Effects of some metal ions on human erythrocyte glutathione reductase: an in vitro study

In this study, we investigated inhibitory effects of some metal ions on human erythrocyte glutathione reductase. For this purpose, initially human erythrocyte glutathione reductase was purified 1051-fold in a yield of 41% by using 2', 5'-ADP Sepharose 4B affinity gel and Sephadex G-200 gel filtration chromatography. SDS polyacrylamide gel electrophoresis was done in order to control the purification of enzyme. SDS polyacrylamide gel electrophoresis showed a single band for enzyme. A constant temperature (4 degrees C) was maintained during the purification process. Enzyme activity was determined with the Beutler method by using a spectrophotometer at 340 nm. Hg(2+), Cd(2+), Pb(2+), Cu(2+), Fe(3+) and Al3+ exhibited inhibitory effects on the enzyme in vitro. K(i) constants and IC(50) values for metal ions were determined by Lineweaver-Burk graphs and plotting activity % vs. [I]. IC(50) values of Pb(2+), Hg(2+), Cu(2+), Cd(2+), Fe(3+) and Al(3+) were 0.011, 0.020, 0.0252, 0.0373, 0.209 and 0.229 mM, and the Ki constants 0.0254+/-0.0027, 0.0378+/-0.0043, 0.0409+/-0.0048, 0.0558+/-0.0083, 0.403+/-0.043 and 1.137+/-0.2 mM, respectively. While Pb(2+), Hg(2+), Cd(2+) and Fe(3+) showed competitive inhibition, others displayed noncompetitive inhibition.     

Influence of ligand structure on Fe(II) spin-state and redox rate in cytotoxic tripodal chelators

The Fe coordination chemistry of several tripodal aminopyridyl hexadentate chelators is reported along with cytotoxicity toward cultured Hela cells. The chelators are based on cis, cis-1,3,5-triaminocyclohexane (tach) with three pendant -CH2-2-pyridyl groups where 2-pyridyl is R-substituted thus are named tach-x-Rpyr where x=3, R=Me; x=3, R=MeO; x=6; R=Me. The structures of [Fe(tach-3-Mepyr)]Cl2 and [Fe(tach-3-MeOpyr)](FeCl4) are reported and their metric parameters indicate strongly bound, low-spin Fe(II). The structure of [Fe(tach-6-Mepyr)](ClO4)2 implies steric effects of 6-Me groups push donor Npy's away so one Fe-Npy bond is substantially longer at 2.380(3)A vs. 2.228(3)A for the others, and Fe(II) in the high-spin-state. Accordingly, anions X(-)=Cl or SCN afford [Fe(tach-6-Mepyr)(X)]+ from [Fe(tach-6-Mepyr)]2+ (UV-vis spectroscopy). Consistent with a biological cytotoxicity involving Fe chelation, chelators of low-spin Fe(II) have greater toxicity in the order [IC50(72 h) is in parentheses then the spin-state SS=H (high) or L (low)]: tachpyr=tach-3-Mepyr (6 microM, SS=L) greater, similar tach-3-MeOpyr (12microM, SS=L)>tach-6-Mepyr (>200 microM, SS=H). Iron-mediated oxidative dehydrogenation with O2 oxidant removes hydrogens from coordinated nitrogen and the adjacent CH2, converting aqueous [Fe(tach-3-Rpyr)]2+ (R=H, Me and MeO) into a mix of low-spin imino- and aminopyridyl-armed complexes, but [Fe(tach-6-Mepyr)]2+ does not react (NMR and ESI-MS spectroscopies). The difference of IC(50) for chelators at different time points (delta IC50=[IC50(24h)-IC50(72 h)]) is used to compare rate of cytotoxic action to qualitative rate of oxidation in the Fe-bound chelator, giving the order, from rapid to slow oxidation and cell killing of: [Fe(tach-3-Mepyr)]2+ (delta IC50=5 microM)>[Fe(tachpyr)]2+ (delta IC50=16 microM)>[Fe(tach-3-MeOpyr)]2+ (delta IC50=118 microM). Thus, those chelators whose Fe(II) complexes undergo rapid oxidation kill cells faster, and those that bind Fe(II) as low-spin are far more cytotoxic.     

