FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2078542822> ?p ?o ?g. }

• W2078542822 endingPage "19342" @default.
• W2078542822 startingPage "19334" @default.
• W2078542822 abstract "Glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) is an integral membrane protein in the protozoan parasite Trypanosoma brucei. Enzyme activity appears to be suppressed in T. brucei, although the polypeptide is readily detectable. The basis for the apparent quiescence of GPI-PLC is not known. Protein oligomerization was investigated as a possible mechanism for post-translational regulation of GPI-PLC activity. An equilibrium between monomers, dimers, and tetramers of purified GPI-PLC was detected by molecular sieving and shown to be perturbed with specific detergents. Homotetramers dominated in Nonidet P-40, and dimers and monomers of GPI-PLC were the major species in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The detergents were exploited as tools to study the effect of oligomerization on enzyme activity. Tetrameric GPI-PLC was 3.6–20-fold more active than the monomeric enzyme. Tetramer existence was confirmed by chemical cross-linking. In vivo cross-linking revealed the oligomeric state of GPI-PLC during latency and after enzyme activation. During quiescence, monomers were the predominant species inT. brucei. Assembly of tetrameric GPI-PLC occurred when parasites were subjected to conditions known to activate the enzyme. InLeishmania where heterologous expression of GPI-PLC causes a GPI deficiency, the enzyme existed as a tetramer. Hence, oligomerization of GPI-PLC is associated with high enzyme activity bothin vivo and in vitro. Glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) is an integral membrane protein in the protozoan parasite Trypanosoma brucei. Enzyme activity appears to be suppressed in T. brucei, although the polypeptide is readily detectable. The basis for the apparent quiescence of GPI-PLC is not known. Protein oligomerization was investigated as a possible mechanism for post-translational regulation of GPI-PLC activity. An equilibrium between monomers, dimers, and tetramers of purified GPI-PLC was detected by molecular sieving and shown to be perturbed with specific detergents. Homotetramers dominated in Nonidet P-40, and dimers and monomers of GPI-PLC were the major species in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The detergents were exploited as tools to study the effect of oligomerization on enzyme activity. Tetrameric GPI-PLC was 3.6–20-fold more active than the monomeric enzyme. Tetramer existence was confirmed by chemical cross-linking. In vivo cross-linking revealed the oligomeric state of GPI-PLC during latency and after enzyme activation. During quiescence, monomers were the predominant species inT. brucei. Assembly of tetrameric GPI-PLC occurred when parasites were subjected to conditions known to activate the enzyme. InLeishmania where heterologous expression of GPI-PLC causes a GPI deficiency, the enzyme existed as a tetramer. Hence, oligomerization of GPI-PLC is associated with high enzyme activity bothin vivo and in vitro. variant surface glycoprotein 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate dimethyl sulfoxide deoxycholate disuccinimidyl suberate dithiobis-succinimidyl propionate dithiothreitol lauryl dimethyl amine oxide m-maleimidobenzoyl-N-hydroxysuccinimide ester phosphate-buffered saline glycosylphosphatidylinositol-specific phospholipase C Trypanosoma brucei GPI-PLC binding protein endoplasmic reticulum polyacrylamide gel electrophoresis mannose glucosamine phosphatidylinositol Trypanosoma brucei is the causative agent of sleeping sickness in humans. In the bloodstream of a vertebrate host, the plasma membrane of the parasite is covered with a variant surface glycoprotein (VSG),1 a glycosylphosphatidylinositol (GPI)-anchored molecule. These trypanosomes contain a GPI-specific phospholipase C (1.Hereld D. Krakow J.L. Bangs J.D. Hart G.W. Englund P.T. J. Biol. Chem. 1986; 261: 13813-13819Abstract Full Text PDF PubMed Google Scholar, 2.Fox J.A. Duszenko M. Ferguson M.A. Low M.G. Cross G.A.M. J. Biol. Chem. 1986; 261: 15767-15771Abstract Full Text PDF PubMed Google Scholar, 3.Bülow R. Overath P. J. Biol. Chem. 1986; 261: 11918-11923Abstract Full Text PDF PubMed Google Scholar) that can cleave the VSG GPI, although the physiological function of the enzyme remains unclear (4.Webb H. Carnall N. Vanhamme L. Rolin S. Van den Abbeele J. Welburn S. Pays E. Carrington M. J. Cell Biol. 1997; 139: 103-114Crossref PubMed Scopus (85) Google Scholar, 5.Ochatt C.M. Butikofer P. Navarro M. Wirtz E. Boschung M. Armah D. Cross G.A. Mol. Biochem. Parasitol. 