DOPE、DOPC对重组去脂肌质网Ca~(2+)-ATPase活力和构象的影响

经磷脂酶Ａ２ 去脂的肌质网Ｃａ２ ＋ － ＡＴＰａｓｅ 重组于不同比例的二油酰磷脂酰胆碱（Ｄｉｏｌｅｏｙｌｐｈｏｐｈａｔｉｄｙｌｃｈｏｌｉｎｅ,ＤＯＰＣ） 和二油酰磷脂酰乙醇胺（Ｄｉｏｌｅｏｙｌｐｈｏｐｈａｔｉｄｙｌｅｔｈａｎｏｌａｍｉｎｅ,ＤＯＰＥ） 形成脂酶体,研究了不同磷脂环境中Ｃａ２ ＋ － ＡＴＰａｓｅ 的ＡＴＰ 水解和Ｃａ２ ＋ 转运活力。结果表明,ＤＯＰＣ 和ＤＯＰＥ 分别有利于ＡＴＰ 水解和Ｃａ２ ＋ 的转运,ＤＯＰＥ 可以增强Ｃａ２ ＋ － ＡＴＰａｓｅ 的ＡＴＰ水解和Ｃａ２ ＋ 转运之间的偶联效率。利用内源荧光、荧光淬灭及Ｆｏｒｓｔｅｒ 能量转移原理测定Ｃａ２ ＋ －ＡＴＰａｓｅ 相应的构象变化, 发现随着ＤＯＰＥ／ ＤＯＰＣ 比例的改变使Ｃａ２ ＋ － ＡＴＰａｓｅ 构象发生相应的变化。     

不同磷脂和糖脂对莱氏衣原体膜上Mg~（2＋）－ATPase活性的影响

莱氏衣原体膜上Ｍｇ~（２＋）－ＡＴＰａｓｅ用ＤＯＣ溶解后,经Ｓｅｐｈａｒｏｓｅ－６Ｂ和ＤＥＡＥ－ＣｅｌｌｕｌｏｓｅＤＥ－５２离子交换柱,得到了部分纯化的Ｍｇ~（２＋）ＡＴＰａｓｅ,并将此ＡＴＰａｓｅ与不同极性头部的磷脂和膜糖脂重组,研究了不同的极性头部的磷脂和膜糖脂对ＡＴＰａｓｅ活性的影响。此酶的活性不依赖酸性磷脂,ＰＧ、ＤＰＧ、大豆磷脂等明显抑制酶活性,中性磷脂ＤＭＰＣ、ＰＥ、ＰＣ则能增加酶活性,其中尤以非双层脂ＰＥ的作用最为明显。从莱氏衣原体膜上提取的糖脂（ＭＧＤＧ,ＤＧＤＧ）单独和ＡＴＰａｓｅ重组时,酶活性增加并不明显,当ＭＧＤＧ和ＤＧＤＧ以等比例混合时,能大大地增加酶活性。这表明Ｍｇ~（２＋）－ＡＴＰａｓｅ的活性很大程度上与磷脂的表面电荷及磷脂的组成相关。     

水分逆境对吊兰叶片脂质组成的影响

测定了吊兰（ Ｃｈｌｏｒｏｐｈｙｔｕｍ ｃｏｍｏｓｕｍ） 在干旱、正常浇水和渍水三种供水条件下叶片的磷脂组成、膜脂和总磷脂的脂肪酸组成,以及磷脂中４ 种主要组分ＰＧ、ＰＥ、ＰＣ 和ＰＩ的脂肪酸组成,观察到干旱使磷脂中ＰＥ 的相对含量增加,ＰＥ 脂肪酸中１６ ：０ 明显减少,而膜脂、总磷脂和ＰＣ、ＰＩ中饱和脂肪酸增加,但ＰＧ脂肪酸组成变化很小     

