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PubMed1999

Separation and partial characterization of three distinct intracellular GLUT4 compartments in rat adipocytes. Subcellular fractionation without homogenization.

Insulin recruits GLUT4 from an intracellular location to the plasma membrane in rat adipocytes. The process involves multiple intracellular compartments and multiple protein functions, details of which are largely unknown partly due to our inability to separate individual GLUT4 compartments. Here, by hypotonic lysis, differential centrifugation, and glycerol density gradient sedimentation, we separated intracellular GLUT4 compartments in rat adipocytes into three fractions: plasma membrane-containing fraction T and plasma membrane-free fractions H and L. The GLUT4 contents in fractions T, H, and L were approximately 25, 56, and 18% of total GLUT4, respectively, in basal adipocytes and 55, 42, and 3-4% in insulin-stimulated adipocytes. The plasma membrane GLUT4 contents estimated separately further revealed that intracellular GLUT4 in fraction T amounts to approximately 20% in both basal and insulin-stimulated adipocytes. Organelle-specific marker and membrane traffic-related protein distribution data suggested that intracellular GLUT4 in fraction T represents sorting endosomes, whereas GLUT4 in fractions H and L represents storage endosomes and exocytic vesicles, respectively. The subcellular fractionation without homogenization described here should be useful in identifying the role of the individual GLUT4 compartments and the associated proteins in insulin-induced GLUT4 recruitment in rat adipocytes.

Adipocytes↗

PubMed1983

Analytical subcellular fractionation of rat cortex: resolution of serotonergic nerve endings and receptors.

An analytical procedure for the subcellular fractionation of rat brain cortex is presented; it consists of a two-step procedure involving a differential centrifugation using the five-fraction scheme and an isopycnic centrifugation in continuous sucrose gradients. All fractions obtained were analyzed for their content of various constituents, such as receptor binding, uptake, and several marker enzymes. Special attention was paid to the subcellular distribution of the serotonin S2 receptors; they were mainly recovered in the microsomal P fraction, but a significant amount was also associated with the mitochondrial (M and L) fractions. After equilibration in density gradients, serotonin S2 receptors revealed two peaks, which were similarly affected after treatment with amitriptyline and/or yohimbine. There is no evidence to suggest that serotonin S2 receptors are associated with nerve endings containing the neurotransmitter serotonin. Although three main profiles, a microsomal, a mitochondrial, and a mixed one, clearly appear from the differential centrifugation, subgroups of these main profiles were also found. For instance, the microsomal distribution patterns of serotonin S2 receptors and 5'-nucleotidase are very similar, but differ from that of UDP-galactosyltransferase. Similarly, the mitochondrial profiles of cytochrome oxidase and 5-HT (serotonin) uptake are different. An analytical approach for brain fractionation, when performed with appropriate measurements (cytochrome oxidase, amine uptake, 5'-nucleotidase, and receptor binding), is rapid and clearly differentiates pre- and postsynaptic constituents.

Animals↗

PubMed2003

Study of chromium-containing proteins in subcellular fractions of rat liver by enriched stable isotopic tracer technique and gel filtration chromatography.

Ten male Wistar rats were intravenously injected with a single approximately physiological dose of enriched stable isotopic Cr-50 tracer solution (200 ng (50)Cr(3+)/100 g body wt). The fundamental distribution patterns of the chromium-containing proteins in the nucleic, mitochondrial, lysosomal, microsomal, and cytosolic subcellular fractions of the rat liver were investigated by means of Sephadex G-100 gel chromatography combined with neutron activation analysis via (50)Cr (n, gamma) (51)Cr reaction. In total, nine kinds of Cr-containing proteins were found in the five subcellular fractions, whose relative molecular masses were 96.6+/-6.2, 68.2+/-1.4, 57.9+/-4.7, 36.6+/-1.2, 24.2+/-1.8, 14.0+/-1.5, 8.8+/-0.6, 6.9+/-0.4, and 4.2+/-0.4 kDa. Approximately 64.5% of Cr proteins accumulated in the cytosolic fraction. The second enriched part was the nucleic fraction; about 12.2% Cr proteins were stored in this section. The 4.2-kDa molecular mass might contain the so-called low molecular weight chromium-containing substance; however, in this research, it was only observed in the mitochondria, lysosome, and microsome. In the mitochondrial fraction, most of the Cr proteins were present as relatively low molecular weight substances: about 56% of chromium-containing proteins had molecular masses < or =6.9 kDa. Nevertheless, more than 69% of Cr-containing proteins were observed with molecular masses > or =57.9 kDa in the liver cytosolic fraction.

