Site-specific substituted cobalt(II) horse liver alcohol dehydrogenases. Preparation and characterization in solution, crystalline and immobilized state.

The specific substitution, using highly selective techniques, of catalytic and/or noncatalytic zinc ions by cobaltous ions in horse liver alcohol dehydrogenase (EC 1.1.1.1) has been studied with dissolved, crystalline and agarose-immobilised enzyme, in order to examine the effect of protein structure on the specificity of the metal exchange. The different binding sites can be clearly distinguished by the absorption spectra of their cobalt derivatives. In solution an anaerobic column chromatographic method made it possible to exchange half of the zinc in the enzyme by cobalt ions in a much shorter time than previous procedures. By raising the temperature in the exchange step, even the slowly exchanging zinc ions were substituted by cobalt, yielding products similar to cobalt alcohol dehydrogenases described earlier. Treatment of crystal suspensions of the enzyme with chelating agents (preferentially dipicolinic acid) gave an inactive protein with two zinc ions remaining bound. The enzyme could be reactivated by treatment of the crystalline protein with 5 mM zinc or cobaltous ions or by dialysis of dissolved inactive protein against 20 microM zinc or 1 mM cobaltous ions. Higher metal concentrations led to denaturation but the inactive protein could be crystallized from solution and then reactivated completely at higher metal concentrations. The preparation and absorption spectrum show that cobalt is bound specifically at the catalytic sites. Since metal substitution at these sites critically depends on the maintenance of the correct tertiary and quaternary structure, these must be preserved in the crystal lattice and partially altered in solution when the catalytic zinc ions are removed (or when excess of metal ions is applied), thus demonstrating the structure-stabilizing role of the catalytic metal ions. The enzyme immobilised on agarose, with unchanged content of active sites [Schneider-Bernl?hr et al. (1978) Eur. J. Biochem. 41, 475--484], was treated like the crystal suspensions. Although half of the zinc was removed, some activity remained. After reactivation with cobaltous ions, a loss of about 30% active sites was measured. Thus the apparently homogenous bound enzyme was rather heterogeneous in the properties of its catalytic metal binding sites. These results are taken as further proof for the dependence of the metal substitution on the proper tertiary and quaternary structure which is strained by multiple interactions in the covalently immobilised enzyme.     

Intracellular Peptidase of Bacillus subtilis

An electrophoretically pure preparation of aminopeptidase was isolated from the cells of a strain of Bacillus subtilis which secreted saccharifying α-amylase. The purified peptidase was active only in the presence of manganese and cobaltous ions. Both the metallic ions were effective to a similar degree for the enzyme on most of the peptides used as the substrate. However, for hydrolysis of leucine amide by the enzyme was effective only cobaltous ion and, in this case, manganese ion showed to act as a competitive inhibitor. On the other hand, for hydrolysis of certain other peptides such as glycyl proline, quite the opposite relationship was observed between those metallic ions. In the present paper is also described a new micro-assay method of amidase activity shown by the peptidase.     

Kinetic and physical properties of Co2+ enolase

The activation of yeast enolase by cobaltous ion in 0.1 M KCl is characterized by an activation constant of 1 microM and an inhibition constant of 18 microM. Measurements of binding of Co2+ to the apoenzyme show that a maximum of four Co2+ ions are bound per dimer in the presence or absence of substrate although binding is far tighter in the presence of substrate. Ultraviolet spectral titrations show evidence for a conformational change due exclusively to the binding of the first two ions of Co2+. Both visible and EPR spectra confirm that the environment of the first pair of cobalt ions ("conformational sites") is markedly different from that of the second pair in the "catalytic" sites. Cobalt at the conformational site appears to be a tetragonally distorted octahedral complex while the second pair of metal ions appears to be in a more regular tetrahedral symmetry. Addition of either Mg2+ or substrate to the enzyme with only one pair of cobalt ions per dimer causes striking changes in the metal ion environment. The conformational metal sites appear sufficiently shielded from solvent to be inaccessible to oxidation by H2O2, in contrast to the second pair of cobaltous ions whose ready oxidation by H2O2 inactivates the enzyme. Comparison of kinetic and binding data suggests that only one site of the dimeric enzyme can be active, since activity requires more than two metals bound per dimer and inactivation results from the binding of the fourth ion per dimer.     

Complexes of metal phthalocyanines with globin as the models of heme proteins.