Ca~（2＋）与含β－桐酸磷脂脂质体的相互作用

通过测定含β－桐酸（β－ＥＳＡ）的双棕榈酰磷脂酰胆碱（ＤＰＰＣ）脂质体在加入Ｃａ（２＋）后浊度,粒度及内包荧光物释放的变化,研究了Ｃａ（２＋）与ＤＰＰＣ／β－ＥＳＡ脂质体的相互作用,结果表明,ＤＰＰＣ／β－ＥＳＡ脂质体是一类对外加Ｃａ（２＋）敏感的脂质体,Ｃａ（２＋）的作用首先是引起脂质体间的集聚然后使脂质体融合;此时加速脂质体内包荧光物的释放。     

胆固醇/磷脂比例的变化对Gs与AC偶联功能的影响

利用生物膜的拆离与重建方法将纯化的激活型ＧＴＰ结合蛋白（Ｇｓ）和腺苷酸环化酶（ＡＣ）重组于不同比例的胆固醇与大豆磷脂的脂质体中,研究了胆固醇对Ｇｓ与ＡＣ的偶联功能的影响。结果提示,类似生理条件的胆固醇含量增加其偶联功能,Ｇｓ对ＡＣ的激活活力为对照的１．２倍,Ｇｓ对ＧＴＰγＳ的结合活力则增加１７％;而类似病理条件的高含量的胆固醇则明显抑制其偶联功能,Ｇｓ对ＡＣ的激活活力降低６２％,Ｇｓ对ＧＴＰγＳ的结合活力则降低４４％。进一步用ＤＰＨ,ｎ－ＡＦ系列荧光探剂进行稳态偏振和荧光寿命的测试表明,胆固醇可能通过影响膜脂表层的物理状态,从而调节Ｇｓ与ＡＣ的偶联功能。所得结果对于阐明某些胆固醇代谢紊乱性疾病,如动脉粥样硬化的信号跨膜转导变化的分子机理具有重要的启示作用     

GENUS ANTROPHYUM KAULF.FROM CHINA AND NEIGHBORING REGIONS

ＧＥＮＵＳＡＮＴＲＯＰＨＹＵＭＫＡＵＬＦ．ＦＲＯＭＣＨＩＮＡＡＮＤＮＥＩＧＨＢＯＲＩＮＧＲＥＧＩＯＮＳＸ．Ｃ．ＺＨＡＮＧ（Ｔｈｅｈｅｒｂａｒｉｕｍ（ＰＥ）,ＩｎｓｔｉｔｕｔｅｏｆＢｏｔａｎｙ,ＣｈｉｎｅｓｅＡｃａｄｅｍｙｏｆＳｃｉｅｎｃｅｓ,Ｂｅｉ...     

t-PA cDNA的克隆及其在毕赤酵母中的表达

应用ＰＣＲ方法,在ｔ－ＰＡ ｃＤＮＡ５＇端引入合适的限制酶位点和酵母分泌信号肽,与酵母表达载体ｐＰＩＣ９重组,构建表达质粒ｐＳＴＥ－Ｙ,利用ＬｉＣｌ转化法转化酵母菌ＹＳ１０８,在ＭＭ和ＭＤ平板上筛选表型,ＰＣＲ筛选ｔ－ＰＡ ｃＤＮＡ与酵母染色体整合而形成的阳性克隆,阳性克隆经甲醇诱导表达后,用ＳＤＳ－ＰＡＧＥ证明表达产物的分子量为６ｋＤａ左右,用酪蛋白板溶圈法测定ｔ－ＰＡ的活性。Ｍｕｔ＾ｓ表型菌表     

一种改进的纯化和鉴定牛脑皮层Gs蛋白的方法

用１％胆酸钠和１５％孢和硫酸铵相结合的方法,从牛脑皮层细胞膜中抽提得到主要含激活型Ｇ－蛋白和腺苷酸环化酶两种蛋白组分的制剂,然后通过Ｓｅｐｈａｒｏｓｅ６Ｂ柱将两者分开,将含Ｇｓ高活力的级分用庚胺－Ｓｅｐｈａｒｏｓｅ４Ｂ柱进一步分离,即可获得高活力的Ｇｓ,ＳＤＳ－ＰＡＧＥ显示为分子量４５０００和３６０００的两条蛋白带,该法具简便,快速,重复性好、产率高等优点,且可同时获得无Ｇｓ污染的ＡＣ。用无Ｇｓ污     

