Decrease of Hormone Binding Capacity of Estrogen Receptor by Calcium

Abstract

We have adressed the question as to whether calcium may modify the [³H]estradiol ([³H]E₂) binding properties of the estrogen receptor (ER). A human recombinant full length ER (yER) expressed in yeast was used to limit the potential interference of ER-associated proteins and proteases present in the target tissues.

Ca⁺⁺ (0.1–10 mM) always produced an important loss of [³H]E₂ binding capacity without any effect on the hormone binding affinity of residual receptors. This loss was reflected in a decrease of immunoreactivity for monoclonal antibodies raised against the hormone binding domain. An ER recombinant expressing solely this domain confirmed that the ion operated at this level. Binding of [¹²⁵I]Z-17 α-(2-iodovinyl)-11β-chloromethyl estradiol-17β (an compound with very high selectivity for ER) as well as [¹²⁵I]tamoxifen aziridine were similarly affected. Size-exclusion chromatography failed to reveal the emergence of any ER isoforms of low molecular weight rejecting the hypothesis of a Ca⁺⁺- induced proteolysis. In agreement with this conclusion, EDTA reversed the loss of [³H]E₂ binding capacity. Phosphoamino acids (PY, PT and PS) partly antagonized the effect of Ca⁺⁺ suggesting its interaction with phosphoamino acid residues. Worthy of note, the effect of Ca⁺⁺ appeared more marked when assessed by DCC than HAP assay. The phosphocalcic nature of the HAP matrix may explain this phenomenon which was observed with cytosolic ER from various origins.     

Dual Effects of Ascorbate on Serotonin and Spiperone Binding in Rat Cortical Membranes

Abstract: Ascorbate-induced lipid peroxidation, as measured by malonyldialdehyde (MDA) production, caused irreversible decreases in B_max of both [³H]5-HT and [³H]spiperone binding. Cacl₂ (4mM) inhibited ascorbateinduced MDA formation at ascorbate concentrations >0.57 mM, but not at ≤ 0.57 mM. Under the standard assay conditions (5.7 mM ascorbate and 4mM CaCl₂), Cacl₂ inhibited the MDA production casued by ascorbate by 88%, and the loss in [³H]5-HT binding by 57%. Ascorbate still decreased [³H]5-HT binding by 57%. Ascorbate still decreased [³H]5-HT binding when lipid peroxidation was completely inhibited by EDTA. This additional effect of ascorbate was reversible after washing the membranes. Other reducing agents (dithiothreitol, glutathione, and metabisulfite) also decreased the binding of [³H]serotonin. In contrast, [³H]spiperone binding was not affected by ascorbate in the absence of lipid peroxidation or by other reducing agents. These experiments demonstrate that ascorbate has a dual and differential effect on serotonin binding sites. First, ascorbate-induced lipid peroxiation irreversibly inactivates both [³H]5-HT and [³H]spiperone binding. Second, independent of lipid peroxidation, there is a direct, reversible effect of ascorbate on [³H]serotonin but not on [³H]spiperone binding, which is probably due to the difference in the biochemical nature of the two serotonin binding sites.     

Characterization of BflI – A Thermostable,Co++-Requiring Isoschizomer of BsiYI from Anoxybacillus Flavithermus

A thermophile, isolated from geothermal areas in the northern Himalayan region of India, was identified by partial 16S rDNA sequence (GenBank accession # AF482430) analysis as Anoxybacillus flavithermus. The isolate produced BflI (REBASE # 4910), a Type II restriction endonuclease, which recognized the sequence 5′-CCNNNNN/NNGG-3′ and was the isoschizomer of BsiYI. The enzyme was purified to homogeneity by passing through Cibacron Blue F3GA agarose, DEAE-cellulose, heparin-agarose and MonoQ FPLC. The purified enzyme (MW 36 kDa) worked best at 60 °C in Promega's buffer C and preferentially required Co⁺⁺(0.4 mM) as cofactor followed by Mg⁺⁺(10 mM) and Mn⁺⁺(1 mM). The enzyme showed high specific activity and worked in the presence of high concentrations of β-mercaptoethanol (200 mM), Triton-X-100 (25%), urea (30%), formamide (6%) and guanidine (40 mM) and showed no star activity in the presence of 40% glycerol. In the absence of any stabilizing agent, BflI retained t _1/2 for at least 96 h at 37 °C, 6 h at 60 °C and 6 months at 4 °C. N-terminal sequencing showed that its first 10 amino acid residues were DFHEDKTIAR. This revised version was published online in July 2006 with corrections to the Cover Date.     

