FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2000392436> ?p ?o ?g. }

• W2000392436 endingPage "16107" @default.
• W2000392436 startingPage "16099" @default.
• W2000392436 abstract "The enzymatic activity of the peripheral membrane protein, phosphatidylinositol-specific phospholipase C (PI-PLC), is increased by nonsubstrate phospholipids with the extent of enhancement tuned by the membrane lipid composition. For Bacillus thuringiensis PI-PLC, a small amount of phosphatidylcholine (PC) activates the enzyme toward its substrate PI; above 0.5 mol fraction PC (XPC), enzyme activity decreases substantially. To provide a molecular basis for this PC-dependent behavior, we used fluorescence correlation spectroscopy to explore enzyme binding to multicomponent lipid vesicles composed of PC and anionic phospholipids (that bind to the active site as substrate analogues) and high resolution field cycling 31P NMR methods to estimate internal correlation times (τc) of phospholipid headgroup motions. PI-PLC binds poorly to pure anionic phospholipid vesicles, but 0.1 XPC significantly enhances binding, increases PI-PLC activity, and slows nanosecond rotational/wobbling motions of both phospholipid headgroups, as indicated by increased τc. PI-PLC activity and phospholipid τc are constant between 0.1 and 0.5 XPC. Above this PC content, PI-PLC has little additional effect on the substrate analogue but further slows the PC τc, a motional change that correlates with the onset of reduced enzyme activity. For PC-rich bilayers, these changes, together with the reduced order parameter and enhanced lateral diffusion of the substrate analogue in the presence of PI-PLC, imply that at high XPC, kinetic inhibition of PI-PLC results from intravesicle sequestration of the enzyme from the bulk of the substrate. Both methodologies provide a detailed view of protein-lipid interactions and can be readily adapted for other peripheral membrane proteins. The enzymatic activity of the peripheral membrane protein, phosphatidylinositol-specific phospholipase C (PI-PLC), is increased by nonsubstrate phospholipids with the extent of enhancement tuned by the membrane lipid composition. For Bacillus thuringiensis PI-PLC, a small amount of phosphatidylcholine (PC) activates the enzyme toward its substrate PI; above 0.5 mol fraction PC (XPC), enzyme activity decreases substantially. To provide a molecular basis for this PC-dependent behavior, we used fluorescence correlation spectroscopy to explore enzyme binding to multicomponent lipid vesicles composed of PC and anionic phospholipids (that bind to the active site as substrate analogues) and high resolution field cycling 31P NMR methods to estimate internal correlation times (τc) of phospholipid headgroup motions. PI-PLC binds poorly to pure anionic phospholipid vesicles, but 0.1 XPC significantly enhances binding, increases PI-PLC activity, and slows nanosecond rotational/wobbling motions of both phospholipid headgroups, as indicated by increased τc. PI-PLC activity and phospholipid τc are constant between 0.1 and 0.5 XPC. Above this PC content, PI-PLC has little additional effect on the substrate analogue but further slows the PC τc, a motional change that correlates with the onset of reduced enzyme activity. For PC-rich bilayers, these changes, together with the reduced order parameter and enhanced lateral diffusion of the substrate analogue in the presence of PI-PLC, imply that at high XPC, kinetic inhibition of PI-PLC results from intravesicle sequestration of the enzyme from the bulk of the substrate. Both methodologies provide a detailed view of protein-lipid interactions and can be readily adapted for other peripheral membrane proteins. Bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) 2The abbreviations used are: PI-PLCphosphatidylinositol-specific phospholipase CPIphosphatidylinositolPLCphospholipase CAF488Alexa Fluor 488 C5CSAchemical shift anisotropyDLSdynamic light scatteringfc-P-NMRhigh resolution field cycling 31P NMR spectroscopyFCSfluorescence correlation spectroscopyLUVlarge unilamellar vesiclesPAdioleoylphosphatidic acidPC1-palmitoyl-2-oleoylphosphatidylcholinePGdioleoylphosphatidylglycerolPMedioleoyl-phosphatidylmethanolPSdioleoylphosphatidylserineSUVsmall unilamellar vesicleTTeslaGPIglycosylphosphatidylinositol.2The abbreviations used are: PI-PLCphosphatidylinositol-specific phospholipase CPIphosphatidylinositolPLCphospholipase CAF488Alexa Fluor 488 C5CSAchemical shift anisotropyDLSdynamic light scatteringfc-P-NMRhigh resolution field cycling 31P NMR spectroscopyFCSfluorescence correlation spectroscopyLUVlarge unilamellar vesiclesPAdioleoylphosphatidic acidPC1-palmitoyl-2-oleoylphosphatidylcholinePGdioleoylphosphatidylglycerolPMedioleoyl-phosphatidylmethanolPSdioleoylphosphatidylserineSUVsmall unilamellar vesicleTTeslaGPIglycosylphosphatidylinositol. enzymes aid in organism infectivity (1Zenewicz L.A. Wei Z. Goldfine H. Shen H. J. Immunol. 