甲基单胞菌Methylomonas sp.GYJ3中甲烷单加氧酶还原酶组分的纯化和性质

Ｍｅｔｙｌｏｍｏｎａｓｓｐ．ＧＹＪ３菌的甲烷单加氧酶（ＭＭＯ）粗酶提取液经ＤＥＡＥ－ＳｅｐｈａｒｏｓｅＣＬ－６Ｂ阴离子交换层析、ＳｅｐｈａｄｅｘＧ－１００凝胶过滤层析和ＤＥＡＥ－ＴＳＫｇｅｌＨＰＬＣ分离纯化出ＭＭＯ还原酶组分．经ＨＰＬＣ分析,纯度大于９５％,纯化倍数为４．４,加入至ＭＭＯ羟基化酶和调节蛋白Ｂ的体系中表现比活为２２８ｎｍｏｌ环氧丙烷每分钟毫克蛋白．ＳＤＳ－ＰＡＧＥ电泳表明还原酶由一种亚基组成,分子量４２ｋＤ．ＩＣＰ－ＡＥＳ测定还原酶的Ｆｅ含量为１．８３ｍｏｌＦｅ每ｍｏｌ蛋白．ＵＶ－Ｖｉｓ光谱表明还原酶除２８０ｎｍ蛋白质特征峰外在４６０ｎｍ有最大吸收峰,且Ａ２８０ｎｍ／Ａ４６０ｎｍ为２．５０,与其它黄素一铁硫蛋白相似,推测还原酶可能含一个ＦＡＤ辅基和Ｆｅ２Ｓ２中心．在厌氧条件下,还原酶能够和ＮＡＤＨ作用,ＵＶ－Ｖｉｓ光谱分析表明还原酶４６０ｎｍ处特征吸收峰消失,说明在ＭＭＯ催化过程中还原酶接受ＮＡＤＨ的电子．ＤＥＡＥ－ＳｅｐｈａｒｏｓｅＣＬ－６Ｂ阴离子交换层析分离出调节蛋白Ｂ,部分纯化的调节蛋白Ｂ的分子量大约在２０ｋＤ,它能够提高ＭＭＯ比活性４０倍,ＭＭＯ还原酶和调节蛋白Ｂ单独存在时不具有ＭＭＯ     

人主动脉硫酸乙酰肝素蛋白聚糖对培养的人血管壁细胞生长的作用

通过培养的人主动脉平滑肌细胞（ｈＡＳＭＣ）及脐静脉内皮细胞（ｈＵＶＥＣ）,应用３Ｈ－ＴｄＲ参入、Ｎｏｒｔｈｅｒｎｂｌｏｔ分析、逆转录多聚酶链反应（ＲＴ－ＰＣＲ）、放射免疫分析（ＲＩＡ）、和紫外比色法等技术观察了人主动脉中硫酸乙酰肝素蛋白聚糖（ＨＳＰＧ）对ｈＡＳＭＣ和ｈＵＶＥＣＤＮＡ合成的作用及对血小板源生长因子（ＰＤＧＦ）、ＰＤＧＦ受体、转化生长因子β（ＴＧＦ－β）、内皮素－１（ＥＴ－１）或碱性成纤维细胞生长因子（ｂＦＧＦ）基因表达和肾素－血管紧张系统（ＲＡＳ）的影响,结果显示,ＨＳＰＧ明显抑制培养的ｈＡＳＭＣ基础的ＤＮＡ合成（ｃｐｍ值为：１０３８５±３２６３ｖｓ,２５５４１±６４２１,Ｐ＜０．０１）及外源性ＰＤＧＦ诱导的ＤＮＡ合成（ｃｐｍ值为：９８７８±１９４７ｖｓ．１３４８１±４４ｌ０,Ｐ＜０．０５）;抑制ＰＤＧＦＡ链、ＴＧＦ－Ｂｐ和ＥＴ－１ｍＲＮＡ表达,提高ＰＤＧＦａ和β受体ｍＲＮＡ的表达;显著降低ｈＡＳＭＣ培养液中血管紧张素Ⅱ（ＡｎｇⅡ）的浓度和血管紧张素转换酶（ＡＣＥ）的活性,推测ＨＳＰＧ抑制ＰＤＧＦＡ链、ＴＧＦ－β及ＥＴ－１ｍＲＮＡ表达,降低ＡＣＥ活性及ＡｎｇⅡ浓度是其抑制ｈＡＳＭＣ增殖的重要机     

