scholarly journals Purification and characterization of membrane-bound semicarbazide-sensitive amine oxidase (SSAO) from bovine lung

1998 ◽  
Vol 331 (1) ◽  
pp. 69-78 ◽  
Author(s):  
José M. LIZCANO ◽  
Keith F. TIPTON ◽  
Mercedes UNZETA
Keyword(s):  
Amine Oxidase ◽  
Gel Filtration ◽  
Integral Membrane Protein ◽  
Significant Activity ◽  
Bovine Plasma ◽  
Sds Page ◽  
Catalytically Active ◽  
Cibacron Blue 3Ga ◽  
Membrane Bound ◽  
Triton X

Semicarbazide-sensitive amine oxidase (SSAO) has been purified from bovine lung microsomes in a form which is catalytically active and stable to storage. The enzyme, an integral membrane protein, was solubilized with Triton X-100 and purification was achieved, in the presence of detergent, by chromatography with Cibacron Blue 3GA-agarose, hydroxylapatite, Lens culinaris-agarose, Resource Q-FPLC and gel filtration on Superdex 200 HR-FPLC. This is the first reported procedure for the extensive purification of a membrane-bound SSAO. The purified enzyme had an apparent Mr of 400000 but exhibited microheterogeneity with SDS/PAGE and isoelectric focusing, probably as a result of its glycoprotein nature. It behaved as a tetramer with subunits with apparent Mr values of 100. Antibodies raised towards the purified enzyme cross-reacted with the enzymes from human lung and bovine plasma. Redox-cycling staining and reaction with carbonyl reagents were consistent with the presence of a quinone cofactor, possibly topa quinone. The enzyme was also shown to contain two mol of Cu/mol of enzyme and removal of half of this bound copper resulted essentially in complete inhibition of enzyme activity. In contrast to the reported behaviour of the SSAO enzymes from plasma, the bovine lung enzyme was relatively insensitive to inhibition by cyanide, copper-chelating agents and amiloride. The specificity of the bovine lung enzyme was also narrower than reported for soluble SSAO. It catalysed the oxidative deamination of benzylamine, methylamine, 2-phenylethylamine and histamine but had no significant activity towards dopamine, 5-hydroxytryptamine, tryptamine or tyramine.

Studies On Glycocalicin And Glycoprotein Gp Ib

1981 ◽  
Author(s):  
N O Solum ◽  
T Sletbakk ◽  
I Hagen ◽  
G Gogstad
Keyword(s):  
Fast Moving ◽  
Gel Filtration ◽  
Integral Membrane Protein ◽  
Human Platelets ◽  
Characteristic Change ◽  
Extraction Buffer ◽  
Sds Page ◽  
Slow Moving ◽  
Triton X ◽  
Two Peaks

Crossed immunoelectrophoresis (CIE) of extracts of human platelets in 1% Triton X-100 using antiserum to purified glycocalicin shows an immunopre- cipitate consisting of two peaks. Previous experiments have shown the small fast-moving peak to represent free glycocalicin whereas the slow-moving one corresponds to a larger amphiphilic protein, probably the integral membrane protein GP Ib. Glycocalicin is probably derived from the latter secondary to an activation of a calsium-dependent protease during platelet lysis. Further studies on these problems are presented. A gradual reduction of the concentration of Triton X-100 in the extraction buffer (tris-glycine, pH 8.7, 135 mOsM) gradually reduced the area of the slow-moving peak and increased that of the fast-moving one untill all was present as free glycocalicin. Reduction of the concentration of Triton in an extract already prepared with 1% Triton X-100 by adsorption to Bio-Beads SM-2 had no such effect. The presence of the protease inhibitor leupeptin during extraction at a low concentration of Triton (0.2%) reduced the peak corresponding to free glycocalicin. GP Ib was purified from Triton extracts by precipitation with con A, affinity chromatography of the supernatant on WGA-Sepharose and elution with NAGA, and gel filtration of the eluate on Ultrogel AcA 22. Triton X-100, EDTA and sodium azide were present at all steps. The characteristic change in mobility of GP Ib on SDS PAGE comparing unreduced to reduced sampLes deduced from studies on whole platelet proteins, was confirmed with the purified material, as was the correspondence between the GP Ib band on SDS and the slow-moving component of the CIE.