A four-iron, four-sulfide ferredoxin with high thermostability from Clostridium thermoaceticum.

A ferredoxin containing only one [Fe4S4] cluster was purified from Clostridium thermoaceticum. It has a molecular weight of about 7,300, a partial specific volume of 0.67, and an isoelectric point of 3.25. Its absorption spectrum has two maxima at 390 nm (epsilon = 16.8 X 10(3)M-1cm-1) and at 280 nm (epsilon = 24.2 X 10(3)M-1cm-1). The absorption at 390 nm is almost half that of other clostridial ferredoxins, which have two [Fe4S4] clusters, and is similar to other ferredoxins with only one [Fe4S4] cluster. The ferredoxin had high thermal stability and retained over 50% of its activity after treatment at 80 degrees C. It functions in the transfer of electrons from pyruvate to nicotinamide adenine dinucleotide phosphate (NADP), which indicates the presence of pyruvate:ferredoxin oxidoreductase and reduced ferredoxin-NADP reductase in C, thermoaceticum. NADPH is used in the synthesis of acetate from CO2 in this organism.     

甲状旁腺激素和转铁蛋白N端半分子融合蛋白在毕赤酵母中的表达

用重叠PCR技术将PTH（parathyroid hormone, 甲状旁腺激素）基因与TFN（transferrin N_terminal half_molecule, 转铁蛋白N端半分子）基因在体外融合,融合基因克隆至真核表达载体pPIC9中,转化毕赤酵母GS115。转化子经甲醇诱导后,融合蛋白得到了表达并分泌到发酵上清液中。经 SP Sepharose F F阳离子交换层析、Phenyl Sepharose Fast Flow疏水层析纯化获得了纯度大于95％的PTH_TFN样品。Western blot分析及腺苷酸环化酶实验证明融合蛋白中的PTH具有与抗PTH抗体结合能力及刺激腺苷酸环化酶的活性,铁饱和实验证明融合蛋白中的TFN和单独的TFN具有相同铁结合能力。因而TFN可望作为PTH的天然运输载体。     

Application of superparamagnetic nanoparticles in purification of plasmid DNA from bacterial cells

The aim of this study was to develop a simple and rapid method for purification of ultrapure supercoiled plasmid DNA with high yields from bacterial cultures. Nanosized superparamagnetic nanoparticles (Fe3O4) were prepared by chemical precipitation method using Fe2+, Fe3+ salt, and ammonium hydroxide under a nitrogen atmosphere. The surface of Fe3O4 nanoparticles was modified by coating with the multivalent cationic agent, polyethylenimine (PEI). The nanoparticles were characterized by transmission electron microscopy, X-ray diffraction, Fourier transformation infrared spectroscopy and superconducting quantum interference device magnetometer. The PEI-modified magnetic nanobeads were employed to simplify the purification of plasmid DNA from bacterial cells. We demonstrated a useful plasmid, pRSETB-EGFP, encoding the green fluorescent protein with T7 promoter, was amplified in DE3 strain of Escherichia coli. The loaded nanobeads are recovered by magnetically driven separation and regenerated by exposure to the elution buffer with optimal ionic strength (1.25 M) and pH (9.0). Up to approximately 35 microg of high-purity (A260/A280 ratio=1.87) plasmid DNA was isolated from 3ml of overnight bacterial culture. EGFP expression was detected by fluorescent microscopy in the transformed E. coli cells, indicating the biological activities of DNA fragments were retained after purified from magnetic nanobeads. The protocol, starting from the preparation of bacterial lysate and ending with purified plasmids takes less than 10 min. Thus, the separation and purification qualities of PEI-modified magnetic nanobeads as well as its ease of use surpass those of conventional anion-exchange resins.     

