The intracellular localization of glycollate oxidoreductase in Euglena gracilis

1. Lowering of the concentration of carbon dioxide in air available to phototrophically growing Euglena cultures from 5% to the normal value (0.03%) resulted in an increased specific activity of glycollate oxidoreductase. 2. The effects of chloramphenicol and cycloheximide suggested that this increase in activity was due to enzyme synthesis de novo on cytoplasmic ribosomes. 3. The Km for glycollate oxidation by the enzyme in crude cell extracts was 3.0×10−3m. 4. Differential centrifugation established that glycollate oxidoreductase present in phototrophically grown Euglena is a particulate enzyme. The enzyme was partially solubilized by the non-ionic detergent Triton X-100. 5. Sucrose-density-gradient centrifugation achieved the separation of the particulate glycollate oxidoreductase from chloroplasts and mitochondria. 6. Glutamate–glyoxylate aminotransferase was associated with particulate glycollate oxidoreductase.

Download Full-text

Purification of the enzyme NADPH: protochlorophyllide oxidoreductase

Biochemical Journal ◽

10.1042/bj1950083 ◽

1981 ◽

Vol 195 (1) ◽

pp. 83-92 ◽

Cited By ~ 38

Author(s):

N S Beer ◽

W T Griffiths

Keyword(s):

Specific Activity ◽

Gradient Centrifugation ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

High Specific Activity ◽

Sucrose Density Gradient ◽

Sucrose Density Gradient Centrifugation ◽

Triton X ◽

Etioplast Membranes

A procedure for the purification of the enzyme NADPH:protochlorophyllide oxidoreductase is described. This involves fractionation of sonicated oat etioplast membranes by discontinuous-sucrose-density-gradient centrifugation, which gives membranes in which the enzyme is present at a high specific activity. The enzyme is solubilized from the membranes with Triton X-100, followed by gel filtration of the extract; enzyme activity is eluted in fractions corresponding to a mol.wt of approx. 35000. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the enzyme-containing fractions from gel filtration shows two peptides, of mol.wts. approx. 35000 and 37000.

Download Full-text

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

Thrombosis and Haemostasis ◽

10.1055/s-0038-1665326 ◽

1983 ◽

Vol 50 (04) ◽

pp. 848-851 ◽

Cited By ~ 11

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.

Download Full-text

Aminoacyl-tRNA synthetase complex in Saccharomyces cerevisiae

Biochemical Journal ◽

10.1042/bj3090321 ◽

1995 ◽

Vol 309 (1) ◽

pp. 321-324 ◽

Cited By ~ 7

Author(s):

C L Harris ◽

C J Kolanko

Keyword(s):

Saccharomyces Cerevisiae ◽

Density Gradient Centrifugation ◽

Gradient Centrifugation ◽

Sucrose Density Gradient ◽

Trna Synthetase ◽

Synthetase Activity ◽

Aminoacyl Trna Synthetase ◽

Sucrose Density Gradient Centrifugation ◽

Cell Extracts ◽

Molecular Masses

The size distribution of aminoacyl-tRNA synthetase activity was investigated in cell extracts prepared from Saccharomyces cerevisiae. Bio-Gel A-5M chromatography of 105,000 g supernatants separated isoleucyl-tRNA synthetase activity into three peaks, with apparent molecular masses (Da) of about 100,000, 350,000 and 10(6) or greater. Similar results were obtained with synthetases specific for glutamic acid, serine and tyrosine. Sucrose-density-gradient centrifugation of yeast supernatants also provided evidence for the existence of synthetase complexes. These data provide the first evidence for the existence of a high-molecular-mass aminoacyl-tRNA synthetase complex in yeast, perhaps similar to those reported in higher eukaryotes.

Download Full-text

5-ENE-3β-HYDROXYSTEROID DEHYDROGENASE OF HUMAN AND BOVINE ADRENOCORTICAL ENDOPLASMIC RETICULUM: SOLUBILIZATION AND FRACTIONATION

Journal of Endocrinology ◽

10.1677/joe.0.0720225 ◽

1977 ◽

Vol 72 (2) ◽

pp. 225-233 ◽

Cited By ~ 14

Author(s):

A. R. EASTMAN ◽

A. M. NEVILLE

Keyword(s):

