Polyribosomes in rat-liver preparations.
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ABSTRACT:
SUBMITTER: Franklin TJ
PROVIDER: S-EPMC1264872 | biostudies-other | 1966 Feb
REPOSITORIES: biostudies-other
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Project description:1. The activity of the enzyme protein disulphide-isomerase (EC 5.3.4.1) was used to measure the binding of ribosomal fractions to reticular membranes from the liver of unstarved male rats. 2. Membranes degranulated by treatment with KCl plus puromycin and washed by centrifugation through 0.5 M-KCl bound oligoribosomes but not monoribosomes. 3. Substitution of 0.25 M-KCl for 0.5 M-KCl in the wash solution produced membranes that bound monoribosomes and 60 S subunits in addition to oligoribosomes. 4. The binding of all classes of ribosomes to smooth and 'lightly granulated' rough membranes was activated by oestradiol. The hormone activation was much greater with polyribosomes from fed rats than for monoribosomes or oligoribosomes from starved rats. 5. Exposure of rough membranes to centrifugal forces caused them to undergo partial degranulation, as demonstrated by unmasking of protein disulphide-isomerase activity.
| S-EPMC1185668 | biostudies-literature
Project description:BackgroundTissue hypoperfusion and inflammation in sepsis can lead to organ failure including kidney and liver. In sepsis, mortality of acute kidney injury increases by more than 50%. Which type of volume replacement should be used is still an ongoing debate. We investigated the effect of different volume strategies on inflammatory mediators in kidney and liver in an early sepsis model.Material and methodsAdult male Wistar rats were subjected to sepsis by cecal ligation and puncture (CLP) and assigned to three fluid replenishment groups. Animals received 30mL/kg of Ringer's lactate (RL) for 2h, thereafter RL (75mL/kg), hydroxyethyl starch (HES) balanced (25mL/kg), containing malate and acetate, or HES saline (25mL/kg) for another 2h. Kidney and liver tissue was assessed for inflammation. In vitro rat endothelial cells were exposed to RL, HES balanced or HES saline for 2h, followed by stimulation with tumor necrosis factor-? (TNF-?) for another 4h. Alternatively, cells were exposed to malate, acetate or a mixture of malate and acetate, reflecting the according concentration of these substances in HES balanced. Pro-inflammatory cytokines were determined in cell supernatants.ResultsCytokine mRNA in kidney and liver was increased in CLP animals treated with HES balanced compared to RL, but not after application of HES saline. MCP-1 was 3.5fold (95% CI: 1.3, 5.6) (p<0.01) and TNF-? 2.3fold (95% CI: 1.2, 3.3) (p<0.001) upregulated in the kidney. Corresponding results were seen in liver tissue. TNF-?-stimulated endothelial cells co-exposed to RL expressed 3529±1040pg/mL MCP-1 and 59±23pg/mL CINC-1 protein. These cytokines increased by 2358pg/mL (95% CI: 1511, 3204) (p<0.001) and 29pg/ml (95% CI: 14, 45) (p<0.01) respectively when exposed to HES balanced instead. However, no further upregulation was observed with HES saline. PBS supplemented with acetate increased MCP-1 by 1325pg/mL (95% CI: 741, 1909) (p<0.001) and CINC-1 by 24pg/mL (95% CI: 9, 38) (p<0.01) compared to RL. Malate as well as HES saline did not affect cytokine expression.ConclusionWe identified HES balanced and specifically its component acetate as pro-inflammatory factor. How important this additional inflammatory burden on kidney and liver function is contributing to the sepsis-associated inflammatory burden in early sepsis needs further evaluation.
| S-EPMC4795752 | biostudies-literature
Project description:Subcellular fractions containing microsomes prepared from rat livers homogenized in the absence of EDTA catalysed the oxidation of cholesterol to 7alpha-hydroxycholesterol, 7-oxocholesterol, 7beta-hydroxycholesterol and 5alpha-cholestane-3beta,5,6beta-triol. These reactions required native protein, molecular oxygen and NADPH. It is suggested that these compounds are formed by a peroxidation analogous to the peroxidation of fatty acids catalysed by liver microsomal preparations. Incubations of [4-(14)C]cholesterol with microsomal preparations from rat liver homogenized in the presence of EDTA gave 7alpha-hydroxy[(14)C]cholesterol as the main product. This reaction required molecular oxygen and NADPH, and was inhibited by CO. The mass of 7alpha-hydroxycholesterol formed during the incubation was measured by a double-isotope-derivative dilution procedure. This procedure was used to assay the activity of cholesterol 7alpha-hydroxylase and to measure low concentrations of endogenous 7alpha-hydroxycholesterol in liver.