A metalloproteinase extracellularly released byCrithidia deanei

2003 ◽  
Vol 49 (10) ◽  
pp. 625-632 ◽  
Author(s):  
Claudia Masini d'Avila-Levy ◽  
Rodrigo F Souza ◽  
Rosana C Gomes ◽  
Alane B Vermelho ◽  
Marta H Branquinha
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Residual Activity ◽  
Major Protein ◽  
Motile Cells ◽  
Sds Page ◽  
Triton X

Actively motile cells from a cured strain of Crithidia deanei released proteins in phosphate buffer (pH 7.4). The molecular mass of the released polypeptides, which included some proteinases, ranged from 19 to 116 kDa. One of the major protein bands was purified to homogeneity by a combination of anion-exchange and gel filtration chromatographs. The apparent molecular mass of this protein was estimated to be 62 kDa by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE). The incorporation of gelatin into SDS–PAGE showed that the purified protein presented proteolytic activity in a position corresponding to a molecular mass of 60 kDa. The enzyme was optimally active at 37 °C and pH 6.0 and showed 25% of residual activity at 28 °C for 30 min. The proteinase was inhibited by 1,10-phenanthroline and EDTA, showing that it belonged to the metalloproteinase class. A polyclonal antibody to the leishmanial gp63 reacted strongly with the released C. deanei protease. After Triton X-114 extraction, an enzyme similar to the purified metalloproteinase was detected in aqueous and detergent-rich phases. The detection of an extracellular metalloproteinase produced by C. deanei and some other Crithidia species suggests a potential role of this released enzyme in substrate degradation that may be relevant to the survival of trypanosomatids in the host.Key words: endosymbiont, trypanosomatid, extracellular, proteinase.

scholarly journals Evidence for the involvement of a 66 kDa membrane protein in the synthesis of sterolglucoside in Saccharomyces cerevisiae.

1995 ◽  
Vol 42 (2) ◽  
pp. 269-274 ◽  
Author(s):  
U Lenart ◽  
J Haplova ◽  
P Magdolen ◽  
V Farkas ◽  
G Palamarczyk
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Mass ◽  
Deae Cellulose ◽  
Yeast Saccharomyces Cerevisiae ◽  
Sds Page ◽  
Nonionic Detergent ◽  
Membrane Bound ◽  
Labeled Probe ◽  
Triton X ◽  
Cellulose Column

The membrane-bound sterolglucoside synthase from the yeast Saccharomyces cerevisiae has been solubilized by nonionic detergent, Nonidet P-40, Triton X-100, and partially purified by DEAE-cellulose column chromatography and ammonium sulfate fractionation. SDS/PAGE of the purified fraction revealed the presence of two protein bands of molecular mass 66 kDa and 54 kDa. In an attempt to identify further the polypeptide chain of sterolglucoside synthase, the partially purified enzyme was treated with [di-125I]-5-[3-(p-azidosalicylamide)]allyl-UDPglucose, a photoactive analogue of UDP glucose, which is a substrate for this enzyme. Upon photolysis the 125I-labeled probe was shown to link covalently to the 66 kDa protein. The photoinsertion was competed out by the presence of unlabeled UDPglucose thus suggesting that this protein contains substrate binding site for UDPglucose. Since photoinsertion of the probe to protein of 66 kDa correlates with the molecular mass of the protein visualized upon enzyme purification we postulate that the 66 kDa protein is involved in sterolglucoside synthesis in yeast.

scholarly journals Purification and characterization of N-ethylmaleimide-insensitive phosphatidic acid phosphohydrolase (PAP2) from rat liver

1995 ◽  
Vol 308 (3) ◽  
pp. 983-989 ◽  
Author(s):  
I N Fleming ◽  
S J Yeaman
Keyword(s):  
Phosphatidic Acid ◽  
Rat Liver ◽  
Lysophosphatidic Acid ◽  
Gel Filtration ◽  
Purification And Characterization ◽  
Sds Page ◽  
Chromatography Columns ◽  
Triton X ◽  
Second Peak

