalase, 1.045) and mitochondria (succinate dehydrogenase, 1.081). In sucrose alkaline phosphatase had a bimodal particulate distribution (1.120, 1.187) distinct from that of 5' nucleotidase (1.160) and of gamma-glutamyl transferase (1.173). Other modal densities were: endoplasmic reticulum (1.187), lysosomes (1.227, 1.200), peroxisomes (1.213) and mitochondria (1.187). Further resolution was achieved by homogenisation in digitonin which disrupted lysosomes and, in sucrose, selectively increased the densities of the plasma membrane components. Both procedures therefore achieved distinct but quite different resolutions of organelles and should prove valuable for investigating subcellular pathology.

Animals↗
PubMed2025

Nascent and Mature RNA Profiling by Subcellular Fractionation in Human Cells.

Transcription and RNA decay determine steady-state RNA levels in cells available for translation and RNA-mediated regulatory functions. Both processes can be assessed by various techniques, for majority, based on RNA labelling or chromatin immunoprecipitation, but require a high level of expertise. Here, we describe a cost-effective, fast, and simple protocol that enables the profiling of nascent and mature RNA in the cytoplasm, nucleoplasm, and chromatin through subcellular fractionation. The workflow can include α-amanitin inhibition of RNA Polymerase II to assess nascent RNAs as a proxy of transcriptional activity, or it can be used without this treatment to investigate distribution of partially processed or mature transcripts across distinct subcellular compartments. It is applicable for studying any of RNA biotypes, including small and long noncoding RNAs, mRNAs, and their splice variants, on both transcript-specific and transcriptome-wide scales. Nascent or mature RNAs isolated from each fraction can be further analyzed by any technique of choice (northern blot, reverse transcription, RNA sequencing).

Humans↗
PubMed1990

Subcellular fractionation of prohormone processing products in the bag cell neurons.

Multiple biologically active peptides arising from a common prohormone are sorted into distinct classes of dense core vesicles within the bag cell neurons of Aplysia californica. In this study, pulse-chase analysis, combined with subcellular fractionation on Percoll gradients, are used to define the location of the prohormone processing events within the secretory pathway. Initial cleavage of the prohormone occurs in a light cellular compartment associated with the Golgi apparatus. The amino-terminal processing intermediate then accumulates in a denser compartment containing small dense cores enclosed in membranous sacs, as well as larger immature vesicles. After 4 h, amino-terminal products are found primarily in a much denser compartment which consists of large and small dense core vesicles. These large and small vesicles can be separated from each other using Percoll gradient centrifugation and are found to be enriched in amino- and carboxy-terminal products, respectively. Lastly, membrane association experiments suggest differential binding to membranes, or integral membrane proteins, as a possible mechanism for sorting of amino- and carboxy-terminal products.

Animals↗
PubMed1994

Localization of RC3 (neurogranin) in rat brain subcellular fractions.

Previous studies have shown that RC3 (neurogranin) is a postsynaptic, protein kinase C (PKC)/calmodulin-binding substrate that accumulates throughout the perikaryal and dendritic cytoplasm and is often closely associated with the postsynaptic density (PSD) in dendritic spines of neostriatal neurons. Here Western immunoblotting studies of rat brain subcellular fractions confirm that RC3 is predominantly a cytosolic protein but is found in lower amounts in membrane-enriched microsomes and synaptosomes. Solubilization of synaptosomes suggests that RC3 may only be loosely associated with the PSD.

Animals↗
PubMed1986

Phospholipids in chromatin: incorporation of [32P]O4(2-) in different subcellular fractions of hepatocytes.