Animals↗

PubMed1973

Metabolism of androst-4-ene-3,17-dione by subcellular fractions of rat adrenal tissue with particular reference to microsomal C19-steroid 5alpha-reductase.

The location and some characteristics of rat adrenal C(19)-steroid 5alpha-reductase were investigated by using [7alpha-(3)H]androst-4-ene-3,17-dione and [7alpha-(3)H]testosterone as substrates. The enzymes system was shown to be NADPH-dependent and associated with the microsomal fraction. In addition, some evidence was also obtained for the existence of a separate NADH-dependent system in the soluble fraction. Further investigation of androst-4-ene-3,17-dione metabolism by subcellular fractions indicated the presence of NADH-dependent 3alpha- and 3beta-hydroxy steroid dehydrogenase systems in the microsomal pellet. This pellet also appeared to contain an NADH-dependent 17beta-hydroxy steroid dehydrogenase system, and a similar though separate system was detected in the cytosol. Malate (20mm) effectively inhibited the microsomal C(19)-steroid 5alpha-reductase, which showed similar values for K(m) and V(max.) when either androst-4-ene-3,17-dione or testosterone was used as substrate. Cytochrome c was added to all incubation mixtures used for the determination of these values to inhibit the formation of metabolites other than 5alpha-androstane-3,17-dione and 5alpha-dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one) respectively. It was also found that corticosterone did not inhibit the 5alpha-reduction of androst-4-ene-3,17-dione under these conditions, indicating that separate enzymes exist for the 5alpha-reduction of C(19)- and C(21)-steroids in the rat adrenal.

17-Ketosteroids↗

PubMed1986

Subcellular fractionation of Trypanosoma cruzi; isolation and characterization of plasma membranes from epimastigotes.

A procedure is described for the isolation of subcellular fractions from epimastigotes of Trypanosoma cruzi. The method could separate most of the nuclei, mitochondria and microsomes. The plasma membranes were purified by discontinuous density gradient centrifugation in alkaline buffer containing sucrose and magnesium. The yield of plasma membrane was 3.7 mg of protein per 10(9) epimastigotes, accounting for approximately 4.2% of the total cell proteins. The plasma membrane obtained from the 34-50% interface of sucrose density gradients was shown, by electron microscopy, to be completely free of other organella and was homogeneous according to enzymatic marker criteria. The specific activity of the 5'-nucleotidase and acid phosphatase were 96- and 5.5-fold, respectively, higher than that in the total cells, suggesting that the enzymes can be considered as good plasma membrane markers for T. cruzi. The results confirmed the possibility of the presence of membrane-bound acid phosphatase of T. cruzi.

Acid Phosphatase↗

PubMed1981

Distribution of six synaptic membrane antigens in subcellular fractions of rat brain cortex.

The subcellular distribution in rat brain cortex of six synaptic membrane antigens (56K, 58K, 62K, 63K, 64K, 66K) was studied by rocket immunoelectrophoresis, using antiserum to a highly purified synaptic plasma membrane fraction. Initial analysis of the insoluble portion of subcellular fractions showed that these antigens were also present in smooth microsomes, rough microsomes, and synaptic vesicles; that only traces were present in synaptic junctions; and that none was present in nuclei, mitochondria, and myelin. A trace amount of activity was also present in synaptic vesicle cytosol, but none in whole brain cytosol. Quantitative measurements of synaptic plasma membranes, smooth microsomes, and synaptic vesicles showed that all six antigens were present in synaptic plasma membranes and smooth microsomes, but that the 66K antigen was absent from synaptic vesicles. The 56K, 58K, 62, 63K, and 64K antigens were present in highest concentration in synaptic plasma membranes, whereas the 66K antigen content was highest in smooth microsomes. Only the 58K, 62K, and 63K antigen were detectable in the membrane fraction of whole brain. Their enrichments in synaptic plasma membranes were 10.9, 5.4, and 5.9, respectively. We conclude that the 56K, 58K, 62K, 63K and 64K antigens are primary components of synaptic plasma membranes. The presence of synaptic plasma membrane antigens in smooth microsomes and synaptic vesicles probably represents material being actively transported, consistent with the hypothesis that proteins of synaptic plasma membranes and synaptic vesicles are hypothesis that proteins of synaptic plasma membranes and synaptic vesicles are transported via smooth endoplasmic reticulum.