The reaction between iron and cobalt tetrasulfonated phthalocyanines and globin results in the formation of the green complexes, as has been proved by difference spectroscopy. Spectrophotometric titration data indicate the formation of those complexes at the molar ratio 1:1. The complexes of ferrous, ferric and cobaltous tetrasulfonated phthalocyanines with globin have been isolated from the reaction mixtures by separation on Sephadex G-50 and precipitation of the protein fractions with ammonium sulfate. The visible spectra of these complexes are characterised by the main intensive peak at 641 nm, 678 nm, and 675 nm for ferric, ferrous and cobaltous derivatives, respectively. The new globin complexes have the property of reversible combination with oxygen and coordination with cyanide ions. It is evidence from the results of the spectrophotometric titrations of hemoglobin and methemoglobin with cobaltous tetrasulfonated phthalocyanine that iron protoporphyrins are displaced by this cobalt derivative; this suggests that phthalocyanine and porphyrin are bonded in a similar manner.     

Bacterial degradation of the herbicide bromoxynil byAgrobacterium radiobacter in biofilm

The bromoxynil-degrading soil microorganism Agrobacterium radiobacter was used for degradation of the herbicide under nonsterile batch and continuous conditions. From four types of carrier particles, beech shavings showed the best stability of the bacterial biofilm, bromoxynil concentration in a column reactor decreasing to 65% after 5 d. The efficacy of degradation was enhanced by addition of ferrous, cobaltous or cupric ions.     

Histofluorescence studies of monoamine neurons in the rat brain after cobaltous acetate intoxication

The aim was to study changes in brain monoamine neurons in an experimental animal model with an extrapyramidal motor syndrome of the parkinsonian type. The neurological signs were observed in rats after acute cobaltous acetate intoxication under mild ischemic conditions. Histofluorescence studies showed a decrease in catecholamine fluorescence (which signifies a decrease in the amine content) in the hypothalamus and mesencephalic reticular formation, but not in the substantia nigra or basal ganglia. Serotonin fluorescence was increased in nerve cell bodies of the dorsal and median raphe nuclei and in nerve terminals in some thalamic and preoptic regions. Histological staining of sections adjacent to the fluorescent ones showed no neuronal loss and some pathology of myelin. The disturbing effect of cobaltous ions on the neuronal transmission, and/or the imbalance between dopamine and serotonin in the extrapyramidal motor syndrome observed in poisoned rats have been discussed.     

Polarographic Behavior of Thiolated Natural and Synthetic Polymers

Polarographic protein waves were studied by using model samples. Two samples were prepared by thiolation of natural and synthetic polymers which show no catalytic wave in the ammoniacal cobalt buffer. The one was made from bacterial α-amylase by thiolation with N-acetylhomocysteine and the other was made from polyvinylalcohol by esterification with thioglycolic acid. These thiolated polymers showed typical double waves similar to protein waves both in cobaltous and cobaltic media though minute differences were present between the waves of thiolated polyvinylalcohol and those of proteins.     

Identification of a new protein involved in the regulation of the anticoagulant activity of activated protein C. Protein S-binding protein

The apparent molecular weight of functional protein S in citrated plasma was observed to be between 115,000 and 130,000 as measured by sedimentation equilibrium in the air-driven ultracentrifuge. The molecular weight of the functional protein decreased to approximately 62,000 when copper ions were added to the plasma. This suggested the presence of a protein S-binding protein in plasma, which was confirmed by gel filtration experiments. Frontal analysis of plasma indicated that functional protein S could exist in as many as three forms. Addition of copper ions to plasma reduced the number of forms to one. In order to isolate the binding protein, plasma was fractionated first on a column of immobilized iminodiacetic acid that had been equilibrated with copper ions. The proteins that eluted in a 0.6 M NaCl wash were passed over a column of protein S immobilized on agarose beads. A protein, eluted in the 0.6 M NaCl wash, was observed to bind to protein S in gel filtration experiments. When added to plasma depleted of both protein S and the binding protein, the binding protein was observed to enhance the anticoagulant activity of activated protein C only in the presence of protein S. Protein S-binding protein was also observed to enhance the rate of factor Va inactivation by activated protein C and protein S.     