2005; 174: 8011-8016Crossref PubMed Scopus (25) Google Scholar), whereas the structurally homologous mammalian PLCδ1 and related enzymes are required for phosphoinositide metabolism and are important for intracellular signaling (2Downes C.P. Gray A. Lucocq J.M. Trends Cell Biol. 2005; 15: 259-268Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 3Rhee S.G. Annu. Rev. Biochem. 2001; 70: 281-312Crossref PubMed Scopus (1219) Google Scholar). These peripheral membrane proteins often have distinct binding modes for substrates and for other lipids that either anchor the protein to the surface or adjust its conformation to enhance catalysis (4Mulgrew-Nesbitt A. Diraviyam K. Wang J. Singh S. Murray P. Li Z. Rogers L. Mirkovic N. Murray D. Biochim. Biophys. Acta. 2006; 1761: 812-826Crossref PubMed Scopus (157) Google Scholar). However, using traditional methods, it has been difficult to determine how lipid composition affects phospholipase binding and, conversely, how protein binding alters the lipid environment, particularly in multicomponent vesicles. Current methods for monitoring peripheral membrane protein binding to mixed component lipid vesicles, including centrifugation, gel filtration, and NMR, can provide information on bulk protein partitioning but do not usually provide insight into how the properties of the individual phospholipids change when protein is bound. Most of these methods also require protein concentrations well above the amounts used in enzyme kinetics. Therefore, even when binding to multicomponent vesicles can be measured, it has been difficult to separate how substrates (or substrate analogues) and activators interact with enzymes, such as PI-PLC, that catalyze interfacial reactions. phosphatidylinositol-specific phospholipase C phosphatidylinositol phospholipase C Alexa Fluor 488 C5 chemical shift anisotropy dynamic light scattering high resolution field cycling 31P NMR spectroscopy fluorescence correlation spectroscopy large unilamellar vesicles dioleoylphosphatidic acid 1-palmitoyl-2-oleoylphosphatidylcholine dioleoylphosphatidylglycerol dioleoyl-phosphatidylmethanol dioleoylphosphatidylserine small unilamellar vesicle Tesla glycosylphosphatidylinositol. phosphatidylinositol-specific phospholipase C phosphatidylinositol phospholipase C Alexa Fluor 488 C5 chemical shift anisotropy dynamic light scattering high resolution field cycling 31P NMR spectroscopy fluorescence correlation spectroscopy large unilamellar vesicles dioleoylphosphatidic acid 1-palmitoyl-2-oleoylphosphatidylcholine dioleoylphosphatidylglycerol dioleoyl-phosphatidylmethanol dioleoylphosphatidylserine small unilamellar vesicle Tesla glycosylphosphatidylinositol. Protein binding to lipid vesicles or cells greatly retards protein translational diffusion. For fluorescently labeled proteins, this change can easily be monitored using fluorescence correlation spectroscopy (FCS) (5Magde D. Elson E.L. Webb W.W. Biopolymers. 1974; 13: 29-61Crossref PubMed Scopus (1110) Google Scholar, 6Rhoades E. Ramlall T.F. Webb W.W. Eliezer D. Biophys. J. 2006; 90: 4692-4700Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 7Rusu L. Gambhir A. McLaughlin S. Rädler J. Biophys. J. 2004; 87: 1044-1053Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). FCS uses low protein concentrations (<1–50 nm), comparable with what is used in kinetic assays, and experiments can be performed in minutes. Thus, FCS allows us to screen different phospholipid compositions and to identify conditions that can be studied in detail by other techniques aimed at probing how protein binding alters the lipid environment. One such technique is high resolution field cycling 31P NMR spectroscopy (fc-P-NMR) (8Roberts M.F. Redfield A.G. J. Am. Chem. Soc. 2004; 126: 13765-13777Crossref PubMed Scopus (65) Google