scholarly journals ANALYTICAL FRACTIONATION OF HOMOGENATES FROM CULTURED RAT EMBRYO FIBROBLASTS

1974 ◽  
Vol 63 (2) ◽  
pp. 383-401 ◽  
Author(s):  
Paul Tulkens ◽  
Henry Beaufay ◽  
André Trouet
Keyword(s):  
Plasma Membrane ◽  
Cytochrome C ◽  
Cytochrome Oxidase ◽  
Equilibrium Density ◽  
Acid Hydrolases ◽  
Rat Embryo ◽  
Cytochrome C Reductase ◽  
Inosine Diphosphatase ◽  
Embryo Fibroblasts ◽  
Nadh Cytochrome

Homogenates of cultured rat embryo fibroblasts have been assayed for acid phosphatase, N-acetyl-ß-glucosaminidase, cathepsin D, acid deoxyribonuclease, cytochrome oxidase, NADH cytochrome c reductase, 5'-nucleotidase, inosine diphosphatase, acid pyrophosphatase, neutral pyrophosphatase, esterase, catalase, cholesterol, and RNA. The validity of the assay conditions was checked. Neutral pyrophosphatase is a readily soluble enzyme. Acid hydrolases, except acid pyrophosphatase, are particle-bound enzymes, which exhibit a high degree of structural latency. They are activated and solubilized in a parallel fashion by mechanical treatments and tensio-active agents. Catalase is also particle-bound and latent; activating conditions stronger than those for hydrolases are required to activate the enzyme. Acid pyrophosphatase, 5'-nucleotidase and inosine diphosphatase are firmly particle-bound, but not latent; they are not easily solubilized. In differential and isopycnic centrifugation, the latent hydrolases, cytochrome oxidase and catalase dissociate largely from each other; this suggests the occurrence of lysosomes and peroxisome-like structures besides mitochondria. The distribution patterns of 5'-nucleotidase and cholesterol are largely similar; digitonin influences their equilibrium density to the same extent; these two constituents are thought to be related to the plasma membrane. Inosine diphosphatase and acid pyrophosphatase are also partially associated with the plasma membrane, although some part of these enzymic activities probably belongs to other structures. NADH cytochrome c reductase is associated partly with the endoplasmic reticulum, partly with mitochondria.

scholarly journals GTP- and inositol 1,4,5-trisphosphate-induced release of 45Ca2+ from a membrane store co-localized with pancreatic-islet-cell plasma membrane

1988 ◽  
Vol 253 (1) ◽  
pp. 67-72 ◽  
Author(s):  
M E Dunlop ◽  
R G Larkins
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Cytochrome C ◽  
Succinate Dehydrogenase ◽  
Close Association ◽  
Receptor Activation ◽  
Pancreatic Islet Cell ◽  
Cytochrome C Reductase ◽  
Inositol 1,4,5 Trisphosphate ◽  
Nadh Cytochrome

Inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], arising from hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], is proposed as the link between membrane-receptor activation and mobilization of Ca2+ from intracellular sites in hormone-secreting cells. The location of Ins(1,4,5)P3-sensitive membranes was investigated in cultured neonatal beta-cells. Membranes were obtained after lysis of cells attached to positively charged Sephadex. After lysis the presence of the enzyme markers 5′-nucleotidase, glucose-6-phosphatase, NADH-cytochrome c reductase, UDP-galactosyltransferase and succinate dehydrogenase indicated the mixed nature of the preparation. After sonication, however, UDP-galactosyltransferase and succinate dehydrogenase activities were undetectable, but 4.8% of total cellular glucose-6-phosphatase and 3.4% of total cellular NADH-cytochrome c reductase remained with 5′-nucleotidase in the preparation, indicating endoplasmic-reticulum association. ATP-dependent 45Ca2+ accumulation was shown in this preparation (410 +/- 24 pmol/mg of protein at 150 nM free Ca2+) and was inhibited by vanadate (100 microM). Ca2+ release was effected by Ins(1,4,5)P3, with half-maximal release at 0.5 +/- 0.14 microM-Ins(1,4,5)P3, t1/2 11.2 +/- 1.1 s. GTP- and guanosine 5′-[beta gamma-imido]triphosphate (p[NH]ppG)-promoted release of 45Ca2+ was demonstrated in this preparation, but the kinetics of release (half-maximal Ca2+ release at 5.4 +/- 0.7 microM, with t1/2 77.3 +/- 6.9 s, and at 51.1 +/- 4.2 microM, with t1/2 19.0 +/- 2.2 s, for GTP and p[NH]ppG respectively), and the ability of neomycin sulphate to block p[NH]ppG-induced release only, are indicative of separate release mechanisms after treatment with these agents. A close association between plasma membrane and elements of the endoplasmic reticulum is indicated in this model, providing a possible mechanism for local alterations in free Ca2+ in the sub-plasma-membrane region.

