Yuan‐Hao Hsu

Phospholipases represent one of the earliest enzyme activities to be identified and studied, and the phospholipase A2 superfamily traces its roots to the identification of lytic actions of snake venom at the end of the 19th century. Both electrostatic and hydrophobic interactions contribute to the interfacial binding of sPLA2 to anionic phospholipid membranes. The interaction between basic residues on the binding surface with anionic vesicles plays an important role in interfacial binding. The major functions will be summarized below and include the ability to kill Gram-positive and Gram-negative bacteria, thereby affecting host defense against bacterial infections. sPLA2 may be involved in the pathogensis of inflammatory bowel disease including Crohn's disease and ulcerative colitis. GIIA sPLA2 protein and mRNA were detected in Paneth cells of the small intestinal mucosa in the intestine in Crohn's disease patients.

Hyaluronic acid (HA) is a glycosaminoglycan that was first isolated and identified from the vitreous body of a bull's eye. HA is ubiquitous in the soft connective tissues of animals and therefore has high tissue compatibility for use in medication. Because of HA's biological safety and water retention properties, it has many ophthalmology-related applications, such as in intravitreal injection, dry eye treatment, and contact lenses. Due to its broad range of applications, the identification and quantification of HA is a critical topic. This review article discusses current methods for analyzing HA. Contact lenses have become a widely used medical device, with HA commonly used as an additive to their production material, surface coating, and multipurpose solution. HA molecules on contact lenses retain moisture and increase the wearer's comfort. HA absorbed by contact lenses can also gradually release to the anterior segment of the eyes to treat dry eye. This review discusses applications of HA in ophthalmology.

Group VIA calcium-independent phospholipase A(2) (GVIA iPLA(2)) has recently emerged as a novel pharmaceutical target. We have now explored the structure-activity relationship between fluoroketones and GVIA iPLA(2) inhibition. The presence of a naphthyl group proved to be of paramount importance. 1,1,1-Trifluoro-6-(naphthalen-2-yl)hexan-2-one (FKGK18) is the most potent inhibitor of GVIA iPLA(2) (X(I)(50) = 0.0002) ever reported. Being 195 and >455 times more potent for GVIA iPLA(2) than for GIVA cPLA(2) and GV sPLA(2), respectively, makes it a valuable tool to explore the role of GVIA iPLA(2) in cells and in vivo models. 1,1,1,2,2,3,3-Heptafluoro-8-(naphthalene-2-yl)octan-4-one inhibited GVIA iPLA(2) with a X(I)(50) value of 0.001 while inhibiting the other intracellular GIVA cPLA(2) and GV sPLA(2) at least 90 times less potently. Hexa- and octafluoro ketones were also found to be potent inhibitors of GVIA iPLA(2); however, they are not selective.

Abstract Chronic inflammation and concomitant oxidative stress can induce mitochondrial dysfunction due to cardiolipin (CL) abnormalities in the mitochondrial inner membrane. To examine the responses of mitochondria to inflammation, macrophage-like RAW264.7 cells were activated by Kdo2-Lipid A (KLA) in our inflammation model, and then the mitochondrial CL profile, mitochondrial activity, and the mRNA expression of CL metabolism-related genes were examined. The results demonstrated that KLA activation caused CL desaturation and the partial loss of mitochondrial activity. KLA activation also induced the gene upregulation of cyclooxygenase (COX)-2 and phospholipid scramblase 3, and the gene downregulation of COX-1, lipoxygenase 5, and Δ-6 desaturase. We further examined the phophatidylglycerol (PG) inhibition effects on inflammation. PG supplementation resulted in a 358-fold inhibition of COX-2 mRNA expression. PG(18:1) 2 and PG(18:2) 2 were incorporated into CLs to considerably alter the CL profile. The decreased CL and increased monolysocardiolipin (MLCL) quantity resulted in a reduced CL/MLCL ratio. KLA-activated macrophages responded differentially to PG(18:1) 2 and PG(18:2) 2 supplementation. Specifically, PG(18:1) 2 induced less changes in the CL/MLCL ratio than did PG(18:2) 2 , which resulted in a 50% reduction in the CL/MLCL ratio. However, both PG types rescued 20–30% of the mitochondrial activity that had been affected by KLA activation.