Nutrients determine the spatial architecture of Paracoccus sp. biofilm

Bacterial biofilms adapt and shape their structure in response to varied environmental conditions. A statistical methodology was adopted in this study to empirically investigate the influence of nutrients on biofilm structural parameters deduced from confocal scanning laser microscope images of Paracoccus sp.W1b, a denitrifying bacterium. High concentrations of succinate, Mg⁺⁺, Ca⁺⁺, and Mn⁺⁺ were shown to enhance biofilm formation whereas higher concentration of iron decreased biofilm formation. Biofilm formed at high succinate was uneven with high surface to biovolume ratio. Higher Mg⁺⁺ or Ca⁺⁺ concentrations induced cohesion of biofilm cells, but contrasting biofilm architectures were detected. Biofilm with subpopulation of pillar-like protruding cells was distributed on a mosaic form of monolayer cells in medium with 10 mM Mg⁺⁺. 10 mM Ca⁺⁺ induced a dense confluent biofilm. Denitrification activity was significantly increased in the Mg⁺⁺- and Ca⁺⁺-induced biofilms. Chelator treatment of various biofilm ages indicated that divalent cations are important in the initial stages of biofilm formation.     

The Characteristics of Arylamine N-Acetyltransferase in Pseudomonas aeruginosa

N-Acetyltransferase (NAT), responsible for bioactivation and detoxification of arylamines, has been demonstrated to be widely distributed in many organisms ranging from humans to microorganisms. Using high performance liquid chromatography (HPLC) to analyze NAT activity in bacteria, the authors found that Pseudomonas aeruginosa exhibited high NAT activity with 2-aminofluorene (2-AF) as substrate. Characteristics of this bacterial NAT were further investigated. The N-acetylation catalyzed by this enzyme is an acetyl coenzyme A (AcCoA)-dependent reaction. As the concentration of AcCoA in the reaction mixture was increased, the apparent K _m and V _max for 2-AF increased. The K _m and V _max were 0.504 ± 0.056 mM and 31.92 ± 3.23 nmol/min/mg protein, respectively, for the acetylation of 2-AF with 0.5 mM AcCoA. The optimum pH for the enzyme activity was estimated to be around 8.5. It was active at a temperature range from 5°C to 55°C, with maximum activity at 37°C. The enzyme activity was inhibited by divalent metal ions including Cu⁺⁺, Fe⁺⁺, Zn⁺⁺, Ca⁺⁺, Co⁺⁺, Mn⁺⁺, and Mg⁺⁺, suggesting that a sulfhydryl group is involved in the N-acetylation activity. The three chemical modification agents, iodoacetamide, phenylglyoxal, and diethylpyrocarbonate, all exhibited a dose-, time-, and temperature-dependent inhibition effect. Preincubation of the NAT with AcCoA provided significant protection against the inhibition of iodoacetamide and diethylpyrocarbonate, but only partial protection against the inhibition of phenylglyoxal. These results indicate that cysteine, histidine, and arginine residues are essential for this bacterial enzyme activity, and the first two are likely to reside on the AcCoA binding site, but arginine residue may be located only near the AcCoA binding site. Our data demonstrate that P. aeruginosa possesses highly active N-acetyltransferase which shares a similar catalytic mechanism as that of higher organisms. These findings are very helpful for further investigating the role of arylamine NAT in this bacterial species. Received: 29 August 1997 / Accepted: 23 December 1997     

