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According to a previous report, only the smaller anions, like chlorides,that readily fit into the anion binding site of alpha-amylase can cause an increased stability (relative to enzymes in aqueous solution), and the anions that are too large to fit into the binding site should have no effect on the enzyme. Even though the results on large benzoate ions are consistent with the above postulate, much larger citrate ions from sodium and potassium citrate show stabilization at moderate salt concentrations and follow an expected trend of low stability only at large salt concentrations. The citrate ions from ammonium citrate exhibit very little to almost no stabilization. In addition, low to moderate concentrations of NaCl that provide a large stability to the enzyme show almost no stability in the presence of EDTA. We put forward an inactivation model that involves a reversible dissociation of the anion bound to the protein, followed by a reversible inactivation step of calcium ion dissociation and an irreversible denaturation step of apoenzyme.
It has previously been found that a process based on solubilization at pH 2.7 gives high yields of herring muscle proteins with good functionality. In this study, the development of lipid oxidation during acid processing of herring mince was studied. It was tested how modifications of the process conditions and/or additions of antioxidants could prevent lipid oxidation during the actual process and then during ice storage of the protein isolates. Processing parameters evaluated were prewash of the mince, exposure time to pH 2.7, inclusion or exclusion of a high-speed centrifugation, and addition of antioxidants. Antioxidants tested were erythorbate (0.2%, 9.3 mM), sodium tripolyphosphate (STPP; 0.2%, 5.4 mM), ethylenediaminetetraacetic acid (EDTA; 0.044%, 1.5 mM), and milk proteins (4%). The first three antioxidants were added in the prewash or during the homogenization step, whereas milk proteins were added to the final precipitate. At time 0, all isolates were analyzed for pH, moisture content, and thiobarbituric reactive substances (TBARS). Selected isolates were also analyzed for lipid and protein content. Stability during ice storage was followed in terms of odor, TBARS, and color (a/b values). Extensive lipid oxidation took place using the "control" process without high-speed centrifugation. This was not significantly (p < or = 0.05) affected by a prewash or varied exposure time to pH 2.7. Including high-speed centrifugation (20 min, 10,000g) significantly (p < or = 0.05) reduced TBARS values, total lipids, a values and b values. Erythorbate alone, or in combination with STPP/EDTA, significantly (p < or = 0.05) reduced lipid oxidation during processing if added in the prewash or homogenization step. During ice storage, better stability was gained when antioxidants were added in both of these steps and when EDTA was used instead of STPP.
The ability of rapeseed oil and/or different antioxidants (alpha- and gamma-tocopherol mixture, ascorbyl palmitate, and EDTA) to protect fish-oil-enriched milk emulsions against oxidation was investigated. Tocopherol isomers in concentrations similar to those found in natural rapeseed oil were added to rapeseed oil stripped of natural tocopherols. The rapeseed oil with added tocopherols significantly inhibited oxidation in the fish-oil-enriched milk emulsions. In contrast, the emulsions with only fish oil and added alpha- and gamma-tocopherol were less stable than the emulsions with fish oil alone. When added individually, the gamma-tocopherol seemed to inhibit oxidation more efficiently than alpha-tocopherol. Ascorbyl palmitate (AP) almost completely retarded oxidation in the fish-oil-enriched milk emulsions, as determined by PV, volatile oxidation products, and sensory evaluation. AP also prevented the otherwise prooxidant effect of tocopherols added to fish oil before emulsification. No interactions between AP, tocopherols, and EDTA were observed, and EDTA added alone to fish oil did not show antioxidant properties in the milk emulsions. Overall, the results showed that addition of AP or rapeseed oil containing natural tocopherols to fish oil was equally efficient in inhibiting oxidation in the fish-oil-enriched milk emulsions.
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After we modified the protocol of purification, monkey metallothionein-1 (mkMT-1) and its mutant at position 33 (C33M mutant) were efficiently expressed and purified by using the glutathione-S-transferase fusion protein system. The protein yield has been considerably improved (8 mg/L culture for mkMT-1 and 10 mg/L culture for C33M mutant). The recombinant MT-1 and C33M mutant were characterized by ESI-MS, UV, and CD spectra. The reactions of MI-1 and C33M mutant with 5,5'-dithiobis(2-nitrobenzoic acid) and EDTA also have been carefully studied. The pH titration of MT-1 and C33M mutant has been studied by UV and CD spectra. The mutation of cysteine-to-methionine at position 33 mostly maintains the alpha-domain structure similar to that in wild-type mkMT-1, but the C33M mutant has significant loss of stability and cooperative properties of the domain.
