Cholesterol-dependent insertion of glycosylphosphatidylinositol-anchored enzyme

Evidence is now accumulating that the plasma membrane is organized in different lipid and protein subdomains. Thus, glycosylphosphatidylinositol (GPI)-anchored proteins are proposed to be clustered in membrane microdomains enriched in cholesterol and sphingolipids, called rafts.By a detergent-mediated method, alkaline phosphatase, a GPI-anchored enzyme, was efficiently inserted into the membrane of sphingolipids- and cholesterol-rich liposomes as demonstrated by flotation in sucrose gradients. We have determined the enzyme extraluminal orientation. Using defined lipid components to assess the possible requirements for GPI-anchored protein insertion, we have demonstrated that insertion into membranes was cholesterol-dependent as the cholesterol addition increased the enzyme incorporation in simple phosphatidylcholine liposomes.     

Cholesterol-dependent tetanolysin damage to liposomes

Tetanolysin caused membrane damage, resulting in release of trapped glucose from liposomes containing cholesterol. Maximum glucose release occurred from liposomes that contained 50 mol% cholesterol. At higher or lower levels of cholesterol, glucose release was reduced and glucose release did not occur at all below 40 mol% cholesterol. The apparent activity of tetanolysin was not influenced by temperature (24°C compared to 32°C) or by liposomal phospholipid fatty acyl chain length. We conclude that tetanolysin caused cholesterol-dependent lysin-mediated damage to liposomes, possibly by means of a pore consisting of a complex of toxin and cholesterol.     

Reaction characteristics and stability of a membrane-bound enzyme reconstituted in bilayers of liposomes

The effects ensuing from the interaction between membrane-bound sarcosine dehydrogenase and the surrounding lipids as well as the effects of membrane fluidity were described in this study. A 25-fold activation was observed upon the reconstitution of the enzyme in bilayers of SUVs made of DMPC. The considerable decrease in K(m) and increase in V(max) suggest the induction of favorable conformational changes in both the substrate-binding site and the catalytic site of the enzyme due to the lipid-protein interaction. In SUVs of negatively charged phospholipids, the enzyme retained its initial activity over 1 month. The break point in the Arrhenius plot of the activity of reconstituted enzyme was found at temperatures close to the gel-liquid crystalline transition point of the phospholipid showing that the activity is sensitive to the physical state of membrane phospholipids. Further, immobilization of the reconstituted enzyme by use of ENT prepolymer resulted in a high activity, whereas no remarkable activity was detected with the immobilized enzyme without reconstitution.     

Anticodons,Frameshifts, and Hidden Periodicities in tRNA Sequences

Abstract

Fourier analysis of the short-range periodicities for the complete set of sequences coding for tRNA genes in genome of Bacillus subtilis proves that periodicities with periods p = 2, 3, 4, and 6 sites are the inherent properties of tRNAs. The related periodicities should be understood in a broad statistical sense and their identifying needs the elaborate statistical methods. To improve the statistics, the analysis of significant periodicities was performed for the binary R-Y, S-W, and K-M sequences. Generally, such short-range periodicities are produced via biased positioning of particular nucleotides rather than via the tandem multiplication and subsequent modifications of repeats, though the latter mechanism may also be realized. Quasi-coherently piercing long segments of tRNA, the short-range periodicities create the effective long-range structural coupling between the acceptor stem and the anticodon loop and may participate in the mechanisms of molecular recognition. The periodicities with p = 2 and 4 provide the natural ground for the translation with spontaneous or programmed frameshifting and are present in tRNAs decoding the most frameshift-prone codons. The observation of short-range periodicities suggests that the mechanisms of amino-acylation of tRNAs and codon-anticodon pairing are not independent. Their study may also provide the important information related to the origin and evolution of the genetic code.     

