scholarly journals Subcellular distribution of agonist-stimulated phosphatidylinositol synthesis in 1321 N1 astrocytoma cells

1993 ◽  
Vol 290 (2) ◽  
pp. 381-387 ◽  
Author(s):  
D J Sillence ◽  
C P Downes
Keyword(s):  
Endoplasmic Reticulum ◽  
Subcellular Distribution ◽  
Reductase Activity ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Astrocytoma Cells ◽  
Astrocytoma Cell ◽  
Membrane Compartment ◽  
Nadph Cytochrome C Reductase

In an inositol-depleted 1321 N1 astrocytoma cell line, propranolol at 0.5 mM concentration and carbachol in the presence of Li+ induce a large increase (30-60-fold) in the amount of CMP-phosphatidate, the lipid substrate of PtdIns synthase. The actions of both agents on CMP-phosphatidate accumulation were reversed by co-incubation with 1 mM inositol. In cells grown in the presence of 40 microM inositol the propranolol- and carbachol-mediated CMP-phosphatidate accumulation was much smaller (2-4-fold). Propranolol- and carbachol-mediated increases in CMP-phosphatidate accumulation were at least additive in both inositol-replete and -depleted cells. The subcellular distribution of accumulated CMP-phosphatidate was investigated by sucrose-density-gradient centrifugation of a lysate of inositol-depleted cells. There were two coincident peaks of carbachol-stimulated [3H]CMP-phosphatidate and PtdIns synthase activity, respectively. The first peak of accumulated [3H]CMP-phosphatidate and PtdIns synthase activity is characteristic of a ‘light vesicle’ fraction, since it sediments at sucrose densities similar to that of endocytosed 125I-transferrin. The later peak, containing both carbachol-stimulated [3H]CMP-phosphatidate and PtdIns synthase activity, has a distribution in the gradient that is similar to NADPH-cytochrome c reductase activity, an endoplasmic-reticulum marker. By contrast, propranolol-stimulated [3H]CMP-phosphatidate accumulates in membranes which sediment as a single peak corresponding to the endoplasmic-reticulum marker. These observations suggest that agonist-stimulated PtdIns synthesis occurs in the endoplasmic reticulum and in at least one additional membrane compartment which is insensitive to propranolol, an inhibitor of endoplasmic-reticulum phosphatidate phosphohydrolase.

scholarly journals Incorporation of [14C]glucosamine and [14C]leucine into mouse kidney β-glucuronidase induced by gonadotrophin

1970 ◽  
Vol 117 (1) ◽  
pp. 161-167 ◽  
Author(s):  
Keitaro Kato ◽  
Hiroyuki Ide ◽  
Tsuranobu Shirahama ◽  
William H. Fishman
Keyword(s):  
Endoplasmic Reticulum ◽  
Microsomal Fraction ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Mouse Kidney ◽  
Differential Centrifugation ◽  
Freezing And Thawing ◽  
Sucrose Density Gradient Centrifugation ◽  
Glucuronidase Activity

Male BALB/C mice were injected intraperitoneally with 2.5 i.u. of gonadotrophin. After the injection, increase of β-glucuronidase activity was first observed in the microsomal fraction. By 36h 45–50% of the total homogenate activity was found in the microsomal fraction compared with 20–25% in the control microsomal fraction. From 36 to 80h not only microsomal β-glucuronidase but also lysosomal β-glucuronidase increased progressively. After 69h stimulation with 2.5 i.u. of gonadotrophin, d-[1-14C]glucosamine or l-[U-14C]leucine was injected intraperitoneally. After a further 3h the kidneys were homogenized and five particulate fractions were prepared by differential centrifugation. The β-glucuronidase in the microsomal and lysosomal fractions was released respectively by ultrasonication and by freezing and thawing treatment. The enzyme was purified by organic-solvent precipitation and by sucrose-density-gradient centrifugation. The results demonstrated the incorporation of these two labels into the mouse renal β-glucuronidase. The microsomal β-glucuronidase was much more radioactive than the lysosomal enzyme and approx. 80% of the newly synthesized enzyme appeared in microsomes and approx. 20% of that was found in lysosomes at this period. These results suggest that the mouse renal β-glucuronidase is a glycoprotein and that the newly synthesized enzyme is transported from endoplasmic reticulum to lysosomes.

scholarly journals Derlin-3 Is Required for Changes in ERAD Complex Formation under ER Stress

