Analysis of the proposed Fe-SX binding region of Photosystem 1 by site directed mutation of PsaA in Chlamydomonas reinhardtii

The psaA and psaB genes of the chloroplast genome in oxygenic photosynthetic organisms code for the major peptides of the Photosystem 1 reaction center. A heterodimer of the two polypeptides PsaA and PsaB is thought to bind the reaction center chlorophyll, P700, and the early electron acceptors A₀, A₁ and Fe-S_X. Fe-S_X is a 4Fe4S center requiring 4 cysteine residues as ligands from the protein. As PsaA and PsaB have only three and two conserved cysteine residues respectively, it has been proposed by several groups that Fe-S_X is an unusual inter-peptide center liganded by two cysteines from each peptide. This hypothesis has been tested by site directed mutagenesis of PsaA residue C575 and the adjacent D576. The C575D mutant does not assemble Photosystem 1. The C575H mutant contains a photoxidisable chlorophyll with EPR properties of P700, but no other Photosystem 1 function has been detected. The D576L mutant assembles a modified Photosystem 1 in which the EPR properties of the Fe-S_A/B centers are altered. The results confirm the importance of the conserved cysteine motif region in Photosystem 1 structure.Dedicated to the memory of Daniel I. Arnon.     

Introduction of a [4Fe-4S (S-cys)4]+1,+2 iron-sulfur center into a four-alpha helix protein using design parameters from the domain of the Fx cluster in the Photosystem I reaction center.

We describe the insertion of an iron-sulfur center into a designed four alpha-helix model protein. The model protein was re-engineered by introducing four cysteine ligands required for the coordination of the mulinucleate cluster into positions in the main-chain directly analogous to the domain predicted to ligand the interpeptide [4Fe-4S (S-cys)4] cluster, Fx, from PsaA and PsaB of the Photosystem I reaction center. This was achieved by inserting the sequence, CDGPGRGGTC, which is conserved in PsaA and PsaB, into interhelical loops 1 and 3 of the four alpha-helix model. The holoprotein was characterized spectroscopically after insertion of the iron-sulfur center in vitro. EPR spectra confirmed the cluster is a [4Fe-4S] type, indicating that the cysteine thiolate ligands were positioned as designed. The midpoint potential of the iron-sulfur center in the model holoprotein was determined via redox titration and shown to be -422 mV (pH 8.3, n = 1). The results support proposals advanced for the structure of the domain of the [4Fe-4S] Fx cluster in Photosystem I based upon sequence predictions and molecular modeling. We suggest that the lower potential of the Fx cluster is most likely due to factors in the protein environment of Fx rather than the identity of the residues proximal to the coordinating ligands.     

The effect of Arg306-->Ala and Arg506-->Gln substitutions in the inactivation of recombinant human factor Va by activated protein C and protein S.

Factor Va (fVa) is inactivated by activated protein C (APC) by cleavage of the heavy chain at Arg306, Arg506, and Arg679. Site-directed mutagenesis of human factor V cDNA was used to substitute Arg306-->Ala (rfVa306A) and Arg506-->Gln (rfVa506Q). Both the single and double mutants (rfVa306A/506Q) were constructed. The activation of these procofactors by alpha-thrombin and their inactivation by APC were assessed in coagulation assays using factor V-deficient plasma. All recombinant and wild-type proteins had similar initial cofactor activity and identical activation products (a factor Va molecule composed of light and heavy chains). Inactivation of factor Va purified from human plasma (fVaPLASMA) in HBS Ca2+ +0.5% BSA or in conditioned media by APC in the presence of phospholipid vesicles resulted in identical inactivation profiles and displayed identical cleavage patterns. Recombinant wild-type factor Va (rfVaWT) was inactivated by APC in the presence of phospholipid vesicles at an overall rate slower than fVaPLASMA. The rfVa306A and rfVa506Q mutants were each inactivated at rates slower than rfVaWT and fVaPLASMA. Following a 90-min incubation with APC, rfVa306A and rfVa506Q retain approximately 30-40% of the initial cofactor activity. The double mutant, rfVa306A/506Q, was completely resistant to cleavage and inactivation by APC retaining 100% of the initial cofactor activity following a 90-min incubation in the presence of APC. Recombinant fVaWT, rfVa306A, rfVa506Q, and rfVa306A/506Q were also used to evaluate the effect of protein S on the individual cleavage sites of the cofactor by APC. The initial rates of rfVaWT and rfVa306A inactivation in the presence of protein S were unchanged, indicating cleavage at Arg506 is not affected by protein S. The initial rate of rfVa506Q inactivation was increased, suggesting protein S slightly accelerates the cleavage at Arg306. Overall, the data demonstrate high specificity with respect to cleavage sites for APC on factor Va and demonstrate that cleavages of the cofactor at both Arg306 and Arg506 are required for efficient factor Va inactivation.     

