Kinetics of formation of antibody-ferric porphyrin complex with peroxidase activity

The antibody 2B4 combines with ferric mesoporphyrin to form an antibody-ferric mesoporphyrin complex which has a peroxidase activity. Formation of the complex was investigated by measuring the absorption in the Soret region after mixing the antibody and ferric mesoporphyrin. A rapid increase and a gradual decrease in the absorption were observed, and the respective first-order rate constants were obtained. From the dependence of values of the rate constants on the concentration of ferric mesoporphyrin, the complex formation was explained by a plausible mechanism, in which the antibody associated with ferric mesoporphyrin to form the first complex followed by a conformational change to the second complex. The first complex had almost the same peroxidase activity as that of the second complex. Our results suggests that the antibody acquires the peroxidase activity as soon as ferric mesoporphyrin is incorporated into its binding site, and that there will be no protein ligand to the iron center of ferric mesoporphyrin in the complex.     

Congenital deficiency of a 20-kDa subunit of mitochondrial complex I in fibroblasts.

The first component of the mitochondrial electron-transport chain is especially complex, consisting of 19 nuclear and seven mitochondrion-encoded subunits. Accordingly, a wide range of clinical manifestations are produced by the various mutations occurring in human populations. In this study, we analyze the subunit structure of complex I in fibroblasts from two patients who have distinct clinical phenotypes associated with complex I deficiency. The first patient died in the second week of life from overwhelming lactic acidosis. Severe complex I deficiency was evident in her fibroblasts, since alanine oxidation was markedly reduced whereas succinate oxidation was normal. Absence of a 20-kDa subunit was demonstrable when newly synthesized proteins were immunoprecipitated from pulse-labeled fibroblasts by anti-complex I antibody. Disordered assembly or decreased stability of the complex was suggested by deficiency of multiple subunits on Western immunoblots. The second patient exhibited a milder clinical phenotype, characterized by moderate lactic acidosis and developmental delay in childhood and by onset of seizures at 8 years of age. Oxidation studies demonstrated expression of the complex I deficiency in fibroblasts, but no subunit abnormalities were detected by immunoprecipitation or Western immunoblotting. This report demonstrates the utility of cultured fibroblasts in studying mutations affecting synthesis and assembly of complex I.     

Complex formation of 70-kDa heat shock protein with acidic glycolipids and phospholipids

A new property of a heat-inducible heat shock protein (Hsp) 70.1 that it forms a complex with acidic lipids was first demonstrated. Based on the behaviors of the complexes on the native PAGE, the acidic lipid/Hsp70.1 complexes are categorized into two groups. The first group is the sulfatide-induced large-sized complex, which stays on the gel top on the native PAGE. Only the N-terminal ATPase domain is responsible for the complex formation. The second group is the ganglioside-induced complex, which is diffused in the resolution gel on the native PAGE. Both the N-terminal ATPase and the C-terminal peptide-binding domains are involved in the complex formation. No complex is formed by neutral glyco- and phospholipids. The complex formation with the acidic glyco- and phospholipids implicates the various functions of Hsp70 on the membrane surfaces.     

Distribution of histone F1 on calf thymus nucleohistone DNA.

A method of large-scale preparation of the histone F1-DNA complex by removing all other proteins from calf thymus nucleohistone was established. This involved gel filtration of nucleohistone through a column containing a band of sodium dodecyl sulfate. The F1-DNA complex obtained had the original amount of F1 and no other. The F1-DNA complex exhibited distinct two-step melting on thermal denaturation. The first step was apparently attributable to naked DNA regions and the second step, about 30 deg. C higher than the first step, to the regions covered with F1. Buoyant density experiments with the complex after fixation with formalin revealed that F1 was distributed fairly evenly over DNA fragments of an average molecular weight of about 4 × 10⁶. Electron microscopic examination of the complex after various degrees of denaturation with formalin indicated that the longest stretch of unbound DNA was about 0·3 μm.     