N-Ethylmaleimide-insensitive phosphatidic acid phosphohydrolase (PAP; EC 3.1.3.4) was purified 5900-fold from rat liver. The enzyme was solubilized from membranes with octylglucoside, fractionated with (NH4)2SO4, and purified in the presence of Triton X-100 by chromatography on Sephacryl S300, hydroxyapatite, heparin-Sepharose and Affi-Gel Blue. Silver-stained SDS/PAGE indicated that the enzyme was an 83 kDa polypeptide. Sephacryl S-300 gel filtration also produced a second peak of enzyme activity, which was eluted from all of the chromatography columns at a different position from the purified enzyme. SDS/PAGE indicated that it contained three polypeptides (83 kDa, 54 kDa and 34 kDa), and gel filtration suggested that it was not an aggregate of the purified enzyme. Both forms were sensitive to inhibition by amphiphilic amines, Mn2+ and Zn2+, but not by N-ethylmaleimide. Purified PAP required detergent for activity, but was not activated by Mg2+, fatty acids or phospholipids. The enzyme was able to dephosphorylate lysophosphatidic acid or phosphatidic acid, and was inhibited by diacylglycerol and monoacylglycerol. No evidence was obtained for regulation of PAP by reversible phosphorylation.

scholarly journals Characterization of a secretase activity which releases angiotensin-converting enzyme from the membrane

1993 ◽  
Vol 292 (2) ◽  
pp. 597-603 ◽  
Author(s):  
S Y Oppong ◽  
N M Hooper
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Human Kidney ◽  
Soluble Form ◽  
Converting Enzyme ◽  
Ph Optimum ◽  
Secretase Activity ◽  
Sds Page ◽  
Microsomal Membranes ◽  
Membrane Bound ◽  
Triton X

Angiotensin-converting enzyme (ACE; EC 3.4.1.15.1) exists in both membrane-bound and soluble forms. Phase separation in Triton X-114 and a competitive e.l.i.s.a. have been employed to characterize the activity which post-translationally converts the amphipathic, membrane-bound form of ACE in pig kidney microvilli into a hydrophilic, soluble form. This secretase activity was enriched to a similar extent as other microvillar membrane proteins, was tightly membrane-associated, being resistant to extensive washing of the microvillar membranes with 0.5 M NaCl, and displayed a pH optimum of 8.4. The ACE secretase was not affected by inhibitors of serine-, thiol- or aspartic-proteases, nor by reducing agents or alpha 2-macroglobulin. The metal chelators, EDTA and 1,10-phenanthroline, inhibited the secretase activity, with, in the case of EDTA, an inhibitor concentration of 2.5 mM causing 50% inhibition. In contrast, EGTA inhibited the secretase by a maximum of 15% at a concentration of 10 mM. The inhibition of EDTA was reactivated substantially (83%) by Mg2+ ions, and partially (34% and 29%) by Zn2+ and Mn2+ ions respectively. This EDTA-sensitive secretase activity was also present in microsomal membranes prepared from pig lung and testis, and from human lung and placenta, but was absent from human kidney and human and pig intestinal brush-border membranes. The form of ACE released from the microvillar membrane by the secretase co-migrated on SDS/PAGE with ACE purified from pig plasma, thus the action and location of the secretase would be consistent with it possibly having a role in the post-translational proteolytic cleavage of membrane-bound ACE to generate the soluble form found in blood, amniotic fluid, seminal plasma and other body fluids.

scholarly journals Activation of a phospholipase Cβ2 deletion mutant by limited proteolysis

1998 ◽  
Vol 330 (1) ◽  
pp. 461-468 ◽  
Author(s):  
Petra SCHNABEL ◽  
Montserrat CAMPS
Keyword(s):  
Deletion Mutant ◽  
Limited Proteolysis ◽  
Gel Filtration ◽  
Cell System ◽  
Heterotrimeric G Proteins ◽  
Sds Page ◽  
Catalytically Active ◽  
Heterodimeric Complex ◽  
High Degree ◽  
Hydrolysis Of