干扰素与转铁蛋白融合蛋白在毕赤酵母中的表达及鉴定

利用重叠 PCR 技术将干扰素 (interferon , IFN) 基因与转铁蛋白 N 端半分子 (transferrin N-terminal half-molecule , TFN) 基因在体外融合,融合基因和单独的 TFN 基因分别克隆至真核表达载体 pPIC9 中,转化毕赤酵母 GS115 ,得到的转化子经诱导表达后在发酵上清中均获得了表达 . 经 SP Sepharose Fast Flow 阳离子交换层析、 Phenyl Sepharose Fast Flow 疏水层析纯化,获得了纯度大于 93 ％的重组融合蛋白 IFN-TFN 和纯度大于 95 ％的重组 TFN 样品 . 生物活性实验证明融合蛋白 IFN-TFN 具有抗病毒活性 . 铁饱和实验证明融合蛋白 IFN-TFN 和单独的 TFN 具有相同的铁结合能力 . 因而 TFN 可望作为 IFN 的天然运输载体 .     

An Integrated Approach to Aggregate Control for Therapeutic Bispecific Antibodies Using an Improved Three Column Mab Platform-Like Purification Process

Single chain variable fragment-IgGs (scFv-IgG) are a class of bispecific antibodies consisting of two single chain variable fragments (scFv) that are fused to an intact IgG molecule. A common trend observed for expression of scFv-IgGs in mammalian cell culture is a higher level of aggregates (10%–30%) compared to mAbs, which results in lower purification yields in order to meet product quality targets. Furthermore, the high aggregate levels also pose robustness risks to a conventional mAb three column platform purification process which uses only the polishing steps (e.g., cation exchange chromatography [CEX]) for aggregate removal. Protein A chromatography with pH gradient elution, high performance tangential flow filtration (HP-TFF) and calcium phosphate precipitation were evaluated at the bench scale as means of introducing orthogonal aggregate removal capabilities into other aspects of the purification process. The two most promising process variants, namely Protein A pH gradient elution followed by calcium phosphate precipitation were evaluated at pilot scale, demonstrating comparable performance. Implementing Protein A chromatography with gradient elution and/or calcium phosphate precipitation removed a sufficient portion of the aggregate burden prior to the CEX polishing step, enabling CEX to be operated robustly under conditions favoring higher monomer yield. From starting aggregate levels ranging from 15% to 23% in the condition media, levels were reduced to between 2% and 3% at the end of the CEX step. The overall yield for the optimal process was 71%. Results of this work suggest an improved three-column mAb platform-like purification process for purification of high aggregate scFv-IgG bispecific antibodies is feasible. © 2018 The Authors. Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers. Biotechnol. Prog., 35: e2720, 2019     

Accumulation of ferrous iron in Chlamydomonas reinhardtii. Influence of CO2 and anaerobic induction of the reversible hydrogenase

The green alga, Chlamydomonas reinhardtii, can photoproduce molecular H(2) via ferredoxin and the reversible [Fe]hydrogenase enzyme under anaerobic conditions. Recently, a novel approach for sustained H(2) gas photoproduction was discovered in cell cultures subjected to S-deprived conditions (A. Melis, L. Zhang, M. Forestier, M.L. Ghirardi, M. Seibert [2000] Plant Physiol 122: 127-135). The close relationship between S and Fe in the H(2)-production process is of interest because Fe-S clusters are constituents of both ferredoxin and hydrogenase. In this study, we used M?ssbauer spectroscopy to examine both the uptake of Fe by the alga at different CO(2) concentrations during growth and the influence of anaerobiosis on the accumulation of Fe. Algal cells grown in media with (57)Fe(III) at elevated (3%, v/v) CO(2) concentration exhibit elevated levels of Fe and have two comparable pools of the ion: (a) Fe(III) with M?ssbauer parameters of quadrupole splitting = 0.65 mm s(-1) and isomeric shift = 0.46 mm s(-1) and (b) Fe(II) with quadrupole splitting = 3.1 mm s(-1) and isomeric shift = 1.36 mm s(-1). Disruption of the cells and use of the specific Fe chelator, bathophenanthroline, have demonstrated that the Fe(II) pool is located inside the cell. The amount of Fe(III) in the cells increases with the age of the algal culture, whereas the amount of Fe(II) remains constant on a chlorophyll basis. Growing the algae under atmospheric CO(2) (limiting) conditions, compared with 3% (v/v) CO(2), resulted in a decrease in the intracellular Fe(II) content by a factor of 3. Incubating C. reinhardtii cells, grown at atmospheric CO(2) for 3 h in the dark under anaerobic conditions, not only induced hydrogenase activity but also increased the Fe(II) content in the cells up to the saturation level observed in cells grown aerobically at high CO(2). This result is novel and suggests a correlation between the amount of Fe(II) cations stored in the cells, the CO(2) concentration, and anaerobiosis. A comparison of Fe-uptake results with a cyanobacterium, yeast, and algae suggests that the intracellular Fe(II) pool in C. reinhardtii may reside in the cell vacuole.     