2020 ◽  
Vol 21 (17) ◽  
pp. 6146
Author(s):  
Yuka Eura ◽  
Toshiyuki Miyata ◽  
Koichi Kokame
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Dependent Manner ◽  
Dynamic Nature ◽  
Sucrose Density Gradient Centrifugation

Endoplasmic reticulum (ER)-associated protein degradation (ERAD) is a quality control system that induces the degradation of ER terminally misfolded proteins. The ERAD system consists of complexes of multiple ER membrane-associated and luminal proteins that function cooperatively. We aimed to reveal the role of Derlin-3 in the ERAD system using the liver, pancreas, and kidney obtained from different mouse genotypes. We performed coimmunoprecipitation and sucrose density gradient centrifugation to unravel the dynamic nature of ERAD complexes. We observed that Derlin-3 is exclusively expressed in the pancreas, and its deficiency leads to the destabilization of Herp and accumulation of ERAD substrates. Under normal conditions, Complex-1a predominantly contains Herp, Derlin-2, HRD1, and SEL1L, and under ER stress, Complex-1b contains Herp, Derlin-3 (instead of Derlin-2), HRD1, and SEL1L. Complex-2 is upregulated under ER stress and contains Derlin-1, Derlin-2, p97, and VIMP. Derlin-3 deficiency suppresses the transition of Derlin-2 from Complex-1a to Complex-2 under ER stress. In the pancreas, Derlin-3 deficiency blocks Derlin-2 transition. In conclusion, the composition of ERAD complexes is tissue-specific and changes in response to ER stress in a Derlin-3-dependent manner. Derlin-3 may play a key role in changing ERAD complex compositions to overcome ER stress.

Purification and partial characterization of proline dehydrogenase from Clostridium sporogenes

1981 ◽  
Vol 27 (9) ◽  
pp. 942-948 ◽  
Author(s):  
Daniel J. Monticello ◽  
Ralph N. Costilow
Keyword(s):  
Dehydrogenase Activity ◽  
Reductase Activity ◽  
Gradient Centrifugation ◽  
Sodium Dodecyl ◽  
Protein Band ◽  
Sucrose Density Gradient ◽  
Proline Dehydrogenase ◽  
Sucrose Density Gradient Centrifugation ◽  
Clostridium Sporogenes ◽  
Enzyme Preparations

A proline dehydrogenase which catalyzes the nicotinamide adenine dinucleotide (NAD) dependent oxidation of proline and the NADH-dependent reduction of Δ1-pyrroline 5-carboxylic acid (PCA) was purified from extracts of Clostridium sporogenes. Following purification, only one protein band was found on analytical polyacrylamide disc gels and on sodium dodecyl sulfate (SDS) – polyacrylamide disc gels. Sucrose density gradient centrifugation and SDS–gel electrophoresis indicated that the enzyme has a molecular weight of approximately 217 000 and consists of two subunits of equal size. During purification of proline dehydrogenase on hydroxylapatite the ratio of dehydrogenase activity to reductase activity decreased significantly, and a similar change in ratio was brought about by storage of partially purified enzyme preparations in low ionic strength buffers. Subsequent purification did not change the ratio. The dehydrogenase activity of proline dehydrogenase was inhibited by L-glutamate (Ki = 0.32 mM at pH 7.4 and Ki = 0.65 mM at pH 10.2). However, the reductase activity of the purified enzyme was not affected by 100 mML-glutamate.

Acetylcholinesterase: a useful marker for the isolation of sarcolemma from the bivalve (Modiolus demissus demissus) myocardium

1978 ◽  
Vol 34 (1) ◽  
pp. 193-208
Author(s):  
J.A. Watts ◽  
S.K. Pierce
Keyword(s):  
Cell Surface ◽  
Reductase Activity ◽  
Gradient Centrifugation ◽  
Surface Membrane ◽  
Sucrose Density Gradient ◽  
Electron Microscopic ◽  
Sucrose Density Gradient Centrifugation ◽  
Cardiac Muscle Cells ◽  
Succinate Cytochrome C Reductase ◽  
Enzymic Assay

The presence of cholinesterase activity in M. demissus hearts was demonstrated by light- and electron-microscopic histochemistry and by enzymic assay. The enzyme proved to be acetylcholinesterase (AChE) since acetylthiocholine was the preferred substrate, and eserine or BW284C5I inhibited the enzyme activity, while isoOMPA was without effect. The AChE was localized and uniformly distributed along the cell surface membranes of the cardiac muscle cells. A fraction 8-fold enriched in AChE was isolated from pooled ventricles by a combination of differential and sucrose density gradient centrifugation. This sarcolemmal fraction contained little mitochondrial contamination as determined by electron microscopy and by succinate cytochrome c reductase activity. In addition, this fraction stained uniformly for AChE, indicating that it was free of other membrane types (for example sarcoplasmic reticulum which did not stain for AChE). Therefore, this fraction contained purified cell surface membrane free of contamination by other membranous organelles.