1999; 103: 35-48Crossref PubMed Scopus (18) Google Scholar). GPI-PLC is highly specific for GPIs (k cat, 2.92 × 103min−1; K m, 360 nm) (1.Hereld D. Krakow J.L. Bangs J.D. Hart G.W. Englund P.T. J. Biol. Chem. 1986; 261: 13813-13819Abstract Full Text PDF PubMed Google Scholar, 6.Mensa-Wilmot K. Morris J.C. Al-Qahtani A. Englund P.T. Methods Enzymol. 1995; 250: 641-655Crossref PubMed Scopus (39) Google Scholar). Turnover of the enzyme-substrate complex is regulated by thio-myristoylation and palmitoylation of the enzyme (7.Armah D.A. Mensa-Wilmot K. J. Biol. Chem. 1999; 274: 5931-5939Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Efficient substrate recognition by GPI-PLC requires a glucosaminylinositol moiety on the substrate (8.Morris J.C. Ping-Sheng L. Shen T.Y. Mensa-Wilmot K. J. Biol. Chem. 1995; 270: 2517-2524Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). GPI-PLC activity is detectable only in developmental stages of the parasite where VSG is present. As revealed by immunoelectron microscopic analysis, GPI-PLC is associated with vesicular structures on the cytoplasmic side of intracellular membranes (9.Bülow R. Griffiths G. Webster P. Stierhof Y.-D. Opperdoes F.R. Overath P. J. Cell Sci. 1989; 93: 233-240Crossref PubMed Google Scholar, 10.Grab D.J. Webster P. Ito S. Fish W.R. Verjee Y. Lonsdale-Eccles J.D. J. Cell Biol. 1987; 105: 737-746Crossref PubMed Scopus (41) Google Scholar). Little or no cleaved VSG is released from healthy cells (11.Black S.J. Hewett R.S. Sendashonga C.N. Parasite Immunol. (Oxf.). 1982; 4: 233-244Crossref PubMed Scopus (49) Google Scholar). While T. brucei divides every 6–8 h, the half-life of cell-associated VSG is 32–34 h (11.Black S.J. Hewett R.S. Sendashonga C.N. Parasite Immunol. (Oxf.). 1982; 4: 233-244Crossref PubMed Scopus (49) Google Scholar, 12.Bülow R. Nonnengasser C. Overath P. Mol. Biochem. Parasitol. 1989; 32: 85-92Crossref PubMed Scopus (79) Google Scholar, 13.Seyfang A. Mecke D. Duszenko M. J. Protozool. 1990; 37: 546-552Crossref PubMed Scopus (89) Google Scholar). Clearly, VSG is not released actively by GPI-PLC. This situation is in sharp contrast to the phenotype ofLeishmania major or Trypanosoma cruzi that have been stably transfected with a cDNA for the T. bruceiGPI-PLC (14.Mensa-Wilmot K. Garg N. McGwire B.S. Lu H.G. Zhong L. Armah D.A. LeBowitz J.H. Chang K.P. Mol. Biochem. Parasitol. 1999; 99: 103-116Crossref PubMed Scopus (30) Google Scholar, 15.Mensa-Wilmot K. LeBowitz J.H. Chang K.P. Al-Qahtani A. McGwire B.S. Tucker S. Morris J.C. J. Cell Biol. 1994; 124: 935-947Crossref PubMed Scopus (56) Google Scholar). In Leishmania promastigotes (extracellular insect-stage form), gp63, the major GPI-anchored protein, is secreted constitutively into the culture medium due to a GPI deficiency (15.Mensa-Wilmot K. LeBowitz J.H. Chang K.P. Al-Qahtani A. McGwire B.S. Tucker S. Morris J.C. J. Cell Biol. 1994; 124: 935-947Crossref PubMed Scopus (56) Google Scholar). Replication of Leishmania amastigotes (the intracellular mammalian stage) is severely inhibited by GPI-PLC expression (14.Mensa-Wilmot K. Garg N. McGwire B.S. Lu H.G. Zhong L. Armah D.A. LeBowitz J.H. Chang K.P. Mol. Biochem. Parasitol. 1999; 99: 103-116Crossref PubMed Scopus (30) Google Scholar). A similar phenotype has been noted in T. cruzi where division of the cell nucleus is blocked as a result of a GPI deficiency (16.Garg N. Tarleton R.L. Mensa-Wilmot K. J. Biol. Chem. 1997; 272: 12482-12491Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar), and differentiation of amastigotes to trypomastigotes is inhibited (17.Garg N. Postan M. Mensa-Wilmot K. Tarleton R.L. Infect. Immun. 