Effects of lipids on the transport activity of the reconstituted glucose transport system from rat adipocyte

The glucose transport system, isolated from rat adipocyte membrane fractions, was reconstituted into phospholipid vesicles. Vesicles composed of crude egg yolk phospholipids, containing primarily phosphatidylcholine (PC) and phosphatidylethanolamine (PE), demonstrated specific d-glucose uptake. Purified vesicles made of PC and PE also supported such activity but PC or PE by themselves did not. The modulation of this uptake activity has been studied by systematically altering the lipid composition of the reconstituted system with respect to: (1) polar headgroups; (2) acyl chains, and (3) charge. Addition of small amounts (20 mol%) of PS, phosphatidylinositol (PI), cholesterol, or sphingomyelin significantly reduced glucose transport activity. A similar effect was seen with the charged lipid, phosphatidic acid. In the case of PS, this effect was independent of the acyl chain composition. Polar headgroup modification of PE, however, did not appreciably affect transport activity. Free fatty acids, on the other hand, increased or decreased activity based on the degree of saturation and charge. These results indicate that glucose transport activity is sensitive to specific alterations in both the polar headgroup and acyl chain composition of the surrounding membrane lipids.     

Chlorpromazine interaction with glycerophospholipid liposomes studied by magic angle spinning solid state (13)C-NMR and differential scanning calorimetry

Phosphatidylserine (PS) extracted from pig brain and synthetic dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) were used to make DPPC/DMPC and DPPC/PS large unilamellar liposomes with a diameter of approximately 1 microm. Chlorpromazine-HCl (CPZ), an amphipathic cationic psychotropic drug of the phenothiazine group, is known to partition into lipid bilayer membranes of liposomes with partition coefficients depending on the acyl chain length and to alter the bilayer structure in a manner depending on the phospholipid headgroups. The effects of adding CPZ to these membranes were studied by differential scanning calorimetry and proton cross polarization solid state magic angle spinning (13)C-nuclear magnetic resonance spectroscopy (CP-MAS-(13)C-NMR). CP-MAS-(13)C-NMR spectra of the DPPC (60%)/DMPC (40%) and the DPPC (54%)/DMPC (36%)/CPZ (10%) liposomes, show that CPZ has low or no interaction with the phospholipids of this neutral and densely packed bilayer. Conversely, the DPPC (54%)/PS (36%)/CPZ (10%) bilayer at 25 degrees C demonstrates interaction of CPZ with the phospholipid headgroups (PS). This CPZ interaction causes about 30% of the acyl chains to enter the gauche conformation with low or no CPZ interdigitation among the acyl chains at this temperature (25 degrees C). The DPPC (54%)/PS (36%)/CPZ (10%) bilayer at a sample temperature of 37 degrees C (T(C)=31.2 degrees C), shows CPZ interdigitation among the phospholipids as deduced from the finding that approximately 30% of the phospholipid acyl chains carbon resonances shift low-field by 5-15 ppm.     

Interaction of rat brain cytidylate cyclase with phospholipids

The interaction of rat brain cytidylate cyclase with some phospholipids such as L-alpha-phosphatidylcholine (PC), L-alpha-phosphatidylserine (PS), L-alpha-phosphatidylethanolamine (PE) and L-alpha-phosphatidic acid (PA) was studied. Cytidylate cyclase activity of Triton X-100 - solubilized fraction was inhibited by PS, PE and PA, but not with PC. The addition of PC to the incubation mixture containing PS, PE or PA dose - dependently reversed the inhibition of enzyme activity by these phospholipids. Phospholipids showed similar effect on the intact membrane - bound enzyme. PC could reactivate the enzyme which was inactivated by deoxycholate treatment, suggesting that PC may be an important factor to reconstitute an active conformation of the enzyme. These findings indicate that cytidylate cyclase could be regulated by phospholipids constituting its microenvironment of the membrane.     

