雪花莲凝集素基因转化小麦及转基因小麦抗蚜性的研究

雪花莲凝集素对具有刺吸式口器的同翅目害虫具有毒杀作用。用基因枪法将1个新的雪花莲凝集素(GNA)基因转入普通春小麦品种中-60634和生产上正在推广的冬小麦高产品种——豫麦66中,分别获得了转基因小麦植株。抗蚜实验证明,转化gna基因的小麦植株对我国北方冬麦区的主要麦蚜——麦长管蚜和禾谷缢管蚜的抗性效果不尽相同。对禾谷缢管蚜,在接种当代即表现出明显的毒杀作用。对麦长管蚜,则表现为虫体发育减缓并且降低了其所生产的若蚜成活率。在自然放养条件下,gna基因则对这两种麦蚜的取食均起到了一定的抑制作用。     

GNA成熟蛋白基因亚克隆及其原核表达载体构建

雪花莲外源凝集素（GNA）对刺吸式昆虫和某些咀嚼式昆虫以及多种线虫均有毒性,从含GNA前体蛋白基因的质粒中亚克隆出GNA的成熟蛋白基因MGNA,将MGNA基因插入大肠杆菌表达载体pET22b的不同位点,再经测序验证,得到了三种不同表达形式的GNA原核表达载体;22bG1(分泌型融合GNA蛋白）,22bG2(包涵体型GNA蛋白）,22bG3(分泌型天然GNA蛋白）,这为进一步在大肠杆菌中表达GNA和将GNA制成生物农药奠定了基础。     

Purification and Characterization of a Major Cell Surface Glycoprotein in Zajdela Ascites Hepatoma Cells Which Displays a Potent Concanavalin A Receptor Activity

A major cell surface sialoglycoprotein with Concanavalin A receptor activity has been isolated from rat Zajdela ascites hepatoma cells. The sialic acid residues of the plasma membrane glycoproteins were specifically labeled by oxidation with NaIO₄ followed by reduction with NaB³H₄. Surface-labeled glycoproteins were released by short incubations with TPCK-trypsin at 37°C and then separated by gel filtration on Sepharose 6B column. The predominantly labeled fraction, GP II₂, was then purified by chromatography on DEAE-cellulose equilibrated with 0.05 M phosphate buffer, pH 7.5, and eluted with increasing molarities of NaCl. It was shown to be homogeneous by protein and carbohydrate staining on SDS-polyacrylamide gels, isoelectric focusing, rechromatography on DEAE-cellulose and immunoelectrophoresis. It has an apparent molecular weight of 110,000 daltons. The location of GP II₂on the cell surface was confirmed by the fact that it could be labeled metabolically with, D-(³H) glucosamine and externally through the nonpenetrating periodate-NaB³H₄ system. GP II₂could not be removed from the cell surface by high salt concentrations, chelator, or chaotropic agents but was released from the membrane by detergents. This suggests that GP II₂could be an integral protein. Analysis of the carbohydrate composition of GP II₂ revealed galactose, N-acetylglucosamine, N-acetylgalactosamine, and sialic acid as major constituents and mannose as a minor one. This suggests that it contains carbohydrate chains both O- and N-linked to the polypeptide chain, most of them being O-linked. Finally, GP II₂has a potent Concanavalin A receptor activity. It inhibits the interaction between Concanavalin A and hepatoma cells and suppresses its effects on hepatoma cell proliferation.     

Alteration of surfactant protein A expression in renal cell carcinoma

Surfactant protein-A (SP-A) belongs to a family of collagen-containing C-type lectins called collectins. SP-A is expressed by renal tubule epithelial cells. We investigated the distribution of SP-A in renal cell carcinomas (RCC) using immunohistochemical techniques and western blotting. We used 35 formalin fixed, paraffin embedded (FFPE) RCC tissue samples. We compared results with clinico-pathological parameters of RCC including age, sex, Fuhrman grade, tumor volume, tumor node metastasis (TNM) and clinical stage. SP-A was localized in the glomerulus and renal tubule epithelium in nontumor tissue and strong SP-A immunoreactivity was observed in tumor tissue. SP-A was expressed in the RCC tumor cells (64%) and nontumor cells (34%) in males and RCC tumor cells (90%) and nontumor cells (30%) in females. There was a significant correlation between SP-A immunoreactivity in tumor cells and gender, age, tumor diameter, Fuhrman grade and tumor diameter. Western blot analysis supported the immunohistochemical findings. We present evidence for involvement of SP-A in RCC and suggest that increased SP-A expression in RCC is associated with favorable prognosis.     

