Activity and Structure Changes of Arginine Kinase from Shrimp Feneropenaeus chinensis Muscle in Trifluoroethanol Solutions

Trifluoroethanol has often been used in protein folding studies. The changes in activity and unfolding of arginine kinase from shrimp Feneropenaeus Ghinensis muscle during denaturation in different concentrations of trifuoroethanol were investigated using far-ultraviolet circular dichroism and fluorescence emission spectra. Arginine kinase was inactivated in trifluoroethanol solutions. The tertiary and secondary structures of arginine kinase were also destroyed in the trifluoroethanol solutions. The unfolding and inactivation courses were measured and compared. Inactivation occurred prior to unfolding, which suggests that the arginine kinase active site is more easily damaged by the denaturant than the enzyme as a whole. The result also indicates that the arginine kinase active site is situated in a limited and flexible region of the enzymemolecule.     

Kinetics of Inhibition of Polyphenol Oxidase Obtained from Tobacco Nicotiana Tobacum

In this study, the kinetics of inhibition of polyphenol oxidase by L-cysteine has been investigated. The inhibition of tobacco polyphenol oxidase was studied using the progress-of-substrate-reaction method proposed by Tsou, following the substrate reaction during irreversible inhibition of the enzyme activity. Analysis of the inhibition kinetics shows that inhibition occurs by an irreversible and non-complexation reaction. The microscopic rate constants were determined for reaction of the inhibitor both with the free enzyme and with the enzyme-substrate complex. The results show that the presence of the substrate has a significant protective effect of the enzyme against inactivation by L-cysteine.     

Molecular mechanisms of serpin deficiencies

The study of serpin deficiency is currently one of the most active areas in basic medical research. Recently, three hypotheses concerning serpin deficiency have been proposed, which are referred to as the conformational disturbance hypothesis (CDH) , loop-sheet polymerisation hypothesis (LSPH) and multiple binding site hypothesis (MB-SH) . CDH was put forward to explicit serpin deficiency due to conformational change of reactive loop of serpins as a result of mutations occurring away from the reactive site residues and LSPH was to explain deficient serpins due to the formation of polymers. MBSH was proposed to explain the mechanism of the formation of stable enzyme-serpin complex via more than one binding site and blockage or mutation in any of the sites resulting in serpin deficiency. A combination of these mechanisms may be critical in understanding the roles of the many documented mutations and autoimmunities which result in qualitative and quantitative serpin deficiency.     

Protein tyrosine phosphatase is possibly involved in cellular signal transduction and the regulation of ABA accumulation in response to water deficit in Maize L.coleoptile

Water deficit-induced ABA accumulation is an ideal model or “stimulus-response”system to investigate cellular stress signaling in plant cels,using such a model the cellular stress signaling triggered by water deficit was investigated in Maize L.coleoptile.Water deficit-induced ABA accumulation was sensitively blocked by NaVO3,a potent inhibitor both to plasma membrane H^ -ATPase(PM-H^ -ATPase)and protein tyrosine phosphatase(PTPase).However,while PM-H^ -ATPase activity was unaffected under water deficit and PM-H^ -ATPase activator did not induce an ABA accumulation instead of water deficit,water deficit induced an increase in the protein phosphatase activity,and furthermore,ABA accumulation was inhibited by PAO,a specific inhibitor of PTPase.These results indicate that protein phosphtases may be involved in the cellular signaling in response to water deficit.Further studies identifiled at least four species of protein phosphtase as assayed by using pNPP as substrate,among which one component was especially sensitive to NaVO3.The NaVO3-sensitive enzyme was purified and finally showed a protein band about 66kD on SDS/PAGE.The purified enzyme showed a great activity to some specific PTPase substrates at pH 6.0.In addition to NaVO3,the enzyme was also sensitive to some other PTPase inhibitors such as Zn^2 and MO3^3 ,but not to Ca^2 and Mg^2 ,indicating that it might be a protein tyrosine phosphatase.Interestingly,the purified enzyme could be deactivated by some reducing agent DTT.which was previously proved to be an inhibitor of water deficit-induced ABA accumulation.This result further proved that PTPase might be involved in the cellular signaling of ABA accumulation in response to water deficit.     