Transbilayer diffusion of phospholipids: dependence on headgroup structure and acyl chain length

The kinetics and thermodynamics of the transmembrane movement (flip-flop) of fluorescent analogs of phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylcholine (PC), and phosphatidylethanolamine (PE) were investigated to determine the contributions of headgroup composition and acyl chain length to phospholipid flip-flop. The phospholipid derivatives containing n-octanoic, n-decanoic or n-dodecanoic acid in the sn-1 position and 9-(1-pyrenyl)nonanoic acid in the sn-2 position were incorporated at 3 mol% into sonicated single-bilayer vesicles of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC). The kinetics of diffusion of the pyrene-labeled phospholipids from the outer and inner monolayers of the host vesicles to a large pool of POPC acceptor vesicles were monitored by the time-dependent decrease of pyrene excimer fluorescence. The observed kinetics of transfer were biexponential, with a fast component due to the spontaneous transfer of pyrenyl phospholipids in the outer monolayer of labeled vesicles and a slower component due to diffusion of pyrenyl phospholipid from the inner monolayer of the same vesicles. Intervesicular transfer rates decreased approx. 8-fold for every two carbons added to the first acyl chain. Correspondingly, the free energy of activation for transfer increased approx. 1.3 kcal/mol. With the exception of PE, the intervesicular transfer rates for the different headgroups within a homologous series were nearly the same, with the PC derivative being the fastest. Transfer rates for the PE derivatives were 5-to 7-fold slower than the rates observed for PC. Phospholipid flip-flop, in contrast, was strongly dependent on headgroup composition with a smaller dependence on acyl chain length. At pH 7.4, flip-flop rates increased in the order PC less than PG less than PA less than PE, where the rates for PE were at least 10-times greater than those of the homologous PC derivative. Activation energies for flip-flop were large, and ranged from 38 kcal/mol for the longest acyl chain derivative of PC to 25 kcal/mol for the PE derivatives. Titration of the PA headgroup at pH 4.0 produced an approx. 500-fold increase in the flip-flop rate of PA, while the activation energy decreased 10 kcal/mol. Increasing acyl chain length reduced phospholipid flip-flop rates, with the greatest change observed for the PC analogs, which exhibited an approx. 2-fold decrease in flip-flop rate for every two methylene carbons added to the acyl chain at the sn-1 position.(ABSTRACT TRUNCATED AT 400 WORDS)     

Enhanced phospholipase B activity and alteration of phospholipids and neutral lipids in Saccharomyces cerevisiae exposed to N-nitrosonornicotine

A tobacco-specific nitrosamine (TSNA), N-nitrosonornicotine (NNN), is a potent carcinogen present in cigarette smoke, and chronic exposure to it can lead to pulmonary cancer. NNN causes changes in phospholipid metabolism and the mechanism is yet to be elucidated. Exposure of Saccharomyces cerevisiae to 50 μM NNN leads to a substantial decrease in phosphatidylserine (PS) by 63%, phosphatidylcholine (PC) by 42% and phosphatidylethanolamine (PE) by 36% with a concomitant increase in lysophospholipids (LPL) by 25%. The alteration in phospholipid content was dependent on increasing NNN concentration. Reduced phospholipids were accompanied with increased neutral lipid content. Here we report for the first time that NNN exposure, significantly increases phospholipase B (PLB) activity and the preferred substrate is PC, a major phospholipid responsible for a series of metabolic functions. Furthermore, NNN also promotes the alteration of fatty acid (FA) composition; it increases the long chain fatty acid (C18 series) in phospholipids specifically phosphatidylethanolamine (PE) and PS; while on the contrary it increases short chain fatty acids in cardiolipin (CL). NNN mediated degradation of phospholipids is associated with enhanced PLB activity and alteration of phospholipid composition is accompanied with acyl chain remodelling. Understanding the altered phospholipid metabolism produced by NNN exposure is a worthwhile pursuit because it will help to understand the toxicity of tobacco smoke.     