The Group VIA-2 Ca2+-independent phospholipase A2 (GVIA-2 iPLA2) is composed of seven consecutive N-terminal ankyrin repeats, a linker region, and a C-terminal phospholipase catalytic domain. No structural information exists for this enzyme, and no information is known about the membrane binding surface. We carried out deuterium exchange experiments with the GVIA-2 iPLA2 in the presence of both phospholipid substrate and the covalent inhibitor methyl arachidonoyl fluorophosphonate and located regions in the protein that change upon lipid binding. No changes were seen in the presence of only methyl arachidonoyl fluorophosphonate. The region with the greatest change upon lipid binding was region 708–730, which showed a >70% decrease in deuteration levels at numerous time points. No decreases in exchange due to phospholipid binding were seen in the ankyrin repeat domain of the protein. To locate regions with changes in exchange on the enzyme, we constructed a computational homology model based on homologous structures. This model was validated by comparing the deuterium exchange results with the predicted structure. Our model combined with the deuterium exchange results in the presence of lipid substrate have allowed us to propose the first structural model of GVIA-2 iPLA2 as well as the interfacial lipid binding region. The Group VIA-2 Ca2+-independent phospholipase A2 (GVIA-2 iPLA2) is composed of seven consecutive N-terminal ankyrin repeats, a linker region, and a C-terminal phospholipase catalytic domain. No structural information exists for this enzyme, and no information is known about the membrane binding surface. We carried out deuterium exchange experiments with the GVIA-2 iPLA2 in the presence of both phospholipid substrate and the covalent inhibitor methyl arachidonoyl fluorophosphonate and located regions in the protein that change upon lipid binding. No changes were seen in the presence of only methyl arachidonoyl fluorophosphonate. The region with the greatest change upon lipid binding was region 708–730, which showed a >70% decrease in deuteration levels at numerous time points. No decreases in exchange due to phospholipid binding were seen in the ankyrin repeat domain of the protein. To locate regions with changes in exchange on the enzyme, we constructed a computational homology model based on homologous structures. This model was validated by comparing the deuterium exchange results with the predicted structure. Our model combined with the deuterium exchange results in the presence of lipid substrate have allowed us to propose the first structural model of GVIA-2 iPLA2 as well as the interfacial lipid binding region. The Group VIA phospholipase A2 is a member of the phospholipase A2 superfamily that cleaves fatty acids from the sn-2 position of phospholipids (1.Six D.A. Dennis E.A. Biochim. Biophys. Acta. 2000; 1488: 1-19Crossref PubMed Scopus (1215) Google Scholar, 2.Burke J.E. Dennis E.A. Cardiovasc. Drugs Ther. 2009; 23: 49-59Crossref PubMed Scopus (276) Google Scholar). The human Group VIA PLA2 3The abbreviations used are: PLA2phospholipase A2GVIAGroup VIAGIVAGroup IVAGIAGroup IAiPLA2Ca2+-independent phospholipase A2DXMSdeuterium exchange coupled with mass spectrometryPAPC1-palmitoyl-2-arachidonoyl-sn-phosphatidylcholineMAFPmethyl arachidonyl fluorophosphonateMOPS4-morpholinepropanesulfonic acidHPLChigh pressure liquid chromatography.3The abbreviations used are: PLA2phospholipase A2GVIAGroup VIAGIVAGroup IVAGIAGroup IAiPLA2Ca2+-independent phospholipase A2DXMSdeuterium exchange coupled with mass spectrometryPAPC1-palmitoyl-2-arachidonoyl-sn-phosphatidylcholineMAFPmethyl arachidonyl fluorophosphonateMOPS4-morpholinepropanesulfonic acidHPLChigh pressure liquid chromatography. gene yields multiple splice variants, including GVIA-1, GVIA-2, GVIA-3 PLA2, GVIA Ankyrin-1, and GVIA Ankyrin-2 (3.Larsson P.K. Claesson H.E. Kennedy B.P. J. Biol. Chem. 1998; 273: 207-214Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 4.Larsson Forsell P.K. Kennedy B.P. Claesson H.E. Eur. J. Biochem. 