Nuclear, chromatin and microsomal fractions were isolated from hepatocytes prepared from rats injected with [32P]O4(2-) and killed subsequently at times between 1 and 48 h. Specific activities of the total phospholipids (PL) were determined for each subcellular fraction. The major points noted were the initial specific activity of the chromatin PL was half that of both nuclear and microsomal PL at 1 h; the first peak of labelling occurred at 6 h in both nuclear and microsomal PL, but was 3 h later (9h) in the chromatin PL; and a second peak of labelling occurred in the chromatin and microsomal PL, but not in those of the nuclei. On fractionation of the PL, the major and most metabolically active components were phosphatidylcholine + phosphatidylethanolamine, whilst sphingomyelin accounted for only about 8 per cent of the total PL. The chromatin and microsomal fractions were somewhat similar in their labelling patterns though with a delayed peaking of activity in the chromatin. This is indicative of a synthesis and transport of PL from the microsomes to the chromatin.

Animals↗
PubMed1989

Enhancement of Epstein-Barr virus/C3d receptor (EBV/C3dR or CR2) and nuclear p120 ribonucleoprotein phosphorylation by specific EBV/C3dR ligands in subcellular fractions of the human B lymphoma cell line, Raji.

We present herein the first evidence that interaction of specific EBV/C3dR ligands, as human C3bi/C3d and anti-EBV/C3dR MoAb, with EBV/C3dR enhanced significantly, in a dose dependent process, phosphorylation of EBV/C3dR and p120 RNP present in subcellular fractions, as purified plasma membranes and nuclei, of the human B lymphoma cell line, Raji. The use of kinase effectors allowed to detect some of the kinases involved in these phosphorylations. Pp60src-like phosphotyrosine kinase and protein kinase C were involved in the phosphorylation of plasma membrane or nuclear EBV/C3dR. An additional calcium/calmodulin-dependent kinase was also involved in nuclear EBV/C3dR phosphorylation. P120 RNP phosphorylation was under the control of protein kinase C and of CaCl2/Calmodulin-dependent kinase but also of casein kinase II.

Burkitt Lymphoma↗
PubMed1983

Hepatic enzymes, CoASH and long-chain acyl-CoA in subcellular fractions as affected by drugs inducing peroxisomes and smooth endoplasmic reticulum.

1. The activities of acyl-CoA hydrolase, catalase, urate oxidase and peroxisomal palmitoyl-CoA oxidation as well as the protein content and the level of CoASH and long-chain acyl-CoA were measured in subcellular fractions of liver from rats fed diets containing phenobarbital (0.1% w/w) or clofibrate (0.3% w/w). 2. Whereas phenobarbital administration resulted in increased microsomal protein, the clofibrate-induced increase was almost entirely attributed to the mitochondrial fraction with minor contribution from the light mitochondrial fraction. 3. The specific activity of palmitoyl-CoA hydrolase in the microsomal fraction was only slightly affected while the mitochondrial enzyme was increased to a marked extent (3-4-fold) by clofibrate. 4. Phenobarbital administration mainly enhanced the microsomal palmitoyl-CoA hydrolase. 5. The increased long-chain acyl-CoA and CoASH level observed after clofibrate treatment was mainly associated with the mitochondrial, light mitochondrial and cytosolic fractions, while the slight increase in the levels of these compounds found after phenobarbital feeding was largely of microsomal origin. 6. The findings suggest that there is an intraperoxisomal CoASH and long-chain acyl-CoA pool. 7. The specific activity of palmitoyl-CoA hydrolase, catalase and peroxisomal palmitoyl-CoA oxidation was increased in the lipid-rich floating layer of the cytosol-fraction. 8. The changes distribution of the peroxisomal marker enzymes and microsomal palmitoyl-CoA hydrolase after treatment with hypolipidemic drugs may be related to the origin of peroxisomes.

Acyl Coenzyme A↗
PubMed1992

Isolation and subcellular fractionation analysis of acini from rabbit lacrimal glands.