Hydrophobic membrane thickness and lipid-protein interactions of the leucine transport system of Lactococcus lactis

The effect of the phospholipid acyl chain carbon number on the activity of the branched-chain amino acid transport system of Lactococcus lactis has been investigated. Major fatty acids identified in a total lipid extract of L. lactis membranes are palmitic acid (16:0), oleic acid (18:1) and the cyclopropane-ring containing lactobacillic acid (19 delta). L. lactis membrane vesicles were fused with liposomes prepared from equimolar mixtures of synthetic phosphatidylethanolamine (PE) and phosphatidylcholine (PC) with cis mono-unsaturated acyl chains. The activity of the branched-chain amino acid carrier is determined by the bulk properties of the membrane (Driessen, A.J.M., Zheng, T., In 't Veld, G., Op den Kamp, J.A.F. and Konings, W.N. (1988) Biochemistry 27, 865-872). PE acts as an activator and PC is ineffective. Counterflow and protonmotive-force driven transport of leucine is sensitive to changes in the acyl chain carbon number of both phospholipids and maximal with dioleoyl-PE/dioleoyl-PC. Above the gel to liquid-crystalline phase transition temperature of the lipid species, membrane fluidity decreased with increasing acyl chain carbon number. Our data suggest that the carbon number of the acyl chains of PE and PC determine to a large extent the activity of the transport system. This might be relevant for the interaction of PE with the transport protein. Variations in the acyl chain composition of PC exert a more general effect on transport activity. The acyl chain composition of phospholipids determines the membrane thickness (Lewis, B.A. and Engelman, D.M. (1983) J. Mol. Biol. 166, 211-217). We therefore propose that the degree of matching between the lipid-bilayer and the hydrophobic thickness of the branched-chain amino acid carrier is an important parameter in lipid-protein interactions.     

Importance of phosphatidylethanolamine for association of protein kinase C and other cytoplasmic proteins with membranes.

Biological membranes exhibit an asymmetric distribution of phospholipids. Phosphatidylserine (PS) is an acidic phospholipid that is found almost entirely on the interior of the cell where it is important for interaction with many cellular components. A less well understood phenomenon is the asymmetry of the neutral phospholipids, where phosphatidylcholine (PC) is located primarily on exterior membranes while phosphatidylethanolamine (PE) is located primarily on interior membranes. The effect of these neutral phospholipids on protein-phospholipid associations was examined using four cytoplasmic proteins that bind to membranes in a calcium-dependent manner. With membranes containing PS at a charge density characteristic of cytosolic membranes, protein kinase C and three other proteins with molecular masses of 64, 32, and 22 kDa all showed great selectively for membranes containing PE rather than PC as the neutral phospholipid; the calcium requirements for membrane-protein association of the 64- and 32-kDa proteins were about 10-fold lower with membranes containing PE; binding of the 22-kDa protein to membranes required the presence of PE and could not even be detected with membranes containing PC. Variation of the PS/PE ratio showed that membranes containing about 20% PS/60% PE provided optimum conditions for binding and were as effective as membranes composed of 100% PS. Thus, PE, as a phospholipid matrix, eliminated the need for membranes with high charge density and/or reduced the calcium concentrations needed for protein-membrane association. A surprising result was that PKC and the 64- and 32-kDa proteins were capable of binding to neutral membranes composed entirely of PE/PC or PC only. The different phospholipid headgroups altered only the calcium required for membrane-protein association. For example, calcium concentrations at the midpoint for association of the 64-kDa protein with membranes containing PS, PE/PC, or PC occurred at 6, 100, and 20,000 microM, respectively. Thus, biological probes detected major differences in the surface properties of membranes containing PE versus PC, despite the fact that both of these neutral phospholipids are often thought to provide "inert" matrices for the acidic phospholipids. The selectivity for membranes containing PE could be a general phenomenon that is applicable to many cytoplasmic proteins. The present study suggested that the strategic location of PE on the interior of the membranes may be necessary to allow some membrane-protein associations to occur at physiological levels of calcium and PS.     