1999; 262: 575-585Crossref PubMed Scopus (117) Google Scholar). At least two isoforms, GVIA-1 and GVIA-2 iPLA2, are active. Our laboratory purified and characterized the first mammalian iPLA2, the 85-kDa GVIA-2 iPLA2 (5.Ackermann E.J. Kempner E.S. Dennis E.A. J. Biol. Chem. 1994; 269: 9227-9233Abstract Full Text PDF PubMed Google Scholar), which became the first cloned iPLA2 (6.Balboa M.A. Balsinde J. Jones S.S. Dennis E.A. J. Biol. Chem. 1997; 272: 8576-8580Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). This enzyme can hydrolyze the sn-2 fatty acyl bond of phospholipids and also has potent lysophospholipase and transacylase activity (7.Lio Y.C. Dennis E.A. Biochim. Biophys. Acta. 1998; 1392: 320-332Crossref PubMed Scopus (84) Google Scholar). GVIA iPLA2 is involved in cell proliferation (8.Roshak A.K. Capper E.A. Stevenson C. Eichman C. Marshall L.A. J. Biol. Chem. 2000; 275: 35692-35698Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), apoptosis (9.Balsinde J. Pérez R. Balboa M.A. Biochim. Biophys. Acta. 2006; 1761: 1344-1350Crossref PubMed Scopus (80) Google Scholar, 10.Pérez R. Balboa M.A. Balsinde J. J. Immunol. 2006; 176: 2555-2561Crossref PubMed Scopus (41) Google Scholar, 11.Bao S. Li Y. Lei X. Wohltmann M. Jin W. Bohrer A. Semenkovich C.F. Ramanadham S. Tabas I. Turk J. J. Biol. Chem. 2007; 282: 27100-27114Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), bone formation (12.Ramanadham S. Yarasheski K.E. Silva M.J. Wohltmann M. Novack D.V. Christiansen B. Tu X. Zhang S. Lei X. Turk J. Am. J. Pathol. 2008; 172: 868-881Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), sperm development (13.Bao S. Miller D.J. Ma Z. Wohltmann M. Eng G. Ramanadham S. Moley K. Turk J. J. Biol. Chem. 2004; 279: 38194-38200Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), and glucose-induced insulin secretion (14.Bao S. Bohrer A. Ramanadham S. Jin W. Zhang S. Turk J. J. Biol. Chem. 2006; 281: 187-198Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 15.Bao S. Song H. Wohltmann M. Ramanadham S. Jin W. Bohrer A. Turk J. J. Biol. Chem. 2006; 281: 20958-20973Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), so its function may vary by cell and tissue. phospholipase A2 Group VIA Group IVA Group IA Ca2+-independent phospholipase A2 deuterium exchange coupled with mass spectrometry 1-palmitoyl-2-arachidonoyl-sn-phosphatidylcholine methyl arachidonyl fluorophosphonate 4-morpholinepropanesulfonic acid high pressure liquid chromatography. phospholipase A2 Group VIA Group IVA Group IA Ca2+-independent phospholipase A2 deuterium exchange coupled with mass spectrometry 1-palmitoyl-2-arachidonoyl-sn-phosphatidylcholine methyl arachidonyl fluorophosphonate 4-morpholinepropanesulfonic acid high pressure liquid chromatography. The human GVIA-2 iPLA2 (806 amino acids), the form of the enzyme studied here, contains seven ankyrin repeats (residues 152–382), a linker region (residues 383–474) with the eighth repeat disrupted by a 54-amino acid insert (16.Sedgwick S.G. Smerdon S.J. Trends Biochem. Sci. 1999; 24: 311-316Abstract Full Text Full Text PDF PubMed Scopus (661) Google Scholar), and a catalytic domain (residues 475–806). The active site serine of the GVIA iPLA2 lies within a lipase consensus sequence (Gly-X-Ser519-X-Gly) (1.Six D.A. Dennis E.A. Biochim. Biophys. Acta. 2000; 1488: 1-19Crossref PubMed Scopus (1215) Google Scholar). The activity of GVIA iPLA2 has been reported to be regulated through several mechanisms. A caspase-3 cleavage site at the N terminus of the enzyme has been identified that is clipped in vitro (17.Atsumi G. Murakami M. Kojima K. Hadano A. Tajima M. Kudo I. J. Biol. Chem. 2000; 275: 18248-18258Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). This truncated form of the enzyme was hyperactive and reduced cell viability when overexpressed in HEK293 cells (17.Atsumi G. Murakami M. Kojima K. Hadano A. Tajima M. Kudo I. J. Biol. Chem. 2000; 275: 18248-18258Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Another possible control mechanism is through ATP binding on the 485GXGXXG motif (18.Hemmer W. McGlone M. Tsigelny I. Taylor S.S. J. Biol. Chem. 1997; 272: 16946-16954Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The activity of phospholipases depends critically on the interaction of the protein with phospholipid membranes. In vitro, GVIA iPLA2 does not have any specificity for the fatty acid in the sn-2 position of substrate phospholipids (5.Ackermann E.J. Kempner E.S. Dennis E.A. J. Biol. Chem. 1994; 269: 9227-9233Abstract Full Text PDF PubMed Google Scholar). GVIA-2 iPLA2 was found to be membrane-associated when overexpressed in COS-7 cells, and this was further confirmed in rat vascular smooth muscle cells (4.Larsson Forsell P.K. Kennedy B.P. Claesson H.E. Eur. J. Biochem. 