藻红蓝蛋白裂合异构酶E基因突变体的构建、表达与酶活性检测

通过诱变分别得到PecE的N端,C端和中间位置缺失突变的一系列突变基因的编码产物,测定了这5种缺失蛋白质的酶活性的大小。实验结果显示：来自层理鞭枝藻(M．laminosus,UTEX 1931)的PecE[简称PecE(UTEX 1931)]与来自层理鞭枝藻(M．laminosus,．PCC 7603)的PecE[简称：PecE(PCC 7603)]有81％的同源性。PecE(UTEX1931)／PeeF(PCC 7603)的酶催化活性与PecE(PCC 7603)／PecE(PCC 7603)的酶催化活性相当。PecE(UTEX 1931)的N端缺失22个氨基酸的蛋白质显示出了约20％的催化活性;而N端缺失36个氨基酸、56个氨基酸的蛋白质,C端缺失23个氨基酸的蛋白质和PecE(PCC 7603)中间缺失27个氨基酸的蛋白质均丧失活性,结果对酶的结构以及对酶活性的影响提供了重要信息。     

藻红蓝蛋白α-亚基脱辅基蛋白与藻蓝胆素的重组

利用在大肠杆菌中表达的藻红蓝蛋白α－亚基脱辅基蛋白与藻蓝胆素PCB重组,吸收光谱、荧光光谱和高效可逆光化学性质分析表明,藻红蓝蛋白α－亚基脱辅基蛋白与藻蓝胆素直接重组,生成的胆素蛋白中辅基色素仍为藻蓝胆素;而藻红蓝蛋白α-亚基脱辅基蛋白与藻蓝胆素在藻红蓝蛋白α－亚基重组酶（pecE和pecF基因的表达产物）催化下重组,生成的胆素蛋白中辅基色素转变为藻紫胆素,并具有高效可逆光化学特性。     

藻胆蛋白

本文扼要介绍了藻胆蛋白的种类、组成和性质,讨论了藻胆蛋白及其超分子聚集体－藻胆体的结构与功能的关系,并就藻胆蛋白的分子生物学近况和合成调控进行了综述,最后提出了藻胆体组分的分子进化模式。     

藻胆蛋白研究

藻胆蛋白是大量出现于红藻 (Ｒｈｏｄｏｐｈｙ ｔａ)、蓝绿藻 (Ｃｙａｎｏｐｈｙｔａ)和隐藻 (Ｃｒｙｐｔｏｐｈｙｔａ)中的捕光色素蛋白 ,主要包括藻红蛋白、藻蓝蛋白和别藻蓝蛋白三种。藻胆蛋白把捕获的光能高效地传递给叶绿素 ,从而使海藻的光合作用得以发生[1] 。细菌、藻类和高等植物的光合作用的共同特征是具有很多“天线分子” ,这些“天线分子”吸收光能并通过非放射性过程将激发能传递到含有叶绿素的“反应中心” ,在红藻、蓝绿藻和隐藻中 ,藻胆蛋白就充当这种“天线分子”的角色。因此 ,最初的藻胆蛋白研究主要集中在探讨其光合作…     

藻胆蛋白短肽性质的研究

在不同温度、ｐＨ条件下,测定了藻胆蛋白短肽中天然色素基团的稳定性,确定了最适保存条件。研究了样品中乙醇含量、微量元素及稳定剂β－胡萝卜素和维生素Ｃ对藻胆蛋白短肽的影响。找出了可使其更稳定的添加剂。     

藻胆蛋白复合物的合成及其分子内能量传递

通过偶联剂3-(2-吡啶联巯基)丙酸N-羟基琥珀亚胺酯(SPDP)及改变配料比, 合成了两个R-藻红蛋白(R-PE)与C-藻蓝蛋白(C-PC)的复合物A和B.利用吸收光谱确定了分子内R-PE与C-PC的摩尔比为6∶1和2∶1. 通过荧光光谱, 观察到能量传递现象, 并计算出能量传递效率为63%和88%.证明分子内能量传递效率很高. 二硫苏糖醇(DTT)还原连接R-PE与C-PC的二硫桥键后, 能量传递被阻断. 这一现象进一步证明复合物中存在分子内能量传递.     

藻胆蛋白功能的研究

藻胆蛋白在藻类细胞中的主要功能是作为光合作用捕光色素系统。在蓝藻和红藻中,不同的藻胆蛋白形成高度有序的超分子复合体──藻胆体作为捕获光能的功能单位。另外,藻胆蛋白在细胞内还可以作贮存蛋白,以使细胞在缺氮的环境中能生存。由于藻胆蛋白与植物光敏素在形态、结构、组成以及光谱特征等方面均十分相似,而且在体外,可以诱导藻胆蛋白具有类似于植物光敏素的光化学特征。因此,我们推测藻胆蛋白与光敏素之间在功能上可能存在着某种关系,它们可能起源于同一祖先蛋白,在长期的进化中,两者的功能发生改变,但在一定的条件下,藻脂蛋白可能会行使类似于植物光敏素的功能。     

