Physicochemical ProPerties and binding-site amino acid residues of galactoside-binding Protein of human Placenta

The galactose-binding lectin of human Placenta has been Purified to homogeneity by affinity chromatograPhy on asialo-fetuin column. The Protein, extractable from the tissue only with lactose is aPParently membrane-bound. Molecular weight determination of native Protein and subunit indicated a dimer of l3.4 kDa subunits. Inhibition of haemagglutination with various saccharides indicate that thiodigalactoside is the best inhibitor followed by lactose. However,P-nitroPhenyl-and 1-O-methyl derivatives of galactose showed that α-anomers inhibited slightly better than β-anomer. Modification of amino acid residues indicated involvement of arginine, lysine and histidine residues at the saccharidebinding site. Cysteine residue modificatioin also abolished haemagglutinating activity. Amino acid comPosition of the lectin is also Presented.     

Dicarboxylic acid anhydrides as dissociating agents of protein-containing structures

Dissociation of protein-containing structures by modification of protein amino groups with dicarboxylic acid anhydrides is a mild procedure which, in some cases, offers advantages over treatment with alternative dissociating agents, such as urea, guanidine hydrochloride, detergents, high ionic strength, and extremes of pH: In addition to dissociating multimeric proteins and protein aggregates, dicarboxylic acid anhydrides are effective dissociating agents for membrane-bound proteins and nucleoprotein particles. With most dicarboxylic acid anhydrides reviewed, the introduced reagent residues can be eliminated under moderate acid conditions, which allows the purification of unmodified individual components, and the use of disassembly-reconstitution systems valuable for investigating the structural and functional roles played by the individual components of complex particles:Each reagent can be suitable for a particular purpose, depending on the required specificity of the modification and stability of the modified groups: The stability of the acylated amino groups ranges from the very stable succinylated amino groups to the very labile acylation obtained with dimethylmaleic anhydride: Between these extremes, the stability of the modified amino groups decreases stepwise in the following order: maleic, exo-cis-3,6-endoxo-⁴-tetrahydrophthalic, citraconic, and 3,4,5,6-tetrahydrophthalic anhydride. With respect to the selectivity of the produced modification, little or no modification of hydroxyamino acid and cysteine residues has been observed with dimethylmaleic, exo-cis-3,6-endoxo-⁴-tetrahydrophthalic, and 3,4,5,6-tetrahydrophthalic anhydrides: With the other reagents, the extent of modification of hydroxyamino acid residues increases in the order citraconic, maleic and succinic anhydride: Citraconic and maleic anhydrides can produce irreversible modification of cysteine residues, the reactivity of sulfhydryl groups being higher with maleic anhydride:     

The chemical modification of cysteine-69 of rat liver fatty acid-binding protein (FABP): a fluorescence approach to FABP structure and function

Summary Hepatic-FABP was labelled at cysteine-69 with the fluorescent environmentally sensitive reporter group AEDANS. The labelled protein had an emission maximum at 465 nm indicating that cysteine-69 was buried in a non-polar environment. The modified protein was still able to bind ligands such as oleic acid, oleoyl CoA and haem. The affinity of AEDANS-FABP for haem was unaltered as compared with the native protein indicating that cysteine-69 must be remote from the ligand binding site. The binding of oleic acid did not significantly perturb the fluorescence emission spectrum of the fluorescent reporter group suggesting that there are not large conformational changes in the region of cysteine-69 on fatty acid binding. The binding of stoichiometric amounts of oleoyl CoA was accompanied by a small fluorescence enhancement which suggests that fatty acyl CoAs may interact with other regions of the FABP molecule not involved in fatty acid binding.Abbreviations FABP Fatty Acid-Binding Protein - AEDANS 5-[2-(Acetyl)aminoEthyl]Aminonaphthalene-1-Sulphonic Acid - AAEDANS 5-[2-(Iodoacetyl-AminolEthyl] aminonaphthalene-1-Sulphonic Acid - DTNB 5,5Dithiobis-(2-Nitrobenzoic acid)     

Chemical modification of active sites in relation to the catalytic mechanism of F1