Animals↗

PubMed2000

[Effect of potential beta-adrenolytics on markers of enzyme activity in subcellular fractions of the cardiac muscle].

The paper investigates the effect of two potential beta-adrenolytical agents, 1-[4-(3-isopropylamino-2-hydroxypropoxy)-3-(propoxymethyl)phenyl]etan on (FA33) and 1-[4-(3-isopropylamino-2-hydroxypropoxy)-3-(propoxymethyl)phenyl]prop anan-1-on (FP33), in the form of salts with fumaric acid on the enzymatic activities of the markers of subcellular fractions of the rabbit cardiac muscle (in vitro). In the mitochondria, both substances inhibit the specific activity of cytochromoxidase (FA33 by 37.14% and FP33 by 35.88%). In the sarcolemma, substance FA33 inhibits the specific activity of Na(+)-K(+)-ATPase by 23.02%, while substance FP33 possesses an activating effect increased by 23.24%. In the sarcoplasmatic reticule both substances inhibit the specific activity of Ca(2+)-Mg(2+)-ATPase (FA33 by 14.96% and FP33 by 8.79%). The different effects of substances FA33 and FP33 on the activities of the markers of the subcellular fractions of the cardiac muscle can be connected with different inotropism of both substances.

Acetophenones↗

PubMed1979

Myocardial subcellular fractionation studies on cardiomyopathic Syrian hamsters.

Myocardial homogenates from control animals and from hamsters with hereditary cardiomyopathy were subjected to analytical subcellular fractionation and enzymic microanalysis. Animals without ventricular hypertrophy or overt heart failure were used in this study. The principal subcellular organelles were characterised by density gradient centrifugation. Apart from evidence of enhanced lysosomal and peroxisomal fragility, probably secondary to the intracellular oedema, the intracellular organelles investigated in this study were unaffected by the myopathic process. Highly significant increases in 5'-nucleotidase activity, a marker for the sarcolemma, and an increased equilibrium density of this organelle were found in the myopathic tissue. Ultrastructural studies revealed patchy myocytolysis associated with lysosomes and with more extensive invaginations of the sarcolemma. It is suggested that a primary defect in membrane composition, leading to increased cation permeability, is the underlying abnormality in the myopathic hamster.

Animals↗

PubMed1982

[Role of age in the interaction of tetracycline with the subcellular fractions of rat liver homogenate].

The effect of the rat age on the binding of tetracyclines to the subcellular fractions of liver homogenates was studied. The tetracyclines significantly lost their activity after the contact with the cell organoids. The level of the binding was lower in the newborn and old rats, the complex of tetracycline with mitochondria being less stable. The greater part of the antibiotic reduced its activity after washing of the organoids. The deposition of the antibiotics in the cells was the most pronounced in the pubertal animals.

Aging↗

PubMedDate not supplied

[Study of the effect of dioxidine on the subcellular fractions of microbial cells].

Penetration of dioxidine to the cells of Staphylococcus aureus and the drug effect on subcellular fractions were examined. High pressure liquid chromatography demonstrated that after penetration to the cells of Staphylococcus aureus dioxidine was found unchanged in the cytoplasmic fraction. Differences were disclosed in the biochemical action pattern of the drug on the growing and stationary culture. Exposure of the growing culture to dioxidine resulted in an appreciable decrease in the protein and especially in nucleic acid levels, in significant changes in the composition of the intracellular protein complex. The drug produced structural alterations within the cell wall. These alterations increased with concentrations and were characterized by the loosening of the wall and by the membrane release.

Bacterial Proteins↗

PubMedDate not supplied

[Alcohol dehydrogenase activity of the subcellular fractions of Torulopsis candida yeasts grown on glucose and hexadecane].

The activity of alcohol dehydrogenase was compared in subcellular fractions of Torulopsis candida grown on glucose ("glucose cells") and on hexadecane ("hexadecane cells"). Two groups of alcohol dehydrogenases (ADH) were found to be present in the cells: (1) soluble ADH located in the cytosol and (2) ADH bound to the membrane structures and located in the mitochondria and other cell structures. Soluble ADH of "glucose cells" actively dehydrated all alcohols tested from C2 to C16. Ln "hexadecane cells", the activity was low or absent. ADH bound to the cell structures dehydrated mainly C5--C16 alcohols; the activity of these ADH was almost by an order of magnitude higher in the "hexadecane cells" as compared to the "glucose cells" (in contrast to soluble ADH). The character of changes in the activity of ADH toward various alcohols in the course of physiological adaptation of the yeast to oxidation of glucose and hexadecane suggests that both soluble and bound ADH contain at least two ADH, one of which is specific to lower alcohols and the other to higher alcohols.

