scholarly journals The subcellular location, maturation and response to increased plasma glucagon of Ruthenium Red-insensitive calcium-ion transport in rat liver

1978 ◽  
Vol 174 (3) ◽  
pp. 1021-1030 ◽  
Author(s):  
Fyfe L. Bygrave ◽  
Charmaine J. Tranter
Keyword(s):  
Rat Liver ◽  
Microsomal Fraction ◽  
Adult Life ◽  
Subcellular Location ◽  
Maximal Activity ◽  
Mitochondrial Fraction ◽  
Ruthenium Red ◽  
Maximal Response ◽  
Transport Activity ◽  
Adult Rats

1. The subcellular distribution and maturation of Ruthenium Red-insensitive Ca2+ transport activity were determined in livers of rats ranging in age from 3 days pre-term to 10 weeks of adult life and compared with those of glucose 6-phosphatase, 5′-nucleotidase and Ruthenium Red-sensitive Ca2+ transport. Initial rates of Ruthenium Red-insensitive Ca2+ transport were highest in those fractions enriched in glucose 6-phosphatase, i.e. the microsomal fraction; this fraction was devoid of Ruthenium Red-sensitive Ca2+ transport activity. Although the heaviest fraction (nuclear) contained significant amounts of 5′-nucleotidase activity it was devoid of Ruthenium Red-insensitive Ca2+ transport activity. 2. Foetal rat liver contain minimal amounts of Ruthenium Red-insensitive Ca2+ transport activity, glucose 6-phosphatase and 5′-nucleotidase activities. These begin to be expressed concomitantly soon after birth; Ruthenium Red-insensitive Ca2+ transport is maximal by 3 to 4 days and remains so for up to at least 10 weeks of adult life. Glucose 6-phosphatase also reaches a peak at 3–4 days, but then rapidly decreases to approach adult values. Maximal activity of 5′-nucleotidase in the microsomal and nuclear fractions is seen about 4–6 days after birth; this enzyme activity remains increased for up to about 10 days and then falls, but not as rapidly as glucose 6-phosphatase. It is tentatively suggested that the bulk of the Ruthenium Red-insensitive Ca2+ transport is attributable to the system derived from the endoplasmic reticulum. 3. Administration of glucagon to adult rats enhances by 2–3-fold the initial rate of Ruthenium Red-insensitive Ca2+ transport in the intermediate but not the microsomal fraction. The hormone-induced effect is fully suppressed by co-administration of puromycin, is dose-dependent with half-maximal response at approx. 1μg of glucagon/100g body wt. and time-dependent exhibiting a half-maximal response about 1h after administration of the hormone. 4. Ruthenium Red-insensitive Ca2+ transport in the post-mitochondrial fraction of foetal liver also responds to the administration in situ of glucagon. The response, which also is prevented by co-administration of puromycin, is maximal in those foetuses nearing term. The suggestion is made that these effects of the hormone on Ruthenium Red-insensitive Ca2+ transport are an integral part of the physiological network in the liver cell.

scholarly journals Glucagon stimulation of ruthenium red-insensitive calcium ion transport in developing rat liver

1981 ◽  
Vol 194 (2) ◽  
pp. 541-549 ◽  
Author(s):  
P H Reinhart ◽  
F L Bygrave
Keyword(s):  
Endoplasmic Reticulum ◽  
Cyclic Amp ◽  
Rat Liver ◽  
Adult Life ◽  
Ruthenium Red ◽  
Enzyme Analysis ◽  
Marker Enzyme ◽  
Transport Activity ◽  
Electron Microscopic ◽  
Metabolic Role