Scholar, 9Roberts M.F. Redfield A.G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17066-17071Crossref PubMed Scopus (41) Google Scholar). Bacterial PI-PLCs recognize the headgroups of substrate, substrate analogue, and activator phospholipids, making 31P NMR an obvious choice for studying protein-lipid interactions. Measurements of dipolar relaxation rates could, in principle, determine the effects of protein binding on headgroup orientation with respect to the bilayer normal as well as correlation times for different headgroup motions. However, in a typical modern spectrometer, the slow rotational diffusion time of lipid vesicles, the dominance of chemical shift anisotropy (CSA) relaxation, and a plethora of faster motions at higher fields preclude obtaining information on lipid structure and dynamics from relaxation rates. This limitation is avoided by fc-P-NMR spin lattice relaxation techniques that resolve the dynamics of each phospholipid component in mixed component vesicles by using the high field to separate resonances but allowing nuclei to relax at defined lower fields. To cycle the magnetic field, the sample is mechanically shuttled between the center high field region of the commercial NMR magnet's probe and a substantially lower magnetic field located above the probe before and after the delay times normally used in conventional NMR relaxation sequences (8Roberts M.F. Redfield A.G. J. Am. Chem. Soc. 2004; 126: 13765-13777Crossref PubMed Scopus (65) Google Scholar). Analysis of the field dependence of spin lattice relaxation rates over a wide field range (0.002–11.7 T) provides several correlation times (τ) for each phospholipid molecule on time scales ranging from ps to μs and also allows us to separate dipolar and CSA interactions (8Roberts M.F. Redfield A.G. J. Am. Chem. Soc. 2004; 126: 13765-13777Crossref PubMed Scopus (65) Google Scholar, 9Roberts M.F. Redfield A.G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17066-17071Crossref PubMed Scopus (41) Google Scholar). Of particular interest is the intermediate 5–10 ns τc that appears to be dominated by the wobbling of each phospholipid in the membrane (10Klauda J.B. Roberts M.F. Redfield A.G. Brooks B.R. Pastor R.W. Biophys. J. 2008; 94: 3074-3083Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). This τc provides a direct probe of lipid dynamics in these multicomponent membranes. A longer correlation time in the μs range, τv, provides information on how vesicle tumbling and lateral diffusion affect the phosphorus nuclei for each type of phospholipid in a mixed vesicle. We have used FCS and fc-P-NMR to probe the interactions of Bacillus thuringiensis PI-PLC with small unilamellar vesicles (SUVs). This enzyme and related, secreted bacterial PI-PLCs are important for bacterial virulence, and explicit measurements of their interactions with lipids could aid in inhibitor design. Bacterial PI-PLCs are also structurally homologous to the catalytic domain of mammalian PLCδ1 and thus provide a good model for assessing how interactions of this domain with membrane components could modulate enzyme activity. B. thuringiensis PI-PLC has a solvent-accessible active site inserted into a relatively rigid (αβ)-barrel (11Heinz D.W. Ryan M. Bullock T.L. Griffith O.H. EMBO J. 1995; 14: 3855-3863Crossref PubMed Scopus (147) Google Scholar). The enzyme exhibits higher specific activity toward small unilamellar vesicles (∼300 Å diameter) over larger vesicles with diameters of ≥1000 Å (12Qian X. Zhou C. Roberts M.F. Biochemistry. 1998; 37: 6513-6522Crossref PubMed Scopus (28) Google Scholar). For soluble substrates, the presence of phosphatidylcholine (PC) in micelles or vesicles activates the enzyme by decreasing the apparent Km and increasing kcat for both the phosphotransferase and phosphodiesterase steps (13Zhou C. Wu Y. Roberts M.F. Biochemistry. 1997; 36: 347-355Crossref PubMed Scopus (61) Google Scholar). PC activation of PI cleavage, the interfacial reaction, also occurs with vesicle assay systems (12Qian X. Zhou C. Roberts M.F. Biochemistry. 