食用菌灭活原生质体电融合及属间融合产物的鉴定分析

食用菌因其显著的营养、保健、药用及综合利用价值受到世人青睐。原生质体融合技术则为食用菌的育种和遗传学研究提供了一条很好的途径。食用菌的种内、种间乃至属间、目间的原生质体融合均已有过报道,但这些工作大多利用营养缺陷型标记的亲株,采用化学融合法完成。这种融合的效率较低,对亲株的标记繁琐费时,甚至会对菌株性状产生不良影响。最近,一种具有较高融合效率的物理融合法——电融合法以及非遗传性标记法分别被一些研究者所采用。本研究将原生质体电融合法与灭活标记法相结合来获得食用菌的属间融合产物,并采用包括同工酶分析和RAPD(或AP-PCR)方法在内的分子生物学方法,对食用菌属间杂交后代的遗传学特性进行初步探讨。1 材料和方法     

亚洲飞蝗在中国新疆维吾尔自治区的发生与防治

从亚洲飞蝗在新疆发生基地的生境特点;形成和消退过程,分析亚洲飞蝗灾害发生消退的原因。自然条件变化和人们经济活动常改变亚洲飞蝗发生基地的生态条件及环境,并诱发或抑制飞蝗灾害的发生。文章指出根治亚洲飞蝗灾害的有效途径是贯彻“改治并举、根除蝗”的方针;加强蝗情监测;适时防治以及通过水利建设和农田开垦,改变飞蝗发生基地和生态条件等。     

Spatial relationships between the anterior centriole and the mitotic center during interphase in the amoebae of the myxomycete Physarum polycephalum

Amoebae of the Myxomycete Physarum polycephalum in the interphase state typically contain only one proflagellar apparatus in which the anterior kinetosome (anterior centriole) is attached to the microtubule organizing center 1 (mtoc 1). We built strains possessing more than one mtoc 1 and a variable number of anterior centrioles to allow the appearance of new structures. In 8% of the amoebae of these strains, the 1:1 attachment between the anterior centriole and the mtoc 1 is not always respected. In nine cases studied using tridimensional reconstructions from ultrastructural thin sections, the pattern of attachment was more complex. A mtoc 1 could be linked to several anterior centrioles, and/or reciprocally an anterior centriole could be linked to several mtoc 1. In one case, an anterior centriole was not linked to a mtoc 1 and in three cases, a single centriole exhibited anterior and posterior characteristics. These observations suggest that (1) each pair of centrioles constitutes a morphological and physiological entity that is distinct from the mitotic center (mtoc 1); (2) the attachment of the anterior centriole to the mtoc 1 occurs at the end of each mitosis; (3) there is an inductory process during the morphogenesis of the link between the anterior centriole and the mtoc 1; (4) the anterior characteristics of a centriole can be present in the absence of the link with the mtoc 1; (5) the anterior and posterior characteristics of a centriole are not exclusive of each other, ruling out the existence of a lineage corresponding to the anterior centriole and a lineage corresponding to the posterior centriole; and (6) the differences between anterior and posterior centrioles result from a maturation process.     

Identity of the Photosystem II reaction center polypeptide

A Photosystem-II (PS-II)-enriched chloroplast submembrane fraction has been subjected to non-denaturing gel-electrophoresis. Two chlorophyll a (Chl a)-binding proteins associated with the core complex were isolated and spectrally characterized. The Chl protein with apparent apoprotein mass of 47 kDa (CP47) displayed a 695 nm fluorescence emission maximum (77 K) and light-induced absorption characteristics indicating the presence of the reaction center Chl, P-680, and its primary electron acceptor, pheophytin. A Chl protein of apparent apoprotein mass of 43 kDa (CP43) displayed a fluorescence emission maximum at 685 nm. We conclude that CP43 serves as an antenna Chl protein and the PS II reaction center is located in CP47.     

Electron transfer in reaction centers of Rhodopseudomonas sphaeroides. I. Determination of the charge recombination pathway of D+QAQ−B and free energy and kinetic relations between Q−AQB and QAQ−B

The electron-transfer reactions and thermodynamic equilibria involving the quinone acceptor complex in bacterial reaction centers from R. sphaeroides were investigated. The reactions are described by the scheme: We found that the charge recombination pathway of D⁺Q_AQ^?_B proceeds via the intermediate state D⁺Q^?_AQ_B, the direct pathway contributing less than approx. 5% to the observed recombination rate. The method used to obtain this result was based on a comparison of the kinetics predicted for the indirect pathway (given by the product k_AD-times the fraction of reaction centers in the Q^?_AQ_B state) with the observed recombination rate, k^obs_{D⁺ →D}. The kinetic measurements were used to obtain the pH dependence (6.1 ? pH ? 11.7) of the free energy difference between the states Q^?_AQ_B and Q_AQ^?_B. At low pH (less than 9) Q_AQ^?_B is stabilized relative to Q^?_AQ_B by 67 meV, whereas at high pH Q^?_AQ_B is energetically favored. Both Q^?_A and Q^?_B associate with a proton, with pK values of 9.8 and 11.3, respectively. The stronger interaction of the proton with Q^?_B provides the driving force for the forward electron transfer.