Large-scale production and purification of recombinant Galanthus nivalis agglutinin (GNA) expressed in the methylotrophic yeast Pichia pastoris

The gene coding for agglutinin from Galanthus nivalis (GNA) was expressed in, and secreted by, the methylotrophic yeast, Pichia pastoris. Transformants of P. pastoris were selected and a process to produce and purify gram quantities of recombinant GNA was developed. GNA was secreted at approximately 80 mg l^–1 at the 200 l scale and was purified to 95% homogeneity using hydrophobic interaction chromatography. The recombinant protein was similar to the protein synthesised in plant with respect to structure and biological activity.     

Specific and Common Epitopes in Mating Pheromones of Euplotes raikovi Revealed by Monoclonal Antibodies

Polypeptide mating pheromones Er-1 and Er-2, purified from the supernatant of Euplotes raikovi cultures of mating type I and mating type II, respectively, were used to immunize mice and obtain monoclonal antibodies. Five hybridoma clones producing antibodies specific to the mating pheromones were selected. They were analyzed for immunospecificity by immunoperoxidase assay, immunoblotting, and for their efficacy in inhibition of mating pheromone activity. Monoclonal antibodies from two hybridoma clones recognized only the mating pheromone used as antigen; those from the other three clones reacted, to comparable extents, with both mating pheromones. On the basis of these results it was assumed that two immunogenic sites exist in Er-1 and Er-2, one specific and the other common to both mating pheromones.     

Concanavalin A-mediated phagocytosis of yeast by Biomphalaria glabrata hemocytes in vitro: Effects of temperature and lectin concentration

Exposure of either Biomphalaria glabrata granulocytes or yeast to concanavalin A (Con A) prior to being incubated together in a “microhemadsorption assay” affected the number of yeast attached to or endocytosed by the granulocytes after 60 min. The yeast-granulocyte interaction was sensitive to temperature, Con A concentration, and the cell treated. Numbers of associated (attached + endocytosed) and endocytosed yeast either increased or decreased, as compared with nontreated controls, depending on the protocol. The effect of a given protocol was not necessarily similar for attachment and endocytosis. By separately enhancing these two stages of phagocytosis, Con A may be a useful tool for studying this process of molluscan granulocytes.     

Inhibition of auxin-induced elongation and xyloglucan breakdown in azuki bean epicotyl segments by fucose-binding lectins

Auxin-induced elongation of epicotyl segments of azuki bean ( Vigna angularis Ohwi and Ohashi cv. Takara) was suppressed by fucose-binding lectins from Tetragonolobus purpureus Moench and Ulex europaeus L. These lectins also inhibited auxin-induced cell wall loosening (decrease in the minimum stress-relaxation time of the cell walls) of segments. Auxin caused a decrease in molecular mass of xyloglucans extracted with 24% KOH from the cell walls. The lectins inhibited auxin-induced changes in molecular mass of the xyloglucans. The autolytic release of xylose-containing products from the pectinase-treated cell walls was also suppressed by the lectins. Fucose-binding lectins pretreated with fucose exhibited little or no inhibitory effect on auxin-induced elongation, cell wall loosning, or breakdown of xyloglucans. These results support the view that the breakdown of xyloglucans is involved in the cell wall loosening responsible for auxin-induced elongation in dicotyledons.     

Proliferating cell nuclear antigen (PCNA) as a marker of cell proliferation in the marine dinoflagellate <Emphasis Type="BoldItalic">Prorocentrum donghaiense</Emphasis> Lu and the green alga <Emphasis Type="BoldItalic">Dunaliella salina</Emphasis> Teodoresco

Proliferating cell nuclear antigen (PCNA) was detected in Prorocentrum donghaiense Lu and Dunaliella salina Teodoresco by enhanced chemiluminescence techniques with one mono-antibody. The observed band detected on western blots of P. donghaiense and D. salina had a molecular weight of 36–33 kDa rather than a PCNA-like protein with a size of 55 kDa reported in the dinoflagellates Crypthecodinium cohnii Biecheler and Gymnodinium catenatum Brav. The abundance of PCNA proteins was growth-stage dependent. Whole-cell immunoflurescence labeling showed that the PCNA antibody specifically stained the target proteins in P. donghaiense and D. salina, and PCNA is only present in the nucleus during the cell cycle. Synchronized cells of P. donghaiense show a cell cycle specific expression pattern with the highest expression in S phase and little expression in the G₁ and G₂/M phases. The results demonstrated that the PCNA-like proteins could be a marker for the estimation of marine phytoplankton growth rates. The different sizes of the PCNA-like proteins observed in dinoflagellates could be related to the variety of dinoflagellate chromosomal structure.     