It is demonstrated that Antimycin A (AA), a respiratory inhibitor produced by Streptomyces bacteria, forms lipophylic complexes with Fe(III) ions. Spectroscopic titration indicates that Fe(III) ions interact with 2AA molecules. At growth-limiting Fe concentrations, AA mediates Fe uptake and promotes growth and chlorophyll synthesis better than other Fe chelators in the halotolerant alga Dunaliella salina. It is proposed that AA enhances Fe bioavailability in hypersaline solutions by formation of lipophylic Fe-AA complexes which are taken-up and utilized by the algae. The results suggest that the respiratory inhibitor AA can affect Fe metabolism in microorganisms.
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OBJECTIVE: To investigate the relationship between blood pressure (BP), renal haemodynamics, anthropometric measures of obesity and urinary albumin excretion in hypertension and in a control group. METHODS: Urinary albumin, BP and anthropometric measurements were carried out in patients attending the hypertension clinic of the University Hospital of the West Indies. A randomised stratified sample was then selected for renal haemodynamic assessment. A normoalbuminuric control group without hypertension or diabetes was also selected. Renal haemodynamics was assessed by measuring glomerular filtration rate using 51-chromium edetic acid (51Cr-EDTA) and renal blood flow using 125-iodohippurate (125-PAH). RESULTS: Urinary albumin excretion was positively and significantly correlated with systolic pressure (beta = 0.011, P < 0.003, R = 0.22), current body weight (beta = 0.014, P < 0.04, R = 0.15) and the presence of diabetes (beta = 0.9, P < 0.001, R = 0.3). In the sample selected for renal haemodynamics, patients with microalbuminuria had lower age-adjusted corrected renal blood flow (P < 0.006), effective renal plasma flow (P < 0.006) and higher filtration fraction (P < 0.006) when compared with patients without microalbuminuria. Glomerular filtration rate in patients with microalbuminuria was not different from those without. Urinary albumin excretion was positively and significantly correlated with systolic pressure (beta = 0.016, P < 0.003, R = 0.40) and inversely related to corrected renal blood flow (beta = -1.13, P < 0.0002, R = 0.46). Waist:hip ratio was inversely related to corrected renal blood flow (beta = -1.74, P < 0.02, adjusted R = 0.48). CONCLUSION: Systolic BP, diabetes and body weight were significant predictors of albuminuria in our patients. Microalbuminuria and body fat distribution as assessed by waist:hip ratio were important determinants of renal haemodynamics in this population.
Chromium(VI) reduction was studied in a system composed of reduced nicotinamide adenine dinucleotide phosphate-cytochrome P450 oxidoreductase (NADPH-P450 reductase) and different iron chelators and iron sources. In an aerobic phosphate buffer containing iron(II), chromium(VI) was not reduced by the Fe2+ probably because of spontaneous autoxidation of Fe2+, but freshly made Fe2+, added directly to a CrVI-containing buffer, reduced CrVI. Under anaerobic conditions, iron(II) reduced chromium(VI) stoichiometrically. A systemic containing ethylenediaminetetraacetic acid (EDTA)-Fe3+, NADPH-P450 reductase and NADPH effectively reduced chromium(VI) anaerobically. Under aerobic conditions this reaction was inhibited by about 45%. Adenosine diphosphate (ADP)-Fe3+, which is a poor acceptor of electrons from NADPH-P450 reductase, reduced chromium(VI) only marginally, Mannitol slightly increased the aerobic CrVI reduction. Addition of superoxide dismutase and catalase, which both regenerate some O2, led to inhibition of CrVI reduction. Ferritin, NADPH-P450 reductase and the iron chelators, EDTA and citrate, reduced CrVI, indicating mobilization of Fe2+ from ferritin. Low levels of EDTA (55 mumol l-1) and citrate (100 mumol l-1) in contrast to high levels (5 mmol l-1) did not increase CrVI reduction in microsomes. Using 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid buffer instead of phosphate buffer, the CrVI-reducing activity was increased.
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Methods are described for the first reported successful isolation of the soluble form of the membrane-bound myo-inositol dehydrogenase(s) from Acetomonas oxydans. Conditions for optimum yields of active enzyme in a crude membrane-free protein extract were established. The relevant conditions are (a) cell rupture by solid-shearing, (b) solubilization of the complete 60000g pellet with sodium deoxycholate at pH7.2, (c) rapid separation of the released protein from sodium deoxycholate by gel chromatography.