Nitrate does not result in iron inactivation in the apoplast of sunflower leaves

It has been hypothesized that nitrate (NO(3)(-)) nutrition might induce iron (Fe) deficiency chlorosis by inactivation of Fe in the leaf apoplast (H.U. Kosegarten, B. Hoffmann, K. Mengel [1999] Plant Physiol 121: 1069-1079). To test this hypothesis, sunflower (Helianthus annuus L. cv Farnkasol) plants were grown in nutrient solutions supplied with various nitrogen (N) forms (NO(3)(-), NH(4)(+) and NH(4)NO(3)), with or without pH control by using pH buffers [2-(N-morpholino)ethanesulfonic acid or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid]. It was shown that high pH in the nutrient solution restricted uptake and shoot translocation of Fe independently of N form and, therefore, induced Fe deficiency chlorosis at low Fe supply [1 micro M ferric ethylenediaminedi(O-hydroxyphenylacetic acid)]. Root NO(3)(-) supply (up to 40 mM) did not affect the relative distribution of Fe between leaf apoplast and symplast at constant low external pH of the root medium. Although perfusion of high pH-buffered solution (7.0) into the leaf apoplast restricted (59)Fe uptake rate as compared with low apoplastic solution pH (5.0 and 6.0, respectively), loading of NO(3)(-) (6 mM) showed no effect on (59)Fe uptake by the symplast of leaf cells. However, high light intensity strongly increased (59)Fe uptake, independently of apoplastic pH or of the presence of NO(3)(-) in the apoplastic solution. Finally, there are no indications in the present study that NO(3)(-) supply to roots results in the postulated inactivation of Fe in the leaf apoplast. It is concluded that NO(3)(-) nutrition results in Fe deficiency chlorosis exclusively by inhibited Fe acquisition by roots due to high pH at the root surface.     

Cytochrome b5 and NADH-cytochrome-b5 reductase from sipunculan erythrocytes; a methemerythrin reduction system from Phascolopsis gouldii

We report the purification and characterization of a soluble cytochrome b5 from coelomic erythrocytes of the sipunculan worm, Phascolopsis gouldii. We also report the isolation and purification of a membrane-bound NADH-cytochrome-b5 reductase from these erythrocytes. The non-heme iron protein, hemerythrin (Hr), is known to be the oxygen carrier in these erythrocytes. The aforementioned purified cytochrome b5 and reductase together catalyze the reduction of P. gouldii [Fe(III),Fe(III)]metHr to [Fe(II),Fe(II)deoxyHr by NADH. EPR spectroscopy demonstrates that a redox process involving formation of the intermediate [Fe(II),Fe(III)]semi-metHr occurs within intact sipunculan erythrocytes as well as in the system of purified components. The rhombic g-tensor of the EPR signal in both cases resembles that of (semi-met)RHr, the form obtained by one-electron reduction of metHr. These observations suggest that cytochrome b5 and NADH-cytochrome-b5 reductase in sipunculan erythrocytes function to counteract autoxidation of oxyHr. The sequence of electron flow in the system of purified components is: NADH----NADH-cytochrome-b5 reductase----cytochrome b5----metHr. At pH 7.5, the reduction of metHr in this system occurs in two phases, only the first of which is dependent on concentration of cytochrome b5. From an analysis of the kinetics and the EPR time-course, we propose that the two phases represent sequential reduction of met- to semi-metHr and reduction of semi-metHr to deoxyHr. This report represents the first demonstration of a physiological system for reduction of metHr.     