Effect of Residue Mutation on the Electrostatic Potential in EcCIC

The effect of mutation of strongly conserved porelining residues in the chloride channel EcClC on the electrostatic potential and binding free energy of the chloride ion was studied using explicit protein-membrane structures. Electrostatic potential distribution and binding free energy of the chloride ion at different binding sites in the wild-type and mutated EcClC were calculated with APBS. The potential data reveal that the electrostatic potential around the selectivity filter, especially around the site Sext and Scen becomes more negative as the residue R147 was mutated to C147. The electrostatic binding free energy shows that the binding free energy of the chloride ion at all binding sites becomes more positive as R147 was mutated. It follows that mutation of R147 decreases ion stabilization at binding sites and affects channel＇s gating.     

Extraction and Purification of Matrix Protein from the Nacre of Pearl Oyster Pinctada fucata

A soluble matrix protein P14 with an apparent molecular mass of 14.5 kDa was isolated from fragmented nacre of pearl oysters （Pinctada fucata） treated with 10% NaOH solution to investigate the nacre matrix proteins and their effect on the CaCO3 crystal. The protein was characterized by gel exclusion chromatography and reversed-phase high performance liquid chromatography after demineralization by 10% acetic acid. The X-ray diffraction pattern of P14 crystals indicates that P14 plays an important role in nacre biomineralization. P14 can induce aragonite formation, stimulate CaCO3 crystal formation, and accelerate aragonite precipitation. Heating of the acid insoluble nacre residue, which was named conchiolin, in 10% sodium dodecyl sulfate solution supplemented with 10% β-mercaptoethanol solution for 10-20 min at about 100℃ gave two other soluble proteins having molecular masses of 19.4 kDa and 25.0 kDa. The present study suggests that these two proteins are linked to the insoluble organic matrix by disulfide bridges because the extraction yield increases when β-mercaptoethanol is added to the medium.     

Effect of the microstructure of Al 7050-T7451 on anodic oxide formation in sulfuric acid

The effect of the microstructure of an Al 7050-T7451 substrate on the anodic oxide formation in sulfuric acid was studied in this article. The microstructure of the substrate was assessed by optical microscope (OM) and transmission electron microscope (TEM). The surface and cross-section morphologies of the oxide films were examined by scanning electron microscope (SEM). The chemical composition of intermetallic particles in the alloys and films was investigated using energy dispersive spectroscope (EDS). The roles of intermetallic phases and grain or subgrain boundaries on the oxide film formation were researched using the potentiodynamic and potentiostatic polarization technique in sulfuric acid solution. The results show that the transition of coarse intermetallic particles or grain (subgrain) boundaries at the surface of Al alloys can be characterized by potentiodynamic polarization curves. The surface and cross-section micrographs of the anodic layer seem to preserve the microstructure of the substrate. Large cavities in the anodic films are caused by the preferential dissolution of coarse AItCuMg particles and the entrance of Cu-rich remnants into the electrolyte during anodizing. The Al₇Cu₂Fe particles tend to be occluded in the oxide layer or lose from the oxide surface because of peripheral trenching. Small pores in the films are induced by the dissolution of precipitates in grain or subgrain boundaries. The film surface of recrystallized grain bodies is smooth and homogeneous.     