Roles of leukotrienes in two rat allergic inflammatory models; IgE-mediated and IgG-antigen complex-induced pleurisies

Rat IgE pleurisy was induced by the injection of di-nitrophenol-conjugated bovine serum albumin (DNP-BSA) 48 hours after the intrapleural injection of rat anti-DNP-IgE serum. IgG-BSA complex pleurisy was also induced by the intrapleural injection of IgG-BSA complexes produced at the optimum ratio in vitro. Plasma exudation was markedly increased in the first 20 minutes, but not observed thereafter, in IgE pleurisy, whereas marked plasma exudation in the first 20 minutes was followed by weak exudation at three and five hours in IgG-BSA complex pleurisy. Leukotrienes (LTs) E4 (100 ng/rat), D4 (32) and B4 (16) were detected on HPLC in the pleural exudate in the first 20 minutes of IgG-BSA complex pleurisy, but less (9 ng/rat) LTE4 alone was detected in the five-hour exudate. The first 20-minute pleural exudate contained 13 ng/rat of LTE4 in IgE pleurisy. The plasma was completely inhibited by simultaneous treatment of rats with pyrilamine (2.5 mg/kg, i.p.) and methysergide (3 mg/kg, i.p.), as it was in compound 48/80-induced pleurisy. In IgG-BSA complex pleurisy, 90% of the pleural exudate for the first 20 minutes was inhibited by the same treatment, and the rest was completely suppressed by simultaneous treatment with an intrapleural injection of AA-1777, a selective 5-lipoxygenase inhibitor. AA-1777 alone did not reduce the plasma exudation significantly. The 5-lipoxygenase inhibitor was also very effective in reducing the migrating numbers of polymorphonuclear and mononuclear leukocytes to half, without affecting the eosinophils of mast cells.     

Relationship between the rate of reductase-cytochrome P450 complex formation and the rate of first electron transfer.

Substrate has recently been shown to affect (a) the high spin content of cytochrome P450 (b) the rate of first electron transfer when LM2 (P450 2B4) and reductase were in a preformed complex, and (c) the rate of functional complex formation between NADPH-cytochrome P450 reductase and cytochrome P450 LM2. When comparing the effect of substrate on each of these parameters, the strongest correlation was demonstrated between the rate of first electron transfer through the preformed complex and the rate of functional complex formation (W.L. Backes and C.S. Eyer, 1989, J. Biol. Chem. 264, 6252-6259). The relationship among high spin content, reduction rate, and the rate of functional complex formation was examined using a number of different cytochrome P450 isozymes. The goal of this study was to determine if the previously established relationship between reduction rate and the rate of reductase-P450 complex formation was a feature only of LM2, or a general characteristic of the cytochrome P450 system. Substrate addition caused an increase in first electron transfer for each of the isozymes examined, with high spin content being increased with cytochromes P450 2B1 (PBRLM5) and P450 2B2 (PBRLM6). Substrate addition to cytochrome P450 2C6 (PBRLM4) resulted in a small decrease in high spin content. P450 2B1 and P450 2B2 showed a positive correlation between substrate-mediated stimulation of reduction and high spin content, whereas P450 2C6 showed a negative correlation between these variables. Substrate also increased the rate of reductase-P450 association for each of the isozymes examined. When compared to the degree of stimulation of reduction through a preformed complex, a strong positive correlation was obtained with each isozyme examined. These results demonstrate that the increase in both the rate of functional reductase-P450 complex formation and the rate of first electron transfer is not simply a property of LM2, but appears to be a general characteristic of many cytochrome P450 isozymes.     

Two-dimensional gel electrophoresis of the membrane-bound protein complexes, including photosystem I, of thylakoid membranes in the presence of sodium oligooxyethylene alkyl ether sulfate/dimethyl dodecylamine oxide and sodium dodecyl sulfate