Cholesterol as activator of ADP-ATP exchange in reconstituted liposomes and in mitochondria

The influence of cholesterol on ADP-ATP exchange activity was measured in the reconstituted system, submitochondrial (sonic) particles and mitoplasts (isolated inner mitochondrial membranes). In the reconstituted system, cholesterol markedly enhanced the nucleotide-uptake rate, when added to membranes of various compositions i.e., pure phosphatidylcholine, phosphatidylcholine/phosphatidylethanolamine mixtures and crude egg yolk phospholipids. The stimulation was linearly dependent on the amount of incorporated cholesterol up to 7–13% added sterol, depending on the type of phospholipids. Cholesterol influenced neither the amount of actively reconstituted carrier proteins nor the affinity of the carrier towards nucleotides nor the breakpoint of temperature dependence in the Arrhenius plot. The stimulation could be correlated with an increase in the molecular activity of the carrier protein. The influence of cholesterol was also measured in the natural environment of the carrier protien, i.e., the inner mitochondrial membrane. Both with submitochondrial particles from beef heart and especially with mitoplasts from rat liver, incorporation of cholesterol by fusion with sterol-containing liposomes led to a stimulation of ADP-ATP exchange activity, comparable to the effect in the reconstituted system. These results are discussed in relation to the absence of cholesterol in the inner mitochondrial membrane and in the view of the generally accepted ordering effect of cholesterol on phospholipid bilayers.     

脂质体重组和脂蛋白体在植物生物膜研究中的应用

脂质体是磷脂在一定条件下在水中形成的由脂质双分子层组成的内部为水相的闭合囊泡。在推动生物膜的研究进展中,它作为模式系统起着非常重要的作用,能用于研究膜蛋白的性质和功能;膜脂和膜蛋白的相互关系;膜的电化学性质等。近年来脂质体重组技术开始引入到植物学研究领域,用于对植物膜蛋白的研究。本文简要介绍了脂质体的制备和脂酶体重组的方法及其在植物生物膜研究中的应用。     

Insertion of a Glycosylphosphatidylinositol-Anchored Enzyme into Liposomes

Incorporation of alkaline phosphatase (AP), a glycosylphosphatidylinositol (GPI)-anchored protein, into liposomes containing detergent, followed by detergent removal with hydrophobic resin was performed. Incorporation media were collected during different steps of detergent removal and were analyzed by flotation in sucrose gradient. The presence of protein was checked by measuring enzymatic activity, while the presence of ³H-radio-labelled liposomes was followed by determination of the radioactivity. The incorporation yield of the protein into liposomes increased with incubation time in presence of hydrophobic resin. Protein was also incorporated at different protein/lipid ratios. At the highest protein lipid ratio, our data showed that 260 molecules of GPI-linked AP (AP-GPI) could be associated with one liposome, corresponding to 65% vesicle coverage. Finally, observations by electron cryomicroscopy indicated (i) that the protein seemed exclusively associated with the lipid bilayer via the GPI-anchor, as shown by the distance—about 2.5 nm—between the protein core and the liposome membrane; (ii) that the AP-GPI distribution was heterogeneous on the liposome surface, forming clusters of protein. Abbreviations: AP, alkaline phosphatase; AP-GPI, glycosylphosphatidylinositol-linked alkaline phosphatase; EM, electron microscopy; EPA, egg phosphatidic acid; GPI, glycosylphosphatidylinositol; OctGlc, n-octyl -D-glucoside; PtdCho, egg yolk phosphatidylcholine; PtdIns-PLC, glycosylphosphatidylinositol-specific phospholipase C. Enzymes: Alkaline phosphatase, orthophosphoric-monoester phosphohydrolase (EC 3.1.3.1); glycosylphosphatidylinositol-specific phospholipase C (EC 3.1.4.10).     

Phosphorylation and phosphate-ATP exchange catalyzed by the ATP synthase isolated from Wolinella succinogenes

The ATP synthase, isolated from Wolinella (formerly Vibrio) succinogenes could be fully incorporated into liposomes without significant cleavage of the enzyme or loss of activity. These proteoliposomes, but not the isolated enzyme, catalyzed phosphate-ATP exchange and the phosphorylation of ADP which was driven by an artificially imposed across the liposomal membrane. Phosphorylation driven by light was catalyzed by proteoliposomes containing also bacteriorhodopsin. The three activities were similarly sensitive to protonophores or dicyclohexylcarbodiimide. This sensitivity was similar to that of the electron-transport-driven phosphorylation catalyzed by bacterial membrane vesicles. With a value of 280 mV to drive phosphorylation the turnover number of the enzyme was in the same order of magnitude as that measured in the electron-transport-driven phosphorylation catalyzed by the bacterial membrane. When the was below 150 mV, the phosphorylation activity of the incorporated enzyme was two orders of magnitude slower, and was about as fast as light-driven phosphorylation or as the exchange reaction.     