The subcellular localization of transglutaminase in normal liver and in glucagon-treated and partial hepatectomized rats

1983 ◽  
Vol 3 (12) ◽  
pp. 1085-1090 ◽  
Author(s):  
Sally E. Bruce ◽  
Timothy J. Peters
Keyword(s):  
Plasma Membrane ◽  
Subcellular Distribution ◽  
Specific Activity ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Normal Liver ◽  
Sucrose Density Gradient ◽  
Subcellular Fractionation ◽  
Sucrose Density Gradient Centrifugation ◽  
Significant Difference

Rat liver was homogenized in isotonic sucrose and subjected to analytical subcellular fractionation by sucrose density-gradient centrifugation. Transglutaminase, when assayed with putrescine and dimethylcasein as substrates, showed three distinct localizations, cytosol (73%), plasma membrane (20%), and nuclei (7%). The distribution was unaffected by homogenization in the presence of potassium chloride, indicating that the particulate activity was not due to adsorbed cytosolic enzyme. The specific activity and subcellular distribution of transglutaminase in rats which had received intra-peritoneal glucagon, stimulating endocytosis; or which had been subjected to sub-total hepatectomy 2, 16, or 32 h previously, showed no significant difference from control animals.

The Combining Ability of Glycoproteins IIb, IIIa and Ca2+ in EDTA-Treated Nonaggregable Platelets

1983 ◽  
Vol 50 (04) ◽  
pp. 848-851 ◽  
Author(s):  
Marjorie B Zucker ◽  
David Varon ◽  
Nicholas C Masiello ◽  
Simon Karpatkin
Keyword(s):  
Density Gradient ◽  
Combining Ability ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Electrophoretic Pattern ◽  
Sucrose Density Gradient ◽  
Irreversible Loss ◽  
Sucrose Density Gradient Centrifugation ◽  
Crossed Immunoelectrophoresis ◽  
Triton X

SummaryPlatelets deprived of calcium and incubated at 37° C for 10 min lose their ability to bind fibrinogen or aggregate with ADP when adequate concentrations of calcium are restored. Since the calcium complex of glycoproteins (GP) IIb and IIIa is the presumed receptor for fibrinogen, it seemed appropriate to examine the behavior of these glycoproteins in incubated non-aggregable platelets. No differences were noted in the electrophoretic pattern of nonaggregable EDTA-treated and aggregable control CaEDTA-treated platelets when SDS gels of Triton X- 114 fractions were stained with silver. GP IIb and IIIa were extracted from either nonaggregable EDTA-treated platelets or aggregable control platelets with calcium-Tris-Triton buffer and subjected to sucrose density gradient centrifugation or crossed immunoelectrophoresis. With both types of platelets, these glycoproteins formed a complex in the presence of calcium. If the glycoproteins were extracted with EDTA-Tris-Triton buffer, or if Triton-solubilized platelet membranes were incubated with EGTA at 37° C for 30 min, GP IIb and IIIa were unable to form a complex in the presence of calcium. We conclude that inability of extracted GP IIb and IIIa to combine in the presence of calcium is not responsible for the irreversible loss of aggregability that occurs when whole platelets are incubated with EDTA at 37° C.

scholarly journals Molecular heterogeneity of mouse duodenal alkaline phosphatase. Association of lipids and peptides

1974 ◽  
Vol 141 (1) ◽  
pp. 93-101 ◽  
Author(s):  
P. R. V. Nayudu ◽  
Fraser B. Hercus
Keyword(s):  
Alkaline Phosphatase ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Buoyant Density ◽  
Sucrose Density Gradient ◽  
Molar Ratio ◽  
Molecular Forms ◽  
Partial Specific Volume ◽  
Molecular Heterogeneity ◽  
Sucrose Density Gradient Centrifugation

