Introduction of a high-resolution cytochemical method for studying the distribution of phospholipids in biological tissues

A novel cytochemical method for the in situ, ultrastructural localization of phospholipids in biological tissues is reported. The method is based on the enzyme-gold approach (M. Bendayan: J. Histochem. Cytochem. 29, 531, 1981). Phospholipase A2 from bee venom was adsorbed on colloidal gold particles (PLA2-gold) and applied for the specific labeling of its substrate, sn3-glycerophospholipids. The binding and enzymic competence of the PLA2-gold complex were confirmed by in vitro, preembedding experiments with erythrocytes and a crude lung surfactant preparation. The substrate specificity of the probe was assessed by labeling Epon thin sections of pure phospholipids. To test the potential applications of the PLA2-gold complex, lung and pancreatic tissues were fixed with glutaraldehyde-osmium and embedded in Epon for transmission electron microscopy (TEM). They were also prepared for critical-point-drying fracture-label (CPD-FL) replicas and thin-section fracture-label (TS-FL) specimens. On TEM thin sections incubated with PLA2-gold, all cellular membranes were labeled. The labeling density over each membrane compartment, as quantitated in lung type II pneumocytes, was classified in order of magnitude as follows: a) nuclear membranes; b) outer mitochondrial membrane and rough endoplasmic reticulm (RER); and c) Golgi complex, mitochondrial cristae and plasma membranes. In lung alveoli, the phospholipid-rich surfactant material was intensely labeled. Labeling of lung thin sections from chlorphentermine-treated rats (phospholipidosis-inducing drug) further demonstrates the reliability of PLA2-gold to label phospholipids. CPD-FL replicas and TS-FL specimens further extended the TEM observations: nuclear membranes and RER were more intensely labeled than plasma membranes. In exocrine pancreatic cells, two distinct labeling patterns were found for secretory granule membranes: sparse and dense. The specificity and reliability of the labeling were confirmed through several control experiments. The studies performed thus demonstrate the great potential of the PLA2-gold technique as a new approach to the high-resolution study of phospholipid distribution and density among biological structures.     

Heterogeneous distribution of phospholipids in membranes along the secretory pathway in pancreatic B-cells

The membrane content in phospholipids along the secretory pathway in rat pancreatic B-cells was studied in situ by high-resolution cytochemistry, applying the recently introduced phospholipase A2-gold technique. The gold particles were mostly associated with cell membranes, and the various types of membranes were labeled to a different extent. Quantitation of the labeling over these membranes revealed a heterogeneous distribution of the labeling across the secretory pathway. This heretogeneity occurred mainly as a progressive, decreasing gradient in the first half of this pathway, between the rough endoplasmic reticulum and the mi-cisternae of the Golgi apparatus. The labeling density remained at a lower level in the trans-most Golgi cisternae and immature secretory granule membranes, to increase in the mature secretory granule membrane, where it reached the value found in the plasma membrane. These results provide evidence that the functional heterogeneity existing across the membrane forming the secretory pathway is parallelled by substantial changes in their phospholipid content.     

Role of phospholipase A inhibition in amiodarone pulmonary toxicity in rats

Amiodarone is effective in the treatment of ventricular and supraventricular arrhythmias. In man its clinical use is associated with the accumulation of phospholipid-rich multilamellar inclusions in various tissues including lung, liver and others. This report presents evidence showing that amiodarone is a potent inhibitor of lysosomal phospholipase A from rat alveolar macrophages, J-744 macrophages and rat liver. When compared with other cationic amphiphilic agents which are known to produce phospholipidosis, amiodarone is one of the most potent inhibitors yet discovered. The subcellular localization of amiodarone has been determined in lung and its distribution was consistent with a lysosomal localization. It is hypothesized that amiodarone causes cellular phospholipidosis by concentrating in lysosomes and inhibiting phospholipid catabolism.     