GPI-alkaline phosphatase insertion into phosphatidylcholine monolayers: phase behavior and morphology changes

GPI-anchored proteins are localized on the outer layer of plasma membranes and clustered in microdomains generally called lipid rafts. To study the interactions between the lipidic GPI-anchor of the protein and phospholipids, we used phosphatidylcholine monolayers at the air-water interface as a biomimetic membrane system and GPI-alkaline phosphatase prepared from bovine intestinal mucosa (GPI-BIAP) as an GPI-anchored protein model. The monolayer technique allowed us to define GPI-BIAP interaction with DPPC and POPC, lipids differing only by the presence of one unsaturation in their acyl chains. Meanwhile the exclusion pressures were similar for the two phospholipids, the comparison of the Langmuir isotherms (i.e., pressure/area diagrams) indicates that GPI-BIAP interacted differently with DPPC and POPC monolayers. BAM images, acquired in order to visualize the interface organization induced by GPI-BIAP incorporation, confirm these differences.     

Secretion of the Hormoconis resinae glucoamylase P enzyme from Trichoderma reesei directed by the natural and the cbh1 gene secretion signal

Abstract We have isolated two alkaline phosphatases (H-AP and L-AP, for high and low molecular mass, respectively) from Pseudomonas aeruginosa PA01. These two enzymes were found to differ in mobility on sodium dodecyl sulphate polyacrylamide gels (H-AP, M _r = 51 000 and L-AP, M _r = 39 500), amino-terminal amino acid sequence and did not cross-react. Both enzymes were active as phosphomonoesterases while only L-AP demonstrated any phosphodiesterase activity. Both enzymes were purified from P. aeruginosa grown in phosphate limiting conditions using the same protocol and were identified in both periplasmic and extracellular locations. A low level of H-AP was produced constitutively whereas L-AP was produced only after induction by reduced phosphate concentration in the growth medium. An L-AP-like enzyme has been previously described, however, this is the first report of a second P. aeruginosa alkaline phosphatase.     

Membrane-anchorage of Cripto protein by glycosylphosphatidylinositol and its distribution during early mouse development

cripto is the original member of the family of EGF-CFC genes, recently recognized as novel extracellular factors essential for vertebrate development. During the early stages of mouse gastrulation, cripto mRNA is detected in mesodermal cells; later, cripto mRNA is detected only in the truncus arteriosus of the developing heart. Here we describe the in vivo distribution of Cripto protein throughout mouse embryo development and show that cripto mRNA and protein colocalize. By means of immunofluorescence analysis and biochemical characterization, we show that Cripto is a membrane-bound protein anchored to the lipid bilayer by a glycosylphosphatidylinositol (GPI) moiety. We suggest that presentation of Cripto on the cell surface via a GPI-linkage is important in determining the spatial specificity of cell–cell interactions that play a critical role in the early patterning of the embryo.     

Nucleotide sequence of the gene for alkaline phosphatase of Thermus caldophilus GK24 and characteristics of the deduced primary structure of the enzyme

The gene encoding Thermus caldophilus GK24 (Tca) alkaline phosphatase was cloned into Escherichia coli. The primary structure of Tca alkaline phosphatase was deduced from its nucleotide sequence. The Tca alkaline phosphatase precursor, including the signal peptide sequence, was comprised of 501 amino acid residues. Its molecular mass was determined to be 54? omitted?760 Da. On the alignment of the amino acid sequence, Tca alkaline phosphatase showed sequence homology with the microbial alkaline phosphatases, 20% identity with E. coli alkaline phosphatase and 22% Bacillus subtilis (Bsu) alkaline phosphatases. High sequence identity was observed in the regions containing the Ser-102 residue of the active site, the zinc and magnesium binding sites of E. coli alkaline phosphatase. Comparison of Tca alkaline phosphatase and E. coli alkaline phosphatase structures suggests that the reduced activity of the Tca alkaline phosphatase, in the presence of zinc, is directly involved in some of the different metal binding sites. Heat-stable Tca alkaline phosphatase activity was detected in E. coli YK537, harboring pJRAP.     