Alcohol Oxidoreductases↗

PubMed1992

The two-step conversion of big endothelin 1 to endothelin 1 and degradation of endothelin 1 by subcellular fractions from human polymorphonuclear leukocytes.

The metabolism of big endothelin 1 (bET) and endothelin 1 (ET-1) by subcellular fractions from human polymorphonuclear leukocytes (PMNs) was investigated by bioassay and reversed-phase high-performance liquid chromatography. More than 80% of endothelin-converting activity was recovered from the cytosolic fraction, which in addition to ET-1 generated other peptides from bET. The processing of bET to all its metabolites including ET-1 was prevented by the serine protease inhibitor 3,4-dichloroisocoumarin (DCI; 50 microM) or the elastase inhibitor ONO-5046 (100 microM) but not by phenylmethylsulfonyl fluoride (PMSF; 143 microM), another serine protease inhibitor. Paradoxically, human leukocyte elastase, despite generating a bET fragmentation pattern similar to that of PMN cytosol, produced very little ET-1. However, subsequent treatment of the elastase-derived metabolites of bET with PMN cytosol in the presence of ONO-5046 dramatically increased the amount of ET-1 formed. The generation of ET-1 following this intervention was inhibited by DCI. The PMN membrane preparation degraded ET-1 to a major metabolite, similar to that produced from ET-1 by elastase, and several minor products, paralleled by a loss of its smooth muscle contracting activity. The degradation of ET-1 by PMN microsomes was prevented by DCI, PMSF, or ONO-5046. Our results suggest that an elastase-initiated serine protease cascade is responsible for the sequential conversion of bET to ET-1 by the PMN cytosol. Elastase also partly accounts for the ET-metabolizing properties of PMN microsomes.

Animals↗

PubMed1985

[Quantitative indicators of gentamicin and sisomicin interaction with isolated subcellular fractions of the liver and kidneys and intracellular distribution of these antibiotics].

Interaction of gentamicin and sisomicin with isolated subcellular fractions of the rat liver and the kidney cortical layer was studied. Distribution of the antibiotics was investigated in a system with the volume ratio of the main components (organoids and cytosol-dissolved drug) simulating the natural volume ratio of the cytoplasm and organoids in the cells. It was shown that lysosomes were the most active elements in binding antibiotics. The microsomes and mitochondria were less active. By the binding capacity the subcellular structures of the kidneys were superior to the analogous structures of the liver. Dissociation of the organoid complexes with the antibiotics was possible, the mobility of the sisomicin complexes being higher than that of the gentamicin complexes. During intracellular distribution the major amount of every of the above aminoglycosides was absorbed by the organoids. Up to 20 per cent of gentamicin and up to 30 per cent of sisomicin remained in the cytoplasm in the active form. The characteristic features of the intracellular distribution of sisomicin (build up of high levels of the free antibiotic in the cytoplasm and relatively easy dissociation of the antibiotic complexes with the organoids of the kidney cortical layer) may define the higher activity of sisomicin as compared to that of gentamicin against intracellularly located microorganisms and its lower potential nephrotoxicity.

Animals↗

PubMed1975

Distribution along the axon and into various subcellular fractions of molecules labeled with (3H)leucine and rapidly transported in the garfish olfactory nerve.