Influence of specimen preparation on the identification of phospholipids by the phospholipase A2-gold method in mineralizing cartilage and bone

The role of phospholipids in biological mineralization has been hypothesized but not fully elucidated. In order to identify phospholipids at the ultrastructural level in the mineralizing extracellular matrix, rat epiphyseal cartilage and metaphyseal bone have been labeled with the phospholipase A₂ (PLA₂)-gold method. The specificity and the efficiency of phospholipid detection have been evaluated by postembedding labeling of sections from epoxy- or hydrophilic resin-embedded samples, and by preembedding labeling of cryosectioned samples. The efficiency of the labeling was higher in cryosections than in hydrophilic resin-embedded specimens, while lower efficiency was found in epoxy resiembedded samples. A 2- to 6-fold increase of the labeling density in calcified with respect to uncalcified areas of cartilage and bone has been found, depending on the specimen preparation used. The labeling intensity was significantly higher, at the periphery of the calcifying nodules in the epiphyseal cartilage matrix and in the calcifying osteoid, while the fully calcified bone matrix presented a weak labeling. Matrix vesicles, which are considered a possible source of extracellular phospholipids, appeared labeled in cryosections and in epoxy resin-embedded samples after a preincubation with PLA₂, which also increased the labeling of the intracellular membranes. The localization of phospholipids in the areas of initial mincralization suggests some hypotheses on the possible involvement of these molecules in the mineralphase deposition process.     

Localization of phospholipase A2 in normal and ras-transformed cells

The cellular localization of phospholipase A2 (PLA2) was examined in normal and ras-transformed rat fibroblasts using immunohistochemical techniques. Polyclonal antibodies were generated against porcine pancreatic PLA2 and were affinity purified for use in this study. The antibodies detected a 16-kD band on immunoblots of total cellular proteins from fibroblasts. In cell-free assays of phospholipase A2 activity, the purified antibodies inhibited the bulk of the enzyme activity whereas control IgG preparations had no effect. Immunofluorescence microscopy indicated that PLA2 was diffusely distributed throughout the cell. Increased concentration of PLA2 was detected under membrane ruffles in normal and ras-transformed cells. Specific immunofluorescence staining was also detected on the outer surface of the normal cells. Immunoelectron microscopy demonstrated the increased accumulation of PLA2 in membrane ruffles and also revealed the presence of the enzyme in microvilli and its association with intracellular vesicles. Ultrastructural localization of PLA2 and the ras oncogene protein, using a double immunogold labeling technique, indicated a spatial proximity between PLA2 and ras proteins in the ruffles of ras-transformed cells. The possible role of PLA2 in the structural rearrangements that underlie membrane ruffling is discussed.     

PLRP2 selectively localizes synaptic membrane proteins via acyl-chain remodeling of phospholipids

The plasma membrane of neurons consists of distinct domains, each of which carries specialized functions and a characteristic set of membrane proteins. While this compartmentalized membrane organization is essential for neuronal functions, it remains controversial how neurons establish these domains on the laterally fluid membrane. Here, using immunostaining, lipid-MS analysis and gene ablation with the CRISPR/Cas9 system, we report that the pancreatic lipase-related protein 2 (PLRP2), a phospholipase A1 (PLA1), is a key organizer of membrane protein localization at the neurite tips of PC12 cells. PLRP2 produced local distribution of 1-oleoyl-2-palmitoyl-PC at these sites through acyl-chain remodeling of membrane phospholipids. The resulting lipid domain assembled the syntaxin 4 (Stx4) protein within itself by selectively interacting with the transmembrane domain of Stx4. The localized Stx4, in turn, facilitated the fusion of transport vesicles that contained the dopamine transporter with the domain of the plasma membrane, which led to the localized distribution of the transporter to that domain. These results revealed the pivotal roles of PLA1, specifically PLRP2, in the formation of functional domains in the plasma membrane of neurons. In addition, our results suggest a mode of membrane organization in which the local acyl-chain remodeling of membrane phospholipids controls the selective localization of membrane proteins by regulating both lipid-protein interactions and the fusion of transport vesicles to the lipid domain.     

Immunoelectron microscopic localization of cell surface antigens on rat hepatocytes detected with monoclonal antibodies (HAM2 and HAM4).