Recent studies of chemically modified F₁-ATPases have provided new information that requires a revision of our thinking on their catalytic mechanism. One of the subunits in F₁-ATPase is distinguishable from the other two both structurally and functionally. The catalytic site and regulatory site of the same subunit are probably sufficiently close to each other, and the interaction between the various catalytic and regulatory sites are probably sufficiently strong to raise the uni-site rate of ATP hydrolysis by several orders of magnitude to that of promoted (multi-site) ATP hydrolysis. Although all three subunits in F₁ possess weak uni-site ATPase activity, only one of them () catalyzes promoted ATP hydrolysis. But all three subunits catalyze ATP synthesis driven by the proton flux. Internal rotation of the ₃₃ or ₃ moiety relative to the remainder of the F₀F₁ complex did not occur during oxidative phosphorylation by reconstituted submitochondrial particles.     

柞蚕抗菌肽D中精氨酸、赖氨酸和色氨酸等氨基酸残基的化学修饰

用不同的化学试剂修饰了柞蚕抗菌肽D分子中的色氨酸、精氨酸和赖氨酸等氨基酸残基。NBS修饰抗菌肽D,以及氨肽酶M对抗菌肽D作用的结果表明色氨酸残基对抗菌肽D抑制E.coli D31的作用影响不大。CHD和MLH对精氨酸和赖氨酸残基的修饰,导致抗菌肽D失去抑制E.coli的作用,但可逆地消除CHD和MLH的修饰作用后,抗菌肽D恢复了对E.coli D31的抑菌作用。这些结果初步认为,抗菌肽D抑菌作用与分子中的荷电性有关,改变了分子的电荷,也就同时失去了其抑菌功能。 此外,对精氨酸残基修饰的结果还表明,抗菌肽D的免疫原性与精氨酸残基有关。但是,抗菌肽D的免疫决定簇与其生物活性中心并不完全平行。     

Developmental Pattern for Deoxyhypusine Hydroxylase in Rat Brain

Deoxyhypusine hydroxylase catalyzes the formation of hypusine from deoxyhypusine in a precursor form of eukaryotic initiation factor 4D (eIF-4D). The enzymatic activity was examined in mammalian brain homogenates and the results were consistent with the existence of deoxyhypusine hydroxylase levels comparable to those occurring in other mammalian tissues. Interspecies differences in the enzyme distribution were quite limited, with the highest specific activity values observed in cow brain (1.82 units/ mg of protein). In the rat the enzyme was found to be unevenly distributed among various brain regions. The parietal cortex contained the highest specific activity (2.1 units/mg of protein). Rat brain deoxyhypusine hydroxylase was mainly present in the postmicrosomal supernatant (81% of the total activity). The highest specific activity (3 units/mg of protein) was observed in the rat brain during the first few days of life. Thereafter the activity started to decline, and continued to do so for 15 days, remaining throughout the rest of life at levels of less than one-half that of newborn.     

To identify impacts in variable systems using anomalous changes: a salt marsh example

A method is proposed to identify impacts of habitat modification in cases where it is difficult to site experimental and control samples. This problem occurs especially in heterogeneous systems, but may pose difficulties in any field experimental situation. The method is relevant to the situation where treated (modified) and untreated sites are spread over a range of habitat types. Types of change are identified and compared to treatments. If a specific change type is associated with a particular treatment then it is likely that the change is causally related to the treatment. There are five stages in the analysis. First, the classes or states of the sample sites, over a period of time, are identified (by numerical classification). Second, for each sample site, the sequence of states is listed. Third, transition matrices are made for each sample site to show the changes which have occurred. Fourth, the transition matrices are classified, to identify types of change. Finally, we use the Chisquared test to indicate whether the treated and untreated sites are associated with particular types of change. As an example, we refer to habitat modification to manage salt-marsh mosquitoes and we evaluate impacts on the environment mainly through changes to the vegetation. We consider that the method has potential to identify changes in heterogeneous systems even though little change was identified in the particular salt marsh studied.     