| S-EPMC1173563 | biostudies-other
Project description:Binding sites for prolactin were identified in a plasma-membrane-enriched fraction isolated from livers of mature female rats. 125I-labelled sheep prolactin prepared by the lactoperoxidase procedure retained the same molecular integrity and binding affinity as the native hormone at physiological pH. The receptors bound prolactin from different species, whereas non-lactogenic hormones were not bound. The binding of 125I-labelled sheep prolactin was activated equally by bivalent and univalent cations, bivalent cations exerting their maximal effect at much lower concentrations. The association of 125I-labelled sheep prolactin with the receptor was a time- and temperature-dependent process. Partial dissociation was detected. The binding of 125I-labelled sheep prolactin was strongly influenced by pH, with an optimum observed at pH 6.5. Receptor activity was destroyed by Pronase and phospholipase C, whereas neuraminidase increased binding. Treatment of the membranes by ribonuclease and deoxyribonuclease did not affect the binding. Binding of 125I-labelled sheep prolactin was inhibited by p-chloromercuribenzoic acid, dithiothreitol and by brief exposure to high temperatures. Scatchard analysis of the binding of 125I-labelled sheep prolactin to receptors indicated that prolactin has a high affinity for its receptor. Binding of prolactin to liver membranes showed some properties different from those observed with mammary cells. Binding by these tissues differed in pH optimum, in effects of ions, and in response to neuraminidase.
| S-EPMC1186575 | biostudies-other
Project description:1. The addition of mitochondria to an incubation system containing the soluble and microsomal fractions of rat liver enhances severalfold the incorporation of each of ethanolamine, phosphorylethanolamine and CDP-ethanolamine into phosphatidylethanolamine. 2. In the presence of microsomal, mitochondrial and soluble fractions, CDP-ethanolamine exhibits the greatest initial rate of incorporation (approx. 6nmol/h per mg of protein), being slightly faster than that of phosphorylethanolamine (approx. 5nmol/h per mg of protein). Incorporation of ethanolamine proceeds very slowly for the first 20min and only after 30min gives rates approaching those of the other two precursors. 3. By using a substrate ;dilution' technique it was shown that in the reconstituted system the affinity of each of the enzymes for their respective substrates is very high: 10mum for ethanolamine, 25mum for phosphorylethanolamine and 5mum for CDP-ethanolamine. 4. Isolation of the mitochondrial and microsomal fractions from the medium after incubation together with phosphorylethanolamine showed that about 70% of the total radioactivity was present in the microsomal fraction and about 30% in the mitochondria after only 20min. Similar experiments with ethanolamine as precursor revealed that after 20min only about 15% of the total radioactivity was present in the mitochondria but that after 40min about 30% was present in this fraction. 5. Heating and phospholipase treatment of mitochondria, but not freeze-thawing, eliminated the stimulatory effect of mitochondria on phospholipid synthesis. 6. The reconstituted system exhibits an absolute requirement for Mg(2+) (2mm gave maximal rates) and is inhibited by very low concentrations of Ca(2+) (100mum-Ca(2+) produced half-maximal inhibition with 3mm-Mg(2+)). Further addition of Mg(2+) overcame the Ca(2+) inhibition, suggesting that the inhibitory effect is readily reversible. 7. The concept that modification of the Mg(2+)/Ca(2+) ratio is a means of controlling the rate of cellular phospholipid synthesis is introduced.
| S-EPMC1165981 | biostudies-other
Project description:3,5-Di-t-butylhydroxytoluene (compound I) was converted into 4-hydroperoxy-4-methyl-2,6-di-t-butylcyclohexa-2,5-dienone (compound II), 4-hydroxy-4-methyl-2,6-di-t-butylcyclohexa-2,5-dienone (compound III) and 2,6-di-t-butyl-4-hydroxymethylphenol (compound IV) by rat liver microsomal preparations in the presence of NADPH and air. The oxidation of compound (I) by m-chloroperbenzoic acid also produced the same compounds. These results suggest that hydroperoxide can be an intermediate in aromatic hydroxylation and that biological oxygenations resemble per-acid reactions.