Molecular Weight ◽

Adrenal Glands ◽

Gradient Centrifugation ◽

Gel Filtration ◽

Hydroxysteroid Dehydrogenase ◽

Sucrose Density Gradient ◽

Sucrose Density Gradient Centrifugation ◽

Microsomal Membranes ◽

Molecular Sizes ◽

Triton X

SUMMARY Protein moieties of various molecular sizes and possessing 5-ene-3β-hydroxysteroid dehydrogenase activity have been successfully solubilized from the microsomal membranes of both bovine and human adrenal glands using a combination of Triton X-100 and sonication. These moieties have been studied by gel filtration, sucrose density gradient centrifugation and isoelectric focusing, and were shown to possess a minimum molecular weight of about 118000, with an isoelectric point between 7·2 and 7·4. The molecular weight was dependent upon the concentration of Triton X-100 used during fractionation. No separation of dehydrogenase activities toward the three steroid substrates, pregnenolone, 17α-hydroxypregnenolone and dehydroisoandrosterone, was observed. Changes in the relative activities for the steroid substrates during fractionation were observed, but have been attributed to the formation of allotypes rather than to the existence of separate dehydrogenases with restricted substrate specificity.

Download Full-text

Changes in the localization of catalase during differentiation of neutrophilic granulocytes

Blood ◽

10.1182/blood.v83.9.2654.2654 ◽

1994 ◽

Vol 83 (9) ◽

pp. 2654-2668 ◽

Cited By ~ 21

Author(s):

CA Ballinger ◽

C Mendis-Handagama ◽

JR Kalmar ◽

RR Arnold ◽

JM Jr Kinkade

Keyword(s):

Gradient Centrifugation ◽

Protein A ◽

Ultrastructural Localization ◽

Sucrose Density Gradient ◽

Sucrose Density Gradient Centrifugation ◽

Cytoplasmic Staining ◽

Cytoplasmic Matrix ◽

Rabbit Polyclonal Antibody ◽

Apparent Shift ◽

Triton X

Abstract The nature of the compartmentalization of catalase in human myeloid cells is an unresolved issue. Using a rabbit polyclonal antibody specific for catalase, indirect immunocytofluorescence of immature leukemic promyelocytes (HL-60 cells) showed a pattern of small, sharp, punctate staining in the cytoplasm of all cells, while mature neutrophils showed a larger diffuse, flocculent pattern of cytoplasmic staining. Differential centrifugation of nitrogen cavitates of HL-60 cells indicated that the putative catalase-containing compartment was relatively fragile compared with the compartment(s) that contained myeloperoxidase (MPO), beta-hexosaminidase, beta-glucuronidase, and lysosomal alpha-mannosidase activities. Parallel studies using dimethylsulfoxide (DMSO)-induced HL-60 cells and mature neutrophils showed that, in the course of differentiation, there was an apparent shift in the localization of catalase from the granule fraction to the cytosolic fraction. Percoll-sucrose density gradient centrifugation of HL-60 cell cavitates showed a catalase-containing compartment with a mean peak density (1.05 g/mL) significantly lower than that of the major myeloperoxidase-containing compartment (1.08 g/mL); in mature neutrophils, catalase activity comigrated with lactate dehydrogenase (LDH) activity. Catalase in isolated fractions was protected from proteolysis in the absence, but not in the presence, of 0.1% Triton X- 100. Digitonin titration experiments confirmed the compartmentalized nature of catalase in immature HL-60 cells and were consistent with a cytosolic localization in mature neutrophils. Ultrastructural localization of catalase by Protein A-gold immunocytochemistry demonstrated four to six catalase-containing compartments in all HL-60 cell profiles. In mature neutrophils, catalase was localized primarily in the cytoplasmic matrix, although in fewer than 2% of the cell profiles, one to two catalase-containing compartments were observed. The changes in catalase localization that occur during myeloid differentiation appear to be similar to the changes that occur during erythroid and megakaryocytic differentiation, and may have potential clinical significance in the classification of acute leukemia and in the development of drug resistance.

Download Full-text

Membrane-associated actin from the microvillar membranes of ascites tumor cells.