Orthorhombic crystal structure of an insulin mutant B9Asp at 0.20 nm resolution

Human B9(Ser~i'Asp) insulin, a fast-absorption insulin analogue, was obtained by site-directed mutagenesis at B9 position. The orthorhombic crystal structure of B9Asp insulin was analyzed by crystallography at 0.20 nm resolution. Although no significant change of its overall conformation was observed, the local conformation flanking B9 site differed greatly from native T6 human insulin. The substitution of serine at B9 position by aspartic acid resulted in obvious alteration of local hydrophobic and hydrophilic interactions. As a result, the insulin dimer became unstable and the capability of the hexamer formation was diminished extensively. All these properties contribute to the fast-absorption of B9Asp, In addition, the open state of N-terminus of B-chain, which differs from T- or R- state, might suggest a new conformational state in the monomer or dimer insulin.     

Equilibrium dialysis study on the interaction between Cu(Ⅱ) and HSA or BSA

The interaction of Cu(Ⅱ) and human serum albumin (HSA) or bovine serum albumin (BSA) at physiological pH is studied by equilibrium dialysis. The successive stability constants are obtained by non-linear least square methods fitting Bjerrum formula. For both the Cu(Ⅱ)-HSA and Cu(Ⅱ)-BSA systems, the order of magnitude of K 1 and K 2 was found to be ≈10 4 mol -1·dm 3. There are about twenty stoichiometry binding sites found in one HSA or BSA molecule. They can be divided into two or three sets. Results of equilibrium dialysis experiments suggest that there exists one strong metal binding site in both Cu(Ⅱ)-HSA and Cu(Ⅱ)-BSA. It is the imidazol group nitrogen atoms of His 3 that are primarily concerned with copper binding site. After reaching dialysis equilibrium, there is the interaction among the different binding sites, the values of K all deviate from the simple statistical effect except for K-1 and K-2 in both Cu(Ⅱ)-HSA and Cu(Ⅱ)-BSA systems, and the positive cooperative effect is found.     

Inactivation of Lactate Dehydrogenase from Pig Heart by o—Phthalaldehyde

IntroductionLactate dehydrogenase( EC1 .1 .1 .2 7,LDH) playsa vital role in the energy flow of higher organisms.It is a tetrameric enzyme that catalyzes thereversible dehydrogenation of lactate,converting itto pyruvate.The enzyme exists in five isozymicforms which are not usually found all together inone tissue or organ.At least three different typesof LDH subunits( M,H and X) are known invertebrates,butthe X subunits are usually presentin only one or at mosta few tissues[1,2 ] .　Variou…     

组氨酸残基在焦磷酸:果糖-6-磷酸1-磷酸转移酶催化功能中的作用

应用焦碳酸二乙酯(DEPC)对焦磷酸:果糖-6-磷酸1-磷酸转移酶(PFP)进行化学修饰时,酶的活性迅速丧失,呈拟一级动力学反应.羟胺的复活作用及修饰后酶的紫外光谱变化表明,酶失活的原因是DEPC与酶的组氨酸残基形成了乙酯基组氨酸复合物.失活动力学表明,酶的活性位点结合1分子DEPC即丧失活性.活性位点的组氨酸残基的完整性是酶活性的必要条件之一.底物F6P、产物果糖1,6-二磷酸(FBP)、P i及激活剂果糖2,6-二磷酸(F2,6BP)均可保护酶免被DEPC失活,底物PP i却没有保护作用.组氨酸残基在酶的催化功能中可能与F6P,FBP及P i的结合过程有关,而与PP i无关.     

Localization of calmodulin and calmodulin-like protein and their functions in biomineralization in P. fucata

Calmodulin (CaM) and calmodulin-like protein (CaLP) are two proteins involved in biomineralization. Their localizations in Pinctada fucata mantle epithelia were studied by Western blot (WB) analysis of the nuclear/cytosol fraction of primary cultured Pinctada fucata mantle cells and immunogold electron microscopy. The results showed a completely different distribution of these two proteins at the subcellular level. CaM was distributed throughout both the nucleus and cytoplasm of the mantle epithelium but CaLP was distributed only in the cytoplasm. The functions of these two proteins in biomineralization were investigated by shell regeneration. During this process, the expressions of CaM and CaLP were greatly enhanced in different organelles of the mantle epithelium. Overexpression of these two proteins and a mutant of calmodulin-like protein (M-CaLP) that lacks an extra C-terminal tail in MC3T3-E1 promoted the mRNA expression of osteopontin, a biomineralization marker for osteoblasts. All of the results indicated that CaM and CaLP have completely different distributions in the mantle epithelium and affect the biomineralization process at different levels. The extra C-terminal tail of CaLP is important for its functions in biomineralization in Pinctada fucata.     