Characterization of Mg-ATP-dependent Ca2+ transport in cat pancreatic microsomes

1983 ◽  
Vol 244 (5) ◽  
pp. G480-G490 ◽  
Author(s):  
A. Kribben ◽  
T. Tyrakowski ◽  
I. Schulz
Keyword(s):  
Plasma Membrane ◽  
Cytochrome C ◽  
Atpase Activity ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Tissue Homogenate ◽  
Ionophore A23187 ◽  
Plasma Membrane Vesicles ◽  
Cytochrome C Reductase ◽  
Nadh Cytochrome

Mg-ATP-dependent 45Ca2+ uptake and Ca2+-ATPase activity have been examined in isolated microsomes obtained by differential centrifugation and in purified subcellular fractions obtained by Ficoll-sucrose density centrifugation in the presence of mitochondrial inhibitors. Mg-ATP-dependent 45Ca2+ uptake increased with increasing EGTA-buffered free [Ca2+], reaching a maximum of 2 nmol 45Ca2+ X 15 min-1 X mg prot-1 at 2 mumol/1 [Ca2+] in the incubation medium. Half-maximal 45Ca2+ uptake was at approximately 0.2 mumol/1 [Ca2+]. Maximal Ca2+ -Mg2+ -ATPase activity was 130 nmol X 15 min-1 X mg prot-1 at 2 mumol/l [Ca2+], with an apparent Km of approximately 0.3 mumol/l [Ca2+]. The Ca2+ ionophore A23187 (10(-6) mol/l), the mercurial compounds mersalyl (10(-5) mol/l) and CH3ClHg (10(-3) mol/l), as well as La3+ (10(-4) mol/l), vanadate (10(-4) mol/l), and saponin (50 micrograms/mg prot), abolished Mg-ATP-promoted 45Ca2+ uptake. In the absence of Mg2+, ATP did not provoke 45Ca2+ uptake. Using the purified smooth membrane fraction (F1) from the Ficoll-sucrose density gradient (enrichment of Na+-K+-ATPase specific activity by ninefold and of NADH-cytochrome c reductase by threefold as compared with total tissue homogenate), Mg-ATP-dependent 45Ca2+ uptake correlated better with Na+-K+-ATPase (r = 0.97) than with the smooth endoplasmic marker NADH-cytochrome c reductase (r = 0.52). No correlation was found with RNA, the marker for rough endoplasmic reticulum. We conclude that pancreatic plasma membranes contain a Ca2+-Mg2+-ATPase that represents the Ca2+ extrusion system from acinar cells. It is also possible that vesicular membrane structures associated with the plasma membrane, or endocytotic plasma membrane vesicles, take up Ca2+ and represent an intracellular Ca2+ pool.

scholarly journals Properties of a cytochrome c-enriched light particulate fraction isolated from the photosynthetic bacterium Rhodopseudomonas spheroides

1978 ◽  
Vol 174 (1) ◽  
pp. 267-275 ◽  
Author(s):  
J Barrett ◽  
C N Hunter ◽  
O T G Jones
Keyword(s):  
Cytochrome C ◽  
Sodium Dodecyl ◽  
Photosynthetic Bacterium ◽  
Particulate Fraction ◽  
Cytochrome C Reductase ◽  
Cytochrome C2 ◽  
Succinate Cytochrome C Reductase ◽  
Bulk Membrane ◽  
Nadh Cytochrome ◽  
Rhodopseudomonas Spheroides

Differential centrifugation of suspensions of French-press-disrupted Rhodopseudomonas spheroides yielded a light particulate fraction that was different in many properties from the bulk membrane fraction. It was enriched in cytochrome c and had a low cytochrome b content. When prepared from photosynthetically grown cells this fraction had a very low specific bacteriochlorophyll content. The cytochrome c of the light particles differed in absorption maxima at 77K from cytochrome c2 attached to membranes; there was pronounced splitting of the alpha-band, as is found in cytochrome c2 free in solution. Potentiometric titration at A552–A540 showed the presence of two components that fitted an n = 1 titration; one component had a midpoint redox potential of +345mV, like cytochrome c2 in solution, and the second had E0′ at pH 7.0 of +110 mV, and they were present in a ratio of approx. 2:3. Difference spectroscopy at 77K showed that the spectra of the two components were very similar. More of a CO-binding component was present in particles from photosynthetically grown cells. Light membranes purified by centrifugation on gradients of 5–60% (w/w) sucrose retained the two c cytochromes; they contained no detectable succinate-cytochrome c reductase or bacteriochlorophyll and very little ubiquinone, but they contained NADH-cytochrome c reductase and some phosphate. Electrophoresis on sodium dodecyl sulphate/polyacrylamide gels showed that the light membranes of aerobically and photosynthetically grown cells were very similar and differed greatly from other membrane fractions of R. spheroides.