The localization of surface antigens and the binding activity of two monoclonal antibodies, HAM2 and HAM4, which recognize the rat major histocompatibility complex (MHC) antigen class I and the rat hepato-renal antigen respectively, on dissociated (free) hepatocytes was examined by light (LM) and electron microscopy (EM), and by radioimmunoassay (RIA). Fixed hepatocytes, fixed before dissociation, and fresh hepatocytes, dissociated by collagenase, were treated by direct staining with HAM2- or HAM4-immunogold complexes (HAM2-gold and HAM4-gold). Some of the directly stained hepatocytes were further mixed with antimouse IgG-gold complex (IgG-gold) to supplement the direct staining. The polarity of the sinusoidal and contiguous faces and the bile canaliculus, i.e. the in situ morphology, was well preserved in the fixed hepatocytes, while the fresh cells had lost the polarity and were round. On the fixed hepatocytes HAM2-gold particles were distributed predominantly on the sinusoidal face, while HAM4-gold particles were localized on both the bile canalicular and sinusoidal faces. No different antigen distribution on the fresh cells was detected with the two antibodies. Supplementation by IgG-gold was noticeable in most cases. The extent of binding activity in both the immunogold and RIA experiments was lower in the fixed cells than in fresh cells. These results suggest that HAM2 and HAM4 are useful monoclonal antibodies for detecting the localization of the MHC class I antigen and the hepato-renal antigen on the hepatocytes, respectively.     

Differential mode of attack on membrane phospholipids by an acidic phospholipase A(2) (RVVA-PLA(2)-I) from Daboia russelli venom

An acidic phospholipase A(2) (RVVA-PLA(2)-I) purified from Daboia russelli venom demonstrated dose-dependent catalytic, mitochondrial and erythrocyte membrane damaging activities. RVVA-PLA(2)-I was non-lethal to mice at the tested dose, however, it affected the different organs of mice particularly the liver and cardiac tissues as deduced from the enzymatic activities measured in mice serum after injection of this PLA(2) enzyme. RVVA-PLA(2)-I preferentially hydrolyzed phospholipids (phosphatidylcholine) of erythrocyte membrane compared to the liver mitochondrial membrane. Interestingly, RVVA-PLA(2)-I failed to hydrolyze membrane phospholipids of HT-29 (colon adenocarcinoma) cells, which contain an abundance of phosphatidylcholine in its outer membrane, within 24h of incubation. The gas-chromatographic (GC) analysis of saturated/unsaturated fatty acids' release patterns from intact mitochondrial and erythrocyte membranes after the addition of RVVA-PLA(2)-I showed a distinctly different result. The results are certainly a reflection of differences in the outer membrane phospholipid composition of tested membranes owing to which they are hydrolyzed by the venom PLA(2)s to a different extent. The chemical modification of essential amino acids present in the active site, neutralization study with polyvalent antivenom and heat-inactivation of RVVA-PLA(2)-I suggested the correlation between catalytic and membrane damaging activities of this PLA(2) enzyme. Our study advocates that the presence of a large number of PLA(2)-sensitive phospholipid domains/composition, rather than only the phosphatidylcholine (PC) content of that particular membrane may determine the extent of membrane damage by a particular venom PLA(2) enzyme.     

热应激时大鼠肺组织中β—肾上腺素受体的变化与膜磷...

To explore the relationship between the change of beta-adrenoceptor and the metabolism of phospholipids in lung tissue from acute heat stressed rats, the Bmax of beta-adrenoceptors, the activity of phospholipase A2 (PLA2), the content of phosphatidylcholine (PC) and phosphatidylserine (PS), and membrane fluidity in lung tissue of normal and heat stressed rats were investigated. The relevant parameter values mentioned above were 479 +/- 94 fmol/mg protein, 78.5 +/- 8.2 U, 53.5 +/- 1.7 mg/g.wet. w. and 425.1 +/- 68.1 micrograms/g.wet. w. respectively. Whereas in the heat stressed rats with rectal temperature raised to 42 degrees C for 15 min, the Bmax of beta-adrenoceptor was decreased by 43% (P less than 0.01), the activity of PLA2 increased by 83% (P less than 0.01), the contents of PC and PS decreased by 50% and 47% (P less than 0.01) respectively. A lower membrane fluidity in lung tissue for heat stressed rats was also demonstrated. The results suggest that the decreased binding sites of beta-adrenoceptor in lung tissue of rat during hyperthermia may be contributed to the activation of PLA2, which then accelerated the catabolism of phospholipids such as PC and PS in the cell plasma membrane, with a consequent alteration of membrane fluidity.     

Asymmetric distribution of phosphoinositides and phosphatidic acid in the human erythrocyte membrane.