The use of the biotinyl estradiol-avidin system for the purification of "nontransformed" estrogen receptor by biohormonal affinity chromatography

Several biotinyl estradiol derivatives have been prepared by coupling estradiol 7 alpha-carboxylic acid to biotin via different linear linkers. All these compounds exhibit a high affinity for the estrogen receptor as determined by competitive binding assays against [3H]estradiol. These compounds also displaced the dye 4-hydroxyazobenzene-2'-carboxylic acid from the biotin-binding sites of avidin free or immobilized on agarose. It was demonstrated that only the derivatives bearing a long spacer chain (greater than 42 A greater than) between estradiol and biotin were able to bind receptor and avidin simultaneously, suggesting some steric hindrance. The biotin-avidin system has been investigated for the purification of the cytosoluble "nontransformed" estrogen receptor stabilized by sodium molybdate. The method relies on: 1) high biohormonal affinity of receptor for biotinyl estradiol derivative; 2) the specific selection by avidin-agarose column of biotinyl estradiol-receptor complexes; and 3) the biohormonal elution step by an excess of radioactive estradiol. Starting from unfractionated cytosol containing molybdate-stabilized nontransformed 8S estrogen receptor with estradiol 7 alpha-(CH2)10-CO-NH-(CH2)2-O-(CH2)2-O-(CH2)2-NH-CO-(CH2)3-NH-biotin, preliminary experiments using avidin-agarose chromatography and then a specific elution step by exchange with free [3H]estradiol, allowed a 500-1,500-fold purification. Further purification of estrogen receptor was obtained by ion exchange chromatography through a DEAE-Sephacel column and led to a congruent to 20% pure protein, assuming one binding site/65,000-Da unit. The hydrodynamic parameters of the purified receptor were essentially identical to those of molybdate-stabilized nontransformed receptor present in crude cytosol. The advantages of this double biotinyl steroid derivative-avidin chromatographic technique over more conventional affinity procedures are discussed and make it applicable to the purification of minute amounts of steroid receptors in a wide variety of tissues.     

Isolation of Eukaryotic Initiation Factor 2 from Rat Brain

Eukaryotic initiation factor 2 (eIF-2) was isolated from salt-washed microsomes of 4-day-old rat brain which show a high rate of protein synthesis. A three-step purification scheme was employed, including heparin-Sepharose, phosphocellulose, and DEAE-cellulose column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the isolated factor revealed three polypeptides with molecular weights of 43,000, 54,000, and 59,000 and 90% purity. The rat brain eIF-2 forms ternary complexes with [3H]methionyl-tRNAi and GTP. In terms of specific activity, the purification does not correspond to that revealed by electrophoretic analysis. During purification there is an apparent loss of additional factors that modulates the activity of eIF-2 and explains the high rate of activity of the crude fraction.     

Novel cytotoxic chelators that bind iron(II) selectively over zinc(II) under aqueous aerobic conditions