The rabbit has been a useful model for in vivo studies of the pharmacologic control of lacrimal gland fluid secretion. However, by contrast with rodent exorbital lacrimal glands, the rabbit lacrimal gland has not been subjected to detailed cellular, subcellular, or biochemical analyses. Procedures were developed to isolate rabbit lacrimal acini by collagenase digestion and mechanical dispersion. The preparations exhibited good morphology, and trypan blue exclusion rates generally exceeded 90%. The isolated acini responded to carbachol by releasing protein and increasing Na+ unidirectional influx rates. The presence of muscarinic cholinergic and beta-adrenergic receptors was indicated by specific binding of the muscarinic cholinergic antagonist, 3H-N-methylscopolamine (3H-NMS; dissociation constant, Kd, 0.55 nmol/l), and the beta-adrenergic antagonist, 3H-CGP12177 (Kd, 0.34 nmol/l). The maximal binding values measured in crude membrane preparations were 79 fmol/mg for 3H-NMS and 40 fmol/mg for 3H-CGP12177. Subcellular fractionation analyses showed various membrane populations, including a series of Golgi-derived populations admixed with a major endoplasmic reticulum-derived population, a population that may represent the basal-lateral plasma membranes, and a series of populations with characteristics suggesting they are involved in the assembly or recycling of basal-lateral membrane constituents. The authors believe the ability to isolate and analyze acinar preparations from the rabbit lacrimal gland will facilitate various studies of acinar cell biochemistry and physiology that would be impractical with the relatively smaller amounts of material that can be obtained from rat or mouse exorbital lacrimal glands.

Animals↗
PubMed2006

Biological significance of metals partitioned to subcellular fractions within earthworms (Aporrectodea caliginosa).

Metal ions in excess of metabolic requirements are potentially toxic and must be removed from the vicinity of important biological molecules to protect organisms from adverse effects. Correspondingly, metals are sequestrated in various forms, defining the accumulation pattern and the magnitude of steady-state levels reached. To investigate the subcellular fractions over which Ca, Mg, Fe, Cu, Zn, Cd, Pb, Ni, and As are distributed, earthworms (Aporrectodea caliginosa) collected from the field were analyzed by isolating metal-rich granules and tissue fragments from intracellular microsomal and cytosolic fractions (i.e., heat-stable proteins and heat-denatured proteins). The fractions showed metal-specific binding capacity. Cadmium was mainly retrieved from the protein fractions. Copper was equally distributed over the protein fraction and the fraction comprising tissue fragments, cell membranes, and intact cells. Zinc, Ca, Mg, and As were mainly found in this fraction as well. Lead, Fe, and Ni were mainly isolated from the granular fraction. To study accumulation kinetics in the different fractions, three experiments were conducted in which earthworms were exposed to metal-spiked soil and a soil contaminated by anthropogenic inputs and, indigenous earthworms were exposed to field soils. Although kinetics showed variation, linear uptake and steady-state types of accumulation patterns could be understood according to subcellular compartmentalization. For risk assessment purposes, subcellular distribution of metals might allow for a more precise estimate of effects than total body burden. Identification of subcellular partitioning appears useful in determining the biological significance of steady-state levels reached in animals.

Animals↗
PubMed1976

Effect of clofibrate treatment on carnitine acyltransferases in different subcellular fractions of rat liver.

The subcellular distribution of carnitine acetyl-, octanoyl-, and palmitoyl- transferase in the livers of normal and clofibrate-treated male rats was studied with isopycnic sucrose density gradient fractionation. In normal liver 48% of total carnitine acetyltransferase activity was peroxisomal, 36% of the activity located in mitochondria and 16% in a membranous fraction containing microsomes. Carnitine octanoyltransferase and carnitine palmitoyltransferase were confined almost totally (77--81%) to mitochondria in normal liver. Clofibrate treatment increased the total activity of carnitine acetyltransferase over 30 times, whereas the total activities of the other two transferases were increased only 5-fold. From the three different subcellular carnitine acetyltransferases the mitochondrial one was most responsive to clofibrate treatment, i.e. the rise in mitochondrial activity was over 70-fold as contrasted to the 6- and 14-fold rises in peroxisomal and microsomal activities, respectively. After treatment mitochondria contained 79% of total activity. It is concluded that the clofibrate-induced increase of carnitine acetyltransferase activity is not due to the peroxisomal proliferation that occurs during clofibrate treatment. The rise in peroxisomal activity contributed only 8% to the total increase. After clofibrate treatment the greatest part of carnitine octanoyl- and palmitoyltransferase activities were located in mitochondria but a considerable amount of both activities was found also in the soluble fraction of liver.