Two-dimensional polyacrylamide gel electrophoresis (PAGE), using a mixture of sodium oligooxyethylene alkyl ether sulfate and dimethyl dodecylamine oxide as detergents (AES-DDAO mixture) in the first dimension and sodium dodecyl sulfate (SDS) in the second dimension, was developed and applied to an analysis of the photosystem I (PS I) complex in thylakoid membranes prepared from spinach chloroplasts. When thylakoid membranes of chloroplasts were solubilized directly in the AES-DDAO mixture and subjected to PAGE in the presence of these detergents as the first dimension, some protein complexes containing chlorophyll were observed. The protein components in these complexes separated into an array of polypeptide spots when the strip of gel after PAGE in the first dimension was subjected to PAGE in the presence of SDS as the second dimension. The main band of protein which separated in the first dimension was demonstrated to be the PS I complex. This complex retained the intrinsic photochemical activity of P700 even after it was subjected to one-dimensional PAGE. These results suggest that certain protein complexes can be separated, with the maintenance of their original structures, by electrophoresis in the presence of the AES-DDAO mixture, and this method appears to have valuable potential for analysis of the components of membrane-bound protein complexes.     

Binding of copper to albumin and participation of cysteine in vivo and in vitro

Albumin, the major copper-binding protein in blood serum, was shown to form different albumin-copper complexes in in vivo and in vitro. Cupric ions added in vitro to control rat serum bound preferentially to mercaptalbumin and the mercaptalbumin-copper complex remained unchanged with time. Cupric ions injected intravenously into the rat first formed the mercaptalbumin-copper complex; this binary complex changed gradually with time to form an albumin-copper-cysteine complex. The participation of cysteine in the formation of this complex was demonstrated in vitro and further suggested that its conversion was an oxidative reaction. Glutathione also participated in forming the complex, but it was not as effective as cysteine. Albumin-copper complexes were separated on a gel filtration column and detected simultaneously by high-performance liquid chromatography-inductively coupled argon plasma-atomic emission spectrometry.     

Roles of leukotrienes in two rat allergic inflammatory models; IgE-mediated and IgG-antigen complex-induced pleurisies

Rat IgE pleurisy was induced by the injection of dinitrophenol-conjugated bovine serum albumin (DNP-BSA) 48 hours after the intrapleural injection of rat anti-DNP-IgE serum. IgG-BSA complex pleurisy was also induced by the intrapleural injection of IgG-BSA complexes produced at the optimum ratio . Plasma exudation was markedly increased in the first 20 minutes, but not observed thereafter, in IgE pleurisy, whereas marked plasma exudation in the first 20 minutes was followed by weak exudation at three and five hours in IgG-BSA complex pleurisy. Leukotrienes (LTs) E₄ (100 ng/rat), D₄ (32) and B₄ (16) were detected on HPLC in the pleural exudate in the first 20 minutes of IgG-BSA complex pleurisy, but less (9 ng/rat) LTE₄ alone was detected in the five-hour exudate. The first 20-minute pleural exudate contained 13 ng/rat of LTE₄ in IgE pleurisy. The plasma exudation for the initial 20 minutes in IgE pleurisy was completely inhibited by simultaneous treatment of rats with pyrilamine (2.5 mg/kg, i.p.) and methysergide (3 mg/kg, i.p.), as it was in compound 48/80-induced pleurisy. In IgG-BSA complex pleurisy, 90% of the pleural exudate for the first 20 minutes was inhibited by the same treatment, and the rest was completely suppressed by simultaneous treatment with an intrapleural injection of AA-1777, a selective 5-lipoxygenase inhibitor. AA-1777 alone did not reduce the plasma exudation significantly. The 5-lipoxygenase inhibitor was also very effective in reducing the migrating numbers of polymorphonuclear and mononuclear leukocytes to half, without affecting the eosinophils or mast cells.     

Inhibition of topoisomerase II catalytic activity by two ruthenium compounds: a ligand-dependent mode of action

The ability of two structurally different ruthenium complexes to interfere with the catalytic activity of topoisomerase II was studied to elucidate their molecular mechanism of action and relative antineoplastic activity. The first complex, [RuCl2(C6H6)(dmso)], could completely inhibit DNA relaxation activity of topoisomerase II and form a drug-induced cleavage complex. This strongly suggests that the drug interferes with topoisomerase II activity by cleavage complex formation. The bi-directional binding of [RuCl2(C6H6)(dmso)] to DNA and topoisomerase II was verified by immunoprecipitation experiments which confirmed the presence of DNA and ruthenium in the cleavage complex. The second complex, Ruthenium Salicylaldoxime, could not inhibit topoisomerase II relaxation activity appreciably and also could not induce cleavage complex formation, though its DNA-binding characteristics and antiproliferation activity were almost comparable to those of [RuCl2(C6H6)(dmso)]. The results suggest that the difference in ligands and their orientation around a metal atom may be responsible for topoisomerase II poisoning by the first complex and not by the second. A probable mechanism is proposed for [RuCl2(C6H6)(dmso)], where the ruthenium atom interacts with DNA and ligands of the metal atom form cross-links with topoisomerase II. This may facilitate the formation of a drug-induced cleavage complex.     