All phosphoinositide-specific phospholipases C (PLC) identified until today exhibit a high degree of similarity within two regions of 170 and 260 residues, respectively, which are designated regions X and Y. The PLCβ family, including four members designated PLCβ1, PLCβ2, PLCβ3 and PLCβ4, is regulated by heterotrimeric G proteins. In order to investigate structure-function relationships of PLCβ2, we expressed PLCβ2Δ, a deletion mutant of PLCβ2 which lacks most of the sequence downstream of region Y, in the baculovirus/insect cell system. The mutant was present in both soluble and particulate fractions of Sf9 cells and was demonstrated to be catalytically active and sensitive to βγ-subunits. Sf9 cytosol containing this mutant was subjected to limited proteolysis by trypsin and S. aureus protease V8, respectively. Immunochemical analysis revealed that both proteases cleaved the enzyme between the regions X and Y. Most interestingly, proteolytic cleavage at this site by both proteases stimulated the catalytic activity of PLC2β2Δ. The proteolytically activated enzyme was still sensitive to βγ-subunits and showed an unchanged concentration dependence on Ca2+. Gel filtration chromatography indicated that the fragments generated by cleavage between the regions X and Y were still connected and formed a functional heterodimeric complex. In order to visualize all fragments generated by protease V8, PLCβ2Δ was purified to homogeneity from Sf9 cytosol. Limited proteolysis of the purified enzyme by S. aureus protease V8 and subsequent SDS/PAGE and silver staining revealed that several cuts take place between the regions X and Y and that the resulting fragments remain intact. We hypothesize that the activating proteolytic cut induces a conformational change of the enzyme which might facilitate hydrolysis of the phospholipid substrate.

scholarly journals MPB70 and MPB83 as Indicators of Protein Localization in Mycobacterial Cells

1998 ◽  
Vol 66 (1) ◽  
pp. 289-296 ◽  
Author(s):  
Morten Harboe ◽  
Harald G. Wiker ◽  
Gunni Ulvund ◽  
Bent Lund-Pedersen ◽  
Åse Bengård Andersen ◽  
...  
Keyword(s):  
Protein Localization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Culture Fluid ◽  
Secreted Proteins ◽  
Enzyme Linked Immunosorbent Assay ◽  
Sds Page ◽  
Shock Proteins ◽  
Triton X

ABSTRACT Culture fluids after growth of Mycobacterium bovis BCG on Sauton medium contain actively secreted proteins and proteins released by bacterial lysis. BCG culture fluids and sonicates ofMycobacterium tuberculosis and Mycobacterium paratuberculosis were tested after separation by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The localization of marker proteins was determined by enzyme-linked immunosorbent assay and Western blotting with selected monoclonal antibodies of known specificities. Soluble secreted proteins (MPB64 and proteins of the antigen 85 complex) and three heat shock proteins (DnaK, GroEL, and GroES) were recovered in a single peak after gel filtration, indicating their occurrence as a free monomer in the culture fluid and cytosol, respectively. Other constituents eluted in two distinct peaks during gel filtration. The first peak corresponded to the void volume, indicating complex formation between several proteins or attachment to lipids in the surface layer or the cytoplasmic membrane; the second peak corresponded to the expected monomer size indicated by SDS-PAGE under conditions that separate proteins from each other during sample preparation. The two-peak group contained constituents with known lipid contents, the 19- and 38-kDa lipoproteins and lipoarabinomannan. The 26-kDa form of MPB83 behaved similarly. After extraction with Triton X-114, these constituents entered into the detergent phase, confirming the lipoprotein nature of 26-kDa MPB83. The MPB83 molecule was shown to be available on the surface of BCG Tokyo bacilli for reaction with monoclonal antibody MBS43 by flow cytometry.

scholarly journals Isolation of a subpopulation of glycoprotein IIb-III from platelet membranes that is bound to membrane actin.

1985 ◽  
Vol 100 (2) ◽  
pp. 652-657 ◽  
Author(s):  
R G Painter ◽  
K N Prodouz ◽  
W Gaarde
Keyword(s):  
High Speed ◽  
High Efficiency ◽  
Gel Filtration ◽  
Insoluble Fraction ◽  
Human Platelets ◽  
Independent Manner ◽  
Platelet Surface ◽  
Sds Page ◽  
Triton X