长毛对虾碱性磷酸酶功能基团的研究

用对－氯汞苯甲酸修饰该酶分子中的巯基，酶活力不受影响，说明该酶不是“巯基酶”．用Ｎ溴代琥珀酰亚胺修饰酶后，活力完全丧失，修饰后的酶在２７５ｎｍ处的紫外吸收峰完全消失，３４０ｎｍ处荧光发射峰也逐渐下降至完全淬灭，说明色氨酸残基是酶活性必需基因之一．用甲醛、醋酸酐修饰氨基，溴代乙酸修饰咪唑基也可以使酶活力完全丧失，说明赖氨酸残基及组氨酸残基也是酶活性功能基因．用乙酰丙酮修饰精氨酸残基，酶活力下降了５０％，精氨酸残基可能与维系酶分子构象有关．     

Cloning and characterization of 37 kD laminin receptor precursor in pearl oyster, Pinctada fucata

A 1063bp cDNA clone encoding a putative 37 kD laminin receptor precursor (37 kD LRP) is isolated from the mantle tissue of pearl oyster, Pinctada fucata. The amino acid sequence predicted from the cDNA sequence is 301 residues long, with a calculated molecular mass of 33.5 kD. RT-PCR analysis shows that 37 kD LRP mRNA is especially highly expressed in the mantle while widely expressed in several tissues. In situ hybridization analysis reveales that 37 kD LRP is expressed in the outer epithelial cells of the mantle edge, suggesting its involvement in cell proliferation and secretion in P. fucata. The identification and characterization of 37 kD LRP in the pearl oyster will help us to further understand the signal transduction in the processes of mantle epithelial cell proliferation and tissue formation.     

合浦珠母贝幼苗的正常生长

调查珍珠养殖合浦珠母贝人工培育幼苗的管养和生长情况,认为合浦珠母贝的幼苗生长正常,不存在“贝种退化”的影响,成活率和生长率的提高,明显是由于幼苗营养方法的改良。     

Cloning, Characterization, and Expression Analysis of Calreticulin from Pearl Oyster Pinctada fucata

Calreticulin is a unique calcium-binding protein with multiple functions mostly located in the sarcoplasmic/endoplasmic reticulum. A large amount of calcium is absorbed from the medium and transported to mineralization sites during biomineralization in pearl oyster. This paper describes the cloning of the full-length cDNA of calreticulin from Pinctada fucata, namely PCRT. PCRT encodes a deduced 414-amino acid protein, which includes a predicted 17- amino acid signal peptide and an endoplasmic reticulum retrieval sequence HDEL. The protein shows 63%-76% sequence identity and shares some common characteristics with calreticulins from other species. Semi-quantitative RT-PCR indicates that PCRT is ubiquitously expressed in all tissues tested with the highest expression in the hemolymph and the mantle. In situ hybridization analysis of PCRT in the mantle showed strong signals in the inner fold, the inner side of middle fold, and the inner side of outer fold of the mantle epithelium, All these results suggest PCRT might be involved in Ca^2＋ transport and storage during oyster biomineralization.     

内切酶xhoI部分氨基酸残基的化学修饰

限制性核酸内切酶xho I能够被巯基试试剂和修饰赖氨酸、精氨酸残基的试剂抑制,酶的抑制作用遵守假一级动力学,底物λ-DNA对酶的抑制作用有强烈地保护作用,这些资料表明xho Ⅰ含有对酶活性至关重要的必须的赖氨酸残基、精氨酸残基和巯基。