scholarly journals Effect of cations on structure-linked sedimentability of lysosomal hydrolases

1968 ◽  
Vol 109 (1) ◽  
pp. 149-154 ◽  
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.

scholarly journals Effect of mercurial compounds on structure-linked latency of lysosomal hydrolases

1967 ◽  
Vol 105 (2) ◽  
pp. 685-690 ◽  
Author(s):  
M. Anthony Verity ◽  
A. Reith
Keyword(s):  
Adult Mouse ◽  
Free Acid ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Enzyme Activation ◽  
Lysosomal Hydrolases ◽  
Irreversible Loss ◽  
Sucrose Density Gradient Centrifugation ◽  
Triton X ◽  
Individual Particles

1. A partially purified lysosomal preparation was obtained from adult mouse livers by sucrose-density-gradient centrifugation of a large-granule fraction. 2. This lysosome-enriched subfraction was contaminated approx. 10% by mitochondrial cytochrome c oxidase and malate dehydrogenase. 3. Free acid phosphohydrolase and β-glucuronidase contributed less than 10% of the total (Triton X-100-solubilized) activity in contrast with approx. 30% free N-acetyl-β-d-glucosaminidase when assayed in an iso-osmotic incubation system. 4. Exposure of the lysosomal preparation to inorganic Hg2+ ions and organic mercurials (p-chloromercuribenzoate, phenylmercuric acetate) induced an irreversible loss of structure-linked latency with resulting enzyme activation. 5. Maximal activation was related to log [Hg2+] and pH. The response was all-or-none for individual particles; the dose–response curve portrayed the variation in particle resistance within the lysosomal population. 6. l-Cysteine and GSH totally prevented Hg2+ ion-induced hydrolase activation. Ascorbate provided approx. 50% protection. 7. The three lysosomal hydrolases were differentially activated at constant [Hg2+], suggesting a different pattern of binding, unique for each enzyme studied.

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 Uridine diphosphate glucose-sterol glucosyltransferase and nucleoside diphosphatase activities in etiolated pea seedlings

1978 ◽  
Vol 169 (2) ◽  
pp. 297-303 ◽  
Author(s):  
M J Staver ◽  
K Glick ◽  
D J Baisted
Keyword(s):  
Density Gradient ◽  
High Speed ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Particulate Fraction ◽  
Partial Recovery ◽  
Sucrose Density Gradient Centrifugation ◽  
Freeze Thaw ◽  
Pea Seedlings ◽  
Nucleoside Diphosphatase

1. UDP-glucose-sterol glucosyltransferase and nucleoside diphosphatases were isolated in a particulate fraction from 7-day-old etiolated pea seedlings. The glucosyltransferase and UDPase (uridine diphosphatase) are stimulated by Ca2+ cation, less so by Mg2+ cation, and inhibited by Zn2+. 2. Each activity has a pH optimum near 8. 3. The glucosyltransferase is specific for UDP-glucose as the glucosyl donor and is inhibited by UDP. Partial recovery from UDP inhibition is effected by preincubation of the enzyme. 4. Freeze-thaw treatment and subsequent sucrose-density-gradient centrifugation of the particulate fraction shows the glucosyltransferase to be widely distributed among cell fractions but to be most active in particles with a density of 1.15 g/ml. UDPase is most active in particulate material with a density of over 1.18 g/ml but an activity peak also appears at 1.15 g/ml. Of several nucleoside diphosphatase activities, UDPase activity is most enhanced by the freeze-thaw and sucrose-density-gradient-fractionation procedures. 5. Detergent treatment with 0.1% sodium deoxycholate allows the partial solubilization of the glucosyltransferase and UDPase. The two activities are similarly distributed between pellet and supernatant after high-speed centrifugation for two different time intervals. 6. A role for UDPase in the functioning of glucosylation reactions is discussed.

scholarly journals Purification of the enzyme NADPH: protochlorophyllide oxidoreductase

1981 ◽  
Vol 195 (1) ◽  
pp. 83-92 ◽  
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.

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.

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

1977 ◽  
Vol 72 (2) ◽  
pp. 225-233 ◽  
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.

scholarly journals Changes in the localization of catalase during differentiation of neutrophilic granulocytes

Blood ◽  
1994 ◽  
Vol 83 (9) ◽  
pp. 2654-2668 ◽  
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.

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

1982 ◽  
Vol 94 (3) ◽  
pp. 624-630 ◽  
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.

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

2008 ◽  
Vol 76 (12) ◽  
pp. 5677-5685 ◽  
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.

scholarly journals The intracellular localization of glycollate oxidoreductase in Euglena gracilis

1971 ◽  
Vol 124 (2) ◽  
pp. 275-281 ◽  
Author(s):  
J. M. Lord ◽  
M. J. Merrett
Keyword(s):  
De Novo ◽  
Specific Activity ◽  
Gradient Centrifugation ◽  
Intracellular Localization ◽  
Sucrose Density Gradient ◽  
Enzyme Synthesis ◽  
Sucrose Density Gradient Centrifugation ◽  
Cell Extracts ◽  
Ionic Detergent ◽  
Triton X

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.

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