The maturation of glucagon-stimulated Ruthenium Red-insensitive Ca2+-transport activity was determined in livers of rats ranging in age from 5 days preterm to 10 weeks of adult life. Previous indications are that this activity is confined to vesicles derived mainly from the endoplasmic reticulum. Perinatal-rat liver contains near-adult values of Ruthenium Red-insensitive Ca2+-transport activity, and exhibits large transient increases in the rate of this activity at two stages of development, immediately after birth, and at 2-5 days after birth. The administration of glucagon to foetal rats, at developmental stages after 19.5 days of gestation (2.5 days before birth), results in a large stable increase (greater than 100%) of Ca2+-transport activity in a subsequently isolated ‘heavy’ microsomal fraction. That this fraction was enriched in vesicles derived from the rough endoplasmic reticulum was indicated by both an electron-microscopic examination and a marker-enzyme analysis of the subcellular fractions. The administration of glucagon into newborn animals only hours old does not enhance further the initial rate of Ca2+-transport activity, and from day 1 to 10 weeks after birth the administration of the hormone results in the moderate enhancement of Ca2+ transport. Experiments with cyclic AMP and inhibitors of phosphodiesterase activity suggest that cyclic AMP plays a key role in the enhancement by glucagon of Ruthenium Red-insensitive Ca2+ transport, and arguments are presented that this transport system has an important metabolic role in the redistribution of intracellular Ca2+ in liver tissue.

scholarly journals Properties of energy-dependent calcium transport by rat liver microsomal fraction as revealed by initial-rate measurements

1978 ◽  
Vol 170 (1) ◽  
pp. 87-91 ◽  
Author(s):  
F L Bygrave
Keyword(s):  
Rat Liver ◽  
Microsomal Fraction ◽  
Initial Rate ◽  
Ruthenium Red ◽  
Transport Activity ◽  
Carbonyl Cyanide ◽  
Liver Microsomal Fraction ◽  
Rapid Transport ◽  
Two Phases ◽  
Energy Dependent

Measurements of the initial rate of Ca2+ transport by rat liver microsomal preparations reveal the existence of two phases of transport activity. The first, a phase of rapid transport, is complete by 3-5 min, at which time the second (slower) phase begins; this remains linear for up to at least 40 min. The initial phase is minimal in the absence of MgATP. The initial rate of Ca2+ transport reaches values as high as 25 nmol/min per mg of protein; the Km for Ca2+total is 1-2 micrometer and that for MgATPtotal about 500 micrometer. Ruthenium Red (3-5 nmol/mg of protein) has little effect on the initial rate of transport, whereas tributylin (2 micrometer) inhibits equally in a KC1- or a KNO3-containing medium. Compunds that collapse components of the proton electrochemical gradient in mitochondria (valinomycin and carbonyl cyanide m-chlorophenylhydrazone) each inhibit by 70-80% the initial rate of microsomal Ca2+ transport.

scholarly journals Subcellular distribution of cytochrome c in rat liver. Methods for its extraction and purification

1967 ◽  
Vol 105 (2) ◽  
pp. 427-442 ◽  
Author(s):  
N. F. González-Cadavid ◽  
P. N. Campbell
Keyword(s):  
Cytochrome C ◽  
Rat Liver ◽  
Subcellular Distribution ◽  
Microsomal Fraction ◽  
Gradient Centrifugation ◽  
Ammonium Phosphate ◽  
Mitochondrial Fraction ◽  
Subcellular Fractions ◽  
Nuclear Fraction ◽  
Extraction And Purification

1. A method for the extraction and purification of cytochrome c from rat liver is described. The method depends on multiple chromatography on Amberlite IRC-50 with elution with ammonium phosphate buffers of differing ionic composition and pH, interspersed with gel filtration with Sephadex G-25. Conditions leading to denaturation are avoided and the product is chromatographically pure. 2. The method may be used for the quantitative analysis of cytochrome c either in unfractionated liver or in subcellular fractions. 3. Two pools of cytochrome c were detected, one extractable at pH4·0 with distilled water and the other extracted from the residues of the first extraction with 0·15m-sodium chloride. 4. For subcellular distribution studies the liver was homogenized in 0·3m-sucrose and a nuclear fraction (washed thoroughly to remove trapped mitochondria), a mitochondrial fraction, a heavy microsomal fraction, a standard microsomal fraction and the cell sap were isolated. The mitochondrial fraction was subfractionated further by density-gradient centrifugation. Each fraction was analysed for protein, RNA, DNA, succinate–neotetrazolium oxidoreductase and glucose 6-phosphatase. 5. A total of 123μg. of cytochrome c was obtained/g. wet wt. of rat liver. 6. Values for the percentage subcellular distribution of cytochrome c are: nuclear fraction, 24·4; mitochondrial fraction, 57·2; heavy microsomal fraction, 5·2; standard microsomal fraction, 10·6; cell sap, 2·7. 7. Three out of the eight mitochondrial subfractions separated by gradient centrifugation contained 76% of the cytochrome c and 85% of the succinate–neotetrazolium oxidoreductase present in the mitochondrial fraction. 8. In unfractionated liver 94% of the cytochrome c was extracted at pH4·0 with water whereas in most of the subcellular fractions the corresponding value was approx. 75–80%.