1998; 37: 6513-6522Crossref PubMed Scopus (28) Google Scholar). Our current model for kinetic activation of B. thuringiensis PI-PLC, based on the crystal structure of an interfacially impaired mutant protein (14Shao C. Shi X. Wehbi H. Zambonelli C. Head J.F. Seaton B.A. Roberts M.F. J. Biol. Chem. 2007; 282: 9228-9235Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) and extensive mutagenesis of surface residues (15Feng J. Wehbi H. Roberts M.F. J. Biol. Chem. 2002; 277: 19867-19875Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 16Feng J. Bradley W.D. Roberts M.F. J. Biol. Chem. 2003; 278: 24651-24657Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 17Guo S. Zhang X. Seaton B.A. Roberts M.F. Biochemistry. 2008; 47: 4201-4210Crossref PubMed Scopus (17) Google Scholar), is that specific binding to PC in the lipid membrane allows key loop residues to penetrate the interface, promoting an enzyme conformation (suggested to be a transient dimer (14Shao C. Shi X. Wehbi H. Zambonelli C. Head J.F. Seaton B.A. Roberts M.F. J. Biol. Chem. 2007; 282: 9228-9235Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 17Guo S. Zhang X. Seaton B.A. Roberts M.F. Biochemistry. 2008; 47: 4201-4210Crossref PubMed Scopus (17) Google Scholar)) with enhanced catalytic activity. However, in all PI vesicle assay systems examined, when the bulk PI is kept constant but the surface concentration decreases, PI-PLC specific activity decreases dramatically above XPC = 0.50 (12Qian X. Zhou C. Roberts M.F. Biochemistry. 1998; 37: 6513-6522Crossref PubMed Scopus (28) Google Scholar, 18Volwerk J.J. Filthuth E. Griffith O.H. Jain M.K. Biochemistry. 1994; 33: 3464-3474Crossref PubMed Scopus (63) Google Scholar). This surface dilution inhibition is often interpreted as the result of the substrate surface concentration decreasing below a two-dimensional Km measured in mole fraction PI (XPI) units (19Carman G.M. Deems R.A. Dennis E.A. J. Biol. Chem. 1995; 270: 18711-18714Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 20Berg O.G. Gelb M.H. Tsai M.D. Jain M.K. Chem. Rev. 2001; 101: 2613-2654Crossref PubMed Scopus (334) Google Scholar). The affinity of the diluent (in this case PC) for the enzyme can also complicate a detailed interpretation of this kinetic effect. To truly understand this PI-PLC, where two discrete phospholipids are critical for optimal catalysis, we need a molecular level description of surface dilution inhibition. Our results clearly indicate a role for lipid dynamics in PI-PLC activation by PC at low XPC and reduced activity of PI-PLC at XPC > 0.5. Specifically, these experiments reveal the following. (i) A relatively small amount of activator PC in the anionic lipid membrane promotes PI-PLC binding to vesicles. (ii) The affinity of the protein for vesicles increases as the amount of PC is increased. Affinities for SUVs reach a maximum for very PC-rich, but not pure PC, bilayers, indicating that both phospholipids contribute to tight vesicle binding (a synergistic effect between activator and active site binding of lipids). (iii) At moderate XPC, 0.1 < XPC < 0.5, the dynamics of both phospholipids are affected similarly, and the restricted, slower motions, which are more or less independent of XPC, parallel the high enzymatic activity. (iv) In the regime of tight binding to PC-rich bilayers, PC dynamics are preferentially slowed, suggesting that this activator lipid sequesters PI-PLC from the bulk of the substrate analogues, leading to significantly lower specific activity than would be expected. This latter observation provides a very specific interpretation of surface dilution inhibition as the result of tight binding of the protein to PC-rich regions that prevents its access to the bulk of the PI. All lipid stock solutions (dioleoyl chains for all of the anionic phospholipids and 1-palmitoyl-2-oleoyl phosphatidylcholine as the PC species) in chloroform were purchased from Avanti Polar Lipids. The chloroform was removed under a stream of nitrogen gas, and the resultant film was lyophilized overnight. The lipid film was rehydrated with phosphate-buffered saline buffer, pH 7.3, for FCS experiments or 50 mm HEPES, pH 7.5, for enzyme assays and for 31P NMR field cycling. Large unilamellar vesicles (LUVs) were prepared by multiple passages of the aqueous lipid solutions through polycarbonate membranes (100-nm pore diameter) using a miniextruder from Avanti. SUVs in phosphate-buffered saline, pH 7.3, were prepared by sonication (Branson Sonifier cell disrupter) of lipid mixtures for 30 min on ice. To introduce a fluorescent lipid into the SUVs, 1:20,000 mol fraction dipalmitoyl-lissamine-rhodamine phosphatidylethanolamine (Avanti) was added into the SUVs, and the mixture was sonicated for another 3 min. Under these conditions, a very small amount of fatty acid may be generated by hydrolysis of the phospholipids. However, no resonances for lyso-phospholipids were observed in a sample of PC/PI (1:1) treated in this fashion, indicating that the amount of lipid hydrolysis in this preparation was less than 0.5 mol %. Previous kinetic experiments have shown that small amounts of fatty acids do not affect enzyme specific activity (13Zhou C. Wu Y. Roberts M.F. Biochemistry. 