scholarly journals Analytical subcellular fractionation of needle-biopsy specimens from human liver

1978 ◽  
Vol 174 (2) ◽  
pp. 435-446 ◽  
Author(s):  
T J Peters ◽  
C A Seymour
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Density Gradient ◽  
Needle Biopsy ◽  
Human Liver ◽  
Resolving Power ◽  
Equilibrium Density ◽  
Marker Enzyme ◽  
Acid Hydrolases ◽  
Biopsy Specimens

1. Fragments (2-20 mg wet wt.) of closed needle-biopsy specimens from human liver were disrupted in iso-osmotic sucrose and subjected to low-speed centrifugation. The supernatant was layered on a linear sucrose-density gradient in the Beaufay small-volume automatic zonal rotor. The following organelles, with equilibrium densities (g/ml) and principal marker enzyme shown in parentheses, were resolved: plasma membrane (1.12-1.14; 5′-nucleotidase); lysosomes (1.15-1.20; N-acetyl-beta-glucosaminidase); mitochondria (1.20; malate dehydrogenase); endoplasmic reticulum (1.17-1.21; neutral alpha-glucosidase); peroxisomes (1.22-1.24; catalase). 2. The distribution of particulate alkaline phosphatase and, to a lesser degree, leucine 2-naphthylamidase followed that of 5′-nucleotidase. gamma-Glutamyltransferase was associated with membranes of significantly higher equilibrium density than was 5′-nucleotidase. 3. The distribution of 12 acid hydrolases was determined in the density-gradient fractions. beta-Glucosidase had a predominantly cytosolic localization, but the other enzymes showed a broad distribution of activity throughout the gradient. Evidence was presented for two populations of lysosomes with equilibrium densities of 1.15 and 1.20 g/ml, but containing differing amounts of each enzyme. Further evidence of lysosomal heterogeneity was demonstrated by studying the distribution of isoenzymes of hexosaminidase and of acid phosphatase. 4. The resolving power of the centrifugation procedure can be further enhanced with membrane perturbants. Digitonin (0.12 mM) selectively disrupted lysosomes, markedly increased the equilibrium density of plasma-membrane components and lowered the density of the endoplasmic reticulum, but did not affect the mitochondria or peroxisomes. Pyrophosphate (15 mM) selectively lowered the equilibrium density of the endoplasmic reticulum.

Endoplasmic reticulum membrane isolated from small-intestinal epithelial cells: enzyme and protein components

1981 ◽  
Vol 52 (1) ◽  
pp. 215-222
Author(s):  
M. Fujita ◽  
H. Ohta ◽  
T. Uezato
Keyword(s):  
Endoplasmic Reticulum ◽  
Epithelial Cells ◽  
Cytochrome C ◽  
Endoplasmic Reticulum Membrane ◽  
Dependent Manner ◽  
Cytochrome C Reductase ◽  
Basolateral Membranes ◽  
Nadh Cytochrome ◽  
Protein Components ◽  
A Minor

Endoplasmic reticulum membrane-rich fraction was obtained by subfractionation of the light microsomes from mouse jejunal mucosal epithelial cells. It was marked by high glucose-6-phosphatase, NADPH-cytochrome c reductase, and NADH-cytochrome c reductase activities and low Na+,K+-ATPase activity. The enrichment of Na+,K+-ATPase was 180-fold higher in the basolateral membranes than in the endoplasmic reticulum membrane-rich fraction relative to glucose-6-phosphatase. The protein peak that was phosphorylated in a Na-dependent manner was prominent in the basolateral membranes while it was a minor peak in the endoplasmic reticulum membrane-rich fraction. Under the electron microscope the fraction was seen to be composed of homogeneous small vesicles with thin smooth membranes.

Ferrocyanide-activated NADH cytochrome c reductase from barley

1980 ◽  
Vol 18 (4) ◽  
pp. 389-393 ◽  
Author(s):  
Ian S. Small ◽  
John L. Wray
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Reductase ◽  
Nadh Cytochrome
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