scholarly journals Subtype identification and functional characterization of ryanodine receptors in rat cerebral artery myocytes

2010 ◽  
Vol 299 (2) ◽  
pp. C264-C278 ◽  
Author(s):  
Thirumalini Vaithianathan ◽  
Damodaran Narayanan ◽  
Maria T. Asuncion-Chin ◽  
Loice H. Jeyakumar ◽  
Jianxi Liu ◽  
...  
Keyword(s):  
Cerebral Artery ◽  
Ryanodine Receptors ◽  
Functional Characterization ◽  
Subcellular Location ◽  
Maximal Activity ◽  
Ruthenium Red ◽  
Voltage Curve ◽  
Subtype Identification ◽  
Steady State Activity

Ryanodine receptors (RyRs) regulate contractility in resistance-size cerebral artery smooth muscle, yet their molecular identity, subcellular location, and phenotype in this tissue remain unknown. Following rat resistance-size cerebral artery myocyte sarcoplasmic reticulum (SR) purification and incorporation into POPE-POPS-POPC (5:3:2; wt/wt) bilayers, unitary conductances of 110 ± 8, 334 ± 15, and 441 ± 27 pS in symmetric 300 mM Cs+ were usually detected. The most frequent (34/40 bilayers) conductance (334 pS) decreased to ≤100 pS when Cs+ was replaced with Ca2+. The predominant conductance displayed 66 bursts/min with at least three open and three closed states. The steady-state activity (NPo)-voltage curve was bell shaped, with NPo drastically decreasing when voltage was switched from −30 to −40 mV. NPo increased when intracellular calcium (Ca2+i) was raised within 0.1–100 μM to abruptly diminish with higher Ca2+i. Thus maximal activity occurred within the Ca2+i range found in rat cerebral artery myocytes under physiological conditions. NPo was reduced by ruthenium red (80 μM), increased monotonically by caffeine (0.1–5 mM) or ryanodine (0.05–5 μM), and unaffected by heparin (2 mg/ml). This phenotype resembles that of cardiac RyR and recombinant RyR2. RT-PCR detected RyR1, RyR2, and RyR3 transcripts in cerebral artery myocytes. However, real-time PCR indicated that RyR2 was 4 and 1.5 times more abundant than RyR1 and RyR3, respectively. Consistently, Western blotting showed that the RyR2 product was very abundant. Immunofluorescence showed that each RyR isoform distributed differentially among subcellular compartments. In particular, RyR2 was drastically stronger in the subplasmalemma than in other compartments, underscoring the predominance of RyR2 in a compartment where SR is abundant. Consistently, RyR from SR-enriched membranes displayed pharmacological specificity typical of RyR2, being activated by digoxin (1 μM), resistant to dantrolene (100 μM), and shifted to a subconductance by neomycin (100 nM). Therefore, RyR2 is the predominant molecular and functional RyR that is expressed in the SR membrane of rat resistance-size cerebral artery myocytes.

scholarly journals Enzymatically inactive forms of acetyl-CoA carboxylase in rat liver mitochondria

1988 ◽  
Vol 251 (3) ◽  
pp. 881-885 ◽  
Author(s):  
J B Allred ◽  
C R Roman-Lopez
Keyword(s):  
Rat Liver ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mitochondrial Fraction ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Outer Mitochondrial Membrane ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa

Biotinyl proteins were labelled by incubation of SDS-denatured preparations of subcellular fractions of rat liver with [14C]methylavidin before polyacrylamide-gel electrophoresis. Fluorographic analysis showed that mitochondria contained two forms of acetyl-CoA carboxylase [acetyl-CoA:carbon dioxide ligase (ADP-forming) EC 6.4.1.2], both of which were precipitated by antibody to the enzyme. When both forms were considered, almost three-quarters of the total liver acetyl-CoA carboxylase was found in the mitochondrial fraction of liver from fed rats while only 3.5% was associated with the microsomal fraction. The remainder was present in cytosol, either as the intact active enzyme or as a degradation product. The actual specific activity of the cytosolic enzyme was approx. 2 units/mg of acetyl-CoA carboxylase protein while that of the mitochondrial enzyme was about 20-fold lower, indicating that mitochondrial acetyl-CoA carboxylase was relatively inactive. Fractionation of mitochondria with digitonin showed that acetyl-CoA carboxylase was associated with the outer mitochondrial membrane. The available evidence suggests that mitochondrial acetyl-CoA carboxylase represents a reservoir of enzyme which can be released and activated under lipogenic conditions.

scholarly journals Ruthenium red-insensitive calcium transport in ascites-sarcoma 180/TG cells

1981 ◽  
Vol 200 (2) ◽  
pp. 343-348 ◽  
Author(s):  
F L Bygrave ◽  
T A Anderson
Keyword(s):  
Rat Liver ◽  
Microsomal Fraction ◽  
Initial Rate ◽  
Liver Microsomes ◽  
Ruthenium Red ◽  
Rat Liver Microsomes ◽  
Sarcoma 180 ◽  
Low Concentrations ◽  
Rat Liver Cells ◽  
Mouse Ascites

1. Ruthenium Red-insensitive Ca2+ transport in the mouse ascites sarcoma 180/TG is enriched in a ‘heavy’ microsomal fraction (microsomes) sedimented at 35 000 g for 20 min. The subcellular distribution of this Ca2+ transport differed from that of Ruthenium Red-sensitive Ca2+ transport and (Na+ + K+)-dependent ATPase activity, but was similar to that of glucose 6-phosphatase. 2. The affinity of this transport system for ‘free’ Ca2+ is high (Km approx. 6 microM) and that for MgATP somewhat lower (Km approx. 100 microM). Ca2+ transport by the tumour microsomes, by contrast with that by liver microsomes, was greatly stimulated by low concentrations of P1. 3. Although incubation of intact ascites cells with glucagon led to an increase in intracellular cyclic AMP, no stable increase in the initial rate of Ca2+ transport in the subsequently isolated ‘heavy’ microsomes could be detected as in similar experiments carried out previously with rat liver cells. Reconstitution experiments suggest that a deficiency exists in the tumour microsomal membrane such that an action of glucagon that is normally present in rat liver microsomes is not evoked.

scholarly journals Phosphatidate biosynthesis in mitochondrial subfractions of rat liver

1969 ◽  
Vol 113 (2) ◽  
pp. 429-440 ◽  
Author(s):  
Elizabeth H. Shephard ◽  
G. Hübscher
Keyword(s):  
Rat Liver ◽  
Mitochondrial Membrane ◽  
Microsomal Fraction ◽  
Mitochondrial Fraction ◽  
Urate Oxidase ◽  
Outer Mitochondrial Membrane ◽  
Short Term ◽  
Conventional Fractionation ◽  
Liver Homogenates ◽  
Nadh Cytochrome

1. After conventional fractionation of rat liver homogenates in 0·88m-sucrose the mitochondrial fraction was subjected to short-term water lysis followed by separation of the resulting membrane preparations. 2. Phosphatidate formation was measured in all subcellular fractions and subfractions and was compared with the distribution of succinate dehydrogenase, monoamine oxidase, rotenone-insensitive NADH cytochrome c reductase, arylsulphatase, urate oxidase, arylesterase and glucose 6-phosphatase. 3. The results obtained indicated that mitochondria were capable of synthesizing phosphatidate, though this activity was only about one-third of the total homogenate activity. 4. Mitochondrial phosphatidate formation was located predominantly in the outer mitochondrial membrane. Although this membrane preparation was found to be significantly contaminated by the microsomal fraction, this contamination was estimated to account for not more than about 20% of the total phosphatidate formation observed in preparations of outer mitochondrial membrane.