Conformation-dependent activation of type II adenylyl cyclase by protein kinase C

Phorbol ester treatment enhanced the catalytic activity of type II adenylyl cyclase overexpressed in insect cells. In cells coexpressing type II adenylyl cyclase and protein kinase C-α, type II adenylyl cyclase catalytic activity was higher even in the absence of phorbol ester treatment; phorbol ester treatment further and markedly enhanced type II adenylyl cyclase catalytic activity. However, this enhancement, either by phorbol ester treatment or by coexpression of protein kinase C-α, was lost following membrane solubilization with detergents. This attenuation was unaffected by phosphatase inhibitor or salts. In contrast, membrane solubilization did not affect forskolin-stimulated type II adenylyl cyclase catalytic activity. Purified type II adenylyl cyclase was stimulated by forskolin and Gsα, but not by protein kinase C-α. Therefore, a specific mammalian protein kinase C isoenzyme can activate type II adenylyl cyclase, but the mechanism clearly differs from that underlying either Gsα- or forskolin-mediated stimulation. J. Cell. Biochem. 64:492–498. © 1997 Wiley-Liss, Inc.     

Metabolism of unusual membrane phospholipids in the marine sponge Microciona prolifera

Sponges are unique in regard to membrane phospholipid composition. Features virtually without parallel in other organisms are the predominance of the C26-C30 polyenoic acids (demospongic acids) in the phosphatidylethanolamines (PE) and the attachment of identical acyl groups to the glycerol moiety. The biosynthesis and disposition of these unusual phospholipids were followed in the marine sponge Microciona prolifera where PE ( delta 5,9-26:2, delta 5,9-26:2) is a major molecular species. Incorporation experiments with radiolabeled fatty acids, bases, and intact phospholipids revealed the de novo biosynthesis of the two major phosphatides, phosphatidylethanolamines (PE) and phosphatidylcholines (PC), via the cytidine pathway as in higher animals, with ethanolamine selectively incorporated into PE( delta 5,9-26:2, delta 5,9-26:2). Methylation of PE and random acyl chain migration across different phospholipid classes were marginal, but the exchange of PC for PE, apparently mediated by the action of phospholipase, was indicated after uptake of the unnatural PC( delta 9-27:1, delta 9-26:1). The present study demonstrates in the most primitive multicellular animals a phospholipid metabolic pattern similar to that in higher organisms, with unique acyl and phosphoethanolamine transferases apparently involved in the biosynthesis of the (demospongic) di-C26-acyl-PE molecular species.     

Structural transitions in short-chain lipid assemblies studied by (31)P-NMR spectroscopy

The self-assembled supramolecular structures of diacylphosphatidylcholine (diC(n)PC), diacylphosphatidylethanolamine (diC(n)PE), diacylphosphatidyglycerol (diC(n)PG), and diacylphosphatidylserine (diC(n)PS) were investigated by (31)P nuclear magnetic resonance (NMR) spectroscopy as a function of the hydrophobic acyl chain length. Short-chain homologs of these lipids formed micelles, and longer-chain homologs formed bilayers. The shortest acyl chain lengths that supported bilayer structures depended on the headgroup of the lipids. They increased in the order PE (C(6)) < PC (C(9)) < or = PS (C(9) or C(10)) < PG (C(11) or C(12)). This order correlated with the effective headgroup area, which is a function of the physical size, charge, hydration, and hydrogen-bonding capacity of the four headgroups. Electrostatic screening of the headgroup charge with NaCl reduced the effective headgroup area of PS and PG and thereby decreased the micelle-to-bilayer transition of these lipid classes to shorter chain lengths. The experimentally determined supramolecular structures were compared to the assembly states predicted by packing constraints that were calculated from the hydrocarbon-chain volume and effective headgroup area of each lipid. The model accurately predicted the chain-length threshold for bilayer formation if the relative displacement of the acyl chains of the phospholipid were taken into account. The model also predicted cylindrical rather than spherical micelles for all four diacylphospholipid classes and the (31)P-NMR spectra provided evidence for a tubular network that appeared as an intermediate phase at the micelle-to-bilayer transition. The free energy of micellization per methylene group was independent of the structure of the supramolecular assembly, but was -0.95 kJ/mol (-0.23 kcal/mol) for the PGs compared to -2.5 kJ/mol (-0.60 kcal/mol) for the PCs. The integral membrane protein OmpA did not change the bilayer structure of thin (diC(10)PC) bilayers.