1999; 262: 575-585Crossref PubMed Scopus (117) Google Scholar, 19.Ma Z. Wang X. Nowatzke W. Ramanadham S. Turk J. J. Biol. Chem. 1999; 274: 9607-9616Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The other active splice variant, GVIA-1, is cytosolic and not specific in targeting membrane surfaces (4.Larsson Forsell P.K. Kennedy B.P. Claesson H.E. Eur. J. Biochem. 1999; 262: 575-585Crossref PubMed Scopus (117) Google Scholar, 19.Ma Z. Wang X. Nowatzke W. Ramanadham S. Turk J. J. Biol. Chem. 1999; 274: 9607-9616Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), indicating two different regulatory mechanisms between these two splice variants. The 54-residue insertion in the eighth ankyrin repeat alters the property of GVIA-2 iPLA2 for membrane association. The ankyrin repeats have been reported to be involved in protein-protein interactions, such as 53BP2-p53, GA-binding protein α-GA-binding protein β, p16INK4a-CDK6, and IκBα-NFκB (16.Sedgwick S.G. Smerdon S.J. Trends Biochem. Sci. 1999; 24: 311-316Abstract Full Text Full Text PDF PubMed Scopus (661) Google Scholar). The ankyrin repeats of GVIA iPLA2 may directly membrane the catalytic domain by does not have activity (3.Larsson P.K. Claesson H.E. Kennedy B.P. J. Biol. Chem. 1998; 273: 207-214Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). the regions of the protein that with the membrane for a of the regulatory mechanisms of the is in GVIA iPLA2 of its in and in is no to on the exchange coupled with mass spectrometry has been used to the of protein-protein Y. C. Taylor S.S. J. Biol. 2004; PubMed Scopus (84) Google Scholar), protein changes C. Li W. Y. Sci. PubMed Scopus Google Scholar, D.A. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar), and protein Sci. 2006; PubMed Scopus Google Scholar), and we have to J.E. Li S. Dennis E.A. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar, J.E. M.J. Li S. Dennis E.A. 2008; PubMed Scopus Google Scholar). are also of with homology to structural information does not Y. J. R. Taylor S. J. Biol. PubMed Scopus Google Scholar). We used deuterium exchange with homology to of the ankyrin repeats based on the and of the catalytic domain based on To the interfacial of GVIA iPLA2, we 1-palmitoyl-2-arachidonoyl-sn-phosphatidylcholine the methyl arachidonyl fluorophosphonate which to the active site and GVIA iPLA2 (7.Lio Y.C. Dennis E.A. Biochim. Biophys. Acta. 1998; 1392: 320-332Crossref PubMed Scopus (84) Google Scholar). to the iPLA2 and structural we were to GVIA iPLA2 with phospholipid membranes. was from arachidonyl fluorophosphonate was from was from other were N-terminal insertion at the of GVIA-2 iPLA2 and GVIA protein were in a of The cell was in and and the was by at for The cell was to the and in and at for the was through a of The protein was in the and The protein was the GVIA iPLA2 was in the protein with at the specific activity of GVIA PLA2, were in composed of at and The were composed of and in a of The was by the of of GVIA PLA2 to and at for the was and the fatty acids were a as D.A. Dennis E.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). was by and for The was and in of on to form The was at for to the in of GVIA iPLA2 inhibitor in methyl was to of exchange of was to of GVIA iPLA2 and at for The binding of to GVIA iPLA2 was by GVIA iPLA2 with at for exchange experiments were by of GVIA iPLA2 of GVIA iPLA2) in protein with of to a of at The of lipid of deuterium was and The were at for The deuterium exchange was by of acid and that the to The were on levels were as reported J.E. Li S. Dennis E.A. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). with were at which was within were at as Y. C. Taylor S.S. J. Biol. 2004; PubMed Scopus (84) Google Scholar). The of is J.E. Li S. Dennis E.A. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). of the of the two N-terminal not any liquid chromatography. The of exchange in was by a of multiple by the Biophys. PubMed Scopus Google is the deuterium N is the and is the of the Z. PubMed Scopus Google Scholar). carried its was to the for in to these to of exchange allowed us to results D.J. Sci. PubMed Scopus Google Scholar, X. Zhang H. J. Sci. PubMed Scopus Google Scholar). The of were and The regions in the were to a in The have and the are exchange are The model of GVIA iPLA2 was by protein of the protein Zhang J. Zhang Z. Miller W. D.J. 1997; PubMed Scopus Google Scholar, S. PubMed Scopus Google Scholar). have homology to the of human M. J. PubMed Scopus Google with of have homology to the of a lipase PubMed Scopus Google with of homologous are for model The which numerous different to protein was used to the catalytic domain of GVIA iPLA2 B. G. J. 2004; PubMed Scopus Google Scholar). was to the of the GVIA protein a model of and a model of the catalytic domain of GVIA based on the predicted the of the and in the in the homology by M.J. L.A. 2008; PubMed Scopus Google Scholar). The of the predicted was by to the of of two protein Y. J. 2004; PubMed Scopus Google Scholar). The a and The model of the catalytic domain has a of The model of the ankyrin repeats has a of We the computational by for between deuterium exchange information and predicted structure. we for regions predicted to have levels of exchange The protein was by and to the in This different which of the protein these different were and for the in the are as in were based on to were on the GVIA PLA2 to for and the activity of well to activity of (7.Lio Y.C. Dennis E.A. Biochim. Biophys. Acta. 1998; 1392: 320-332Crossref PubMed Scopus (84) Google Scholar). exchange experiments were carried out as with and PLA2 J.E. Li S. Dennis E.A. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar, J.E. M.J. Li S. Dennis E.A. 2008; PubMed Scopus Google Scholar, J.E. Li S. Dennis E.A. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). In GVIA PLA2 was with for seven different time from to and the The results of the exchange at seven time from to are in To the results when were several the with the and the were to a specific region of the of the two N-terminal on of the not be to any the these are not in the are in the The the that were and The results of deuterium exchange experiments reported in a only the deuterium exchange levels of regions of the not the In this when we the we are to that was identified in the we the we are to a of the protein for which the deuterium exchange was may may not to the to the results for of the GVIA that were not in in deuteration the time such as and be used to the between two to GVIA-2 PLA2 contains N-terminal region from to of by seven ankyrin repeats from to as well as a linker region from to and a catalytic domain from to the active site at The deuterium exchange in the N-terminal region from to showed levels of exchange at time with no deuteration at of The seven ankyrin repeats and linker region from to numerous regions of at and regions deuteration at of The of the ankyrin and linker region showed levels of exchange to of the N-terminal region. The catalytic region from to has two regions that showed exchange at and regions of in the first of The of the catalytic domain no regions with deuteration at of regions in the catalytic domain between and deuteration in the first of The catalytic domain of exchange from to of experiments were on the GVIA-2 PLA2 in the presence of phospholipid to the enzyme on the surface. experiments were carried out in the presence of with to of the substrate were deuterium exchange and at of enzyme substrate the was experiments were carried out at seven different time from to as in The for these experiments was from the of two experiments of seven time points. regions with changes in exchange we The were by of a and are in The of exchange were and The decrease of the deuteration can be by the in the of in different of in regions of GVIA-2 iPLA2 within a different of of exchange the of in the of in the of in by the of in by in a different regions of the protein showed changes in exchange upon of substrate of these 708–730, and were located on the catalytic and showed decreases in region in the ankyrin repeat and linker region showed a change in exchange and this was in The region a decrease at the time in the presence of The presence of the of exchange to to and a decrease in exchange in the presence of phospholipid This that a region been from at different were in this region. The binding within the region 708–730, and showed a and decrease at This region contains numerous regions and may be in the regions