Factors Affecting the Ascorbate- and Phenolic-dependent Generation of Hydrogen Peroxide in Dulbecco's Modified Eagles Medium

Ascorbate and several polyphenolic compounds have been reported to undergo oxidation in cell culture media to generate hydrogen peroxide (H?0?), but the mechanism underlying this has not been established. We therefore investigated the parameters affecting H?0? production. H?0? gene ration from ascorbate, gallic acid and other phenolic compounds in Dulbecco's Modified Eagles' Medium (DMEM) at 37°C under 95% air - 5% C0? was not significantly inhibited by high (5-10 mM) concentration of EGTA, o-phenanthroline or desferrioxamine, but partial inhibition by EDTA and diethylenetriaminepentaacetic acid (DTPA) was observed. Incubation of DMEM alone at 37°C led to an upward drift of pH, even under an atmosphere of 95% air - 5% C0?. Prevention of this pH rise by increasing the concentration of N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (Hepes) buffer lowered the levels of H?0? generated by ascorbate and phenolic compounds, but there was still substantial H?0? generated at pH 7.4. Mixtures of ascorbate and phenolic compounds led to less H?0? generation than would be expected from the rates observed with ascorbate or phenolic compounds alone. Ascorbate prevented the loss of gallic acid incubated in DMEM. The role of metal ions and other constituents of the culture medium in promoting H?0? generation is discussed.     

Binding of calcium and zinc to the acetylcholine receptor purified from Torpedo Californica

Binding of [⁶⁵Zn⁺⁺] and [⁴⁵Ca⁺⁺] to the acetylcholine (ACh)-receptor, purified from the $\text{Torpedo}$ electric organ, was studied by equilibrium dialysis. Whereas [⁶⁵Zn⁺⁺] bound to 56 nmoles of sites per mg protein with a dissociation constant of 2.5 × 10⁻⁶M, no binding of [⁴⁵Ca⁺⁺] at concentrations up to 10⁻³M could be detected with this method. However, the binding of [acetyl-³H]choline to the receptor was blocked equally by very high Zn⁺⁺ or Ca⁺⁺ concentrations, and the K_i for this low affinity binding was 7 × 10⁻³M. The high affinity binding of [⁶⁵Zn⁺⁺] to the receptor was blocked best by Cd⁺⁺ then Co⁺⁺ and Mn⁺⁺, but least by Mg⁺⁺ and Ca⁺⁺. When the purified ACh-receptor itself was analyzed for the presence of cations by atomic absorption, it was discovered that 4.7% of its weight was due to bound Ca⁺⁺ that could not be removed even by extensive dialysis. When Ca⁺⁺-free solutions (containing 1 mM EDTA) were used during purification, 0.6% of the molecular weight of the receptor was still due to bound Ca⁺⁺. This was equivalent to 15 moles of Ca⁺⁺ for each mole of ACh bound at saturation. It is suggested that the source of this Ca⁺⁺ is endogenous, and that it is tightly bound to the ACh-receptor molecule.     

Different Endothelin Receptor Affinities in Dog Tissues

Abstract

Endothelin (ET) is a long-lasting potent vasoconstrictor-peptide. Here we report different binding affinities of endothelin-1 (ET-1) to ET-receptors of various dog tissues. Crude microsomal fractions were prepared after homogenisation of dog tissues in 50 mM Tris/HCl, 20 mM MnCl₂, 1 mM EDTA, p^H 7.4 by differential centrifugation. Aliquots of microsomal fractions (70 u.g of protein) were incubated at 25°C for 180 min in the presence of 20 p^M125I-ET-1 and various concentrations of cold ET-1. Four different ET-1 receptor binding affinities were found: adrenals, cerebrum, liver, heart, skeletal muscle and stomach microsomal membranes contained high affinity binding sites (Kd 50 – 80 p^M, Bmax 60 – 250 fmol/mg). In cerebellum and spleen medium affinity ET-1 receptors (Kd 350 p^M, Bmax 880 and 1200 fmol/mg respectively) were present. In comparison lung and kidney microsomes contained a low affinity ET-1 receptor (Kd 800 and 880 p^M, Bmax 1600 and 350 fmol/mg). Receptors of even lower affinity were present in heart, intestine and liver microsomes with Kd values of 3 – 6 nM.     