A ferredoxin-type iron-sulfur protein gene,frx B,is expressed in the chloroplasts of tobacco and spinach

Previously, a ferredoxin-type iron-sulfur protein, frx B protein, was identified in a high-salt extract of the purified thylakoid membrane of Chlamydomonas reinhardtii, a unicellular green alga. Polyclonal antibody was raised against a synthetic pentadecameric peptide with an amino acid sequence corresponding to the highly conserved region of the putative frx B proteins of 3 land plants [21]. In this report, protein(s) reacting strongly and specifically with this antibody was detected in the equivalent high-salt extract prepared from purified chloroplast of spinach and tobacco. One strong reaction polypeptide band from tobacco chloroplast was purified from SDS-polyacrylamide gel and subjected to endoproteinase lys C digestion. The resulting polypeptides were separated by reversed-phase chromatography. N-terminal sequencing of 3 purified polypeptides revealed that the protein is encoded by the frxB gene identified from DNA sequence analysis.     

Binding of the insecticidal lectin Concanavalin A in pea aphid, Acyrthosiphon pisum (Harris) and induced effects on the structure of midgut epithelial cells

Concanavalin A (lectin from Canavalia ensiformis L., ConA) has previously been shown to act as a feeding inhibitor for Acyrthosiphon pisum, the pea aphid. In the present study a range of histochemical and biochemical techniques were used to elucidate the target tissues and binding sites of the lectin in the aphid. Diet uptake was evaluated using a radioactive tracer (¹⁴C-methylated inulin) and demonstrated that adults were capable of ingesting high quantities of the toxin (approx. 1 μg over a 48 h period). Electophoretic analysis and enzyme-linked immuno-sorbent assay of honeydew samples confirmed these results and further demonstrated that only small levels of ConA were excreted. Histofluorescence and immunolocalisation studies on nymphs revealed that the stomach was the primary target for ConA. At concentrations up to 400 μg ml⁻¹, lectin binding only occurred in the stomach region, however, at high concentrations (800 μg ml⁻¹) the whole digestive tract was stained, although there was no evidence of binding in either the oesophagus or rectum. In addition to binding, there was evidence to suggest that ConA was also causing systemic effects in that the lectin appeared to cross the intestinal epithelial barrier. Immunohistochemical and electron microscopy studies revealed that ConA induced severe cellular swelling of the epithelial cells, accompanied by hypersecretion and a progressive detachment of the apical membrane; however, the striated border itself did not appear to be directly affected. Furthermore, there was no lysis of the epithelium, nor loss of integrity of the epithelial cells themselves. Our results suggest that ConA interacts with glycosylated receptors at the surface of the stomach epithetial cells, interfering with normal metabolism and cell function, resulting in a rapid feedback response on feeding behaviour. Whilst our results provide a much greater understanding regarding the modes of action of ConA in insects, they suggest that different lectins, including other mannose binding lectins, have different modes of action at the cellular levels, and thus generalizations should be treated with caution.     

Uterine lipid alterations during early pseudopregnancy and following the artificial induction of decidualization by concanavalin A in QS mice

Lipid extracts of whole uterine tissue from mice were examined by gas chromatography-mass spectrometry during days 2, 3, and 4 of pseudopregnancy (day 1 = copulatory plug) and following the artificial induction of the decidual cell reaction (DCR) on day 4. The range of lipids identified during pseudopregnancy and their percentage composition on day 2 included saturated fatty acids (SFA, 38%), monounsaturated fatty acids (MUFA, 20%), polyunsaturated fatty acids (PUFA, 17%), sterols (25%), long chain alcohols (0.12%), and alkylglycerols (0.11%). Of these, the main components were the fatty acids 16:0 (21%), 18:0 (14%), cis18:1n-9 (14%), 18:2n-6 (8.5%), and cholesterol (24%). Although only subtle changes in the composition of uterine lipids occurred through days 2 and 3 of pseudopregnancy, more substantial changes were detected on day 4, at a time when the uterus normally initiates its transient “window of receptivity.” Following induction of the DCR with the lectin Concanavalin A (Con A) at this time, even greater alterations in uterine lipid composition were observed. From 20 to 1,280 min post-Con A-treatment the percentage composition of SFA in the treated left uterine horn changed from 43% to 64%, sterols from 19% to 4%, PUFA from 15% to 10%, while MUFA remained unchanged at 23%. The lipid profile of the untreated right uterine horn of these animals was similar to that of the Con A-treated left uterine horn during the early stages. However, by 1,280 min substantial differences were observed, at a time corresponding with Con A-induced uterine growth. In contrast, differences in the lipid profile of Con A- and saline-treated uteri were observed at 320 min post-treatment, a time preceding Con A-induced uterine growth. Furthermore, the tissue concentration (nmol/mg dry weight) of SFA and sterols in uterine tissue decreased significantly following Con A treatment. The results suggest that uterine lipid changes are implicated in the development of uterine receptivity, and in the remodeling of uterine tissue for successful embryonic invasion and the establishment of pregnancy. © 1996 Wiley-Liss, Inc     