1997; 65: 4055-4060Crossref PubMed Google Scholar). GPI intermediates (e.g. GlcN-PI and Man1–3GlcN-PI) are found on the cytoplasmic side of the ER membrane (18.Vidugiriene J. Menon A.K. J. Cell Biol. 1994; 127: 333-341Crossref PubMed Scopus (97) Google Scholar). These compounds appear to co-localize with GPI-PLC, given the data from the in vivo transfection studies withLeishmania and T. cruzi. Why then in T. brucei are these GPIs spared from cleavage by this potent membrane-bound phospholipase C? It is estimated that there are 2.4 × 104 molecules of glycolipid A (ethanolamine-phospho-Man3-GlcN-Ins-phospho-dimyrisotylglycerol), the prefabricated GPI anchor (19.Doering T.L. Pessin M.S. Hoff E.F. Hart G.W. Raben D.M. Englund P.T. J. Biol. Chem. 1993; 268: 9215-9222Abstract Full Text PDF PubMed Google Scholar), and 3.5 × 104molecules of GPI-PLC (3.Bülow R. Overath P. J. Biol. Chem. 1986; 261: 11918-11923Abstract Full Text PDF PubMed Google Scholar); this represents an excess of the enzyme over the complete GPI anchor. With a turnover number (k cat) of 2920 min−1(6.Mensa-Wilmot K. Morris J.C. Al-Qahtani A. Englund P.T. Methods Enzymol. 1995; 250: 641-655Crossref PubMed Scopus (39) Google Scholar), GPI-PLC could cleave all the glycolipid A inside T. brucei within seconds. Since the parasites remain capable of adding GPIs to VSG, it seems clear that GPI-PLC is somehow prevented from depleting T. brucei of GPI anchors. The absence of the GPI-negative phenotype observed with transgenic T. cruzi andL. major expressing GPI-PLC is indirect evidence for regulation of the enzyme in T. brucei. In lieu of efforts to understand factors that might control activity of GPI-PLC in T. brucei, we characterized the native state of the purified enzyme, and we explored possible contributions of self-association to enzyme activity both in vivo andin vitro. Our observations led us to propose a model for post-translational regulation of GPI-PLC activity in vivowhere prevention of tetramerization might play an important role. Monomorphic T. brucei strain 427 bloodstream form was used in this work. Parasites were grown in rodents and harvested by chromatography on DE52 (20.Cross G.A.M. Parasitology. 1975; 71: 393-417Crossref PubMed Scopus (641) Google Scholar). Superdex 75 HR10/30 column, Superdex 200 HR10/30 column, the fast protein liquid chromatography system, and [35S]methionine were from Amersham Pharmacia Biotech. Lauryl dimethyl amine oxide (LDAO), Nonidet P-40 (protein grade), and transferrin were purchased from Calbiochem. Thesit and aprotinin were obtained from Roche Molecular Biochemicals. GelCode Blue, disuccinimidyl suberate (DSS), dithiobissuccinimidyl propionate (DSP), and m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) were purchased from Pierce. Gel filtration standards were purchased from Bio-Rad. Sodium deoxycholate (DOC), CHAPS, and all other reagents were from Sigma. Purified recombinant GPI-PLC expressed in Escherichia coli (1–2 μg) was preincubated in 20–30 μl of 37.5 mm Tris-HCl, pH 9.3, 0.5% Nonidet P-40, containing one of these reagents as follows: 3–4 m urea, 100 mmdithiothreitol (DTT), 1% Nonidet P-40, or 0.1–2% CHAPS at 27 °C for 5 min. Samples were resolved by continuous glycine non-denaturing polyacrylamide gel electrophoresis (pH 9.5; 5% minigel (Bio-Rad)) (21.Best S. Speicher D.W. Coligan J.E. Dunn B.M. Ploegh H.L. Speicher D.W. Wingfield P.T. Current Protocol in Protein Science. John Wiley & Sons, Inc., New York1995: 10.0.1-10.3.11Google Scholar). To keep track of protein migration, 2 μl of 1% bromphenol blue was added to each sample prior to loading. Gels were run at 16 A for either 30 min or 4–5 h at room temperature. Proteins were visualized with GelCode Blue (Pierce). Standards used were α-lactalbumin (14.2 kDa, pI = 4–5), carbonic anhydrase (29 kDa, pI = 5.4–5.9), ovalbumin (43 kDa, pI = 4.6), bovine serum albumin (66-kDa monomer and 132-kDa dimer, pI = 4.7), and urease (272-kDa trimer and 545-kDa hexamer, pI = 5.0) (Sigma). Seven micrograms of marker proteins were analyzed. Molecular sieving was performed by fast protein liquid chromatography on either Superdex 75 HR10/30 at 5 °C or Superdex 200 HR10/30 at 27 °C. For all runs, a 100-μl sample was loaded, and 500-μl fractions were collected at a flow rate of 1 ml/min. The void volume of the columns was determined with blue dextran, using PBS (140 mm NaCl, 3 mm KCl, 10 mmNa2HPO4, 1.8 mmKH2PO4; pH 7.4) as running buffer. Prior to loading each sample, the columns were equilibrated with 3 column volumes of PBS containing the indicated amounts of detergent (see figure legends). For each running buffer, the columns were calibrated with thyroglobulin (670 kDa), IgG (160 kDa), transferrin (80 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), ribonuclease A (13.7 kDa), and aprotinin (6.5 kDa). Protein peaks were monitored byA 280 absorption. Purified GPI-PLC (620 ng) in 5 μl of 75 mm Tris-HCl, pH 9.3, containing 1% Nonidet P-40 (6.Mensa-Wilmot K. Morris J.C. Al-Qahtani A. Englund P.T. Methods Enzymol. 