Characterization of Dominant Resistance to Cobalt Chloride in DICTYOSTELIUM DISCOIDEUM and Its Use in Parasexual Genetic Analysis

Strains of Dictyostelium discoideum resistant to cobaltous chloride have been isolated at a frequency of approximately 10^-6. The resistant strains have one of three phenotypes, recessive to wild type, dominant to wild type and dominant to wild type but requiring the presence of cobaltous chloride to maintain resistance. Strains carrying a dominant cobaltous chloride resistance mutation and a recessive growth temperature-sensitive mutation can be mixed with wild-type haploid lines and then subjected to selection so that only diploid lines survive. Differential sensitivity to cycloheximide has also been observed. Hypersensitivity to cycloheximide in combination with dominant cobaltous chloride resistance provides a means of selecting diploids without the use of temperature-sensitive mutations.     

Role of metal cations in the regulation of NADP-linked isocitrate dehydrogenase from porcine heart

The regulatory role of divalent metal cations in the NADP-linked isocitrate dehydrogenase (EC 1.1.1.42) from porcine heart was analysed. Saturation curves with respect to the substrate threo-Ds-isocitrate complexed with the metals including manganous, cadmium, cobaltous and zinc ions showed sigmoid relationships characteristic of allosteric enzymes. The Hill's interaction coefficients were 1.90, 1.75, 1.28 and 1.12, respectively. Saturation kinetics of the substrate-metal complexes including magnesium, ferrous and nickel ions exhibited normal hyperbolic curves with Hill's coefficients of 1. The ionic radii of metal cations were closely correlated with the maximal velocity, the enzyme affinity and the Hill's n values for the substrate-metal complexes. Cooperative interactions of metal-substrate complexes with NADP-isocitrate dehydrogenase are discussed in relation to the sites of the enzyme for the binding of the metal-substrate complex.     

Plasma protein binding of APD: role of calcium and transferrin.

The bisphosphonate drug APD (pamidronate, 3-amino-1-hydroxypropylidene-1,1-bisphosphonate) has been shown to bind to human plasma proteins. This was an unexpected observation since this hydrophilic, anionic drug is not typical of molecules that exhibit this characteristic. At a concentration of 5 micrograms/ml the extent of binding of APD to fresh human plasma in vitro was variable between subjects 30.2% +/- 8.5% (mean +/- S.D., n = 10). Binding was not influenced by the time or concentration of APD over the range 0.05-10.0 micrograms/ml. At 20 and 50 micrograms/ml some precipitation of APD occurred. Both calcium and iron play a role in the binding of APD to plasma proteins, addition of calcium to plasma increased the degree of binding of APD, whereas the calcium chelators EDTA and EGTA reduced the binding of APD. Similarly, addition of iron to plasma increased the binding and the inclusion of the iron chelator desferrioxamine diminished the binding of the drug. The effects of iron and desferrioxamine were less pronounced than those of calcium and EDTA, indicating that the majority of the binding involves calcium ions and a smaller contribution is made by ferric ions. The equilibrium dissociation constants (Kd) for APD binding to calcium and iron binding sites on plasma proteins were estimated to be 852 microM and 29 microM, respectively. Calcium binding sites were of high capacity but low affinity and the iron binding sites were of lower capacity and higher affinity. Electrophoresis of plasma proteins following incubation with [14C]APD revealed binding to the transferrin and globulin fractions. However, there was some dissociation of protein bound APD during the electrophoresis. The consequences of hypercalcaemia on the pharmacokinetics of APD are discussed.     

Na+/H+ antiport activity in plasma membrane and tonoplast vesicles isolated from NaCl-treated cucumber roots

Sodium/proton antiporter activity in the plasma membrane and tonoplast of cucumber seedling roots treated with 200 mM NaCl for 24 h was determined. It was observed that plasma membrane and tonoplast antiporter activity was only present in membranes from salt-treated plants. In addition, the plasma membrane antiporter protein was present in membranes after induction with NaCl, whereas tonoplast antiporter protein was observed in control and at elevated level in NaCl-treated plants. Moreover, based on the affinity of studied antiporter proteins to sodium ions, it could be assumed that excess sodium ions are firstly translocated from the cytosol to the vacuole and then excluded to the apoplast through the plasma membrane.     