and showed no changes in exchange at time with the greatest in exchange seen at The changes in region involved the of and to in the presence of This may be by this region phospholipid changes upon substrate binding. The region a showed a the region The region in the ankyrin repeat domain of the protein of at time from to This region showed a from to that the binding of may also the position between ankyrin repeats and the catalytic domain. To the phospholipid binding from the binding we carried out further experiments with iPLA2 at seven time from to at binding no changes in exchange No we identified contains the active site which we to be the site of covalent by To in the of the changes of deuterium exchange due to phospholipid binding a structural model was No exists for the GVIA have homology to the of human and the catalytic domain from to has a homology to the lipase with homology is a for computational and can be as as a Z. Sci. 2006; PubMed Scopus Google Scholar). The results of homology are in In this the of deuterium exchange at is on the structure. The are composed of ankyrin repeat and linker domain from to and the catalytic domain from to No information on the of these two is based on homology To the model we the exchange the predicted and for a between has used deuterium exchange with homology to Y. J. R. Taylor S. J. Biol. PubMed Scopus Google Scholar). of the protein that are composed of and be and regions that are in the have levels of the exchange of the catalytic domain that the homology model well to the exchange The catalytic domain is predicted to be composed of domain with and that showed levels of exchange at of The only two regions with exchange at of are regions and has no predicted and is located at the of the protein. has within this region, are by and are not the of the protein. of the high of exchange in region 708–730, is and the two are only To further this model we the exchange of the catalytic domain of the GVIA PLA2 with the PLA2 at of J.E. Li S. Dennis E.A. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). The exchange of the of GVIA PLA2 and the of PLA2 in both were In PLA2 the region showed The deuteration levels of region in GVIA PLA2 the exchange of the region of The model of the ankyrin repeat and linker regions from to contains the seven ankyrin repeats predicted from sequence homology at Balsinde J. Dennis E.A. Biochim. Biophys. Acta. 2000; 1488: PubMed Scopus Google Scholar), homology and also predicted that the region contains ankyrin of can be the as in these ankyrin homologous repeats may have different from the other seven ankyrin In the repeat regions (residues 152–382), ankyrin repeats and showed ankyrin repeats and The of the ankyrin repeat was at the time The through ankyrin repeats not exchange in The regions and also in these ankyrin The linker region (residues 383–474) with a 54-amino acid insertion in the of the eighth ankyrin repeat showed high of This region is predicted to be a of structures. This that the ankyrin repeats in the linker region are not as well as the seven reported ankyrin The changes in deuteration upon binding phospholipid substrate were on the homology of the catalytic domain and the ankyrin repeat region The change is in region 708–730, which is located the active site of the other regions with decreases in exchange are located on the of the enzyme as and also showed a of a decrease in exchange in with exchange of exchange the model these regions are predicted to be the the active are a of different in the region of two different in the region of different in the region of 708–730, and in the region of that The exchange in the N terminus and the ankyrin repeats not have a change upon binding region of the exchange any change in the ankyrin repeats and are in in the linker region showed a of exchange at indicating that this region became to upon phospholipid membrane homology the for this in exchange is not To the ankyrin repeats binding to and the interaction between the ankyrin repeats and the catalytic we experiments on the protein in the presence and of at two time and The results are that the protein not with We also found that regions and in the linker region of the showed a exchange at the enzyme The linker region of exchange the of the catalytic domain. the changes were only located in the linker region, indicating that the ankyrin repeats not the catalytic domain. The region to the catalytic domain is to the linker region. This is the first structural of the GVIA We carried out deuterium exchange in the presence and of substrate and these exchange a homology model of the two different of the GVIA We that be between the model and the be This model is for the of exchange the model was to the of the enzyme on the membrane surface. This is a of the phospholipase A2 superfamily of and membrane binding deuterium exchange mass spectrometry J.E. Li S. Dennis E.A. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar, J.E. M.J. Li S. Dennis E.A. 2008; PubMed Scopus Google Scholar, J.E. Li S. Dennis E.A. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). The deuterium exchange results showed a to the predicted of the homology exchange has been used with homology to model the of specific protein protein Y. J. R. Taylor S. J. Biol. PubMed Scopus Google Scholar). This used predicted exchange based on to a homology we used a between the exchange of the of GVIA iPLA2 and the of J.E. Li S. Dennis E.A. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). The exchange of the in the iPLA2 model well with the exchange of the in at the time of The of the GVIA iPLA2 levels of deuteration at time the This is due to the that the of the GVIA iPLA2 does not the region including a the active site as in the The active site is in the GVIA iPLA2 with the This the of specificity for the fatty acid in the sn-2 position of GVIA iPLA2 with the specificity of this we are that the catalytic domain is to model the and of membrane binding. The between the PLA2 A. J. H. M. J. 1999; Full Text Full Text PDF PubMed Scopus Google and the GVIA PLA2 structural model is the of any region. The GVIA PLA2 has no specificity for fatty acids in the sn-2 position (5.Ackermann E.J. Kempner E.S. Dennis E.A. J. Biol. Chem. 1994; 269: 9227-9233Abstract Full Text PDF PubMed Google Scholar, J. M. J. Jones S.S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). PLA2 is specific for acid in the sn-2 position L.A. Full Text PDF PubMed Scopus Google Scholar). The in this may be due to the active site of GVIA PLA2 to the in the model of GVIA PLA2 with we have showed that both substrate and the inhibitor in exchange in regions of the PLA2 the region, a in a the active site J.E. Li S. Dennis E.A. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). No in exchange were seen in the catalytic domain of GVIA PLA2 upon substrate binding binding. This that the mechanisms that the activity of GVIA PLA2 are different from that of The results of the GVIA PLA2 binding to a membrane showed changes in exchange in different of these regions was located in the ankyrin repeat linker region of the protein. located in the of the linker region a in exchange at of deuterium exchange experiments were with the ankyrin repeat linker the linker region in exchange with the This that the linker region is in with the catalytic domain. The in exchange at may that upon lipid binding is a change in the of the catalytic domain to the ankyrin repeat linker region. of the changes in deuterium exchange were to the catalytic and were decreases in The decreases in exchange on the catalytic domain in that have numerous different The region with the greatest decrease in exchange is This region a decrease in exchange at the time with only a decrease at This region is the region in the protein in the of The of exchange at of in the presence of lipid that this region is the membrane surface. This region contains and that may the lipid surface. a decrease in exchange at and is The decreases in exchange in this region in as in This region of that is of the catalytic which the of are no regions in this region, and decreases are due to between the membrane and the and changes by substrate binding. and are both to the active site and are on the of the enzyme as the membrane region of of these regions not have regions and are also with the of the membrane Our model of PLA2 membrane binding the of the region from the membrane with regions and with the of the phospholipid This showed the first information on the of the GVIA PLA2 upon binding phospholipid as well as the first structural model of the ankyrin repeat linker region and catalytic domain of GVIA We A. for with