The distribution of molecules labeled with [3H]leucine by fast axoplasmic transport in vivo has been studied in the garfish olfactory nerve after incorporation of the amino acid by the olfactory mucosa. Owing to the size of the nerve, it has been possible to follow the fate of the labeled molecules in 10 different subcellular fractions of 6 consecutive nerve segments. Each segment represents a different part of the profile developed by the transported radioactive molecules. In order to determine the influence of the perikaryon (rate of protein synthesis and rate of protein release into the axon) transport was studied under 3 different conditions: (1) intact nerves (simply labeled with [3H]leucine); (2) nerves cut from the cell bodies 6 h after application of [3H]leucine; and (3) nerves pulse-chase labeled for 1 h. Several conclusions can be drawn. (1) The bulk of the rapidly transported molecules are membranous axonal proteins, as determined by enzyme markers. Most are found in subcellular fractions representing 17% of the total axonal protein. They are synthesized very rapidly in the cell bodies (less than 1 h after isotope deposition) and exhibit the highest specific activities measured. These high specific activities were found in the same axonal membrane fractions in both plateau and crest, suggesting that the membrane precursors are transported as particles rather than as subunits. (2) The majority of these proteins are released into the axon immediately after synthesis; however, at least 30% of the labeled axonal membranous proteins are not released with the fast wave itself but progressively over a long period of time. (3) The majority of the moving material, particularly in membranous fractions, is left behind the fast wave and is deposited in the axon. When the front base of the fast wve has covered 70% of the total nerve length, only 19% of the labeled material of the main axonal membranous fraction appears still to be moving. (4) Proteins with high specific activities are found near the cell bodies and may be the result of early axonal transport of amino acids, diffusing later into the surrounding cells and being incorporated into proteins. Some free amino acids are also transported along the axon.

Acetylcholinesterase↗

PubMed1988

Determination of the intracellular protein thiol distribution of hepatocytes using monobromobimane derivatisation of intact cells and isolated subcellular fractions.

The derivatisation of intact rat hepatocytes with monobromobimane resulted in rapid labelling of accessible protein thiols in several subcellular fractions. The derivatisation procedure did not cause acute cytotoxicity, nor did it alter the buoyant densities of the fractions or their gross protein compositions. Quantitation of the fluorescence irreversibly associated with the fractions demonstrated considerable intracellular heterogeneity in this pool of thiols. Values were highest in cytosol (ca. 90 nmol/mg protein), intermediate in microsomes (ca. 65 nmol/mg protein) and mitochondria (ca. 45 nmol/mg protein) and lowest in a crude fraction containing both nuclei and plasma membrane (ca. 35 nmol/mg protein). Similar values were obtained from microsomes and cytosol derivatised after fractionation but there were significant increases of ca. 100% in corresponding values from isolated mitochondria and the nuclear/plasma membrane fraction. These results are discussed in terms of the dynamic fluxes in monobromobimane protein thiols during fractionation and the applicability of this noninvasive method to studies of the mechanism(s) of toxicity of reactive xenobiotics and the role(s) of protein thiols in normal cellular function.

Animals↗

PubMed1975

Distribution of fibrinogen, and platelet factors 4 and XIII in subcellular fractions of human platelets.

The distribution of fibrinogen, platelet factor 4 (heparin-neutralizing factor) and factor XIII amongst subcellular fractions of human platelets was determined. It was found that fibrinogen and platelet factor 4 peaked sharply in the region of the density gradient previously shown to be heavily enriched with alpha-granules. By contrast, factor XIII, fibrin-stabilizing factor, was found exclusively in the supernatant.

Blood Coagulation Factors↗

PubMed1983

Preparation of representative samples of subcellular fractions for electron microscopy by filtration with dextran.

A simple method is described for obtaining a representative field of subcellular fractions for thin section electron microscopy. The prefixed sample is admixed together with high molecular weight dextran, filtered onto a Millipore filter, and embedded in polymer. Filtration serves to uniformly distribute structures of different sizes and densities. The dextran serves as a nonosmophilic spacer, increasing the space between individual structures and preventing sample compression during filtration onto Millipore filters. Sample aggregation can also readily be assessed by the procedure.

Dextrans↗

PubMedDate not supplied

Immunogenic potential of Aspergillus nidulans subcellular fractions and their polypeptide components.

Cell-free extracts of the ascomycetous fungus Aspergillus nidulans were separated into three subcellular fractions: cell walls, total membranes and cytosol, and two different immunization protocols were used to raise antibodies against them in 12 New Zealand rabbits. The immune response was followed over time by dot and Western blot analyses to determine the immunogenic potential of each individual fraction and their polypeptide components. The IgG fractions, purified from pools of the best sera, were used to analyze in detail the antigenic composition of A. nidulans mycelium. The fast immunization protocol provided a much earlier response and higher sera titres. Cytosols and membranes were more immunogenic than cell walls and, in most cases, a positive correlation was shown between the titre of each serum and the number of detected antigens. The polypeptides of A. nidulans included six major immunodominant antigens of the molecular weights ranging between 13 and 200 kDa.

Animals↗