Studies on Metabolic Pathway of NAD in Yeast

The properties of yeast 5′-nucleotidase, one of NAD-metabolic system in yeast, were studied.

1) The enzyme has optimum pH at 5.8~6.1 for its activity and is most stable at pH 6. It is inactivated completely at 55°C for 6 min, pH 7, but never at 40°C for 6 min. 2) The enzyme hydrolyzes only 5′-nucleotides of guanine, adenine, hypoxanthine, uracil and cytosine, but never splits nicotinamide mononucleotide, thiamine monophosphate, ribose 5-monophosphate and flavin mononucleotide. 3) The enzyme seems to have specially high affinity for 5′-AMP. 4) The enzyme activity is accelerated by addition of Co⁺⁺ and Ni⁺⁺, but inhibited by Ag⁺, Cu⁺⁺, EDTA, I₂ and N-bromosuccimide. Mg⁺⁺, KCN, NaF and thiol reagents except p-chloromercuribenzoate have no effects. 5) Nucleosides have inhibitory effects, among which adenosine is most effective inhibitor. 6) The activity is reduced up to 30% by dialysis against 1 mm EDTA solution, and the reduced activity is completely reactivated by addition of Co⁺⁺ or Ni⁺⁺, but not by Mn⁺⁺ or Mg⁺⁺.     

The active transport of Mg++ and Mn++ into the yeast cell

Certain bivalent cations, particularly Mg⁺⁺ and Mn⁺⁺, can be absorbed by yeast cells, provided that glucose is available, and that phosphate is also absorbed. The cation absorption is stimulated by potassium in low concentrations, but inhibited by higher concentrations. From the time course studies, it is apparent that the absorption rather than the presence of phosphate and the potassium is the important factor. Competition studies with pairs of cations indicate that binding on the surface of the cell is not a prerequisite to absorption. The absorption mechanism if highly selective for Mg⁺⁺ and Mn⁺⁺, as compared to Ca⁺⁺, Sr⁺⁺, and UO₂⁺⁺, whereas the binding affinity is greatest for UO₂⁺⁺, with little discrimination between Mg⁺⁺, Ca⁺⁺, Mn⁺⁺, and Sr⁺⁺. In contrast to the surface-bound cations which are completely exchangeable, the absorbed cations are not exchangeable. It is concluded that Mg⁺⁺ and Mn⁺⁺ are actively transported into the cell by a mechanism involving a phosphate and a protein constituent.     

Ascorbate Transport and Recycling by SH-SY5Y Neuroblastoma Cells: Response to Glutamate Toxicity

Neurons maintain relatively high intracellular concentrations of vitamin C, or ascorbic acid. In this work we studied the mechanisms by which neuronal cells in culture transport and maintain ascorbate, as well as how this system responds to oxidant stress induced by glutamate. Cultured SH-SY5Y neuroblastoma cells took up ascorbate, achieving steady-state intracellular concentrations of 6 mM and higher at extracellular concentrations of 200 μM and greater. This gradient was generated by relatively high affinity sodium-dependent ascorbate transport (K _m of 113 μM). Ascorbate was also recycled from dehydroascorbate, the reduction of which was dependent on GSH, but not on d-glucose. Glutamate in concentrations up to 2 mM caused an acute concentration-dependent efflux of ascorbate from the cells, which was prevented by the anion channel blocker 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid. Intracellular ascorbate did not affect radiolabeled glutamate uptake, showing absence of heteroexchange.     