Triton X-100-insoluble residues, or skeletons, of plasma membrane-rich vesicles obtained from unstimulated human platelets were isolated by high speed centrifugation. About 10-15% of the total surface iodinatable glycoproteins IIb and III (GPIIb and GPIII, respectively) co-isolated with the insoluble fraction. After sonication and centrifugation the solubilized material was further purified by affinity chromatography on Lens culinaris lectin-Sepharose. SDS PAGE analysis of this material revealed the presence of at least three major proteins, which were shown to be GPIIb, GPIII, and membrane actin, as judged by their electrophoretic properties and on the basis of immunological criteria. Antibodies directed against platelet surface glycoproteins and antibodies directed against rabbit actin were able to immunoprecipitate all three proteins, which indicates that they were noncovalently associated with one another. Gel filtration of the Lens lectin-purified Triton-insoluble complex on Ultrogel AcA 22 showed that greater than 85% of the total surface GPIIb and III was associated with an actin-rich peak that eluted in the void volume. In contrast, the form of GPIIb-III present in the Triton-soluble membrane fraction behaved as monomeric species when chromatographed under identical conditions. Finally, the GPIIb-III membrane actin complex bound with high efficiency to rabbit f-actin in vitro in a Ca++-independent manner, whereas the monomeric forms found in the Triton-soluble fraction did not bind to actin. These results indicate that two forms of GPIIb and III exist: one that binds directly to endogenous membrane actin and one that does not.

Membrane-bound carbonic anhydrase in the heart

1992 ◽  
Vol 262 (2) ◽  
pp. H577-H584 ◽  
Author(s):  
W. Bruns ◽  
G. Gros
Keyword(s):  
Carbonic Anhydrase ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Integral Membrane Protein ◽  
Rabbit Heart ◽  
Microsomal Membranes ◽  
Membrane Bound ◽  
The One ◽  
Triton X

Microsomal membranes from bovine heart homogenates were subfractionated by density gradient centrifugation. Fractions with high levels of a sarcolemmal (SL) marker are enriched in specific carbonic anhydrase (CA) activity up to ninefold compared with the microsomes. Fractions with high levels of a sarcoplasmic reticulum marker and a mitochondrial marker, respectively, exhibit specific CA activities that are similar to the one found in the microsomes. Determination of cytosolic markers reveals that the CA activity in the SL fraction is not due to contamination by cytosolic CA, and it is shown by Triton X-114 phase separation that the CA activity is due to an integral membrane protein. In cryosections from rabbit heart the SL region of cardiomyocytes is stained by the fluorescent CA inhibitor dansylsulfonamide. Intracellular staining occurs also, with a pattern suggesting the presence of CA associated with intracellular membranes. Although it cannot be excluded that there is a contribution by endothelial membranes, it appears likely that most CA of the heart is bound to the SL. The possible involvement of the enzyme in extracellular proton buffering is discussed.

scholarly journals Quantification of apolipoprotein B-48 and B-100 in rat liver endoplasmic reticulum and Golgi fractions

1992 ◽  
Vol 285 (1) ◽  
pp. 153-159 ◽  
Author(s):  
I J Cartwright ◽  
J A Higgins
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Smooth Endoplasmic Reticulum ◽  
Protein A ◽  
Subcellular Fractions ◽  
Apo B ◽  
Sds Page ◽  
Membrane Bound ◽  
Triton X ◽  
Quantitative Immunoblotting

We have developed a method for measurement of apolipoprotein (apo) B-48 and apo B-100 in blood and subcellular fractions of rat liver based on SDS/PAGE followed by quantitative immunoblotting using 125I-Protein A. Standard curves were prepared in each assay using apo B prepared from total rat lipoproteins by extraction with tetramethylurea. Subcellular fractions (rough and smooth endoplasmic reticulum and Golgi fractions) were prepared from rat liver and separated into membrane and cisternal-content fractions. For quantification, membrane fractions were solubilized in Triton X-100, and the apo B was immunoprecipitated before separation by SDS/PAGE and immunoblotting. Content fractions were concentrated by ultrafiltration and separated by SDS/PAGE without immunoprecipitation. Quantification of apo B in subcellular fractions and detection of apo B by immunoblotting yielded consistent results. In all fractions apo B-48 was the major form, accounting for approximately three-quarters of the total apo B. By using marker enzymes as internal standards, it was calculated that all of the apo B was recovered in the endoplasmic reticulum and Golgi fractions, with approximately 80% of each form of apo B in the endoplasmic reticulum. More than 90% of the apo B of the rough- and smooth-endoplasmic-reticulum fractions was membrane-bound, whereas approx. 33 and 15% of the apo B of the cis-enriched Golgi fractions and trans-enriched Golgi fractions respectively were membrane-bound.

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