Extracellular glycosylphosphatidylinositol-anchored mannoproteins and proteases of Cryptococcus neoformans

Extracellular proteins of Cryptococcus neoformans are involved in the pathogenesis of cryptococcosis, and some are immunoreactive antigens that may potentially serve as candidates for vaccine development. To further study the extracellular proteome of the human fungal pathogen Cry. neoformans, we conducted a proteomic analysis of secreted and cell wall-bound proteins with an acapsular strain of Cry. neoformans. Proteins were identified from both intact cells and cell walls. In both cases, extracellular proteins were removed with trypsin or beta-glucanase, and then all proteins/peptides were purified by solid-phase extraction, spin dialysis, and HPLC, and identified by liquid chromatography-mass spectrometry. This study identified 29 extracellular proteins with a predicted N-terminal signal sequence and also a predicted glycosylphosphatidylinositol anchor motif in more than half. Among the novel proteins identified were five glycosylphosphatidylinositol-anchored proteins with extensive Ser/Thr-rich regions but no apparent functional domains, a glycosylphosphatidylinositol-anchored aspartic protease, and a metalloprotease with structural similarity to an elastinolytic metalloprotease of Aspergillus fumigatus. This study suggests that Cry. neoformans has the machinery required to target glycosylphosphatidylinositol-anchored proteins to the cell wall, and it confirms the extracellular proteolytic ability of Cry. neoformans.     

Some properties of membrane-bound, solubilized and reconstituted into liposomes H+-ATPase of vacuoles of Saccharomyces carlsbergensis

Vacuoles of yeast grown in peptone medium possessed high ATPase activity (up to 1 mumol X mg protein-1 X min-1). Membrane-bound and solubilized ATPase activities were insensitive to vanadate and azide, but were inhibited by NO-3 . K+ and cyclic AMP stimulated both membrane-bound and solubilized ATPase activities. Dio-9 activated the membrane form of vacuolar ATPase 1.5-2-fold and did not affect the solubilized enzyme. Solubilized and partially purified vacuolar ATPase was reconstituted with soy-bean phospholipids by a freeze-thaw procedure. ATPase activities in native vacuoles and proteoliposomes were stimulated effectively by Dio-9, the protonophore FCCP and ionophores valinomycin and nigericin. ATP-dependent H+ transport into proteoliposomes was also shown by quenching of ACMA fluorescence. Vacuolar and partially purified ATPase preparations possessed also GTPase activity. Unlike ATPase, however, GTPase was not incorporated as a proton pump into liposomes.     

AC voltammetric carbon paste-based enzyme immunosensors

Carbon paste electrodes, previously anodised in a basic media, are the basis for the development of a new voltammetric immunosensor device. Passive adsorption of the appropriate immunochemical reagent was performed onto the electrode surface. Alkaline Phosphatase labelled immunoglobulin was the tracer used in this work, 3-indoxyl phosphate being a very suitable enzymatic substrate for the electrochemical detection of the corresponding affinity reaction. The hydrolysis of this molecule generates indigo dimmer. This product was detected by alternating current voltammetry taking advantage of the adsorptive and inherent electrodic properties that it exhibits. The same electrochemical anodisation was used at the end of one assay to remove the entire protein layer attached to the carbon paste surface, allowing the formation of a new sensing phase and the use of the same support in several consecutive experiments. The methodology was applied to the design of two different immunoassays for the determination of human IgG. Good reproducibility of the electrodic signal and a limit of detection around 10⁻¹⁰ M were achieved.     

怀头鲇早期发育阶段消化酶及碱性磷酸酶活性变化(英文)

采用动物性饵料和人工饲料培育1～10日龄怀头鲇(Silurus soldatovi)仔稚鱼,分析测定了全鱼酸性、碱性蛋白酶、淀粉酶、脂肪酶以及碱性磷酸酶的活性。结果表明:孵化后3天开口期仔鱼已具有较高的碱性蛋白酶活性,5日龄时碱性蛋白酶比活力达到较高值,8日龄时出现低值,总体变化呈波动上升趋势;酸性蛋白酶活性在1～8日龄处于较低水平,8日龄后开始迅速升高;淀粉酶活性在5日龄左右达到最高值,随后酶活性开始下降至较低水平;脂肪酶活性变化波动较大,表现为双峰型,两个峰值分别出现在3～4日龄和6～8日龄。摄食动物性饵料仔稚鱼消化酶活性和碱性磷酸酶活性均高于摄食人工饲料。在整个早期发育过程中,碱性蛋白酶比酸性蛋白酶活性高,碱性蛋白酶、淀粉酶比活力在约8日龄仔稚鱼转变期明显下降,而酸性蛋白酶活性开始迅速升高,这说明消化酶活性的变化与仔稚鱼发育过程中消化机能转换具有相关性。怀头鲇在10日龄内碱性磷酸酶活性呈上升趋势,表明怀头鲇胃肠道功能的逐步发育完善。     