Polyacrylamide-gel electrophoresis and Bio-Gel P-300 molecular-sieve chromatography of mouse duodenal alkaline phosphatase demonstrates its molecular heterogeneity, which, in a kinetic sense, is manifest also in the differential relative velocities of the heterogeneous forms of the enzyme with two substrates, phenylphosphate and β-glycerophosphate. Different treatments that eliminate most of the electrophoretic and chromatographic variability of the enzyme also decrease the velocities with both substrates so that the molar ratio of hydrolysis of one substrate relative to the other is also altered to a low but stable value. Concomitant with these changes, lipids and peptides are dissociated from the enzyme. The lipids are tentatively identified as a sterol and phospholipids. The peptides have an average composition of four to six amino acids and appear to be strongly electropositive. The conditions of dissociation suggest that their binding to the enzyme is non-covalent and predominantly based on hydrophobic and ionic bonding. The concept of lipid and peptide association would suggest prima facie differential molecular weights as a factor in the observed electrophoretic and chromatographic heterogeneity. However, the molecular forms of the enzyme with differences in elution volume equivalent to more than one-half the void volume of the Bio-Gel P-300 column, or even enzyme fractions dissociated from the lipids and peptides compared with undissociated portions, do not show any differences in sedimentation on sucrose-density-gradient centrifugation. This may be because the alterations in molecular weight owing to binding of small molecules are too small to be detected by this method. Alternatively, since lipids are involved, the binding may alter the partial specific volume in such a way that the buoyant density is not significantly altered.

scholarly journals Alteration of hepatic glucocorticoid receptor stability and nuclear binding in vitro by citrate

1983 ◽  
Vol 210 (1) ◽  
pp. 259-263 ◽  
Author(s):  
J Hubbard ◽  
M Kalimi
Keyword(s):  
Glucocorticoid Receptor ◽  
Glucocorticoid Receptors ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Deae Cellulose ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Nuclear Binding ◽  
Wide Range

Citrate greatly stabilized rat hepatic unbound glucocorticoid receptors in cell-free conditions at 4 degrees C with optimal effectiveness at 5-15 mM. Control receptors were inactivated at 4 degrees C with a half-life of less than 12 h. However, in the presence of 10 mM-citrate, unbound receptors were almost completely stabilized for 48 h at 4 degrees C. Citrate at a concentration of 1-2 mM yielded half-maximal stabilization. The stabilizing effect of citrate was rather specific, as succinate, alpha-oxoglutarate, oxaloacetate, malate and pyruvate had no apparent stabilizing action. Citrate stabilized receptors over a wide range of H+ concentrations, with complete protection between pH 6.5 and 8.5. In addition, citrate appeared to have a significant effect on glucocorticoid-receptor complex activation into a nuclear binding form. Thus 5-10 mM-citrate enhanced nuclear binding, with optimal activation achieved at 10 mM concentration. As analysed by sucrose-density-gradient centrifugation and DEAE-cellulose chromatography, no apparent change was observed in the physical characteristics of the glucocorticoid receptor in the presence of citrate.

scholarly journals Thermolability of 28S ribosomal ribonucleic acid from the liver of Crotalus durissus terrificus (Ophidia, Reptilia)

1973 ◽  
Vol 135 (1) ◽  
pp. 73-79 ◽  
Author(s):  
J. F. Giorgini ◽  
F. L. De Lucca
Keyword(s):  
Molecular Weight ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sucrose Density Gradient ◽  
Dimethyl Sulphoxide ◽  
Low Molecular Weight ◽  
28S Rrna ◽  
Sucrose Density Gradient Centrifugation ◽  
Crotalus Durissus Terrificus ◽  
Crotalus Durissus

Instability of 28S rRNA of Crotalus durissus terrificus liver was observed during hotphenol extraction: purified 28S rRNA is converted into an 18S RNA component by heat treatment. It was also found that ‘6S’ and ‘8S’ low-molecular-weight RNA species were released during the thermal conversion. This conversion and the release of the low-molecular-weight species were also induced by 8m-urea and 80% (v/v) dimethyl sulphoxide at 0°C. Evidence is presented that this phenomenon is an irreversible process and results from the rupture of hydrogen bonds. The 18S RNA product was shown to be homogeneous by polyacrylamide-gel electrophoresis and by sucrose-density-gradient centrifugation. The base composition of the 18S RNA products obtained by heat, urea or dimethyl sulphoxide treatments was similar. The C+G content of the 18S RNA product was different from that of the native 18S rRNA, but similar to that of 28S rRNA.

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