Acyltransferases↗
PubMed1999

Relative contributions of mouse liver subcellular fractions to the bioactivation of mitomycin C at various pH levels.

Mitomycin C (MMC) is a clinically active anticancer drug that requires reductive activation to exert its toxicity. The enzymes currently recognized as capable of activating MMC cannot account for all of the toxicity of the drug. These studies were conducted to identify and compare the subcellular compartments where MMC reduction can take place under different physiological conditions. Subcellular fractionation of mouse liver was achieved using differential centrifugation and isopycnic equilibrium gradient centrifugation. Nuclear, mitochondrial, microsomal, lysosomal, peroxisomal, and cytosolic fractions were assayed for their ability to reductively activate MMC at pH 6.0 and 7.4. MMC reductive activation was determined by its ability to generate reactive oxygen species. The results of these studies showed that MMC reductive activation by the various fractions was pH dependent. At pH 7.4, the microsomal fraction accounted for approximately 78% of the total MMC reductive activation. The peroxisomal fraction accounted for 12% and the nuclear and lysosomal fractions each accounted for 5% of the total reductive activation. At pH 6.0, the microsomes accounted for 51% and the peroxisomes for 34% of the total reductive activation. The mitochondrial fraction, which did not reductively activate MMC at pH 7.4, accounted for 9% of the total activation at pH 6.0. These results suggested that peroxisomes may be important in MMC activation at either pH and that at pH 6.0 the mitochondrial fraction may also be important for MMC reductive activation.

Animals↗
PubMed1984

Two distinct mechanisms for taurocholate uptake in subcellular fractions from rat liver.

As part of the enterohepatic circulation, hepatocytes take up bile acids from the intestines via the hepatic portal blood using a sodium-dependent carrier mechanism and resecrete the bile acids into the bile. In order to assess whether intracellular organelles are involved in the transcellular secretion of bile acids, we measured directly the ability of purified subcellular fractions of rat liver to take up taurocholate using a Millipore filtration assay. Two distinct uptake mechanisms can be discerned, one localized in the plasma membranes and the other in the Golgi and smooth microsomal fractions. Plasma membranes prepared by the method of Fleischer and Kervina (Fleischer, S., and Kervina, M. (1974) Methods Enzymol. 31, 6) take up taurocholate in a saturable manner with an apparent Vmax of 2.4 nmol min-1 mg protein-1 and a Km of 190 microM at 37 degrees C. After preincubation of the membranes with K+ ions, a sodium gradient (100 mM outside) stimulates the uptake rate by 90% with the observed Km unchanged. The stimulation is inhibited by phalloidin but not by bromosulfophthalein. Bile canalicular plasma membranes made according to Kramer et al. (Kramer, W., Bickel, U., Buscher, H. P., Gerok, W., and Kurz, G. (1982) Eur. J. Biochem. 129, 13-24) do not take up taurocholate. The transport by Golgi vesicles and smooth microsomes differs from that in the plasma membrane fraction in that it is not stimulated by a sodium gradient, has a Vmax of 12 nmol min-1 mg protein-1 and a Km of 440 microM at 37 degrees C, and is inhibited by bromosulfophthalein but not by phalloidin. Taurocholate uptake into smooth microsomes is abolished by filipin, an antibiotic that complexes with cholesterol to disrupt the membrane. This suggests that taurocholate uptake occurs into a nonendoplasmic reticulum subfraction since endoplasmic reticulum membranes contain negligible amounts of cholesterol. Little uptake was observed using rough microsomes or mitochondria. A model of transhepatic transport compatible with our observations is that taurocholate uptake into the cytoplasm occurs via the plasma membranes on the sinusoidal side of the hepatocyte; taurocholate is then taken up into smooth vesicles and the Golgi complex and is secreted into the bile by exocytosis as the vesicles fuse with the canalicular plasma membranes.