Guanylate cyclase from Dictyosteliumdiscoideum

Guanylate cyclase from crude homogenates of vegetative $\text{Dictyostelium}$$\text{discoideum}$ has been characterized. It has a pH optimum of 8.0, temperature optimum of 25°C and requires 1 mM dithiothreitol for optimal activity. It strongly prefers Mn⁺⁺ to Mg⁺⁺ as divalent cation, requires Mn⁺⁺ in excess of GTP for detectable activity, and is inhibited by high Mn⁺⁺ concentrations. It has an apparent Km for GTP of approximately 517 μM at 1 mM excess Mn⁺⁺.The specific activity of guanylate cyclase in vegetative homogenates is 50–80 pmoles cGMP formed/min/mg protein. Most of the vegetative activity is found in the supernatant of a 100,000 x g spin (S100). The enzyme is relatively unstable. It loses 40% of its activity after 3 hours storage on ice. Enzyme activity was measured from cells that had been shaken in phosphate buffer for various times. It was found that the specific activity changed little for at least 8 hours. Cyclic AMP at 10^?4 M did not affect the guanylate cyclase activity from crude homogenates of vegetative or 6 hour phosphate-shaken cells.     

Production of Alkaline Lipase by Corynebacterium paurometabolum, MTCC 6841 Isolated from Lake Naukuchiatal, Uttaranchal State, India

A moderately psychrophilic bacterium Corynebacterium paurometabolum MTCC 6841 (gram positive, short rod type) producing extracellular alkaline lipase was isolated from Lake Naukuchiatal, Uttaranchal, India. The bacterium was able to grow within a broad range of pH (5–10). Soyabean oil and olive oil served as the best carbon sources for lipase production. The bacterium preferred inorganic nitrogenous compounds, NaNO₃ and KNO₃, over organic nitrogenous compound for its growth. Maximum lipase production occurred at 25°C and 8.5 pH. The enzyme activity was found to be maximum at the same values of temperature and pH. The enzyme was reasonably stable in the presence of various organic solvents. No significant effect of Ca⁺, Cu⁺⁺, Fe⁺⁺, Na⁺, K⁺, Mg⁺⁺, Mn⁺, NH4⁺, Co⁺⁺ ions over enzyme activity was detected. Treatment with EDTA reduced the activity to nearly one half.     

Calcium activation of brain tryptophan hydroxylase.

Using both radioisotopic and fluorometric techniques to measure the activity of midbrain soluble enzyme, we have demonstrated that calcium activates tryptophan hydroxylase. The observed activation apparently results from an increased affinity of the enzyme for both its substrate, tryptophan, and the cofactor 2-amino-4-hydroxy-6-methyl-5,6,7,8-tetrahydropteridine (6-MPH₄). The calcium activation of tryptophan hydroxylase appears to be specific for both enzyme and effector: other brain neurotransmitter biosynthetic enzymes, such as aromatic amino acid decarboxylase(s) and tyrosine hydroxylase, are not affected by calcium (at concentrations ranging from 0.01 mM to 2.0 mM); other divalent cations, such as Ba⁺⁺, Mg⁺⁺, and Mn⁺⁺, have no activating effect on tryptophan hydroxylase. This work suggests that increases in brain serotonin biosynthesis induced by neural activation may be due to influx of Ca⁺⁺ associated with membrane depolarization and resulting activation of nerve ending tryptophan hydroxylase.     

The divalent cation dependence of liver glycogen synthase phosphatase activity

Summary In a previous report it was shown that EDTA inhibition of liver glycogen synthase phosphatase activity in preparations from normal, fed rats could be increased upon glucagon or cAMP treatment. This occurred without a change in the half-maximum inhibitory concentration of EDTA. Glucose administration to animals resulted in decreased EDTA inhibition. The inhibitory action of EDTA has been further characterized by comparing its action with that of other chelators (CDTA and EGTA) and examining the effects of various divalent cations on chelator inhibition. Both CDTA and EDTA which differ structurally were inhibitory at 5 mm concentrations whereas EGTA which is structurally similar to EDTA was not inhibitory at concentrations up to 10 mm. The lack of inhibition by EGTA could be explained by its weak affinity for Mg⁺⁺ in the preparation. A comparison of CDTA and EDTA revealed that CDTA was a more potent inhibitor than EDTA (I_0.5, 0.15 mm vs 0.3 mm). Glucagon and glucose treatment of rats resulted in changes in CDTA inhibition which closely paralleled those of EDTA. A large group of divalent cations were tested but only Mg⁺⁺, Ca⁺⁺, and Mn⁺⁺ both prevented and reversed CDTA or EDTA inhibition. Fifty percent reversal using either chelator occurred at calculated free-metal ion concentrations of approximately 2 µm, 0.08 µm and 0.0004 µm, respectively. Thus, it is clear that EDTA inhibition is due to its chelation effect and is not due to a nonspecific anionic effect.     

