Isolation, fractionation and partial characterization of the tegumental surface from protoscoleces of the hydatid organism, Echinococcus granulosus

Parasitology ◽  
1985 ◽  
Vol 90 (1) ◽  
pp. 111-129 ◽  
Author(s):  
D. P. McManus ◽  
N. J. Barrett
Keyword(s):  
Echinococcus Granulosus ◽  
Surface Membrane ◽  
Qualitative And Quantitative ◽  
Tegumental Surface ◽  
Sds Page ◽  
Pas Staining ◽  
Approximate Molecular Weight ◽  
Membrane Bound ◽  
Triton X

Several approaches were adopted for the disruption and removal of the tegumental surface from protoscoleces of the horse strain of the hydatid organism, Echinococcus granulosus. The effectiveness of each method and the purity of subsequent microthrix-enriched fractions obtained by differential centrifugation were evaluated by electron microscopy, by the amount of protein released and by the degree of enrichment of surface plasma membrane marker enzymes. Incubation in saponin for 10 min produced the purest microtriche preparation, but in low yield; freeze/thawing, incubation in Triton X-100 for 10 mm or in saponin for 20 min produced fractions containing significant amounts of relatively pure microtriches, but mild homogenization was a poor method for surface disruption and subsequent isolation of microtriches. Phosphodiesterase, adenosine triphosphatase (total and ouabain-inhibited), leucine aminopeptidase and glutamyltransferase were active in the protoscoleces but none were enriched in any of the microthrix fractions. In contrast, alkaline phosphatase, acid phosphatase, 5′ nucleotidase and maltase were enriched significantly in all of the isolated microtriche preparations, which suggests that these enzymes are predominantly surface membrane bound. The protein profiles of the microthrix-enriched fractions, following SDS—PAGE, were basically similar, although there were some qualitative and quantitative differences in the proteins released by each isolation procedure. Three major PAS-staining components were present in all the preparations and these probably originated from the glycocalyx. One of these PAS-positive components, with an approximate molecular weight of 110 kDa, may be a glycoprotein specific to the horse strain of E. granulosus.

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 Characterization of the soluble, secreted form of urinary meprin

1996 ◽  
Vol 315 (2) ◽  
pp. 461-465 ◽  
Author(s):  
Robert J. BEYNON ◽  
Simon OLIVER ◽  
Duncan H. L. ROBERTSON
Keyword(s):  
Proteolytic Activity ◽  
Protein Band ◽  
Peptide Sequence ◽  
Soluble Form ◽  
Α Subunit ◽  
Alternative Processing ◽  
Sds Page ◽  
Processing Pathways ◽  
Membrane Bound

A soluble form of the kidney membrane metalloendopeptidase, meprin, is present in urine. Urinary meprin is expressed in BALB/C mice with the Mep-1a/a genotype (high meprin, expressing meprin-α and meprin-β) but not in BALB.K mice of the Mep-1b/b genotype (that only express meprin-β). Western blotting with antisera specific to the meprin-α and the meprin-β subunits established that the only form of meprin present in urine samples was derived from meprin-α. This form of meprin is partially active, and comprises at least three variants by non-reducing SDS/PAGE and by zymography and two protein bands on reducing SDS/PAGE. Sequencing of these two bands established that the N-terminus of the larger protein band begins with the pro-peptide sequence of the α-subunit (VSIKH..), whereas the smaller band possessed the mature meprin N-terminal sequence (NAMRDP..). Trypsin is able to remove the pro-peptide, with a concomitant activation in proteolytic activity. After deglycosylation, the size of the pro- and mature forms of urinary meprin are consistent with cleavage in the region of the X–I boundary. There is a pronounced sexual dimorphism in urinary meprin expression. Females secrete a slightly larger form, and its proteolytic activity is about 50% of that released by males. The urinary meprin is therefore a naturally occurring secreted form of this membrane-bound metalloendopeptidase and is more likely to be generated by alternative processing pathways than by specific release mechanisms.