To achieve cellular iron deprivation by chelation, it is important to develop chelators with selective metal-binding properties. Selectivity for iron has long been the province of certain oxygen-donor chelators such as desferrioxamine, which target Fe(III) and exploit the strength of a relatively ionic Fe(III)-O interaction. We have been studying novel chelators that possess mechanisms to selectively chelate +2 biometals, particularly tachpyr [N,N',N"-tris(2-pyridylmethyl)-1,3,5-cis,cis-triaminocyclohexane] and derivatives from N,N',N"-trialkylation and pyridine ring alkylation. Metal-exchange and metal-binding competition reactions have been conducted at pH 7.4, 37 degrees C and time periods until no further change was observed (generally 24-48 h). Under anaerobic conditions, tachpyr is strongly selective for iron, binding 95+/-5% Fe(II) versus 5+/-5% Zn(II) in the forms [Fe(tachpyr)](2+) and [Zn(tachpyr)](2+) respectively. Under aerobic conditions, tachpyr complexes Fe(II) more effectively than Fe(III), forming iminopyridyl complexes [Fe(tachpyr-ox-n)](2+) (n=2, 4) by O(2)-induced and iron-mediated oxidative dehydrogenation. Complexes [Fe(tachpyr-ox-n)](2+) are also strongly bound forms of iron that are unaffected by an excess of Zn(II) (75 mol zinc:1 mol iron complex). The preference of tachpyr for iron over zinc under aerobic conditions appears to be hindered by oxidation of Fe(II) to Fe(III), such that the proportions bound are 44+/-10% Fe(II) versus 56+/-10% Zn(II), in the respective forms [Fe(tachpyr-ox-n)](2+) and [Zn(tachpyr)](2+). However, upon addition of the reducing agent Na(2)S(2)O(4) that converts Fe(III) to Fe(II), the binding proportions shift to 76+/-10% Fe(II) versus 24+/-10% Zn(II), demonstrating a clear preference of tachpyr for Fe(II) over Zn(II). Iron(II) is in the low-spin state in [Fe(tachpyr)](2+) and [Fe(tachpyr-ox-n)](2+) (n=2, 4), which is a likely cause of the observed selectivity. N-methylation of tachpyr [giving (N-methyl)(3)tachpyr] results in the loss of selectivity for Fe(II), which is attributed to the steric effect of the methyl groups and a resulting high-spin state of Fe(II) in [Fe(N-methyl)(3)tachpyr)](2+). The relationship of chelator selectivity to cytotoxicity in the tach family will be discussed.     

Studies on a phospholipase B from Penicillium notatum. Purification, properties, and mode of action.

1. Phospholipase B which hydrolyzes both the acyl ester bonds of diacylphospholipids (diacyl-hydrolase) and the acyl ester bond of monoacylphospholipids or lysophospholipids, [monoacyl-hydrolase or lysophospholipase, EC 3.1.1.5] was purified from Penicillium notatum about 2000-fold over the crude extract. The final preparation was homogeneous on disc electrophoresis. The apparent molecular weight, determined by gel filtration on Sephadex G-200, was about 116,000. The isoelectric point was pH 4.0. 2. The purified enzyme was a glycoprotein. The carbohydrate content was approximately 30%, consisting of mannose, glucose, and glucosamine. The amino acid composition was also determined. 3. The ratio of monoacyl-hydrolase to diacyl-hydrolase activities was influenced by the physical state of the substrate in the assay system. It was about 1 : 1 or 100 : 1 in the presence of absence of Triton X-100, respectively, and the latter value remained constant throughout the purification procedures. 4. Both enzyme activities had the same pH optimum, 4.0, and were heat-labile. None of the metals tested had any effect on either activity except for Fe2+ and Fe3+. Diisopropyl fluorophosphate at relatively high concentrations completely inhibited both enzyme activities. 5. The Michaelis-Menten constants (Km) of the enzyme for egg lecithin were about 1.5 and 25 mM in the absence and presence of Triton X-100, respectively. The Km value for dicaproyllecithin was 9.8 mM in the absence of Triton X-100. 6. Using a mixture of 1-[14C]stearoyl-lecithin and 2-[14C]oleoyl-lecithin in the presence of Triton X-100 as a substrate, it was found that the P. notatum phospholipase B attacked the acyl ester bonds sequentially, first the 2-acyl and then 1-acyl groups.     

Hemin uptake in Porphyromonas gingivalis: Omp26 is a hemin-binding surface protein.