Electron spin resonance study on the formation of ascorbate free radical from ascorbate: The effect of dehydroascorbic acid and ferricyanide

Summary Ascorbate free radical is considered to be a substrate for a plasma membrane redox system in eukaryotic cells. Moreover, it might be involved in stimulation of cell proliferation. Ascorbate free radical can be generated by autoxidation of the ascorbate dianion, by transition metal-dependent oxidation of ascorbate, or by an equilibrium reaction of ascorbate with dehydroascorbic acid. In this study, we investigated the formation of ascorbate free radical, at physiological pH, in mixtures of ascorbate and dehydroascorbic acid by electron spin resonance spectroscopy. It was found that at ascorbate concentrations lower than 2.5 mM, ascorbate-free radical formation was not dependent on the presence of dehydroascorbic acid. Removal of metal ions by treatment with Chelex 100 showed that autoxidation under these conditions was less than 20%. Therefore, it is concluded that at low ascorbate concentrations generation of ascorbate free radical mainly proceeds through metal-ion-dependent reactions. When ascorbate was present at concentrations higher than 2.5 mM, the presence of dehydroascorbic acid increased the ascorbate free-radical signal intensity. This indicates that under these conditions ascorbate free radical is formed by a disproportionation reaction between ascorbate and dehydroascorbic acid, having aK _equil of 6 × 10^–17 M. Finally, it was found that the presence of excess ferricyanide completely abolished ascorbate free-radical signals, and that the reaction between ascorbate and ferricyanide yields dehydroascorbic acid. We conclude that, for studies under physiological conditions, ascorbate free-radical concentrations cannot be calculated from the disproportionation reaction, but should be determined experimentally.Abbreviations AFR ascorbate free radical - DHA dehydroascorbic acid - EDTA ethylenediaminetetraacetic acid - DTPA diethylenetri-aminepentaacetic acid - TEMPO 2,2,6,6-tetramethylpiperidinoxy     

Identification of Petriella setifera LH and characterization of its crude carboxymethyl cellulase for application in denim biostoning

The phylogenetic tree of the partial elongation factor-1 alpha gene fits better than the partial 18S rDNA for generic classification. From the results of the molecular tree and analysis of morphological characters, Petriella setifera LH was identified. It can be induced to produce carboxymethyl cellulase (CMCase). The crude CMCase only shows a 44.1-kDa band by activity staining after SDS-PAGE. It is optimally active at 55°C and pH 6.0, and is stable from pH 5.0–8.0 and at 45°C or below. The crude CMCase, which is not affected by Co²⁺, is strongly activated in the presence of 10 mM Na⁺, K⁺, Ca²⁺, Mg²⁺, EDTA, and Mn²⁺. It is strongly inhibited by 10 mM Fe²⁺, Pb²⁺, Al³⁺, Zn²⁺, Ag⁺, Fe³⁺, and Cu²⁺. When compared with denim treatment by Novoprime A800 (a commercial neutral cellulase), crude CMCase exhibits a similar fabric weight loss and indigo dye removal. These results indicate that crude CMCase has potential application in denim biostoning.     