1995; 250: 641-655Crossref PubMed Scopus (39) Google Scholar), was added to 95 μl of the indicated running buffer (see figure legends) and incubated at 27 °C for 5 min before loading onto the specified column. The elution profile of GPI-PLC was determined by assaying 5 μl of each fraction for enzyme activity (22.Mensa-Wilmot K. Englund P.T. Mol. Biochem. Parasitol. 1992; 56: 311-322Crossref PubMed Scopus (26) Google Scholar). T. brucei (5 × 108parasites) was lysed hypotonically in 1 ml of hypotonic lysis buffer (10 mm sodium phosphate, 1 mm EDTA, pH 8) containing a protease inhibitor mixture and centrifuged at 14,000 × g at 4 °C for 10 min (7.Armah D.A. Mensa-Wilmot K. J. Biol. Chem. 1999; 274: 5931-5939Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). The membranous pellet obtained was solubilized with 500 μl of PBS containing either 1% Nonidet P-40 or 1% CHAPS. An aliquot of each sample (107cell equivalents for Nonidet P-40 and 108 cell equivalents for CHAPS) was fractionated on the indicated gel filtration column. Two μg of SDS-depleted [3H]myristate-labeled membrane-form variant surface glycoprotein was used as substrate in buffer AB (50 mm Tris-HCl, pH 8.0, 5 mm EDTA, 1% Nonidet P-40). Reaction mixtures were incubated at 37 °C for 15 min, unless otherwise indicated. Cleaved [3H]dimyristoylglycerol was quantitated by liquid scintillation counting (6.Mensa-Wilmot K. Morris J.C. Al-Qahtani A. Englund P.T. Methods Enzymol. 1995; 250: 641-655Crossref PubMed Scopus (39) Google Scholar). Detergent is needed for solubilization of GPI-PLC under all conditions because it is an integral membrane protein. Replacement of Nonidet P-40 in the purified recombinant protein was therefore achieved by serial dilution into the detergent chosen to replace Nonidet P-40. Purified enzyme (6.2 ng) in 5 μl of 75 mm Tris-HCl, pH 9.3, containing 1% Nonidet P-40 (6.Mensa-Wilmot K. Morris J.C. Al-Qahtani A. Englund P.T. Methods Enzymol. 1995; 250: 641-655Crossref PubMed Scopus (39) Google Scholar), was diluted 10-fold into buffer AB lacking Nonidet P-40. A 5-μl aliquot of the diluted enzyme (620 pg) was diluted further into a new detergent by adding 45 μl of buffer AB containing 1% of the detergent. To measure enzyme activity, 5 μl of each sample was assayed in 25 μl of the corresponding buffer AB, in which the detergent being tested had replaced Nonidet P-40. (Residual Nonidet P-40 is now 0.002%.) Enzyme reaction mixtures were incubated for 15 min at either 5 or 27 °C. Purified GPI-PLC (124 ng/μl in 75 mm Tris-HCl, pH 9.3, 1% Nonidet P-40) was diluted 10-fold into PBS to reduce the concentration of Nonidet P-40 and Tris. A 10-μl aliquot (124 ng) of the diluted enzyme was then placed in a microcentrifuge tube containing 11.6 μl of deionized water and 3 μl each of 10× PBS and 10% of the specified detergent. These actions resulted in a 30-fold dilution of the original enzyme sample. After a 5-min preincubation at 2, 15, or 27 °C, cross-linking was initiated by addition of 2.4 μl of 3.125 mm disuccinimidyl suberate (DSS) in dimethyl sulfoxide (Me2SO), and the reactions were incubated under one of these conditions as follows: 2 °C for 40 min, 15 °C for 40 min, or 27 °C for 10 min. The final concentration of DSS was 250 μm. In control experiments, 2.4 μl of Me2SO was added. To quench the reactions, 2.5 μl of 1m Tris-HCl, pH 7.5, was introduced, and after 5 min incubation on ice, 10 μl of 5× SDS-PAGE sample buffer added. Samples were heated at 90 °C for 2–3 min. Proteins were resolved by SDS-PAGE (12% minigel; Bio-Rad), and GPI-PLC was detected by Western blotting (7.Armah D.A. Mensa-Wilmot K. J. Biol. Chem. 1999; 274: 5931-5939Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). T. brucei(2 × 108 cells) was metabolically labeled with [35S]methionine (7.Armah D.A. Mensa-Wilmot K. J. Biol. Chem. 1999; 274: 5931-5939Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) and washed with PBS. Parasites were resuspended in 396 μl of PBS. To initiate cross-linking, 4 μl of 100 mm DSS, DSP, or MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester), all membrane-permeable reagents, was added. In control experiments, 4 μl of Me2SO was introduced. Parasites were incubated at 27 °C for 30 min, after which reactions were quenched by the addition of 100 μl of 1 m Tris-HCl, pH 7.5. Samples were incubated on ice for 5 min followed by centrifugation at 14,000 ×g for 10 min at 4 °C. The pelleted cells were lysed 1ml of in 10 mm sodium phosphate, 1 mm EDTA, pH 8. A membranous pellet was recovered by centrifugation at 14,000 ×g, 10 min at 4 °C, and solubilized in 1 ml of immunoprecipitation dilution buffer ((1% v/v) Triton X-100, 200 mm NaCl, 60 mm Tris-HCl, pH 7.5, 6 mm EDTA, 10 units/ml aprotinin). [35S]Methionine-labeled GPI-PLC was immunoadsorbed to anti-GPI-PLC monoclonal antibody (mc2A6-6) (7.Armah D.A. Mensa-Wilmot K. J. Biol. Chem. 1999; 274: 5931-5939Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Radiolabeled proteins were visualized by phosphorimaging (Personal Molecular Imager FX (Bio-Rad)). Western blotting with anti-BiP antibody (23.Bangs J.D. Uyetake L. Brickman M.J. Balber A.E. Boothroyd J.C. J. Cell Sci. 1993; 105: 1101-1113Crossref PubMed Google Scholar) was performed as described (7.Armah D.A. Mensa-Wilmot K. J. Biol. Chem. 1999; 274: 5931-5939Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) with 10 μl of the detergent-solubilized extract. Leishmania(2.5 × 108 promastigotes) expressing T. brucei GPI-PLC (15.Mensa-Wilmot K. LeBowitz J.H. Chang K.P. Al-Qahtani A. McGwire B.S. Tucker S. Morris J.C. J. Cell Biol. 1994; 124: 935-947Crossref PubMed Scopus (56) Google Scholar) was metabolically labeled with [35S]methionine (15.Mensa-Wilmot K. LeBowitz J.H. Chang K.P. Al-Qahtani A. McGwire B.S. Tucker S. Morris J.C. J. Cell Biol. 1994; 124: 935-947Crossref PubMed Scopus (56) Google Scholar) and washed with PBS. Parasites were resuspended in 990 μl of PBS, pH 7.4. Cross-linking was initiated by the addition of 10 μl of 100 mm MBS. Ten μl of Me2SO was introduced in control experiments. Parasites were incubated at 27 °C for 30 min. Reactions were quenched and analyzed as described under “In Vivo Cross-linking of T. brucei.” T. brucei was metabolically labeled with [35S]methionine, and parasites (2.5 × 108) were lysed hypotonically (7.Armah D.A. Mensa-Wilmot K. J. Biol. Chem. 1999; 274: 5931-5939Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). A membranous pellet obtained by centrifugation (14,000 × g, 10 min, 4 °C) was resuspended in 450 μl of PBS. Cross-linking was initiated by the addition of 50 μl of 2.5 mm DSS, or DSP, followed by incubation at either 27 °C for 5 min or 2 °C for 40 min. In control experiments, 50 μl of Me2SO was added. Reactions were quenched by the addition of 100 μl of 1 mTris-HCl, pH 7.5, followed by incubation on ice for 5 min. Samples were centrifuged at 14,000 × g for 10 min at 4 °C. The pellet was solubilized and analyzed as described under “In Vivo Cross-linking of T. brucei.” Immunoadsorption of GPI-PLC to a monoclonal antibody was performed as described previously (7.Armah D.A. Mensa-Wilmot K. J. Biol. Chem. 1999; 274: 5931-5939Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). A 40-μl aliquot of the cross-linked reaction mixture containing recombinant GPI-PLC (∼120 ng) was analyzed by Western blotting (7.Armah D.A. Mensa-Wilmot K. J. Biol. Chem. 1999; 274: 5931-5939Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). [35S]Methionine-labeled GPI-PLC, from intact cells or lysates, was immunoadsorbed to protein A-Sepharose-mc2A6-6 beads (6.Mensa-Wilmot K. Morris J.C. Al-Qahtani A. Englund P.T. Methods Enzymol. 