The Journal of Cell Biology ◽

10.1083/jcb.94.3.624 ◽

1982 ◽

Vol 94 (3) ◽

pp. 624-630 ◽

Cited By ~ 25

Author(s):

K L Carraway ◽

R F Cerra ◽

G Jung ◽

C A Carraway

Keyword(s):

Membrane Fraction ◽

Density Gradient Centrifugation ◽

Gradient Centrifugation ◽

Gel Filtration ◽

Sucrose Density Gradient ◽

Membrane Preparation ◽

Intermediate Form ◽

Sucrose Density Gradient Centrifugation ◽

Transmission Electron ◽

Triton X

A membrane fraction (MF2) has been purified from isolated microvilli of the MAT-C1 subline of the 13762 rat mammary ascites adenocarcinoma under conditions which cause F-actin depolymerization. This membrane preparation contains actin as a major component, although no filamentous structures are observed by transmission electron microscopy. Membranes were extracted with a Triton X-100-containing actin-stabilizing buffer (S buffer) or actin-destabilizing buffer (D buffer). In D buffer greater than 90% of metabolically labeled protein and glycoprotein was extracted, and 80-90% of these labeled species was extracted in S buffer. When S buffer extracts of MF2 were fractionated by either gel filtration on Sepharose 6 B or rate-zonal sucrose density gradient centrifugation, most of the actin was found to be intermediate in size between G- and F-actin. In D buffer most of the MF2 actin behaved as G-actin. Extraction and gel filtration of intact microvilli in S buffer also showed the presence of the intermediate form of actin, indicating that it did not arise during membrane preparation. When [35S]methionine-labeled G-actin from ascites cells was added to S buffer extracts of MF2 and chromatographed, all of the radioactivity chromatographed as G-actin, indicating that the intermediate form of actin did not result from an association of G-actin molecules during extraction or chromatography. The results of this study suggest that the microvillar membrane fraction is enriched in an intermediate form of actin smaller than F-actin and larger than G-actin.

Download Full-text

Evidence that Membrane Rafts Are Not Required for the Action of Clostridium perfringens Enterotoxin

Infection and Immunity ◽

10.1128/iai.00854-08 ◽

2008 ◽

Vol 76 (12) ◽

pp. 5677-5685 ◽

Cited By ~ 13

Author(s):

Justin A. Caserta ◽

Martha L. Hale ◽

Michel R. Popoff ◽

Bradley G. Stiles ◽

Bruce A. McClane

Keyword(s):

Clostridium Perfringens ◽

Gradient Centrifugation ◽

Sucrose Density Gradient ◽

Membrane Rafts ◽

Cholesterol Depletion ◽

Appreciable Effect ◽

Sucrose Density Gradient Centrifugation ◽

Small Pool ◽

Independent Association ◽

Triton X

ABSTRACT The action of bacterial pore-forming toxins typically involves membrane rafts for binding, oligomerization, and/or cytotoxicity. Clostridium perfringens enterotoxin (CPE) is a pore-forming toxin with a unique, multistep mechanism of action that involves the formation of complexes containing tight junction proteins that include claudins and, sometimes, occludin. Using sucrose density gradient centrifugation, this study evaluated whether the CPE complexes reside in membrane rafts and what role raft microdomains play in complex formation and CPE-induced cytotoxicity. Western blot analysis revealed that the small CPE complex and the CPE hexamer 1 (CH-1) complex, which is sufficient for CPE-induced cytotoxicity, both localize outside of rafts. The CH-2 complex was also found mainly in nonraft fractions, although a small pool of raft-associated CH-2 complex that was sensitive to cholesterol depletion with methyl-β-cyclodextrin (MβCD) was detected. Pretreatment of Caco-2 cells with MβCD had no appreciable effect on CPE-induced cytotoxicity. Claudin-4 was localized to Triton X-100-soluble gradient fractions of control or CPE-treated Caco-2 cells, indicating a raft-independent association for this CPE receptor. In contrast, occludin was present in raft fractions of control Caco-2 cells. Treatment with either MβCD or CPE caused most occludin molecules to shift out of lipid rafts, possibly due (at least in part) to the association of occludin with the CH-2 complex. Collectively, these results suggest that CPE is a unique pore-forming toxin for which membrane rafts are not required for binding, oligomerization/pore formation, or cytotoxicity.

Download Full-text

Subcellular localization and properties of rat liver adenosine diphosphatase

Biochemical Journal ◽

10.1042/bj1920527 ◽

1980 ◽

Vol 192 (2) ◽

pp. 527-535 ◽

Cited By ~ 11

Author(s):

G P Smith ◽

G D Smith ◽

T J Peters

Keyword(s):