A 26-kDa outer membrane protein (Omp26) has been proposed to play a role in hemin acquisition by Porphyromonas gingivalis (T. E. Bramanti and S. C. Holt, J. Bacteriol. 174:5827-5839, 1992). We studied [55Fe]hemin uptake in P. gingivalis grown under conditions of hemin starvation (Omp26 expressed on the outer membrane surface) and hemin excess (Omp26 not expressed on surface). [55Fe]hemin uptake occurred rapidly in hemin-starved cells which incorporated up to 70% of total [55Fe]hemin within 3 min. P. gingivalis grown under hemin-starved conditions or treated with the iron chelator 2,2'-bipyridyl to induce an iron stress took up six times more [55Fe]hemin than hemin-excess-grown cells. Polyclonal monospecific anti-Omp26 antibody added to hemin-starved cells inhibited [55Fe]hemin uptake by more than 50%, whereas preimmune serum had no effect. [55Fe]hemin uptake in hemin-starved P. gingivalis was inhibited (36 to 67%) in the presence of equimolar amounts of unlabeled hemin, protoporphyrin IX, zinz protoporphyrin, and Congo red dye but was not inhibited in the presence of non-hemin-containing iron sources. Heat shock treatment (45 degrees C) of hemin-excess-grown P. gingivalis (which cases translocation of Omp26 to the surface) increased [55Fe]hemin uptake by threefold after 3 min in comparison with cells grown at 37 degrees C. However, no [55Fe] hemin uptake beyond 3 min was observed in either hemin-excess-grown or hemin-starved cells exposed to heat shock. In experiments using heterobifunctional cross-linker analysis, hemin and selected porphyrins were cross-linked to Omp26 in hemin-starved P. gingivalis, but no cross-linking was seen with hemin-excess-grown cells. However, cross-linking of hemin to Omp26 was observed after heat shock treatment of hemin-excess-grown cells. Finally, anti-Omp26 antibody inhibited cross-linked of hemin to Omp26. These findings indicate that hemin binding and transport into P.gingivalis cell mediated by Omp26.     

Electron transfer between hydrogenases and mono- and multiheme cytochromes in Desulfovibrio ssp

 A comparative study of electron transfer between the 16 heme high molecular mass cytochrome (Hmc) from Desulfovibrio vulgaris Hildenborough and the [Fe] and [NiFe] hydrogenases from the same organism was carried out, both in the presence and in the absence of catalytic amounts of cytochrome c ₃. For comparison, this study was repeated with the [NiFe] hydrogenase from D. gigas. Hmc is very slowly reduced by the [Fe] hydrogenase, but faster by either of the two [NiFe] hydrogenases. In the presence of cytochrome c ₃, in equimolar amounts to the hydrogenases, the rates of electron transfer are significantly increased and are similar for the three hydrogenases. The results obtained indicate that the reduction of Hmc by the [Fe] or [NiFe] hydrogenases is most likely mediated by cytochrome c ₃. A similar study with D. vulgaris Hildenborough cytochrome c ₅₅₃ shows that, in contrast, this cytochrome is reduced faster by the [Fe] hydrogenase than by the [NiFe] hydrogenases. However, although catalytic amounts of cytochrome c ₃ have no effect in the reduction by the [Fe] hydrogenase, it significantly increases the rate of reduction by the [NiFe] hydrogenases. Received: 14 April 1998 / Accepted: 25 June 1998     

Relatively stable N-ligated [2Fe2S](2+) clusters with dipyrromethane capping ligands

[2Fe2S] clusters with terminal N-ligation (His, Arg) and unique functions are increasingly recognized in biological systems. In this work three new [2Fe2S] clusters 1-3 with different 1,1'-dipyrrolmethane derivatives as chelating terminal ligands have been prepared and fully characterized, including by X-ray crystallography: (NEt(4))(2)[L(2)Fe(2)(mu-S)(2)] with L=Me(2)C(C(4)H(3)N)(2) (1), Ph(2)C(C(4)H(3)N)(2) (2), (CH(2))(5)C(C(4)H(3)N)(2) (3). These systems represent rare examples of synthetic [2Fe2S] complexes with N-donor capping ligands. While geometric parameters as well as spectroscopic and electrochemical characteristics of the new complexes are as anticipated, the chelating nature of the terminal ligands in 1-3 imparts a relatively high stability that will be advantageous for reactivity studies of the [2Fe2S] core.     