色氨酸残基在磷酸烯醇式丙酮酸羧化酶催化功能中的作用

在pH7.5条件下,用NBS对PEP羧化酶中色氨酸残基进行共价修饰表明,PEP羧化酶中48个色氨酸残基均能被NBS修饰。用邹承鲁图解法求得,其中4个残基为酶表现催化活性所必需的。 PEP羧化酶的变构效应剂G6P、Gly及Mal分别与酶预保温后,再经NBS修饰,前两种处理中,同样浓度的NBS所用修饰的色氨酸残基数和处理后的残存酶活与对照相比有很大的差异,而用Mal处理的,两者与对照相差无几。     

C1 metabolism inParacoccus denitrificans: Genetics ofParacoccus denitrificans

Paracoccus denitrificans is able to grow on the C₁ compounds methanol and methylamine. These compounds are oxidized to formaldehyde which is subsequently oxidized via formate to carbon dioxide. Biomass is produced by carbon dioxide fixation via the ribulose biphosphate pathway. The first oxidation reaction is catalyzed by the enzymes methanol dehydrogenase and methylamine dehydrogenase, respectively. Both enzymes contain two different subunits in an ₂₂ configuration. The genes encoding the subunits of methanol dehydrogenase (moxF andmoxI) have been isolated and sequenced. They are located in one operon together with two other genes (moxJ andmoxG) in the gene ordermoxFJGI. The function of themoxJ gene product is not yet known.MoxG codes for a cytochromec _551i , which functions as the electron acceptor of methanol dehydrogenase. Both methanol dehydrogenase and methylamine dehydrogenase contain PQQ as a cofactor. These so-called quinoproteins are able to catalyze redox reactions by one-electron steps. The reaction mechanism of this oxidation will be described. Electrons from the oxidation reaction are donated to the electron transport chain at the level of cytochromec. P. denitrificans is able to synthesize at least 10 differentc-type cytochromes. Five could be detected in the periplasm and five have been found in the cytoplasmic membrane. The membrane-bound cytochromec ₁ and cytochromec ₅₅₂ and the periplasmic-located cytochromec ₅₅₀ are present under all tested growth conditions. The cytochromesc _551i andc _553i , present in the periplasm, are only induced in cells grown on methanol, methylamine, or choline. The otherc-type cytochromes are mainly detected either under oxygen limited conditions or under anaerobic conditions with nitrate as electron acceptor or under both conditions. An overview including the induction pattern of allP. denitrificans c-type cytochromes will be given. The genes encoding cytochromec ₁, cytochromec ₅₅₀, cytochromec _551i , and cytochromec _553i have been isolated and sequenced. By using site-directed mutagenesis these genes were mutated in the genome. The mutants thus obtained were used to study electron transport during growth on C₁ compounds. This electron transport has also been studied by determining electron transfer rates inin vitro experiments. The exact pathways, however, are not yet fully understood. Electrons from methanol dehydrogenase are donated to cytochromec _551i . Further electron transport is either via cytochromec ₅₅₀ or cytochromec _553i to cytochromeaa ₃. However, direct electron transport from cytochromec _551i to the terminal oxidase might be possible as well. Electrons from methylamine dehydrogenase are donated to amicyanin and then via cytochromec ₅₅₀ to cytochromeaa ₃, but other routes are used also.P. denitrificans is studied by several groups by using a genetic approach. Several genes have already been cloned and sequenced and a lot of mutants have been isolated. The development of a host/vector system and several techniques for mutation induction that are used inP. denitrificans genetics will be described.     

Prospective theoretical and experimental study towards electrochemically grafted poly(N-vinyl-2-pyrrolidone) films on metallic surfaces

Quantum chemistry calculations of equilibrium geometry, atomic charges, dipole moment, and frontier orbital energies are carried out on model N-vinyl-2-pyrrolidone, its radical anion and cation, and three protonated derivatives. Attempts to obtain poly(N-vinyl-2-pyrrolidone) films grafted on nickel and platinum electrodes, respectively by cathodic and anodic polarizations, are reported. A thin, covering and insulating film is obtained on the Pt anode, as indicated by surface characterizations carried out with UPS and IRRAS spectroscopies. The fact that the film remains adherent to the metallic surface in spite of a permanent contact with an acetonitrile solution in which poly(N-vinyl-2-pyrrolidone) is soluble suggests that chemisorption has indeed been achieved.