1995; 250: 641-655Crossref PubMed Scopus (39) Google Scholar) and eluted by heating at 90 °C for 2–3 min in 25 μl of 2.5× SDS-PAGE sample buffer. When preservation of DSP cross-links was required, a modified 2.5× SDS-PAGE sample buffer lacking Tris and β-mercaptoethanol was used. Such samples were only warmed at 37 °C for 5 min. Eluates were separated from the Sepharose beads by centrifugation at 14,000 × g for 3 min at 27 °C. A 30-μl aliquot of each supernatant was subjected to SDS-PAGE (12% minigel) and processed for phosphorimaging or fluorographic detection with BioMaxTM MR film (Eastman Kodak Co.). GPI-PLC is a 39-kDa polypeptide (24.Hereld D. Hart G.W. Englund P.T. Proc. Natl. Acd. Sci. U. S. A. 1988; 85: 8914-8918Crossref PubMed Scopus (49) Google Scholar). In native gel electrophoresis, it was detected as a diffuse band that barely entered a 5% minigel (without DTT or SDS) (Fig.1 A, lane 6). Absence of SDS from the sample and running buffers may explain the diffuse nature of protein bands in this experiment. Urea (up to 4 m) was added in an attempt to disrupt hydrogen bonds and dissociate what appeared to be a complex of unusual shape and/or size. The mobility of GPI-PLC did not change (Fig. 1 A, lane 7; Fig. 1 B, lane 3). Likewise, pretreatment with DTT (100 mm) to reduce disulfide bonds failed to alter mobility of GPI-PLC in the native gel (Fig. 1 B, lane 2). When the enzyme was treated with both DTT (100 mm) and urea (4 m), a minor change occurred in its migration (Fig. 1 B, lane 4). Treatment with 1% SDS caused GPI-PLC to run off the gel (data not presented), most likely due to denaturation and net negative charge introduced by the detergent. CHAPS caused a significant change in the mobility of GPI-PLC, resolving it into two species (Fig. 1 B, lane 5). A titration of the detergent revealed that 0.5% was sufficient for optimal migration of GPI-PLC (Fig. 1 C, lanes 2–5). The major species of GPI-PLC detected during electrophoresis in CHAPS is labeled F (Fig.1 C). Two conclusions can be drawn from these observations. First, the size, shape, or net charge of GPI-PLC can be modulated by CHAPS. (The detergent has no net charge (Fig. 8).) Second, the quaternary structure of GPI-PLC is not dependent on disulfide bonds. One hypothesis to explain the altered mobility of GPI-PLC in the native gel after addition of CHAPS was that the detergent changed the quaternary structure of the enzyme. To examine this proposal, GPI-PLC was analyzed by gel filtration in a buffer containing different detergents, some with structures similar to CHAPS. A Superdex 75 HR10/30 column employed for this purpose had a void volume of 9 ml (fraction 18) and a bed volume of 24 ml (fraction 48). The experiment was performed at 5 °C. Proteins of 100 kDa and higher were excluded from the column. In 0.1% Nonidet P-40, the peak of GPI-PLC activity was in fraction 22, close to the elution position of an 80-kDa standard (Fig.2 A). The migration of enzyme activity is consistent with that of a dimer (78 kDa). Twelve percent of the enzyme activity was detected in the void volume (Fig.2 A), suggesting one of two possibilities. First, GPI-PLC forms complexes (e.g. trimers) larger than 100 kDa. Alternatively, GPI-PLC may be associated with micelles of the detergent that have a molecular mass of 90 kDa such that a monomer-micelle complex will have a mass of 130 kDa, causing it to be excluded from the column. In 1% Nonidet P-40 GPI-PLC was predominantly dimeric (data not shown). To determine whether the hydrophilic head group or hydrophobic tail of Nonidet P-40 contributed to dimerization of GPI-PLC, Thesit (65–68 kDa) whose hydrophilic head is identical to that of Nonidet P-40 was tested. In 1% Thesit, most of GPI-PLC was a dimer and 29% of the enzyme eluted in the void volume (Fig. 2 A). More importantly, detection of dimers suggest that the polymeric oxyethylene ((CH2-CH2-O)9–10) head (present in both Nonidet P-40 and Thesit) may be sufficient to cause oligomerization of purified GPI-PLC. In CHAPS, the majority of the enzyme migrated with a molecular mass greater than the 39-kDa monomer but less than the 80-kDa dimer (Fig.2 B). This finding is consistent with the assignment of a 47-kDa molecular size to the enzyme, after adsorption to a 7-kDa CHAPS micelle (1.Hereld D. Krakow J.L. Bangs J.D. Hart G.W. Englund P.T. J. Biol. Chem. 1986; 261: 13813-13819Abstract Full Text PDF PubMed Google Scholar). Only 23% of the total activity migrated as a dimer (Fig.2 B). About 13% of active enzyme eluted in fraction 19 (Fig.2 B), suggesting the presence of larger oligomers. Analysis of the enzyme