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 The Characterization of Plasma Membrane-Bound Tubulin of Cauliflower Using Triton X-114 Fractionation

1997 ◽  
Vol 115 (3) ◽  
pp. 1001-1007 ◽  
Author(s):  
A. Sonesson ◽  
M. Berglund ◽  
I. Staxen ◽  
S. Widell
Keyword(s):  
Plasma Membrane ◽  
Membrane Bound ◽  
Triton X

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 Subcellular distribution and characterization of acetylcholinesterase activities from sheep platelets: relationships between temperature- dependence and environment

Blood ◽  
1990 ◽  
Vol 76 (4) ◽  
pp. 737-744
Author(s):  
J Sanchez-Yague ◽  
JA Cabezas ◽  
M Llanillo
Keyword(s):  
Acetylcholinesterase Activity ◽  
Break Point ◽  
Molecular Forms ◽  
Kinetic Properties ◽  
Multiple Molecular Forms ◽  
Key Enzyme ◽  
Membrane Bound ◽  
Hill Coefficients ◽  
Triton X

Acetylcholinesterase is a key enzyme in cholinergic neurotransmission for hydrolyzing acetylcholine and has been shown to possess arylacylamidase activity in addition to esterase activity. The enzyme is found at various loci, where its functional significance remains to be clarified, and it exists in multiple molecular forms. Sheep platelets have been shown to exhibit acetylcholinesterase activity associated with plasma membrane (Bp), endoplasmic reticulum (Cp), mitochondria granules (Dp), and soluble (As) fractions. These activities show differences in some physicochemical and kinetic properties. The soluble acetylcholinesterase is the most thermostable, and the enzyme from the Cp fractions shows the lowest affinity for the acetylthiocholine substrate and the strongest inhibition by fluoride. In all cases a noncompetitive inhibition of the enzyme by this ion is found. When membrane-bound acetylcholinesterases were assayed at temperatures between 12 degrees C and 33 degrees C, the Arrhenius plots of all activities exhibited a break point at about 17 degrees C. This discontinuity was abolished by addition of detergent to the assay medium (0.02% Triton X-100, final concentration). Their Hill coefficients were calculated in the presence of fluoride, showing unitary values in all cases, which points to a noncooperative effect and nonallosteric behavior in the particulate enzyme. These results suggest that the sheep platelet acetylcholinesterase associated with membrane-bound systems is modulated by the physical state of its environment, despite the fact that the enzyme might be lipid- or nonlipid-dependent.

Identification and partial characterization of an extracellular acid phosphatase activity of Leishmania donovani promastigotes

1982 ◽  
Vol 2 (1) ◽  
pp. 76-81
Author(s):  
M Gottlieb ◽  
D M Dwyer
Keyword(s):  
Acid Phosphatase ◽  
Leishmania Donovani ◽  
Surface Membrane ◽  
Extracellular Enzyme ◽  
Growth Cycle ◽  
Growth Media ◽  
Ph Optimum ◽  
Membrane Bound ◽  
Extracellular Acid Phosphatase

An extracellular acid phosphatase was detected in the growth media of Leishmania donovani promastigotes. The enzyme was released at all stages of the growth cycle and in amounts which accounted for 90% of the total amount of this enzyme in the culture. The exoenzyme exhibited a pH optimum of 4.5 to 5.0 and was active with a variety of organic phosphates. The enzymatic activity was excluded from Sephacryl S-300 and was retained by ultrafilters with nominal molecular weight cutoffs of up to 300,000. The results of comparative studies indicated that the extracellular enzyme was distinct from a surface membrane-bound acid phosphatase of L. donovani promastigotes which has been previously described.

scholarly journals Isolation and structural characterization of the Leishmania donovani kinetoplastid membrane protein-11, a major immunoreactive membrane glycoprotein

1995 ◽  
Vol 305 (1) ◽  
pp. 307-313 ◽  
Author(s):  
A Jardim ◽  
V Funk ◽  
R M Caprioli ◽  
R W Olafson
Keyword(s):  
Membrane Protein ◽  
Cell Surface ◽  
Leishmania Donovani ◽  
Surface Membrane ◽  
Immunoblot Analysis ◽  
Post Translational Modifications ◽  
Sds Page ◽  
Membrane Molecule