The distribution of phosphoinositides and phosphatidic acid (PA) between the outer and inner layers of the human erythrocyte membrane was investigated by using two complementary methodologies: hydrolysis by phospholipase A2 (PLA2) and immunofluorescence detection with monoclonal antibodies against polyphosphoinositides. The contents of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP) and PA were decreased by 15-20% after 60 min incubation with PLA2, while that of phosphatidylinositol (PI) was increased. Studies with 32P-labelled cells revealed that PLA2 treatment led to indirect effects on the metabolism of these phospholipids. Therefore, the asymmetric distribution of phosphoinositides and PA was inferred from the data obtained in ATP-depleted erythrocytes. In these cells with arrested phosphoinositide metabolism, the asymmetric distribution of the major phospholipids was maintained: PLA2 hydrolyzed approx. 20% of PI, PIP2 and PA (but no PIP) indicating their localization in the outer layer of the membrane. This finding was confirmed by immunofluorescence studies with antibodies specific to each phosphoinositide. External addition of anti-PIP2 but not anti-PIP gave a positive reaction both in control and in ATP-depleted erythrocytes. A pretreatment of cells with PLA2 led to a decrease in the intensity of anti-PIP2 staining. These results demonstrate that significant fractions of PIP2, PI and PA are localized on the outer surface of the erythrocyte membrane.     

Distribution and submicroscopic immunogold localization of cellular prion protein (PrPc) in extracerebral tissues

In transmissible spongiform encephalopathies (TSE), such as scrapie in animals and Creutzfeldt-Jakob disease in humans, the central event is the conversion of a host-encoded amyloidogenic protein (PrPc) into an abnormal isoform (PrPsc) that accumulates as amyloid in TSE brain. PrPc is a membrane sialoglycoprotein synthesized in the central nervous system and elsewhere. We have examined the ultrastructural localization of PrPc in numerous hamster and some human extracerebral tissues, by means of a post-embedding electron-microscopic method combined with immunogold labeling. In stomach, intestine, lung, and kidney from hamsters, and in stomach, kidney, and spleen from humans, immunogold labeling specific for PrPc is observed on various cellular substructures related to secretory pathways: Golgi apparatus, secretory globules, and plasma membrane. In mucous epithelial cells of stomach and intestine, PrPc appears to be concentrated in secretory globules, suggesting a role for PrPc in the secretory function of the digestive tract. The secretory aspect of PrPc may be a key to understanding the physiopathological mechanisms underlying TSE.     

Immuno-localization of peroxidase in pumpkin (Cucurbita ficifolia Bouché) seedlings exposed to high-dose gamma ray

Peroxidases (PODs) were localized in pumpkin tissues by using an immunogold labeling technique in combination with transmission electron microcopy (TEM). Polyclonal antibodies raised in rabbits against horseradish peroxidase were utilized. The localization patterns of gold particles for peroxidases from tissues of both control and gamma-irradiated plants were typically present in the plasma membrane, cytosol, and cell walls. However, particle densities were remarkably increased on the cell corner middle lamellae of parenchyma cells, especially in the petioles. Based on these results, we propose that growth inhibition is associated with cell-wall stiffening, as related to the formation of cross-links among cell wall polymers. In addition, the densities of POD following gamma irradiation vary according to cell and tissue types in pumpkin.     

Phospholipase A2 in the central nervous system: implications for neurodegenerative diseases

Phospholipase A2 (PLA2) belongs to a family of enzymes that catalyze the cleavage of fatty acids from the sn-2 position of phospholipids. There are more than 19 different isoforms of PLA2 in the mammalian system, but recent studies have focused on three major groups, namely, the group IV cytosolic PLA2, the group II secretory PLA2 (sPLA2), and the group VI Ca(2+)-independent PLA2. These PLA2s are involved in a complex network of signaling pathways that link receptor agonists, oxidative agents, and proinflammatory cytokines to the release of arachidonic acid (AA) and the synthesis of eicosanoids. PLA2s acting on membrane phospholipids have been implicated in intracellular membrane trafficking, differentiation, proliferation, and apoptotic processes. All major groups of PLA2 are present in the central nervous system (CNS). Therefore, this review is focused on PLA2 and AA release in neural cells, especially in astrocytes and neurons. In addition, because many neurodegenerative diseases are associated with increased oxidative and inflammatory responses, an attempt was made to include studies on PLA2 in cerebral ischemia, Alzheimer's disease, and neuronal injury due to excitotoxic agents. Information from these studies has provided clear evidence for the important role of PLA2 in regulating physiological and pathological functions in the CNS.     

Cellular localization of group IIA phospholipase A2 in rats.