Rat Liver ◽

Gradient Centrifugation ◽

Intracellular Localization ◽

Sucrose Density Gradient ◽

Subcellular Fractionation ◽

Single Step ◽

Sucrose Density Gradient Centrifugation ◽

Ph Optimum ◽

Outer Membranes ◽

Selective Membrane

ADPase (adenosine diphosphatase) was assayed in rat liver homogenates with [beta-32P]ADP as substrate. The activity had a pH optimum of 8.0 and was strongly activated by Mg2+. The intracellular localization was determined by analytical subcellular fractionation with single-step sucrose-density-gradient centrifugation. Selective membrane perturbants were used to enhance the resolution of the various organelles. ADPase was localized to the mitochondria. Mitochondria were isolated by differential centrifugation and subfractionated by selective disruption of the inner and outer membranes. The intramitochondrial localization of ADPase was compared with various marker enzymes and was shown to be concentrated in the outer-membrane fractions. The effects of various inhibitors on the ADPase activity were determined and the possibility that the activity could be due to known enzyme systems was considered. It is concluded that ADP degradation is due to a hitherto unrecognized mitochondrial enzyme.

Download Full-text

Effect of cations on structure-linked sedimentability of lysosomal hydrolases

Biochemical Journal ◽

10.1042/bj1090149 ◽

1968 ◽

Vol 109 (1) ◽

pp. 149-154 ◽

Cited By ~ 20

Author(s):

M. Anthony Verity ◽

R. Caper ◽

W. Jann Brown

Keyword(s):

High Speed ◽

Gradient Centrifugation ◽

Sucrose Density Gradient ◽

Lysosomal Hydrolases ◽

Freezing And Thawing ◽

Sucrose Density Gradient Centrifugation ◽

Bivalent Cation ◽

Repeated Freezing ◽

Triton X ◽

Total Enzyme

1. A partially purified lysosomal preparation was obtained from mouse liver sucrose homogenates by differential and discontinuous gradient centrifugation. 2. Triton X-100 or repeated freezing and thawing of the lysosomal suspension (subfraction B) allowed comparison of free and activated values for acid phosphohydrolase, β-glucuronidase and N-acetylglucosaminidase in the presence and absence of ascorbate. 3. The distribution of hydrolase activities between supernatant and pellet after high-speed centrifugation was measured and the percentages of total enzyme found in the supernatant were: acid phosphohydrolase, 40·7; β-glucuronidase, 51; N-acetylglucosaminidase, 39·4. 4. Differential rates of elution of the three hydrolases from the membrane fraction occurred with increasing Na+ and K+ concentrations, whereas complex biphasic elution curves were obtained as a function of bivalent cation concentration with Ca2+ and Mg2+. 5. Sucrose-density-gradient centrifugation of frozen-and-thawed subfraction B demonstrated highly significant changes in the protein gradient profile in the presence of a low concentration of bivalent cation, indicating membrane aggregation and enzyme–membrane association. 6. The data provide further evidence for the nature of lysosomal enzyme binding and indicate the presence of different enzyme–membrane bonds conferring structure-linked latency upon individual lysosomal enzymes.

Download Full-text

Isolation and antigen analysis of surface tegument membranes from schistosomula ofSchistosoma mansoni

Parasitology ◽

10.1017/s0031182000056729 ◽

1984 ◽

Vol 89 (3) ◽

pp. 495-510 ◽

Cited By ~ 16

Author(s):

D. W. Taylor ◽

Pamela Z. Wells

Keyword(s):

Specific Activity ◽

Gradient Centrifugation ◽

Polyacrylamide Gel Electrophoresis ◽

Sucrose Density Gradient ◽

Two Dimensional ◽

Sucrose Density Gradient Centrifugation ◽

Western Blots ◽

Peptide Composition ◽

Membrane Antigens ◽

Compare And Contrast

SUMMARYThe outer tegument membranes ofSchistosoma mansonischistosomula have been removed by mechanical disruption in a hypotonic salt solution and partially purified by differential and sucrose density gradient centrifugation. Fractionation was monitored by measuring increase in specific activity of bound [125I] wheat-germ agglutinin (WGA), alkaline phosphatase and calcium-stimulated ATPase. Two-dimensional IEF/SDS polyacrylamide gel electrophoresis was used to analyse the peptide composition of the isolated membranes and to compare and contrast with lactoperoxidase/glucose oxidase surface labelled peptides. At least 35 surface-labelled peptides were resolved on the two-dimensional maps: all were also present in the membrane material recovered from the sucrose gradient. Western blot analysis demonstrated a marked heterogeneity in the antibody response of infected human patients to individual membrane antigens. The antigenic profile of membranes isolated from cercariae, 18 and 96 h schistosomula were compared using Western blots: some minor differences were observed between the three preparations.

Download Full-text