A novel membrane molecule, previously observed to be co-isolated with lipophosphoglycan and called lipophosphoglycan-associated protein, has been detected in Leishmania donovani promastigotes and amastigotes. This kinetoplastid membrane protein (KMP-11) has been purified by preparative SDS/PAGE after organic solvent extraction of promastigote membranes. Isoelectric-focusing experiments indicated that this was an acidic protein with an isoelectric point of 4.8. Immunoblot analysis of subcellular fractions, together with 125I-labelling experiments, showed this molecule to be associated with the promastigote cell surface membrane. KMP-11 was expressed at a copy number similar to that of lipophosphoglycan (1 x 10(6)-2 x 10(6) molecules per cell), making this glycoprotein one of the major features on the parasite cell surface. The primary structure, less a blocked N-terminal region, was determined by automated Edman degradation of peptides derived from CNBr or enzymic fragmentation. Several post-translational modifications were also found during these studies, including an O-linked oligosaccharide and an NG-monomethylarginine functionality which was verified by m.s. Finally, a set of sequential synthetic peptides was made based on the established partial sequence allowing structural determination of two distinct antibody-binding sites for the monoclonal antibodies L98 and L157.

scholarly journals Purification and Characterization of Lipase Produced by Leuconostoc mesenteroides Subsp. mesenteroides ATCC 8293 Using an Aqueous Two-Phase System (ATPS) Composed of Triton X-100 and Maltitol

Molecules ◽  
2018 ◽  
Vol 23 (7) ◽  
pp. 1800 ◽  
Author(s):  
Nurfadhilah Eko Sukohidayat ◽  
Mohammad Zarei ◽  
Badlishah Baharin ◽  
Mohd Manap
Keyword(s):  
Phase System ◽  
Two Phase ◽  
Aqueous Two Phase System ◽  
Process Characteristics ◽  
Sds Page ◽  
Ph Range ◽  
Lipase Purification ◽  
Triton X ◽  
First Time

Purification of lipase produced by L. mesenteroides subsp. mesenteroides ATCC 8293 was conducted for the first time using a novel aqueous two-phase system (ATPS) composed of Triton X-100 and maltitol. The partitioning of lipase was optimized according to several parameters including pH, temperature, and crude load. Results showed that lipase preferentially migrated to the Triton X-100 rich phase and optimum lipase partitioning was achieved in ATPS at TLL of 46.4% and crude load of 20% at 30 °C and pH 8, resulting in high lipase purification factor of 17.28 and yield of 94.7%. The purified lipase showed a prominent band on SDS-PAGE with an estimated molecular weight of 50 kDa. The lipase was stable at the temperature range of 30–60 °C and pH range of 6–11, however, it revealed its optimum activity at the temperature of 37 °C and pH 8. Moreover, lipase exhibited enhanced activity in the presence of non-ionic surfactants with increased activity up to 40%. Furthermore, results exhibited that metals ions such as Na+, Mg2+, K+ and Ca2+ stimulated lipase activity. This study demonstrated that this novel system could be potentially used as an alternative to traditional ATPS for the purification and recovery of enzymes since the purified lipase still possesses good process characteristics after undergoing the purification process.

Characterization of Acetylcholinesterase in Caco-2 Cells

2002 ◽  
Vol 227 (7) ◽  
pp. 480-486 ◽  
Author(s):  
Lauren R. Plageman ◽  
Giovanni M. Pauletti ◽  
Kenneth A. Skau
Keyword(s):  
Active Site ◽  
Substrate Inhibition ◽  
Extracellular Fluid ◽  
Acetylcholinesterase Activity ◽  
Monomeric Form ◽  
Catalytic Center ◽  
Membrane Bound ◽  
Degree Of Differentiation ◽  
Triton X

Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) was solubilized from cultured Caco-2 cells. It was established that this enzyme activity is acetylcholinesterase by substrate specificity (acetylthiocholine, acetyl-β-methylthiocholine>propionylthiocholine>butyrylthiocholine), substrate inhibition, and specificity of inhibitors (BW284c51>iso-OMPA). The acetylcholinesterase activity increased proportional to the degree of differentiation of the cells. Most of the enzyme was membrane bound, requiring detergent for solubilization, and the active site faced the external fluid. Only one peak of activity, which corresponded to a monomeric form, could be detected on linear sucrose density gradients. The sedimentation of this form of the enzyme was shifted depending on whether Triton X-100 or Brij 96 detergent was used. These results indicate that the epithelial-derived Caco-2 cells produce predominantly an amphiphilic, monomeric form of acetylcholinesterase that is bound to the plasma membrane and whose catalytic center faces the extracellular fluid.

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