in 2% LDAO produced similar results. The majority of GPI-PLC behaved as monomer, with about 23% dimers and 11% of oligomers (data not presented). To test the effects of temperature on interactions between GPI-PLC monomers, gel filtration was performed at 27 °C. Initial observations indicated that the peak of enzyme activity was outside the optimal separation range of Superdex 75 HR10/30 column (i.e.larger than 80 kDa) (data not presented). Therefore Superdex 200 HR10/30 with an optimal separation range up to 600 kDa was used. In 0.1% Nonidet P-40, the peak of enzyme activity was found in fraction 22, indicating that GPI-PLC was a tetramer (Fig. 2 C). About 17% of the activity migrated as a larger oligomer in fraction 19 (Fig.2 C). In contrast, CHAPS (2%) converted GPI-PLC into monomers and dimers predominantly (63% of activity) (Fig.2 D). Tetramers comprised 26% of the activity (Fig.2 D). Taken together, these observations indicate that (i) GPI-PLC can oligomerize, and (ii) temperature as well as detergents can influence the distribution between monomers and oligomers of the enzyme. At 5 °C, CHAPS and LDAO promote formation and/or stabilization of monomers, whereas Nonidet P-40 and Thesit enable detection of dimers. At 27 °C, GPI-PLC is a tetramer in Nonidet P-40 but exists primarily as dimers and monomers in CHAPS. Finally, from a comparison of the gel filtration data with that obtained from the native gel analysis, it seems likely that the slow species detected during native gel electrophoresis in Nonidet P-40 (S, Fig. 1 C) represents the tetramer of GPI-PLC. The faster-moving species in CHAPS (F, Fig. 1 C) may correspond to dimers and monomers. Capitalizing on the ability of detergents and temperature to modulate self-association of GPI-PLC, we investigated whether different oligomers of the enzyme possessed varying activity. When an enzyme assay was performed in Nonidet P-40 at 5 °C where GPI-PLC is predominantly dimeric (Fig.2 A), the activity was 1.7-fold higher than the monomer (Fig.3) found in CHAPS (Fig. 2 B). This implies that dimeric GPI-PLC may be slightly more active than the monomer. To compare the activity of dimeric GPI-PLC with that of the tetramer, enzyme was assayed in Nonidet P-40 at 27 °C (Fig. 2 C) and compared with its activity in CHAPS at 27 °C (Fig. 2 D). Tetrameric GPI-PLC was 3.6-fold more active than dimers and monomers (Fig. 3). An attempt to isolate tetramers generated by cross-linking for the purposes of activity determination was not feasible, since DSS, DSP, and MBS were all found to inhibit the activity of the enzyme (data not presented). Replacement of Nonidet P-40 with Thesit or LDAO reduced GPI-PLC activity 2–4-fold at 5 °C (Fig. 3). Deoxycholate (1%) completely inhibited GPI-PLC activity at both 5 and 27 °C (Fig. 3). When the assay was performed at 37 °C in 1% of detergent, GPI-PLC was 20-fold more active in Nonidet P-40 than in CHAPS, Thesit, LDAO, or DOC (Fig. 3). These observations indicate that GPI-PLC activity is increased under conditions where tetramer formation or stabilization is enhanced. Additional evidence for self-association of GPI-PLC was obtained by chemical cross-linking. Purified GPI-PLC migrates normally as a 39-kDa protein after SDS-polyacrylamide gel electrophoresis (Fig.4 A, lane 1). Following cross-linking with DSS, the major product formed was a doublet, most likely tetramers and (possibly) pentamers (Fig. 4 A, lanes 2–6). Interestingly, denaturation of GPI-PLC with SDS inhibits cross-linking by DSS, leaving the monomer as the predominant species (Fig. 4 A, lanes 7 and 8). Aberrant (possibly intramolecular) cross-linking might also have occurred in the presence of SDS, since a ladder of bands with apparent molecular masses of 80 kDa and larger is visible (Fig. 4 A, lanes 7 and8). To test whether intermediates between monomers and tetramers could be detected, cross-linking was performed (i) at lower temperatures, or (ii) in the presence of Tris (16.7 mm). At 2 °C, dimers of GPI-PLC were detected in both Nonidet P-40 and CHAPS (Fig. 4 B, lanes 2 and 3). In addition, a small proportion of trimers was detected. The species of GPI-PLC marked with anasterisk in Fig. 4 B (lanes 2 and3) could arise from intramolecular monomer cross-linking tha" @default.