Influence of 1,4-benzdiazepin-2-ones on rat liver microsomal fraction and hepatocytes

1987 ◽  
Vol 21 (1) ◽  
pp. 5-8
Author(s):  
T. I. Davidenko ◽  
O. V. Sevast'yanov ◽  
L. N. Yakubovskaya
Keyword(s):  
Rat Liver ◽  
Microsomal Fraction ◽  
Liver Microsomal Fraction

scholarly journals The association of the sulphogalactosylglycerolipid of rat brain with myelination

1977 ◽  
Vol 166 (3) ◽  
pp. 421-428 ◽  
Author(s):  
Joanne Pieringer ◽  
G. Subba Rao ◽  
Paul Mandel ◽  
Ronald A. Pieringer
Keyword(s):  
Rat Brain ◽  
Adult Life ◽  
Developmental Pattern ◽  
Adult Rats ◽  
Jimpy Mouse ◽  
Myelin Fraction ◽  
Old Rats

The sulphogalactosylglycerolipid of rat brain is closely associated with the process of myelination, as demonstrated by the following observations. 1. The lipid is barely detectable in rat brain before 10 days of age, accumulates rapidly between age 10 and 25 days, and remains relatively constant in amount (between 0.3 and 0.4μmol per brain) thereafter into adult life. 2. The activity of adenosine 3′-phosphate 5′-sulphatophosphate–galactosyldiacylglycerol sulphotransferase is almost absent before 10 days of age, attains a maximum at age 20 days, and slowly decreases thereafter with increasing age. This developmental pattern correlates well with that of other myelin-specific metabolites. 3. Both the concentration of the sulphogalactosylglycerolipid and the activity of sulphotransferase are greatly decreased in the non-myelinating jimpy mouse. 4. The myelin fraction of rat brain contains most of the sulphogalactosylglycerolipid. The lipid occurs in a diacyl and an alkylacyl form. Determinations of the relative amount of each type in brain showed about a 1:1 mixture in both 21-day-old and adult rats. Rats injected with H235SO4 at 20 days of age lost35S from the diacyl form at a higher rate than from the alkylacyl compound over a 21-day period. These data suggest that the diacyl form has a higher turnover than the alkylacyl derivative. The percentage of the total sulpholipid content of brain contributed by the sulphogalactosylglycerolipid is 16% in 21-day-old rats and 8.4% in adult rats.

scholarly journals The fractionation of nuclei from mammalian cells by zonal centrifugation

1968 ◽  
Vol 109 (1) ◽  
pp. 127-135 ◽  
Author(s):  
I R Johnston ◽  
A P Mathias ◽  
F. Pennington ◽  
D. Ridge
Keyword(s):  
Rat Liver ◽  
Mammalian Cells ◽  
Mouse Liver ◽  
The Other ◽  
Slow Speed ◽  
Adult Rats ◽  
Sucrose Solutions ◽  
Liver Nuclei ◽  
Effect Of Age ◽  
Zonal Rotor

1. Purified liver nuclei from adult rats separate into two main zones when centrifuged in the slow-speed zonal rotor. One zone contains diploid nuclei, the other tetraploid. 2. The effect of age on the pattern of rat liver ploidy was examined. Tetraploid nuclei are virtually absent from young animals. They increase in proportion steadily with age. Partial hepatectomy disturbs the pattern of ploidy. 3. The zonal centrifuge permits the separation of diploid, tetraploid, octaploid and hexadecaploid nuclei from mouse liver. 4. Rat liver nuclei are isopycnic with sucrose solutions of density 1·35 at 5°.

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