It has been known that group II phospholipase A2 (PLA2) mRNA and protein are present in the homogenates of the spleen, lung, liver, and kidney in normal rats, but the cellular origin of this enzyme has not been yet identified. At present, five subtypes of group II PLA2 have been identified in mammals. Antibodies or mRNA probes previously used for detecting group II PLA2 need to be evaluated to identify the subtypes of group II PLA2. In this study we tried to identify group IIA PLA2-producing cells in normal rat tissues by in situ hybridization (ISH) using an almost full-length RNA probe for rat group IIA enzyme. Group IIA PLA2 mRNA was detected in megakaryocytes in the spleen and Paneth cells in the intestine by ISH. These cells were also immunopositive for an antibody raised against group IIA PLA(2) isolated from rat platelets. Group IIA PLA2 mRNA-positive cells were not detected in lung, liver, kidney, and pancreas. Under normal conditions, group IIA PLA2-producing cells are splenic megakaryocytes and intestinal Paneth cells in rats.     

Human group IVC phospholipase A2 (cPLA2gamma). Roles in the membrane remodeling and activation induced by oxidative stress

To create the unique properties of a certain cellular membrane, both the composition and the metabolism of membrane phospholipids are key factors. Phospholipase A(2) (PLA(2)), with hydrolytic enzyme activities at the sn-2 position in glycerophospholipids, plays critical roles in maintaining the phospholipid composition as well as producing bioactive lipid mediators. In this study we examined the contribution of a Ca(2+)-independent group IVC PLA(2) isozyme (cPLA(2)gamma), a paralogue of cytosolic PLA(2)alpha (cPLA(2)alpha), to phospholipid remodeling. The enzyme was localized in the endoplasmic reticulum and Golgi apparatus, as seen using green fluorescence fusion proteins. Electrospray ionization mass spectrometric analysis of membrane extracts revealed that overexpression of cPLA(2)gamma increased the proportion of polyunsaturated fatty acids in phosphatidylethanolamine, suggesting that the enzyme modulates the phospholipid composition. We also found that H(2)O(2) and other hydroperoxides induced arachidonic acid release in cPLA(2)gamma-transfected human embryonic kidney 293 cells, possibly through the tyrosine phosphorylation pathway. Thus, we propose that cPLA(2)gamma is constitutively expressed in the endoplasmic reticulum and plays important roles in remodeling and maintaining membrane phospholipids under various conditions, including oxidative stress.     

An anti-NH2-terminal antibody localizes NBCn1 to heart endothelia and skeletal and vascular smooth muscle cells

The electroneutral sodium bicarbonate cotransporter NBCn1 or NBC3 was originally cloned from rat aorta and from human skeletal muscle. NBCn1 (or NBC3) has been localized to the basolateral membrane of various epithelia, but thus far it has been impossible to detect the protein in these tissues by using anti-COOH-terminal antibodies. Hence an antibody was developed against the NH2-terminus of NBCn1 and was validated by peptide recognition and immunoblotting on positive control tissues and by binding of an approximately 180-kDa protein in the rat kidney, cerebrum, cerebellum, and duodenum. In addition, an approximately 180-kDa immunoreactive band appeared using samples from the aorta, heart ventricles and atria, mesenteric arteries, lung, spleen, liver, pancreas, and epididymis. Immunohistochemical analysis confirmed the previously described labeling in the kidney, duodenum, and the choroid plexus. The anti-NH2-terminal antibody localized NBCn1 to the plasma membrane domains of endothelia and smooth muscle cells in small mesenteric and renal arteries, as well as the capillaries of the heart ventricles, spleen, and salivary glands. NBCn1 was also detected in neuromuscular junctions and vasculature in skeletal muscle. Analysis of variable NBCn1 splicing by RT-PCR revealed that an NH2-terminal sequence, the cassette III, seems absent from cardiovascular NBCn1 and that both cassettes I and III are variable in most epithelia, whereas cassette II is absent from epithelial NBCn1. Thus the development of the NH2-terminal antibody allowed the localization of NBCn1 protein to major cardiovascular tissues where NBCn1 mRNA was previously detected. The NBCn1 is a likely candidate for mediating the reported electroneutral Na+-HCO3(-) cotransport in vascular smooth muscle.     