Lectin-mediated agglutination of murine lymphoma cells. Cell surface deformability and reversibility of agglutination by saccharides

Agglutination of S49 mouse lymphoma cells by Ricinus communis I agglutinin can be reversed by the competing haptenic saccharide, lactose, soon after agglutination, but after further incubation in the absence of lectin the agglutination reaction could not be reversed by lactose and the cells remained as multicell aggregates. The irreversibility of S49 cell agglutination was time, temperature and lectin concentration dependent and its onset correlated with ultrastructurally observed deformation of adjacent cell surfaces and an increase in the proportion of adjacent cell surface areas in close apposition within multicell aggregates. Pretreatment of S49 cells with cytochalasin B or cytochalasin B plus vinblastine enhanced R. communis I agglutinin-mediated agglutination, while vinblastine alone and fluoride plus azide had essentially no effect. When drug-treated cells were agglutinated and then incubated in lectin-free drug-containing media for various times prior to lactose addition, the drug effects were more pronounced. Cytochalasin B alone or with vinblastine inhibited lactose reversal of S49 cell agglutination compared to the drug-free controls, while fluoride plus azide enhanced hapten reversibility. Electron microscopic analysis revealed that the onset of agglutination irreversibility correlated with cell surface deformation in the drug-treated cells. Cell aggregates that were more readily reversible by lactose (fluoride plus azide) were unchanged or less deformed, while S49 aggregates treated with cytochalasin B plus vinblastine were more deformed compared to controls without drugs. These experiments suggest a role for cell surface deformability as an important secondary effect during lectin-mediated cell agglutination of 849 lymphoma cells.     

环孢素A抑制骨水泥颗粒诱导破骨细胞形成及功能

目的:探讨环孢素A对颗粒诱导破骨细胞形成及功能的影响。方法:取SD仔鼠双侧股骨和胫骨的骨髓,以不含血清的α-MEM培养液洗涤并收集骨髓细胞,再将细胞重悬于含10%胎牛血清及10~(-8)mol/L1,25-(OH)_2D_3的α-MEM培养液中,细胞计数后配成1.5×10~7/ml的细胞悬液,加入聚甲基丙烯酸甲酯(PMMA)颗粒和不同浓度的环孢素A(10~((-8)mol/L、10~(-7)mol/L、10~(-6) mol/L)于24孔培养板进行培养,并设置阳性对照组(只加PMMA颗粒)和阴性对照组(PMMA颗粒和CsA均不加),每组均有4孔放置骨磨片1片进行培养。培养2周后,行抗酒石酸(TRAP)染色检测破骨细胞形成;骨磨片行甲苯胺蓝染色观察。结果:PM- MA颗粒能够诱导大量TRAP染色阳性的破骨细胞形成,骨磨片有吸收陷窝形成;用环孢素A(10~(-8)mol/L、10~(-7)mol/L)和PMMA颗粒共同培养下TRAP染色阳性的破骨细胞形成数量明显减少,环孢素A浓度达到10~(-6)mol/L时无TRAP染色阳性的破骨细胞形成;环孢素A浓度在(10~(-8)mol/L、10~(-7)mol/L)时骨磨片有吸收陷窝形成,但少于阳性对照组,在10~(-6)mol/L时骨磨片则无吸收陷窝的形成。结论:环孢素A对PMMA颗粒诱导的破骨细胞的形成有着明显的抑制作用,且呈剂量依赖性。     

Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae

Genetic variation is frequently mediated by genomic rearrangements that arise through interaction between dispersed repetitive elements present in every eukaryotic genome. This process is an important mechanism for generating diversity between and within organisms^1-3. The human genome consists of approximately 40% repetitive sequence of retrotransposon origin, including a variety of LINEs and SINEs⁴. Exchange events between these repetitive elements can lead to genome rearrangements, including translocations, that can disrupt gene dosage and expression that can result in autoimmune and cardiovascular diseases⁵, as well as cancer in humans^6-9.Exchange between repetitive elements occurs in a variety of ways. Exchange between sequences that share perfect (or near-perfect) homology occurs by a process called homologous recombination (HR). By contrast, non-homologous end joining (NHEJ) uses little-or-no sequence homology for exchange^10,11. The primary purpose of HR, in mitotic cells, is to repair double-strand breaks (DSBs) generated endogenously by aberrant DNA replication and oxidative lesions, or by exposure to ionizing radiation (IR), and other exogenous DNA damaging agents. In the assay described here, DSBs are simultaneously created bordering recombination substrates at two different chromosomal loci in diploid cells by a galactose-inducible HO-endonuclease (Figure 1). The repair of the broken chromosomes generates chromosomal translocations by single strand annealing (SSA), a process where homologous sequences adjacent to the chromosome ends are covalently joined subsequent to annealing. One of the substrates, his3-Δ3'', contains a 3'' truncated HIS3 allele and is located on one copy of chromosome XV at the native HIS3 locus. The second substrate, his3-Δ5'', is located at the LEU2 locus on one copy of chromosome III, and contains a 5'' truncated HIS3 allele. Both substrates are flanked by a HO endonuclease recognition site that can be targeted for incision by HO-endonuclease. HO endonuclease recognition sites native to the MAT locus, on both copies of chromosome III, have been deleted in all strains. This prevents interaction between the recombination substrates and other broken chromosome ends from interfering in the assay. The KAN-MX-marked galactose-inducible HO endonuclease expression cassette is inserted at the TRP1 locus on chromosome IV. The substrates share 311 bp or 60 bp of the HIS3 coding sequence that can be used by the HR machinery for repair by SSA. Cells that use these substrates to repair broken chromosomes by HR form an intact HIS3 allele and a tXV::III chromosomal translocation that can be selected for by the ability to grow on medium lacking histidine (Figure 2A). Translocation frequency by HR is calculated by dividing the number of histidine prototrophic colonies that arise on selective medium by the total number of viable cells that arise after plating appropriate dilutions onto non-selective medium (Figure 2B). A variety of DNA repair mutants have been used to study the genetic control of translocation formation by SSA using this system^12-14.     

Phosphorylated Glycans Produced from Nonreducing Mono- and Oligosaccharides by the Action of P2O5 in Dimethyl Sulfoxide,and Their Interactions with Concanavalin A

The action of P₂O₅ in DMSO on methyl α-d-glucopyranoside, sucrose and trehalose afforded nondializable, phosphorylated glycans in 6~34% yields. Polysucrose has a molecular weight of ~9,500. The synthetic glycans consist of carbohydrate (46~59%) and P (11.4~13.1%) and show reducing sugar values (5.0~30.8%). The alkaline hydrolysis of polysucrose was accompanied with a depolymerization and afforded sugar phosphates and oligosaccharides. The periodate oxidation gave formic acid (0.15~0.34 mole) and formaldehyde (0.07~0.17 mole/monosaccharide residue). The methylation study indicated their variously branched structures. 2,3,4,6-Tetra-O-methyl-d-glucose was found in only 0.7 ~3.2% yields, and this is in agreement with their weak precipitation reactions with Con A. It is considered that the glycans are produced from nonreducing mono- and oligosaccharides by dehydration, transglycosidation and esterification with phosphate.     

Regulation of Sealing Ring Formation by L-plastin and Cortactin in Osteoclasts

The aim of this study is to identify the exact mechanism(s) by which cytoskeletal structures are modulated during bone resorption. In this study, we have shown the possible role of different actin-binding and signaling proteins in the regulation of sealing ring formation. Our analyses have demonstrated a significant increase in cortactin and a corresponding decrease in L-plastin protein levels in osteoclasts subjected to bone resorption for 18 h in the presence of RANKL, M-CSF, and native bone particles. Time-dependent changes in the localization of L-plastin (in actin aggregates) and cortactin (in the sealing ring) suggest that these proteins may be involved in the initial and maturation phases of sealing ring formation, respectively. siRNA to cortactin inhibits this maturation process but not the formation of actin aggregates. Osteoclasts treated as above but with TNF-α demonstrated very similar effects as observed with RANKL. Osteoclasts treated with a neutralizing antibody to TNF-α displayed podosome-like structures in the entire subsurface and at the periphery of osteoclast. It is possible that TNF-α and RANKL-mediated signaling may play a role in the early phase of sealing ring configuration (i.e. either in the disassembly of podosomes or formation of actin aggregates). Furthermore, osteoclasts treated with alendronate or αv reduced the formation of the sealing ring but not actin aggregates. The present study demonstrates a novel mechanistic link between L-plastin and cortactin in sealing ring formation. These results suggest that actin aggregates formed by L-plastin independent of integrin signaling function as a core in assembling signaling molecules (integrin αvβ3, Src, cortactin, etc.) involved in the maturation process.     