1997; 36: 347-355Crossref PubMed Scopus (61) Google Scholar). The SUVs had a narrow vesicle size distribution as assessed by dynamic light scattering (21Maulucci G. De Spirito M. Arcovito G. Boffi F. Castellano A.C. Briganti G. Biophys. J. 2005; 88: 3545-3550Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar) using a Protein Solutions DynaProdynamic light scattering instrument from Wyatt Technology (22Schuck P. Biophys. J. 2000; 78: 1606-1619Abstract Full Text Full Text PDF PubMed Scopus (3045) Google Scholar). The anion-rich SUVs were slightly smaller, with a narrower size distribution than PC-rich SUVs. All of the SUVs were used within 1 week of preparation. Recombinant B. thuringiensis PI-PLC was overexpressed in E. coli and purified as described previously (15Feng J. Wehbi H. Roberts M.F. J. Biol. Chem. 2002; 277: 19867-19875Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Protein concentrations were assessed by both 280 nm absorption and Lowry assays. Samples for fc-P-NMR were prepared with 10 mm total (dioleoylphosphatidylmethanol (PMe) plus PC) phospholipid and 0.5 or 3 mg/ml PI-PLC (0.014 or 0.086 mm) in 50 mm HEPES, pH 7.5. EDTA (1 mm) was present to scavenge any paramagnetic ions in solution. Fluorescently labeled PI-PLC was prepared from the N168C mutant protein, constructed using the QuikChange methodology (Stratagene). The altered gene sequence was confirmed by DNA sequencing (Genewiz). N168C was fluorescently labeled using Alexa Fluor 488 C5 maleimide (AF488 maleimide; Invitrogen), according to the manufacturer's protocol. AF488 does not bind to vesicles (7Rusu L. Gambhir A. McLaughlin S. Rädler J. Biophys. J. 2004; 87: 1044-1053Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), and this was confirmed by performing FCS experiments in the presence of free AF488 and SUVs. A labeling ratio of 100 ± 10% was determined by comparing the absorption of the protein at 280 nm with that of the probe at 495 nm. Specific activities of recombinant B. thuringiensis PI-PLC toward PI in SUVs with varying mole fractions of PC were measured by 31P NMR spectroscopy (13Zhou C. Wu Y. Roberts M.F. Biochemistry. 1997; 36: 347-355Crossref PubMed Scopus (61) Google Scholar, 23Volwerk J.J. Shashidhar M.S. Kuppe A. Griffith O.H. Biochemistry. 1990; 29: 8056-8062Crossref PubMed Scopus (94) Google Scholar). Each sample was in 50 mm HEPES, pH 7.5, with 1 mm EDTA and 0.1 mg/ml bovine serum albumin. In parallel to the fc-P-NMR studies, the total phospholipid concentration of the vesicles was 10 mm, with the mole fraction PC varying from 0.0 to 0.9. The amount of enzyme added, 0.2–5 μg (corresponding to concentrations of 5.8–144 nm), and the incubation time were chosen to allow less than 20% PI cleavage for each sample. Samples were incubated for fixed times at 28 °C, and the reaction was quenched by adding 40 μl of acetic acid to a 1-ml sample. Triton X-100 (100 μl) was added to solubilize the phospholipids for analysis by 31P NMR. In these assays, myoinositol 1,2-(cyclic)-phosphate was the only product, and the specific activity was calculated according to integration of the myoinositol 1,2-(cyclic)-phosphate resonance compared with an internal standard (protons were not decoupled in these experiments). FCS experiments were performed using a previously described (24Liu L. Mushero N. Hedstrom L. Gershenson A. Biochemistry. 