Purification and characterization of phosphatidylinositol kinase from porcine liver microsomes

Phosphatidylinositol kinase was solubilized and purified from porcine liver microsomes to apparent homogeneity. The purification procedure includes: solubilization of microsomes by 2% Triton X-100, ammonium sulfate precipitation (20-35% saturation), Reactive blue agarose chromatography, DEAE-Sephacel chromatography and two consecutive hydroxyapatite chromatographies. A total of 4900-fold purification with 8% recovery of enzyme activity was achieved. The molecular weight of the enzyme as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 55000. The enzyme is stimulated in a decreasing order by Mg2+, Fe2+, Mn2+, Fe3+ and Co2+. Ca2+ inhibited Mg2+-stimulated activity with an I50 of 0.4 mM. Apparent Km values for phosphatidylinositol and ATP are 120 and 60 microM, respectively. The enzyme is inhibited by adenosine (I50 = 70 microM), ADP (I50 = 120 microM) and quercetin (I50 = 100 microM). The enzyme is also sensitive to sulfhydryl inhibitors. Using the purified enzyme as an immunogen, we have successfully prepared antibodies for phosphatidylinositol kinase in rabbits. The antibodies appear to recognize an antigen of Mr 55000 on SDS-polyacrylamide gel electrophoresis from various porcine tissues in Western blot analysis.     

Catalytic oxidation of cyclohexane by a binuclear Fe(III) complex biomimetic to methane monooxygenase

A novel binuclear Fe(III) complex [Fe(III)(BPMP)Cl(mu-O)Fe(III)Cl3] (1) was prepared from the reaction between (bis(2-pyridylmethyl)-1,4-piperazine) and [Fe(OH2)6]Cl3, in acetonitrile. The title compound was characterized by spectroscopic, electrochemical and X-ray crystallography analysis. The catalytic activity of the complex was evaluated through cyclohexane oxidation, using hydrogen peroxide as the terminal oxidant. Reaction products were identified by gas chromatography. Conversions up to 19.2% were observed (12.6% and 6.6% yields for cyclohexanol and cyclohexanone, respectively). The catalytic activity exhibited by 1 suggests that it can be considered as a functional biomimetic analog to methane monooxygenase.     

金属离子及保护剂对深绿木霉T155产芥子酶的活性影响及芥子酶的分离纯化

【目的】探求金属离子及保护剂对深绿木霉T155产生的粗芥子酶液活性的影响以及芥子酶的分离纯化方法。【方法】通过添加不同浓度的金属离子及保护剂研究对芥子酶活性的影响并通过细胞破壁、硫酸铵沉淀、透析、Sephadex G-100柱层析、DEAE-52离子交换层析、Sephadex G-200柱层析等方法提取到纯的芥子酶,最后通过电泳检测芥子酶的纯度和分子量。【结果】研究发现Ag+、Zn2+、Pb2+、Mg2+、Hg2+、Fe3+等金属离子在低浓度时对芥子酶均表现为一定的促进作用,但达到一定浓度后则起抑制作用,Ca2+浓度在0.069–17.000 g/L范围内均对酶活性起到促进作用,且促进效果基本与Ca2+浓度成正比;添加1 mmol/L EDTA和3 mmol/L DTT对芥子酶的保护作用最好,4°C保存26 d后芥子酶能够保持85%活性;电泳分析该芥子酶的分子量大约在150 kD左右,并且是一个二聚体。【结论】Ca2+在实验浓度内主要是提高芥子酶活性,而其他金属离子对芥子酶活性主要起抑制作用;EDTA或者EDTA与DTT协同能够较好保持芥子酶活性的稳定性;通过一系列分离纯化步骤得到了纯的二聚体芥子酶,研究结果为挖掘产芥子酶的微生物资源提供了新的途径和思路。