Newborn response to cationic amphiphilic drugs

Administration of various cationic amphiphilic drugs in utero results in induction of a phospholipid storage disorder in many tissues, particularly in lungs. In addition to the phospholipidosis in utero, drug exposure results in toxicity to the offspring; newborn rats die within 48 h of birth. Although drug-induced pulmonary pathological changes appear to be involved in the observed mortality, this relationship remains unclear. In contrast to mammals, administration of cationic amphiphilic drugs to the chick embryo seems not to induce phospholipid storage in the tissues examined. Treatment of newborn rats directly with these drugs also induces phospholipidosis in several tissues including lung and kidney; however, mortality does not occur. Concurrent administration of phenobarbital and chlorphentermine reduces or prevents amphiphilic drug-induced phospholipid storage in newborn rat lung and kidney. Modification of chlorphentermine actions by phenobarbital may be caused by alterations in amphiphilic drug excretion, metabolism, and catabolic phospholipase activity. Evidence thus indicates that regardless of age, animals appear susceptible to the effects of cationic amphiphilic drugs; however, species and tissues examined, as well as specific drug administration, play an important role in the observed qualitative and quantitative responses.     

Characterisation of a potential biomarker of phospholipidosis from amiodarone-treated rats

A novel and relatively simple analytical method for the separation, characterisation and semi-quantitation of phospholipids (PLs) from extracts of complex biological samples has been developed. This methodology allows PL extracts from cells and tissues to be analysed by liquid chromatography (LC) coupled to electrospray ionisation mass spectrometry (ESI-MS). Complex mixtures of PLs were separated on a high-performance liquid chromatography (HPLC) system using 0.5% ammonium hydroxide in methanol/water/hexane/formate mixture with UV detection at 205 nm. Identification and structural characterisation of molecular species were carried out utilising ESI-MS and MS/MS in the negative ion mode.The abnormal accumulation of PLs (phospholipidosis) was induced in male Sprague-Dawley rats by administration of the cationic amphiphilic drug (CAD), amiodarone. Analysis of the PL profile of liver and lung tissues, lymphocytes and serum from treated rats was carried out using this analytical procedure (LC-ESI/MS/MS). Differences in PL profiles between treated and untreated animals were highlighted by principal component analysis (PCA). This led to the selection of a potential metabolic marker of phospholipidosis (PLD) identified as a lyso-bis-phosphatidic acid (LBPA) derivative, also known as bis(monoglycero)phosphate (BMP). This PL was absent in control animals but was present in quantifiable amounts in all samples from amiodarone-treated rats.     

Localization of mitochondrial 60-kD heat shock chaperonin protein (Hsp60) in pituitary growth hormone secretory granules and pancreatic zymogen granules.

We used quantitative immunogold electron microscopy and biochemical analysis to evaluate the subcellular distribution of Hsp60 in rat tissues. Western blot analysis, employing both monoclonal and polyclonal antibodies raised against mammalian Hsp60, shows that only a single 60-kD protein is reactive with the antibodies in brain, heart, kidney, liver, pancreas, pituitary, spleen, skeletal muscle, and adrenal gland. Immunogold labeling of tissues embedded in the acrylic resin LR Gold shows strong labeling of mitochondria in all tissues. However, in the anterior pitutary and in pancreatic acinar cells, Hsp60 also localizes in secretory granules. The labeled granules in the pituitary and pancreas were determined to be growth hormone granules and zymogen granules, respectively, using antibodies to growth hormone and carboxypeptidase A. Immunogold labeling of Hsp60 in all compartments was prevented by preadsorption of the antibodies with recombinant Hsp60. Biochemically purified zymogen granules free of mitochondrial contamination are shown by Western blot analysis to contain Hsp60, confirming the morphological localization results in pancreatic acinar cells. In kidney distal tubule cells, low Hsp60 reactivity is associated with infoldings of the basal plasma membrane. In comparison, the plasma membrane in kidney proximal tubule cells and in other tissues examined showed only background labeling. These findings raise interesting questions concerning translocation mechanisms and the cellular roles of Hsp60.     

Design and synthesis of conformationally restricted phospholipids as phospholipase A2 inhibitors

The use of conformationally restricted phospholipids 1 and 2 has been employed to understand the conformational preference of phospholipase A2 (PLA2) for substrate phospholipids. Inhibition of porcine pancreatic PLA2 with 1 and 2 indicated a two- to fivefold preference for the distal isomer 2 over the proximal isomer 1. Based upon these studies, both side-chains of the substrate phospholipid appear to occupy the lipid binding domains near the active site with the side-chains further apart most preferred by PLA2.