Reconstitution into liposomes of glucose active transport from the rabbit renal proximal tubule. Characteristics of the system

This paper describes the characteristics of Na⁺-dependent d-glucose transport into liposomes made from soybean phospholipids into which have been reconstituted detergent-solubilized components from the rabbit renal proximal tubular brush border membrane. Conditions for optimal and quantitative reconstitution of glucose carriers are defined. Na⁺-dependent d-glucose uptake occurs via a saturable system with a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 0.125–0.135 mM, is responsive to the volume of the internal liposomal space, and shows ‘overshoot’ as seen in natural membranes. The rate of Na⁺-dependent d-glucose uptake and the magnitude of the ‘overshoot’ are proportional to the concentration of protein used in reconstitution.     

A comparative analysis of strategies for isolation of matrix vesicles

Matrix vesicles (MVs) are extracellular organelles involved in the initial steps of mineralization. MVs are isolated by two methods. The first isolation method of MVs starts with collagenase digestion of osseous tissues, followed by two differential centrifugations. The second isolation method does not use proteases but rather starts with differential centrifugation, followed by a fractionation on a sucrose gradient. The first method results in a homogeneous population of MVs with higher cholesterol/lipid content, alkaline phosphatase activity, and mineral formation rate as compared with MVs isolated by the second method. The second method leads to higher protein diversity as compared with MVs isolated according to the first method. Due to their distinct protein composition, lipid-to-protein and cholesterol-to-phospholipid ratios, and differences in rates of mineral formation, both types of isolated MVs are crucial for proteomic analysis and for understanding the regulation of mineralization process at the molecular level.     

Characterization of human placental alkaline phosphatase by activity and protein assays, capillary electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Placental alkaline phosphatase (PLAP) that had been isolated from human placenta was further purified using subsequent ion-exchange chromatography (IEC), affinity chromatography (AC) and centrifugal membrane concentration (CMC). During the process, the PLAP samples from the different stages of purification were characterized regarding purity and activity. This was accomplished by combining Lowry analysis, enzymatic activity assay, capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE) and matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF-MS). The sample obtained after IEC had a rather low specific activity (6.8 U/mg) and appeared to contain several major contaminants, among which was human serum albumin (HSA). AC followed by CMC yielded PLAP with a specific activity of 128 U/mg. The purity and identity of the protein was indicated by MALDI-TOF-MS yielding a spectrum with one major peak at m/z 58 101. Interestingly, CZE of the pure PLAP revealed a cluster of peaks, which probably reflects the presence of various glycoforms and/or oligomers. The same analytical approach was used to characterize commercially available PLAP. This sample showed a moderate specific activity (15 U/mg) and appeared to be highly impure containing various other proteins.     

Synergistic anti-tumor effect of glycosylphosphatidylinositol-anchored IL-2 and IL-12

BACKGROUND: Preclinical and clinical studies have demonstrated that interleukin 2 (IL-2), interleukin 12 (IL-12), and some other cytokines, play important roles in activating host immune responses against tumor growth. However, severe side effects caused by systemic high-dose administration of these cytokines limit their clinical application. In our previous study, local high doses of IL-2 were achieved by a GPI-anchoring technology; therefore, it will be interesting to know if this technology works for other cytokines. METHODS: A fusion gene containing murine IL-12 and the glycosylphosphatidylinositol (GPI) anchor signal sequence was generated and transfected into the murine melanoma tumor cell line B16F0 either alone or together with a vector encoding GPI-anchored IL-2. The GPI-anchored cytokine expression of the selected stable clones was assayed in vitro by ELISA and their anti-tumor effects were analyzed in vivo by tumor lymphocyte infiltration and tumor growth studies. RESULTS: GPI-anchored IL-12 was successfully expressed on the cell surface as indicated by FACS analysis and IL-12 ELISA assay. The GPI-anchored IL-12 enhanced lymphocyte infiltration and significantly inhibited tumor growth. More importantly, when GPI-anchored IL-12 and GPI-anchored IL-2 were co-delivered, a synergistic anti-tumor effect was observed in both subcutaneous and intravenous tumor models. CONCLUSIONS: GPI anchorage of cytokines represents a new approach to locally deliver high doses of cytokines without the severe adverse effects normally accompanied with systematic high-dose administration of these cytokines.