2006; 45: 10865-10872Crossref PubMed Scopus (11) Google Scholar) home-built confocal setup based on an IX-70 inverted microscope (Olympus). Further details on the apparatus and data treatment are provided in the supplemental material. FCS experiments were carried out at 22 °C on 300-μl samples in phosphate-buffered saline, pH 7.3, plus 1 mg/ml bovine serum albumin, to stabilize PI-PLC, in chambered coverglass wells (LabTek). Prior to use, the chambers were coated with 10 mg/ml bovine serum albumin and rinsed with phosphate-buffered saline buffer to prevent protein adhesion to the sides of the wells. For PI-PLC vesicle binding experiments, 3.5 nm labeled protein was titrated with unlabeled vesicles. The substrate, PI, was not used for FCS experiments, because PI cleavage by PI-PLC produces diacylglycerol, leading to vesicle fusion (25Goñi F.M. Alonso A. Biosci. Rep. 2000; 20: 443-463Crossref PubMed Scopus (55) Google Scholar). Thus, the anionic phospholipids PMe, PG, PA, or PS were used as substrate analogues. PG is a PI-PLC substrate and can be cleaved over a period of days by >1 mg/ml concentrations of PI-PLC (18Volwerk J.J. Filthuth E. Griffith O.H. Jain M.K. Biochemistry. 1994; 33: 3464-3474Crossref PubMed Scopus (63) Google Scholar, 26Zhou C. Qian X. Roberts M.F. Biochemistry. 1997; 36: 10089-10097Crossref PubMed Scopus (48) Google Scholar). However, at the low protein concentrations used in the FCS experiments, no detectable hydrolysis of PG occurred. The auto- and cross-correlation, Gj,k(τ), were calculated from the time-dependent fluorescence intensities (27Elson E.L. Magde D. Biopolymers. 1974; 13: 1-27Crossref Scopus (1319) Google Scholar, 28Thompson N.L. Lakowicz J. Topics in Fluorescence Microscopy. Plenum Press, New York1991: 337-378Google Scholar). To measure the average diffusion coefficient of the SUVs, FCS experiments were performed on SUVs containing a small amount (0.5 μm) of rhodamine-labeled PC. Diffusion coefficients of free, AF488-labeled PI-PLC (DPI-PLC) and labeled SUVs (DSUV) were 58 ± 5 μm2 s−1 (corresponding to a diffusion time (τD) = 173 μs) and 10.4 ± 1.0 μm2 s−1 (τD = 1.48 ms), respectively. In the presence of labeled PI-PLC and unlabeled lipid vesicles, FCS auto- and cross-correlations, G(τ), were analyzed using two-component, diffusion-only fits (5Magde D. Elson E.L. Webb W.W. Biopolymers. 1974; 13: 29-61Crossref PubMed Scopus (1110) Google Scholar, 29Takakuwa Y. Pack C.G. An X.L. Manno S. Ito E. Kinjo M. Biophys. Chem. 1999; 82: 149-155Crossref PubMed Scopus (26) Google Scholar),G(τ)=1⟨N⟩{(1−f)((1+ττD,free)1+τS2τD,free)−1+f((1+ττD,bound)1+τS2τD,bound)−1}(Eq. 1) where 〈N〉 is the time-averaged number of PI-PLC molecules in the observation volume, f is the fraction of PI-PLC bound to vesicles, and τD,free and τD,bound are the diffusion times for free and vesicle-bound PI-PLC, respectively. For 488-nm excitation, values of S (related to the observation volume) were between 6.8 and 8. The 31P field cycling spin lattice relaxation rate (R1) experiments were made at 25 °C on a Varian Unityplus 500 spectrometer using a standard 10-mm Varian probe in a custom-built device that moves the sample, sealed in a 10-mm tube, from the sample probe location to a higher position within, or just above, the magnet, where the magnetic field is between 0.06 and 11.7 T (8Roberts M.F. Redfield A.G. J. Am. Chem. Soc. 2004; 126: 13765-13777Crossref PubMed Scopus (65) Google Scholar, 30Roberts M.F. Cui Q. Turner C.J. Case D.A. Redfield A.G. Biochemistry. 2004; 43: 3637-3650Crossref PubMed Scopus (41) Google Scholar). Spin lattice relaxation rates at each field strength were measured using 6–8 programmed delay times and analysis of the data with an exponential function to extract R1 = 1/T1. Although the details of the R1 measurement for SUVs and the analysis to obtain and model R1 as a function of field have been described previously (8Roberts M.F. Redfield A.G. J. Am. Chem. Soc. 2004; 126: 13765-13777Crossref PubMed Scopus (65) Google Scholar, 9Roberts M.F. Redfield A.G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17066-17071Crossref PubMed Scopus (41) Google Scholar), a more detailed discussion of the methodology, data analysis, and associated assumptions is presented in the supplemental material. The field dependence of the 31P relaxation rates was analyzed in two discrete segments. R1 from 0.06 up to 11.7 T was fit with contributions from three terms (8Roberts M.F. Redfield A.G. J. Am. Chem. Soc. 2004; 126: 13765-13777Crossref PubMed Scopus (65) Google Scholar, 30Roberts M.F. Cui Q. Turner C.J. Case D.A. Redfield A.G. Biochemistry. 