藻胆蛋白荧光探针及其标记

藻胆蛋白是一系列新型的荧光标记探针,具有优良的荧光特性,以藻胆蛋白荧光探针标记抗体还可用于血清可溶性抗原（或抗体）的荧光免疫检测,其标记方法可分为直接法和间接法。结合藻胆蛋白的特点,研究藻胆蛋白的标记方法有助于提高荧光免疫检测的灵敏度。     

Chromophore attachment to biliproteins: specificity of PecE/PecF, a lyase-isomerase for the photoactive 3(1)-cys-alpha 84-phycoviolobilin chromophore of phycoerythrocyanin

PecE and PecF, the products of two phycoerythrocyanin lyase genes (pecE and pecF) of Mastigocladus laminosus (Fischerella), catalyze two reactions: (1) the regiospecific addition of phycocyanobilin (PCB) to Cys-alpha 84 of the phycoerythrocyanin alpha-subunit (PecA), and (2) the Delta 4-->Delta 2 isomerization of the PCB to the phycoviolobilin (PVB)-chromophore [Zhao et al. (2000) FEBS Lett. 469, 9-13]. The alpha-apoprotein (PecA) as well PecE and PecF were overexpressed from two strains of M. laminosus, with and without His-tags. The products of the spontaneous addition of PCB to PecA, and that of the reaction catalyzed by PecE/F, were characterized by their photochemistry and by absorption, fluorescence, circular dichroism of the four states obtained by irradiation with light (15-Z/E isomers of the chromophore) and/or modification of Cys-alpha 98/99 with thiol-directed reagents. The spontaneous addition leads to a 3(1)-Cys-PCB adduct, which is characteristic of allophycocyanins and phycocyanins, while the addition catalyzed by PecE and PecF leads to a 3(1)-Cys-PVB adduct which after purification was identical to alpha-PEC. The specificity and kinetics of the chromophore additions were investigated with respect to the structure of the bilin substrate: The 3-ethylidene-bilins, viz., PCB, its 18-vinyl analogue phytochromobilin, phycoerythrobilin and its dimethylester, react spontaneously to yield the conventional addition products (3-H, 3(1)-Cys), while the 3-vinyl-substituted bilins, viz., bilirubin and biliverdin, were inactive. Only phycocyanobilin and phytochromobilin are substrates to the addition-isomerization reaction catalyzed by PecE/F. The slow spontaneous addition of phycoerythrobilin is not influenced, and there is in particular no catalyzed isomerization to urobilin.     

Novel activity of a phycobiliprotein lyase: both the attachment of phycocyanobilin and the isomerization to phycoviolobilin are catalyzed by the proteins PecE and PecF encoded by the phycoerythrocyanin operon

We have previously isolated the efp (estrogen-responsive finger protein) that is required for the normal estrogen-induced cell proliferation. Here, we show the genomic organization of the human efp gene which consists of nine exons. The efp mRNA was expressed in human breast tumors and the estrogen-induced expression of the efp was found in MCF-7 human breast cancer cells. Moreover, efp promoter activity was enhanced through the estrogen-responsive element dependent on estrogen and estrogen receptor. These results suggest that the efp can mediate estrogen actions such as cell growth in human breast cancer as a primary responsive gene.     

Transposon insertion in genes coding for the biosynthesis of structural components of the Anabaena sp. phycobilisome

Anabaena sp. PCC 7120 mutants defective in phycobiliprotein biosynthesis or phycobilisome assembly were generated by transposon mutagenesis. Four mutants with grossly reduced content of the major phycobiliprotein, phycocyanin, were found to have insertions within the cpcBACDEFG1G2G3G4 operon coding for phycocyanin biosynthesis and assembly. The insertion in mutant B646 separated the promoter from the open reading frames and eliminated production of the phycocyanin (CpcA) and (CpcB) subunits. Insertion in cpcC in mutant B642 eliminated production of the L³⁶ _Rlinker polypeptide required for assembly of phycocyanin into the distal discs of the phycobilisome rod substructures. Mutants B64328 and B64407 had insertions, respectively, in cpcE and cpcF, genes coding for the subunits of the heterodimeric lyase which catalyzes the attachment of phycocyanobilin to the phycocyanin apo- subunit. Mutant SB12, often unable to survive under low light, was found to have an insertion in the apcE gene coding for the large core-membrane linker (L¹²⁸ _CM) that provides the scaffold for assembly of the phycobilisome core. DNA sequencing 3 of apcE revealed genes apcABC, coding for the and subunits of allophycocyanin and for the small core linker L^7.8 _C. Amino acid sequence comparisons showed that the ApcA and ApcB proteins are 37% identical and that each of these polypeptides is highly similar to corresponding polypeptides from the distantly related filamentous strains Calothrix sp. PCC7601 and Mastigocladus laminosus.     