• W2078542822 created "2016-06-24" @default.
• W2078542822 creator A5000859439 @default.
• W2078542822 creator A5009417240 @default.
• W2078542822 date "2000-06-01" @default.
• W2078542822 modified "2023-09-30" @default.
• W2078542822 title "Tetramerization of Glycosylphosphatidylinositol-specific Phospholipase C from Trypanosoma brucei" @default.
• W2078542822 cites W1484768876 @default.
• W2078542822 cites W1520718345 @default.
• W2078542822 cites W1534548156 @default.
• W2078542822 cites W1584902893 @default.
• W2078542822 cites W1608333308 @default.
• W2078542822 cites W1939117002 @default.
• W2078542822 cites W1978899653 @default.
• W2078542822 cites W1999805827 @default.
• W2078542822 cites W1999989347 @default.
• W2078542822 cites W2009410139 @default.
• W2078542822 cites W2014621836 @default.
• W2078542822 cites W2020097304 @default.
• W2078542822 cites W2022195743 @default.
• W2078542822 cites W2040708692 @default.
• W2078542822 cites W2047630725 @default.
• W2078542822 cites W2066940178 @default.
• W2078542822 cites W2075843997 @default.
• W2078542822 cites W2077860019 @default.
• W2078542822 cites W2079798453 @default.
• W2078542822 cites W2103391080 @default.
• W2078542822 cites W2129179491 @default.
• W2078542822 cites W2143381397 @default.
• W2078542822 cites W2168228094 @default.
• W2078542822 cites W2169317674 @default.
• W2078542822 doi "https://doi.org/10.1074/jbc.m001798200" @default.
• W2078542822 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10764777" @default.
• W2078542822 hasPublicationYear "2000" @default.
• W2078542822 type Work @default.
• W2078542822 sameAs 2078542822 @default.
• W2078542822 citedByCount "9" @default.
• W2078542822 countsByYear W20785428222022 @default.
• W2078542822 crossrefType "journal-article" @default.
• W2078542822 hasAuthorship W2078542822A5000859439 @default.
• W2078542822 hasAuthorship W2078542822A5009417240 @default.
• W2078542822 hasBestOaLocation W20785428221 @default.
• W2078542822 hasConcept C104317684 @default.
• W2078542822 hasConcept C12927208 @default.
• W2078542822 hasConcept C181199279 @default.
• W2078542822 hasConcept C185592680 @default.
• W2078542822 hasConcept C2778566189 @default.
• W2078542822 hasConcept C2778597717 @default.
• W2078542822 hasConcept C2779502636 @default.
• W2078542822 hasConcept C55493867 @default.
• W2078542822 hasConcept C86803240 @default.
• W2078542822 hasConcept C89423630 @default.
• W2078542822 hasConcept C95444343 @default.
• W2078542822 hasConceptScore W2078542822C104317684 @default.
• W2078542822 hasConceptScore W2078542822C12927208 @default.
• W2078542822 hasConceptScore W2078542822C181199279 @default.
• W2078542822 hasConceptScore W2078542822C185592680 @default.
• W2078542822 hasConceptScore W2078542822C2778566189 @default.
• W2078542822 hasConceptScore W2078542822C2778597717 @default.
• W2078542822 hasConceptScore W2078542822C2779502636 @default.
• W2078542822 hasConceptScore W2078542822C55493867 @default.
• W2078542822 hasConceptScore W2078542822C86803240 @default.
• W2078542822 hasConceptScore W2078542822C89423630 @default.
• W2078542822 hasConceptScore W2078542822C95444343 @default.
• W2078542822 hasIssue "25" @default.
• W2078542822 hasLocation W20785428221 @default.
• W2078542822 hasOpenAccess W2078542822 @default.
• W2078542822 hasPrimaryLocation W20785428221 @default.
• W2078542822 hasRelatedWork W1762755856 @default.
• W2078542822 hasRelatedWork W1975941863 @default.
• W2078542822 hasRelatedWork W2005859080 @default.
• W2078542822 hasRelatedWork W2011609455 @default.
• W2078542822 hasRelatedWork W2031410409 @default.
• W2078542822 hasRelatedWork W2055617588 @default.
• W2078542822 hasRelatedWork W2068224016 @default.
• W2078542822 hasRelatedWork W2169317674 @default.
• W2078542822 hasRelatedWork W2952275338 @default.
• W2078542822 hasRelatedWork W4322742496 @default.
• W2078542822 hasVolume "275" @default.
• W2078542822 isParatext "false" @default.
• W2078542822 isRetracted "false" @default.
• W2078542822 magId "2078542822" @default.
• W2078542822 workType "article" @default.