2004; 43: 3637-3650Crossref PubMed Scopus (41) Google Scholar): (i) dipolar relaxation associated with a correlation time, τc, typically in the 5–10 ns range and Rc(0), the maximum relaxation rate for this correlation time, which is directly proportional to τc and inversely proportional to rP-H6 (Rc(0) = τc (μo/4π)2 (h/2π)2γP2γH2 rPH−6), where rP-H indicates the average distance between the phosphorus and its nearest proton neighbors; (ii) CSA relaxation associated with the same slow correlation time (from which we can extract SC2, an order parameter squared (8Roberts M.F. Redfield A.G. J. Am. Chem. Soc. 2004; 126: 13765-13777Crossref PubMed Scopus (65) Google Scholar)); and (iii) CSA relaxation due to a faster motion (it is this term that dominates relaxation at high fields and is distinguished by a square law dependence). R1 in the field range 0.06–11.7 T is then the sum of these three terms,R1=(Rc(0)/2τc)(0.1J(ωH−ωP)+0.3J(ωP)+0.6J(ωH+ωP))+CLωP2J(ωP)+CHH2(Eq. 2) where J(ω) = 2τc/(1 + ω2τc2), and the field, H, is the magnetic field measured in tesla; the coefficient CL = (1/15)(1 + η2/3)σ2SC2. In these expressions, γ represents the gyromagnetic ratio (for 1H or 31P), and σ and η are the CSA interaction size and asymmetry for phospholipids (8Roberts M.F. Redfield A.G. J. Am. Chem. Soc. 2004; 126: 13765-13777Crossref PubMed Scopus (65) Google Scholar). The last term is for a faster motion and is not of interest in this work. A protein interacting with a given phospholipid would be expected to slow the motion associated with τc as well as increase SC2. A second distinct increase in R1 can be measured at very low fields (<0.03 T). The data in this low field regime are fit to a term added to Rc(0) for dipolar relaxation with a submicrosecond correlation time, τv, and a maximum extrapolated relaxation rate of Rv(0) for this slower motion (9Roberts M.F. Redfield A.G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17066-17071Crossref PubMed Scopus (41) Google Scholar).R1=(Rv(0)/2τv)(0.1J(ωH−ωP)+0.3J(ωP)+0.6J(ωH+ωP))+Rc(0)(Eq. 3) The parameter τv primarily reflects vesicle tumbling, although lateral diffusion also contributes to this value. For two phospholipids in the same vesicle, a change in τv in the presence of protein can provide information on segregation of phospholipids (and faster lateral diffusion of one versus the other). The specific activity (μmol min−1 mg−1) of PI-PLC toward PI-containing SUVs with a fixed enzyme and total lipid concentration (10 mm) and increasing mole fractions of the activator PC, XPC, was determined. To emphasize the initial activation and eventual inhibition, the PI-PLC relative activity (specific activity normalized to the value obtained for pure PI SUVs, 91.3 ± 9.2 μmol min−1 mg−1) is presented in Fig. 1. Although there is considerable error when averaging different series of experiments with SUVs, the trends are always the same. As little as 0.10 XPC increases the enzyme activity toward PI 2–3-fold, and the activity is more or less constant from 0.10 to 0.50 XPC. The magnitude of this activation depends on the concentration of PI and on the PC species; for example, a 7-fold activation was reported previously using 0.2 mol fraction dimyristoylphosphatidylcholine (12Qian X. Zhou C. Roberts M.F. Biochemistry. 1998; 37: 6513-6522Crossref PubMed Scopus (28) Google Scholar). Apparent Km values for pure PI SUVs (derived from treating the system as if it followed Michaelis-Menten kinetics) have been estimated as 3–5 mm (12Qian X. Zhou C. Roberts M.F. Biochemistry. 1998; 37: 6513-6522Crossref PubMed Scopus (28) Google Scholar), which might suggest that the large kinetic effect of PC is from enhanced protein binding (12Qian X. Zhou C. Roberts M.F. Biochemistry. 1998; 37: 6513-6522Crossref PubMed Scopus (28) Google Scholar, 26Zhou C. Qian X. Roberts M.F. Biochemistry. 1997; 36: 10089-10097C" @default.
• W2000392436 created "2016-06-24" @default.
• W2000392436 creator A5003467630 @default.
• W2000392436 creator A5034396249 @default.
• W2000392436 creator A5043473088 @default.
• W2000392436 creator A5047196423 @default.
• W2000392436 creator A5079087907 @default.
• W2000392436 date "2009-06-01" @default.
• W2000392436 modified "2023-09-28" @default.
• W2000392436 title "Correlation of Vesicle Binding and Phospholipid Dynamics with Phospholipase C Activity" @default.
• W2000392436 cites W16780149 @default.
• W2000392436 cites W1917284703 @default.
• W2000392436 cites W1945473274 @default.
• W2000392436 cites W1958742527 @default.
• W2000392436 cites W1973351141 @default.
• W2000392436 cites W1973951124 @default.