Laminin A, B1, and B2 Chain Gene Expression in Transected and Regenerating Nerves: Regulation by Axonal Signals

Abstract: Laminin A, B1, and B2 chain mRNA levels in degenerating and regenerating mouse sciatic nerves were examined using northern blot analysis. In normal intact nerves, B1 and B2 mRNA steady-state levels were high, but when the nerves were crushed, the steady-state levels of B1 and B2 mRNA per milligram wet tissue weight of the distal segments of the nerves increased five- to eightfold over that of control levels as the total RNA and β-actin mRNA levels increased, suggesting that these increases were the consequence of Schwann cell proliferation after axotomy. When the steady-state levels of B1 and B2 mRNA were normalized as the ratio to total RNA or β-actin mRNA levels, however, they drastically decreased to about 20% of the normal nerve levels in the nerve segments distal to both the crush and transaction sites 1 day after injury. In the crushed nerves, B1 and B2 mRNA levels gradually increased as the regenerating nerves arrived at the distal segments and reestablished normal axon–Schwann cell contact, and then returned to normal levels on the 21 st day. In the transected nerves, where Schwann cells continued to be disconnected from axons, both B1 and B2 mRNA levels remained low. Cultured Schwann cells expressed detectable levels of B1 and B2 chain mRNA which significantly increased when the cells were cocultured with sensory neurons. However, mRNA for A chain was not detectable in the normal, axotomized nerves or in cultured Schwann cells. These data indicate that Schwann cells express laminin B1 and B2 chain mRNA that are up-regulated by axonal or neuronal contact, but they do not express A chain mRNA.     

Formation of Scales in Paraphysomonas vestita and the Inhibition of Growth by Germanium Dioxide*

SYNOPSIS Paraphysomonas vestita (Stokes), a colorless flagellate, has silicified external scales. In this study EDAX analysis of the scales confirmed earlier experimental evidence that silicon is the major component of the scales. The scales are formed in the endoplasmic reticulum of the cytoplasm and carried to the plasmalemma in vesicles. The flagellate is euryhaline growing in a range from 2.6X seawater to near distilled water. From the salinity data it appears that Paraphysomonas vestita is the same as Paraphysomonas imperforata Lucas. A GeO₂ concentration of 2.0 mg l^-1 (19.2 μM Ge) results in complete inhibition of growth.     

Inhibition of human gastric and pancreatic lipases by chiral alkylphosphonates. A kinetic study with 1,2-didecanoyl-sn-glycerol monolayer

Enantiomerically pure alkylphosphonate compounds RR′P(O)PNP (R=C_nH_2n+1, R′=OY with Y=C_n′H_2n′+1 with n=n′ or n≠n′; PNP=p-nitrophenoxy) noted (RY), mimicking the transition state occurring during the carboxyester hydrolysis were synthesized and investigated as potential inhibitors of human gastric lipase (HGL) and human pancreatic lipase (HPL). The inhibitory properties of each enantiomer have been tested with the monomolecular films technique in addition to an enyzme linked immunosorbent assay (ELISA) in order to estimate simultaneously the residual enzymatic activity as well as the interfacial lipase binding. With both lipases, no obvious correlation between the inhibitor molar fraction (₅₀) leading to half inhibition, and the chain length, R or Y was observed. (R₁₁Y₁₆)s were the best inhibitor of HPL and (R₁₀Y₁₁)s were the best inhibitors of HGL. We observed a highly enantioselective discrimination, both with the pure enantiomeric alkylphosphonate inhibitors as well as a scalemic mixture. We also showed, for the first time, that this enantioselective recognition can occur either during the catalytic step or during the initial interfacial adsorption step of the lipases. These experimental results were analyzed with two kinetic models of covalent as well as pseudo-competitive inhibition of lipolytic enzymes by two enantiomeric inhibitors.