NADH Oxidase Activity of Plasma Membranes of Soybean Hypocotyls Is Activated by Guanine Nucleotides

The activity of an auxin-stimulated NADH oxidase of the plasma membrane of hypocotyls of etiolated soybean (Glycine max Merr.) seedlings responded to guanine and other nucleotides, but in a manner that differed from that of enzymes coupled to the classic trimeric and low molecular weight monomeric guanine nucleotide-binding proteins (G proteins). In the presence and absence of either auxin or divalent ions, both GTP and GDP as well as guanosine-5[prime]-O-(3-thiotriphosphate) (GTP-[gamma]-S) and other nucleoside di- and triphosphates stimulated the oxidase activity over the range 10 [mu]M to 1 mM. GTP and GTP-[gamma]-S stimulated the activity at 10 nM in the absence of added magnesium and at 1 nM in the presence of added magnesium ions. Other nucleotides stimulated at 100 nM and above. The NADH oxidase was stimulated by 10 [mu]M mastoparan and by 40 [mu]M aluminum fluoride. Neither cholera nor pertussis toxins, tested at a concentration sufficient to block mammalian G protein function, inhibited the activity. Guanosine 5[prime]-O-(2-thiodi-phosphate) (GDP-[beta]-S) did not stimulate activity, suggesting that the stimulation in response to GDP may be mediated by a plasma membrane nucleoside diphosphate kinase through conversion of GDP to GTP. Auxin stimulation of the NADH oxidase was unaffected by nucleotides at either high or low nucleotide concentrations in the absence of added divalent ions. However, pretreatment of plasma membranes with auxin increased the apparent affinity for nucleotide binding. This increased affinity, however, appeared not to be the mechanism of auxin stimulation of the oxidase, since auxin stimulation was similar with or without low concentrations of guanine nucleotides. The stimulation by nucleotides was observed after incubating the membranes with 0.1% Triton X-100 prior to assay. The results suggest a role of guanine (and other) nucleotides in the regulation of plasma membrane NADH oxidase that differs from the interactions with G proteins commonly described for animal models.     

The two alcohol dehydrogenases of Zymomonas mobilis. Purification by differential dye ligand chromatography, molecular characterisation and physiological roles

The two alcohol dehydrogenases found in Zymomonas mobilis have each been purified using dye-ligand chromatography and affinity elution with nucleotides. The isoenzyme with lower electrophoretic mobility (ZADH-1) is a zinc enzyme with properties essentially similar to preparations described elsewhere. The faster isoenzyme (ZADH-2) accounted for some 90% of the ethanol-oxidizing activity in freshly prepared extracts and corresponded to the iron-activated enzyme previously described. This enzyme was inactivated by zinc; activity could only be retained during purification by including either ferrous ions or cobaltous ions in the buffers. ZADH-2 has relatively low acetaldehyde reductase activity; consequently ZADH-1 is responsible for about half of the physiological activity (acetaldehyde reduction) in Zymomonas cells. Kinetic studies showed that ZADH-2 is activated by ethanol in both reaction directions; a hypothesis for the mechanism of activation is presented. Metal ion analyses of ZADH-2 prepared in the presence of iron or cobalt indicated one atom of the relevant metal per subunit, with no significant zinc content. N-terminal sequence analyses showed that the ZADH-1 has some homology with the Bacillus stearothermophilus enzyme, whereas ZADH-2 resembles the yeast enzyme more closely.     

Antimutagenic action of cobaltous chloride on radiation-induced mutations in cultured Chinese hamster cells

The effects of cobaltous chloride on 8-azaguanine (8AG)-resistant mutations induced by gamma-rays or ultraviolet (UV) light in cultured Chinese hamster V79 cells were examined. Cobaltous chloride alone had no significant effects on survival and mutations of V79 cells at concentrations less than 1 x 10(-5) M. Cobaltous chloride at a concentration of 3 x 10(-6) M had a marked effect in reducing 8AG-resistant mutations induced by gamma-rays of 2-6 Gy, when cells were incubated for 6-7 days in the presence of cobaltous chloride after gamma-ray irradiation (posttreatment). The pretreatment of cells with cobaltous chloride for 6 days before gamma-ray irradiation reduced 8AG-resistant mutations induced by gamma-rays. Pre- or post-treatment with cobaltous chloride had no such effect on UV-induced mutations, however. The difference in responsiveness to cobaltous chloride between bacterial and mammalian cell systems is discussed.     

Phosphorylation by Inorganic Phosphate of the Plasma Membrane H-ATPase from Red Beet (Beta vulgaris L.)