Characterization of Active Lentinula edodes Glucoamylase Expressed and Secreted by Saccharomyces cerevisiae

The gene encoding Lentinula edodes glucoamylase (GLA) was cloned into Saccharomyces cerevisiae, expressed constitutively and secreted in an active form. The enzyme was purified to homogeneity by (NH₄)₂SO₄ fractionation, anion exchange and affinity chromatography. The protein had a correct N-terminal sequence of WAQSSVIDAYVAS, indicating that the signal peptide was efficiently cleaved. The recombinant enzyme was glycosylated with a 2.4% carbohydrate content. It had a pH optimum of 4.6 and a pH 3.4–6.4 stability range. The temperature optimum was 50°C with stability ≤50°C. The enzyme showed considerable loss of activity when incubated with glucose (44%), glucosamine (68%), galactose (22%), and xylose (64%). The addition of Mn⁺⁺ activated the enzyme by 45%, while Li⁺, Zn⁺⁺, Mg⁺⁺, Cu⁺, Ca⁺⁺, and EDTA had no effect. The enzyme hydrolyzed amylopectin at rates 1.5 and 8.0 times that of soluble starch and amylose, respectively. Soluble starch was hydrolyzed 16 and 29 times faster than wheat and corn starch granules, respectively, with the hydrolysis of starch granules using 10× the amount of GLA. Apparent K_m and V_max for soluble starch were estimated to be 3.0 mg/ml and 0.13 mg/ml/min (40°C, pH 5.3), with an apparent k_cat of 2.9×10⁵ min⁻¹.     

Ascorbate oxidase of Cucumis melo L. var. reticulatus: purification, characterization and antibody production

Ascorbate oxidase activity and ascorbic acid content were followedduring the development of muskmelon (Cucumis melo L. var. reticulatus)fruits. The enzyme was highly expressed in ovaries and veryyoung fruit tissues, followed by a decrease in 10- and 20-d-oldfruits and an increase in 30- and 35-d-old fruits which coincidedwith early events of fruit ripening. Ascorbic acid content wasnegatively correlated with ascorbate oxidase activity. The enzymewas purified to homogeneity following ion exchange, affinityand gel filtration chromatographic trials. The purified enzymewas a glycoprotein of molecular weight 137 000 composed of twosubunits of molecular weight 68000, and formed by six isoenzymeswith isoelectric points in the range of pH 7.7 to 8.3. Its electronparamagnetic resonance and optical spectra were in agreementwith other copper proteins and the enzyme contained eight copperatoms per dimeric molecule. The K_m of the enzyme for ascorbicacid was 50 µM. Ascorbate oxidase activity was inhibitedby azide and by EDTA, two inhibitors of copper proteins. Optimalconditions for enzyme activity was pH 5.5, and a temperatureof 37 C. Polyclonal antibodies were produced against the purifiedprotein and immunoprecipitated ascorbate oxidase activity. Key words: Cucumis melo, muskmelon, ascorbate oxidase, fruit ripening     

Isolation and growth characteristics of nitrilotriacetate-degrading bacteria

Two bacterial strains that degrade nitrilotriacetate (NTA) were isolated from NTA-acclimatized activated sludge. These bacteria grew well in NTA medium with optimal pH around 7. The growth rate constants of the bacteria, strains N-2 and N-5, were 0.046 h⁻¹ and 0.11 h⁻¹ at the concentration of 0.1% NTA (pH 7.0, 25°C), respectively.The growth of each bacterium was inhibited at high concentrations NTA. The growth rate decreased roughly linearly with increasing concentration of NTA. The strains N-2 and N-5 showed maximal cell growth at the concentrations of 0.2% and 0.25% NTA, respectively. The strain N-2 would not grow at the concentration of 0.5% NTA. On the other hand, the strain N-5 showed a little growth under the same conditions. Also, the bacterial growth was almost completely inhibited when divalent metal ions such as Mg⁺⁺, Ca⁺⁺, and Fe⁺⁺ were omitted from the culture medium, or slightly excess EDTA (1 mM) was added to the medium. These results suggest that the bacterial growth inhibition at high concentration of NTA is caused by the sequestration of metal ions in the medium.