1. No evidence could be found for production of the superoxide radical, O2-, during autoxidation of ascorbic acid at alkaline pH values. Indeed, ascorbate may be important in protection against O2- genat-d in vivo. 2. Oxidation of ascorbate at pH 10.2 was stimulated by metal ions. Stimulation by Fe2+ was abolished by superoxide dismutase, probably because of generation of O2-- during reduction of O2 by Fe2+, followed by reaction of O2-- with ascorbate. EDTA changed the mechanism of Fe2+-stimulated ascorbate oxidation. 3. Stimulation of ascorbate oxidation by Cu2+ was also decreased by superoxide dismutase, but this appears to be an artifact, since apoenzyme or bovine serum albumin showed similar effects.
1. Inhibition of endogenous microsomal NADPH oxidase by CO enables membrane-bound glutathione-insulin transhydrogenase (EC 1.8.4.2) to be assayed conveniently by a linked assay involving NADPH and glutathione reductase (EC 1.6.4.2). 2. The specific activity of the enzyme in rat liver microsomal preparations is of the order of 1 nmol of oxidized glutathione formed/min per mg of membrane protein. 3. The specific activity of the enzyme is comparable in rough and smooth microsomal fractions, and the activity is not affected by treatment with EDTA and the removal of ribosomes from rough microsomal fractions. 4. Membrane-bound glutathione-insulin transhydrogenase is not affected by concentrations of deoxycholate up to 0.5%, whereas protein disulphide-isomerase (EC 5.3.4.1) is drastically inhibited. 5. On these grounds it is concluded that, in rat liver microsomal fractions, glutathione-insulin transhydrogenase and protein disulphide-isomerase activities are not both catalysed by a single enzyme species.
Ferritins are iron-storage proteins that accumulate in plastids during seed formation, and also in leaves during senescence or iron overload. Iron release from ferritins occurs during growth of seedlings and greening of plastids. Depending on the concentration of the reducing agent ascorbate, either an overall iron release or uptake by ferritins from iron(III) citrate may occur. We have designed methods to measure these simultaneous and independent uptake and release fluxes. Each individual step of the exchange was studied using different iron chelates and an excess of ligand. It is shown that: (i) the chelated form of iron, and not ionic Fe3+, is the substrate for iron reduction, which controls the subsequent uptake by ferritin; (ii) iron uptake by ferritins is faster at pH 8.4 than at pH 7 or 6 and is inhibited by an excess of strongly binding free ligands; and (iii) strongly binding free ligands are inhibitory during iron release by ascorbate. When reactions are allowed to proceed simultaneously, the iron chelating power is shown to be a key factor in the overall exchange. The interactions of iron chelating power, reducing capacity and pH are discussed with regard to their influence on the biochemical mobilization of iron.
A synthetic siderophore, O-Trensox (L), has been designed and synthesized to improve iron nutrition of plants. The affinity for iron of this ligand [pFe(III) = 29.5 and pFe(II) = 17.9] is very high compared with EDTA. In spite of its high and specific affinity for iron, O-Trensox was found to be able to prevent, and to reverse, iron chlorosis in several plant species grown in axenic conditions. It also allows the iron nutrition and growth of Acer pseudoplatanus L. cell suspensions. The rate of iron metabolization was monitored by 59Fe radioiron. Ferritins, the iron storage proteins, are shown to be the first iron-labelled proteins during iron metabolization and to be able to further dispatch the metal. Using Fe(III)-Trensox, the rate of iron incorporation into ferritin was found to be higher than when using Fe-EDTA, but slower than with Fe-citrate, the natural iron carrier in xylem. During a plant cell culture, the extracellular concentrations of iron complex and free ligand were measured; changes in their relative amounts showed that the iron complex is dissociated extracellularly and that only iron is internalized. This suggests a high affinity for iron of a putative carrier on the plasmalemma. In contrast with Fe-citrate and Fe-EDTA complexes, Fe(III)-Trensox is not photoreducible. Its ability to induce radical damage as a Fenton reagent was tested using supercoiled DNA as target molecule. Unlike Fe-citrate and Fe-EDTA, Fe(II)-Trensox and Fe(III)-Trensox were proven to be harmless even during ascorbate-driven reduction, while Fe-EDTA and Fe-citrate generate heavy damage to DNA.