Animals↗
PubMed1980

In vitro metabolism of N-nitrosopyrrolidine by rat lung subcellular fractions: alpha-hydroxylation in a non-target tissue.

N-Nitrosopyrrolidine is carcinogenic for the liver but not for the lung. Optimal conditions for the metabolism of N-nitrosopyrrolidine (NO-PYR) by rat lung microsomes and post-microsomal supernatant were determined. Neither lung nor liver subcellular fractions were able to form detectable amounts of 3-hydroxyNO-PYR. Liver and lung microsomes could only alpha-hydroxylate NO-PYR, and even though the rates of lung reactions were considerably slower than those of liver, similar products were produced by the action of both lung and liver supernatants on microsomal products. The apparent Km for the lung microsome plus supernatant reaction was approx. 20 mM as compared with 0.36 mM for the liver system. Since both the target and non-target tissue extracts could metabolize NO-PYR by the same pathways, we speculate that alpha-hydroxylation of NO-PYR may be a necessary, but not sufficient cause for its carcinogenic actions.

Animals↗
PubMed2006

Low-speed subcellular fractionation method for determining noxious stimulus-evoked spinal neurokinin-1 receptor internalization.

Substance P release from nociceptive primary afferents activates post-synaptic neurokinin-1 (NK-1) receptors causing subsequent NK-1 receptor internalization. Fluorescent immunohistochemistry is typically used to quantify NK-1 receptor internalization, an indirect measure of substance P (SP) release. However, this technique entails several limitations that restrict its application. Using simple subcellular fractionation and immunoblotting methods, we demonstrate that intrathecal SP invokes a rapid and dose-dependent increase in dorsal horn cytoplasmic NK-1 receptors. We also show that hind paw compression and noxious thermal stimulation increase cytoplasmic NK-1 receptor, when compared to sham stimulations. Fluorescent immunohistochemistry confirmed that increases in cytoplasmic NK-1 corresponded with increased NK-1 receptor internalization. Herein, we report that low-speed centrifugation and Western immunoblotting provide NK-1 internalization results consistent with those obtained by more traditional methods. These data support previous findings demonstrating a role for spinal NK-1 receptors in nociceptive processing.

Animals↗
PubMed1998

Altered residual ATP content in rat brain cortex subcellular fractions following status epilepticus induced by lithium and pilocarpine.

Changes in residual ATP concentrations were investigated following subcellular fractionation of rat brain cortex after a prolonged period of status epilepticus induced by sequential administration of lithium and pilocarpine. After 2 h of continuous high-amplitude rapid spiking on EEG, we found significantly decreased levels of residual ATP in the homogenate and mitochondria fractions from status epilepticus rat brains compared to matched controls. No difference in residual ATP level was observed in the synaptosomal preparations of status epilepticus animals compared to controls. Inorganic phosphate concentration in the status animals was higher than controls in the cytosolic fraction only. F1-ATPase activity, an enzymatic indicator of mitochondrial ATP synthesis rate, was significantly higher in the status brains, whereas other mitochondrial enzymes were not different in the status and control rat groups. These findings, together with our earlier report of reduced synaptosomal ecto-ATPase activity, suggest that either the corresponding in vivo ATP concentrations were reduced as a result of status epilepticus or other biochemical changes had occurred that facilitated the hydrolysis of ATP following decapitation. Controls for and measurement of such other changes failed to provide an explanation for the observed changes in residual ATP.

Adenosine Triphosphate↗