Partial inhibition of hepatic microsomal aminopyrine N-demethylase by caffeine in partially purified cytochrome P450

Cytochrome P-450 substrate interactions were studied with cytochrome P-450 partially purified from livers of untreated, phenobarbital-treated, benzo[a]pyrene-treated and caffeine-treated rats. Partial inhibition of aminopyrine N-demethylase in presence of in vitro caffeine observed with intact microsomes was further investigated in a reconstituted system composed of partially purified cytochrome c reductase. Caffeine addition (in vitro) to partially purified cytochrome P-450 altered the hexobarbital, aniline and ethylisocyanide induced spectral change, and decreased NADPH oxidation in presence of substrates aminopyrine and acetanilide. NADPH oxidation was found to be increased in presence of aminopyrine and unaltered in presence of acetanilide in reconstituted system having partially purified cytochrome P-450 from caffeine-treated rats. Our studies suggest that caffeine acts as a true modifier of cytochrome P-450 and is possibly responsible for the formation of abortive complexes with aminopyrine.     

Biliprotein chromophore attachment: chaperone-like function of the PecE subunit of alpha-phycoerythrocyanin lyase

Biliproteins are post-translationally modified by chromophore addition. In phycoerythrocyanin, the heterodimeric lyase PecE/F covalently attaches phycocyanobilin (PCB) to cysteine-alpha84 of the apoprotein PecA, with concomitant isomerization to phycoviolobilin. We found that: (a) PecA adds autocatalytically PCB, yielding a low absorbance, low fluorescence PCB.PecA adduct, termed P645 according to its absorption maximum; (b) In the presence of PecE, a high absorbance, high fluorescence PCB.PecA adduct is formed, termed P641; (c) PecE is capable of transforming P645 to P641; (d) When in stop-flow experiments, PecA and PecE were preincubated before chromophore addition, a red-shifted intermediate (P650, tau=32 ms) was observed followed by a second, which was blue-shifted (P605, tau=0.5 s), and finally a third (P638, tau=14 s) that yielded the adduct (P641, tau=20 min); (e) The reaction was slower, and P605 was missing, if PecA and PecE were not preincubated; (f) Gel filtration gave no evidence of a stable complex between PecA and PecE; however, complex formation is induced by adding PCB; and (g) A red-shifted intermediate was also formed, but more slowly, with phycoerythrobilin, and denaturation showed that this is not yet covalently bound. We conclude, therefore, that PecA and PecE form a weak complex that is stabilized by PCB, that the first reaction step involves a conformational change and/or protonation of PCB, and that PecE has a chaperone-like function on the chromoprotein.     

Sodium channel activity of lobster nerve membrane treated with purified phospholipase A2

Sodium channel activity was determined by measuring the veratridine-tetrodotoxin-sensitive sodium influx in reconstituted vesicles prepared from lobster nerve membrane and soybean lipids. The sodium channel activity was abolished by treatment of membranes, prior to reconstitution, with purified phospholipase A₂. When the treatment with phospholipase A₂ was carried out in the presence of bovine serum albumin the channel activity was fully preserved. Electron microscopy revealed that the normal vesicular appearance of the membranes was changed to an amorphous mass by treatment of the membranes with enzyme alone. A population of preserved vesicular structures was observed when bovine serum albumin was present during the enzyme treatment. Analysis of the membrane components indicate that there is no relationship between phospholipid composition and sodium channel activity.     

Crystal structure and molecular mechanism of an E/F type bilin lyase-isomerase

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Biotinylation of transducin and G0 from bovine brain

A method is described for modifying G proteins with biotin. With transducin, better results were obtained with the amino group-specific derivative BXNHS (biotinyl--aminocaproic acid N-hydroxysuccinimide ester) as compared to the -SH-group specific reagent MBB (maleimidobutyrylbiocytin). Modification occurred under conditions preserving functional activity: Interaction of the biotinylated transducin with rod outer segment membranes was shown by its light-dependent association and by a GTPS-binding assay. G₀ from bovine brain was also biotinylated under conditions preserving its activity. Biotinyl-₀ was shown to bind to a streptavidin Sepharose matrix. Biotinyl-G proteins, therefore, are proposed as tools for extracting proteins (receptors and effector systems), which interact under specific conditions with G proteins.     

A fast way to track functional OmpF reconstitution in liposomes: Escherichia coli total lipid extract

A major requirement to perform structural studies with membrane proteins is to define efficient reconstitution protocols that ensure a high incorporation degree and protein directionality and topology that mimics its in vivo conditions. For this kind of studies, protein reconstitution in membrane systems via a detergent-mediated pathway is usually successfully adopted because detergents are generally used in the initial isolation and purification of membrane proteins. This study reports OmpF reconstitution in preformed Escherichia coli liposomes followed by detection of its insertion by analyzing modifications on membrane structure by two different techniques: steady-state fluorescence anisotropy and dynamic light scattering. Another important issue is protein directionality. For OmpF, it is known that interaction with polyamines promotes channel blockage. In this work, the spermine–OmpF interaction was evaluated using surface plasmon resonance, and protein directionality was confirmed.