• W2000392436 cites W1975202544 @default.
• W2000392436 cites W1975317507 @default.
• W2000392436 cites W1987075231 @default.
• W2000392436 cites W1987713423 @default.
• W2000392436 cites W1997241998 @default.
• W2000392436 cites W2001102262 @default.
• W2000392436 cites W2007708814 @default.
• W2000392436 cites W2008264883 @default.
• W2000392436 cites W2009148211 @default.
• W2000392436 cites W2020382054 @default.
• W2000392436 cites W2020994777 @default.
• W2000392436 cites W2039705260 @default.
• W2000392436 cites W2043150057 @default.
• W2000392436 cites W2047438853 @default.
• W2000392436 cites W2050606799 @default.
• W2000392436 cites W2052204832 @default.
• W2000392436 cites W2057323682 @default.
• W2000392436 cites W2058593388 @default.
• W2000392436 cites W2058628523 @default.
• W2000392436 cites W2074953899 @default.
• W2000392436 cites W2085611814 @default.
• W2000392436 cites W2088690308 @default.
• W2000392436 cites W2093750956 @default.
• W2000392436 cites W2095039779 @default.
• W2000392436 cites W2111794178 @default.
• W2000392436 cites W2135290527 @default.
• W2000392436 cites W2149744058 @default.
• W2000392436 cites W2156988770 @default.
• W2000392436 cites W2159190107 @default.
• W2000392436 cites W2163930707 @default.
• W2000392436 cites W2949246524 @default.
• W2000392436 doi "https://doi.org/10.1074/jbc.m809600200" @default.
• W2000392436 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2713506" @default.
• W2000392436 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19336401" @default.
• W2000392436 hasPublicationYear "2009" @default.
• W2000392436 type Work @default.
• W2000392436 sameAs 2000392436 @default.
• W2000392436 citedByCount "25" @default.
• W2000392436 countsByYear W20003924362012 @default.
• W2000392436 countsByYear W20003924362013 @default.
• W2000392436 countsByYear W20003924362014 @default.
• W2000392436 countsByYear W20003924362016 @default.
• W2000392436 countsByYear W20003924362018 @default.
• W2000392436 countsByYear W20003924362019 @default.
• W2000392436 countsByYear W20003924362020 @default.
• W2000392436 countsByYear W20003924362021 @default.
• W2000392436 crossrefType "journal-article" @default.
• W2000392436 hasAuthorship W2000392436A5003467630 @default.
• W2000392436 hasAuthorship W2000392436A5034396249 @default.
• W2000392436 hasAuthorship W2000392436A5043473088 @default.
• W2000392436 hasAuthorship W2000392436A5047196423 @default.
• W2000392436 hasAuthorship W2000392436A5079087907 @default.
• W2000392436 hasBestOaLocation W20003924361 @default.
• W2000392436 hasConcept C121332964 @default.
• W2000392436 hasConcept C12554922 @default.
• W2000392436 hasConcept C130316041 @default.
• W2000392436 hasConcept C145912823 @default.
• W2000392436 hasConcept C185592680 @default.
• W2000392436 hasConcept C24890656 @default.
• W2000392436 hasConcept C2778918659 @default.
• W2000392436 hasConcept C41625074 @default.
• W2000392436 hasConcept C55493867 @default.
• W2000392436 hasConcept C86803240 @default.
• W2000392436 hasConceptScore W2000392436C121332964 @default.
• W2000392436 hasConceptScore W2000392436C12554922 @default.
• W2000392436 hasConceptScore W2000392436C130316041 @default.
• W2000392436 hasConceptScore W2000392436C145912823 @default.
• W2000392436 hasConceptScore W2000392436C185592680 @default.
• W2000392436 hasConceptScore W2000392436C24890656 @default.
• W2000392436 hasConceptScore W2000392436C2778918659 @default.
• W2000392436 hasConceptScore W2000392436C41625074 @default.
• W2000392436 hasConceptScore W2000392436C55493867 @default.
• W2000392436 hasConceptScore W2000392436C86803240 @default.
• W2000392436 hasIssue "24" @default.
• W2000392436 hasLocation W20003924361 @default.
• W2000392436 hasLocation W20003924362 @default.
• W2000392436 hasLocation W20003924363 @default.
• W2000392436 hasLocation W20003924364 @default.
• W2000392436 hasOpenAccess W2000392436 @default.
• W2000392436 hasPrimaryLocation W20003924361 @default.
• W2000392436 hasRelatedWork W1625647566 @default.
• W2000392436 hasRelatedWork W1983699312 @default.

• next