Behavioral contrast of a complex operant

Two experiments were conducted in which pigeons were trained to perform a complex operant consisting of a peck to one key followed by a peck to another key. In the first experment this performance was reinforced on a variable-interval schedule and the birds were then subjected to a multiple schedule in which the variable-interval was alternated with extinction. The first two pigeons trained showed some deterioration of the cohesiveness of the response sequence. After measures were taken to correct this the pigeon gave evidence of behavioral contrast in a condition where sessions with discrimination were alternated rapidly with sessions with no discrimination (variable-interval only). Another two birds were trained on variable-interval followed by discrimination. One of these birds showed evidence of contrast but the results of the other were not clear.Experiment II was a three phase experiment in which animals were first trained to make the complex response. In the second phase they were extinguished by providing noncontingent reinforcement in each of two components. In the third phase reinforcement was removed from one component, providing for a stimulus-reinforcer contingency. In the third phase the complex response reappeared temporarily.The results of these two studies indicate that a complex response may show behavioral contrast and that it may be enhanced by stimulus-reinforcer contingencies if it has already been trained.     

The first nuclear-encoded complex I mutation in a patient with Leigh syndrome.

Nicotinamide adenine dinucleotide (NADH):ubiquinone oxidoreductase (complex I) is the largest multiprotein enzyme complex of the respiratory chain. The nuclear-encoded NDUFS8 (TYKY) subunit of complex I is highly conserved among eukaryotes and prokaryotes and contains two 4Fe4S ferredoxin consensus patterns, which have long been thought to provide the binding site for the iron-sulfur cluster N-2. The NDUFS8 cDNA contains an open reading frame of 633 bp, coding for 210 amino acids. Cycle sequencing of amplified NDUFS8 cDNA of 20 patients with isolated enzymatic complex I deficiency revealed two compound heterozygous transitions in a patient with neuropathologically proven Leigh syndrome. The first mutation was a C236T (P79L), and the second mutation was a G305A (R102H). Both mutations were absent in 70 control alleles and cosegregated within the family. A progressive clinical phenotype proceeding to death in the first months of life was expressed in the patient. In the 19 other patients with enzymatic complex I deficiency, no mutations were found in the NDUFS8 cDNA. This article describes the first molecular genetic link between a nuclear-encoded subunit of complex I and Leigh syndrome.     

A computational study of the interaction between dopamine and DNA/RNA nucleosides

The interaction between protonated dopamine and neutral RNA and DNA nucleosides was studied by means of density functional theory calculations in vacuum and in implicit water. On the most stable complexes formed with each of the nucleosides, the vertical absorption excitation energies were evaluated and compared with the values of separated dopamine and corresponding nucleoside. The most stable complex was formed with guanosine and the spectral changes in this complex resulted in a significant reduction of the oscillator strength of the first dopamine’s transition. In the first guanosine’s transition, a redshift of 0.2 eV was found combined with a reduction of the oscillator strength.     

Assembly of the alpha-subunit of Torpedo californica Na+/K(+)-ATPase with its pre-existing beta-subunit in Xenopus oocytes

The alpha- and beta-subunits of Torpedo californica Na+/K(+)-ATPase were expressed in turn in single oocytes by alternately microinjecting the specific mRNAs for the alpha- and beta-subunits. The mRNA first injected was degraded prior to the injection of the second mRNA by injecting the antisense oligonucleotide specific for the first mRNA. The pre-existing beta-subunit, which had been synthesized by injecting mRNA for the beta-subunit, could assemble with the alpha-subunit expressed later in the single oocytes and the resulting alpha beta complex acquired both ouabain-binding and Na+/K(+)-ATPase activities. On the other hand, formation of the alpha beta complex was not detected when the alpha-subunit was expressed first, followed by the beta-subunit. These data suggest that the beta-subunit acts as a receptor or a stabilizer for the alpha-subunit upon the biogenesis of Na+/K(+)-ATPase.     