| S-EPMC1174016 | biostudies-other
Project description:1. Rat tissue homogenates convert dl-1-aminopropan-2-ol into aminoacetone. Liver homogenates have relatively high aminopropanol-dehydrogenase activity compared with kidney, heart, spleen and muscle preparations. 2. Maximum activity of liver homogenates is exhibited at pH9.8. The K(m) for aminopropanol is approx. 15mm, calculated for a single enantiomorph, and the maximum activity is approx. 9mmumoles of aminoacetone formed/mg. wet wt. of liver/hr.at 37 degrees . Aminoacetone is also formed from l-threonine, but less rapidly. An unidentified amino ketone is formed from dl-4-amino-3-hydroxybutyrate, the K(m) for which is approx. 200mm at pH9.8. 3. Aminopropanol-dehydrogenase activity in homogenates is inhibited non-competitively by dl-3-hydroxybutyrate, the K(i) being approx. 200mm. EDTA and other chelating agents are weakly inhibitory, and whereas potassium chloride activates slightly at low concentrations, inhibition occurs at 50-100mm. 4. It is concluded that aminopropanol-dehydrogenase is located in mitochondria, and in contrast with l-threonine dehydrogenase can be readily solubilized from mitochondrial preparations by ultrasonic treatment. 5. Soluble extracts of disintegrated mitochondria exhibit maximum aminopropanol-dehydrogenase activity at pH9.1 At this pH, K(m) values for the amino alcohol and NAD(+) are approx. 200 and 1.3mm respectively. Under optimum conditions the maximum velocity is approx. 70mmumoles of aminoacetone formed/mg. of protein/hr. at 37 degrees . Chelating agents and thiol reagents appear to have little effect on enzyme activity, but potassium chloride inhibits at all concentrations tested up to 80mm. dl-3-Hydroxybutyrate is only slightly inhibitory. 6. Dehydrogenase activities for l-threonine and dl-4-amino-3-hydroxybutyrate appear to be distinct from that for aminopropanol. 7. Intraperitoneal injection of aminopropanol into rats leads to excretion of aminoacetone in the urine. Aminoacetone excretion proportional to the amount of the amino alcohol administered, is complete within 24hr., but represents less than 0.1% of the dose given. 8. The possible metabolic role of amino alcohol dehydrogenases is discussed.
| S-EPMC1270274 | biostudies-other
Project description:1. A centrifugation method for the fractionation of the postmitochondrial fraction from rat-liver homogenates is described. The technique, in which no detergent is used, may be used as a tool to discriminate between two classes of ribosomes. One class is firmly bound to membranes and the other consists either of free polysomes or of ribosomes attached by weaker forces to the membranes of the endoplasmic reticulum. 2. Electron-micrograph studies revealed that the polysomes were not contaminated with bound ribosomes or with membranous fragments. 3. The separated fractions were characterized by their RNA, protein, ribonuclease and phospholipid content. 4. The influence of starvation on the RNA and protein contents of the different fractions was investigated. 5. Labelling of the various centrifugal fractions in vivo revealed no difference in uptake of radioactive amino acid between the two classes of ribosomes. 6. Incorporation of radioactive leucine in vitro and the polyuridylic acid-directed phenylalanine incorporation were similar for both classes of ribosomes.
| S-EPMC1270382 | biostudies-other
Project description:When investigating the potential for xanthine oxidase (XO)-mediated metabolism of a new chemical entity in vitro, selective chemical inhibition experiments are typically used. Most commonly, these inhibition experiments are performed using the inhibitor allopurinol (AP) and commercially prepared human liver cytosol (HLC) as the enzyme source. For reasons detailed herein, it is also a common practice to perfuse livers with solutions containing AP prior to liver harvest. The exposure to AP in HLC preparations could obviously pose a problem for measuring in vitro XO activity. To investigate this potential problem, an HPLC-MS/MS assay was developed to determine whether AP and its primary metabolite, oxypurinol, are retained within the cytosol for livers that were treated with AP during liver harvest. Differences in enzymatic activity for XO and aldehyde oxidase (AO) in human cytosol that can be ascribed to AP exposure were also evaluated. The results confirmed the presence of residual AP (some) and oxypurinol (all) human liver cytosol preparations that had been perfused with an AP-containing solution. In every case where oxypurinol was detected, XO activity was not observed. In contrast, the presence of AP and oxypurinol did not appear to have an impact on AO activity. Pooled HLC that was purchased from a commercial source also contained residual oxypurinol and did not show any XO activity. In the future, it is recommended that each HLC batch is screened for oxypurinol and/or XO activity prior to testing for XO-mediated metabolism of a new chemical entity.
| S-EPMC3965898 | biostudies-literature
Project description:Free loosely bound and tightly bound polyribosomes were separated from rat liver homogenate by salt extraction followed by differential centrifugation, and several of their structural and functional properties were compared to resolve the existence of loosely bound polyribosomes and verify the specificity of the separation. The free and loosely bound polyribosomes have similar sedimentation profiles and polyribosome contents, their subunit proteins have similar electrophoretic patterns and their products of protein synthesis in vitro show a close correspondence in size and amounts synthesized. In contrast, the tightly bound polyribosomes have different properties from those of the free and loosely bound polyribosomes; their average size is significantly smaller; their polyribosome content is higher; their 60 S-subunit proteins lack two components and contain four or more components not found elsewhere; their products of protein synthesis in vitro differ in size and amounts synthesized. These observations show that rat liver membranes entrap a large fraction of the free polyribosomes at low salt concentrations and that these polyribosomes are similar to those of the free-polyribosome fraction and are different from those of the tightly bound polyribosome fraction in size, structure and function.
| S-EPMC1184110 | biostudies-other