The phosphorylation of plasma membrane proteins from red beet (Beta vulgaris L.) by radioactive inorganic phosphate was studied. Only few proteins were phosphorylated, among them was one polypeptide with an apparent molecular weight of about 100,000. The phosphorylation of this protein was decreased when orthovanadate was present in the reaction mixture, or when the phosphorylated protein was treated with hydroxylamine. These facts suggest that this protein is a transport ATPase which is phosphorylated in a carboxyl group during the catalytic cycle. This protein was identified immunologically as the plasma membrane H⁺-ATPase. The phosphorylation level of this enzyme was enhanced by dimethyl sulfoxide, whereas potassium ions did not have a significant effect on this level unless ATP was present. ATP stimulated the phosphorylation by inorganic phosphate. This stimulation was more apparent in the presence of potassium ions.     

Calcium binding proteins in smooth muscle plasma membrane

Rat myometrium plasma membrane showed a number of 45Ca-binding proteins as identified by gel electrophoresis. An attempt was made to identify these either by studying the inhibition of this binding by several ions or by studying binding of these proteins to calmodulin, A9 an antibody against skeletal muscle Ca-binding proteins and Stains-all. On the basis of the molecular weight, calmodulin binding and La-sensitivity of Ca-binding, the Ca-binding protein at 137 +/- 2 kDa has been identified as the Ca-pump. This protein as judged from Coomassie blue staining forms a very small percentage of the proteins present in the plasma membrane.     

植物镉忍耐的分子机理

Cd是植物非必需的微量元素,对植物有很强的毒性.Cd抑制植物细胞生长,抑制氧化磷酸化,引发氧化胁迫,影响光合作用,损伤核仁和影响质膜ATP酶的活力.一些耐Cd植物通过诱导形成螯合肽、金属硫蛋白、植物应激蛋白等抵御Cd毒,也有的耐Cd植物则通过细胞壁固定、液泡分隔、腺体分泌等途径来抵御Cd毒.植物螯合肽合成酶(PCS)相关的一些基因已得到克隆.金属硫蛋白(MT)的克隆基因导入植物,使植物对Cd毒的抗性增加;植物胁迫蛋白可提高植物对Cd毒的抗性,Zn转运蛋白可运转Cd.修饰基因则通过影响主要基因提高植物对Cd的忍耐能力.野生型植物耐Cd毒是多基因控制的,而植物短期的Cd忍耐,则仅受一个或少数基因控制.     

The ionic regulation of action potentials in the axon of a stretch receptor neuron of the stick insect (Carausius morosus)

Summary The ionic requirements for the action potentials recorded from the axon of the dorsal longitudinal stretch receptor inCarausius morosus have been studied using extracellular electrodes.In the intact preparation prolonged exposure to sodium-free, calcium-free, or magnesium-free salines produces no observable change in the amplitude of action potentials. Similarly, tetrodotoxin (1×10^–6 M) and cobaltous chloride (1×10^–2 M) are both ineffective in blocking the action potentials.In preparations in which the ionic barrier has been disrupted by removal of the nerve sheath the action potentials show sodium dependence. They are sustained in high sodium salines (150 mM) but are reversibly abolished in sodium-free salines. They are also reversibly abolished in 1×10^–6 M TTX, but unaffected by calcium-free or magnesium-free salines, or by cobaltous chloride (1×10^–2 M).It is concluded that the action currents in the axon of the stretch receptor are carried by sodium ions.     

Homocarnosinase: a hog kidney dipeptidase with a broader specificity than carnosinase.

A dipeptidase was isolated from hog kidney; it is the first enzyme described that has the capacity to cleave homocarnosine. It was purified to apparent homogeneity and split carnosine, anserine, and several other dipeptides in addition to homocarnosine. Homocarnosinase had a molecular weight of 57,000 as determined by sodium dodecyl sulfate-gel electrophoresis; it appeared to consist of a single polypeptide chain and did not contain sulfhydryl groups or serine residues essential to its activity. The enzyme was activated by Co²⁺ and by Mn²⁺, cobaltous ions being much more effective than manganous ions. Its isoelectric point was 5.6 and no evidence of isozymes was seen during isoelectric focusing. Homocarnosinase had a broader specificity, higher solubility, lower stability, and different metal ion sensitivity than hog kidney carnosinase (EC 3.4.13.3). Carnosinase was present in most tissues of the rat, whereas homocarnosinase was detected only in kidney, uterus, lung, and liver.