Partial purification of the glucocorticoid receptor from rat liver: a rapid, two-step procedure using DNA-cellulose.

Glucocorticoid receptor from rat liver was purified 1800-fold by a rapid two-step procedure using DNA-cellulose. The procedure is based on increasing the affinity of the glucocorticoid-receptor complex for DNA by heating the complex. During a first chromatography step, unheated glucocorticoid-receptor complex is separated from cytosol proteins that bind to DNA-cellulose with high affinity. During a second chromatographic step, heat-treated glucocorticoid-receptor complex is separated from proteins with low affinity for DNA. The partially purified complex is functionally competent in that it is taken up by isolated rat liver nuclei.     

The kinetics of complex formation of tryptophan containing proteins with pyridinium salts

Upon preincubation with urea, various 3- or 4-substituted N-methylpyridinium salts form charge-transfer complexes with tryptophan containing proteins such as, L-chymotrypsin and lysozyme. The complexes were studied by using the difference spectrophotometric technique. The fluorescence examination showed that tryptophyl residues in protein molecules are engaged in the complex formation process. The complex formation reactions proceed at a considerable rate. The stopped-flow method was used to determine the pseudo first order rate constants. A linear dependence of the pseudo first order rate constants with the donor concentration was found. The second order rate constants were obtained by dividing the mean value of the pseudo first order rate constants by the initial donor concentration for each run. The linear dependence of second order rate constants with the electron affinity of the acceptors can serve as a criterion for the formation of charge-transfer complexes.     

因子分析和聚类分析在玉米品种资源分类上的应用

本研究对201份湖北省玉米品种资源的28个性状进行了因子分析,结果前7个公因子对变异的累计方差贡献率达79.4％,前7个独立的公因子真实地反了所控制的28个性状及其相互关系．以前7个公因子为综合指标进行系统聚类,201份玉米品种聚成12类,其中A,B,H,K类综合性状较好．     

A study of the interaction between promotor DNA and T. thermophilus DNA-dependent RNA polymerase 1,2

As the first step in the process of RNA synthesis, RNA polymerase binds to a specific site (promoter) and forms an open complex. In this process, it is considered that the structure of DNA is changed to an unidentified form. We investigated the structure of this DNA, which consists of an open complex, by means of CD. For this purpose, we used very stable RNA polymerase (of T. thermophilus HB8) and the fd-RF-DNA fragment (Hap-Hga V), which has only one promoter. We have confirmed that the complex of the holo enzyme with Hap-Hga V fragment at 50 degrees C is an open complex. We obtained the CD spectral difference between the open complex and its constituents for the first time. The observed CD difference spectra in the UV region (250-300 nm) were compared with the theoretical difference CD. It was deduced that the DNA of the open complex may be melted around the initiation point over a rather longer range than expected.     

RNA replication: required intermediates and the dissociation of template, product, and Q beta replicase.

Replication complexes containing only one molecule of Q beta replicase and one strand of midivariant RNA (MDV-1 RNA) template were prepared by incubating the replicase with an excess of MDV-1 (-) RNA. In the presence of excess minus strands, these monoenzyme replication complexes were shown to synthesize essentially pure MDV-1 (+) RNA in both the first and second cycles of replication. When an equivalent concentration of mutant MDV-1 (-) RNA was added to this reaction before completion of the first cycle of replication, only wild-type MDV-1 (+) RNA was produced in the first cycle, but both mutant and wild-type MDV-1 (+) RNA were produced in the second cycle of replication. These results demonstrate that a monoenzyme complex is competent to synthesize RNA and, therefore, that a multienzyme replication complex is not a necessary intermediate of replication. The data also imply that after the completion of chain elongation, the product strand is released from the replication complex and that the template and the replicase then dissociate.