Abstract: Kinetic measurements of a novel copper-dependent amine oxidase, purified from rat liver mitochondria matrix, were carried out using various substrates in a large pH (5.6-10.2) and ionic strength range (5-200 mM), in order to study the docking of substrates to the enzyme and, as a consequence, to verify the physicochemical characteristics of the active site. Relatively small changes of V(max) values (approx. 2.5-folds) over the substrates tested, suggest that the rate determining step of the catalysis is only slightly affected by amine chemical structure. In contrast, the strong change of K(M) and k(c)/K(M) values (approx. two orders of magnitude) indicates electrostatic control of the docking process, since the changes of K(M) and k(c)/K(M) values appear due to the presence of positively charged groups in the substrate molecules. These results suggest the presence in the enzyme active site of two negatively charged amino acid residues which seem to interact with positively charged groups of the substrate molecules. Analogies and differences with bovine serum amine oxidase are also described.
Abstract: Copper containing amine oxidases (Cu-AO) represent a heterogeneous class of enzymes classified as EC 1.4.3.6. The present study reports preliminary results on the presence of a novel amine oxidase activity in rat liver mitochondria lysates. Such enzymatic activity was found in the soluble mitochondrial fraction, obtained by simple osmotic shock. The mitochondrial amine oxidase was isolated by affinity chromatography on a newly synthesised spermine-Sepharose. SDS-PAGE showed a single band at about 60kDa. Upon chromatographic purification, the enzymatic activity was very labile. The crude enzyme activity was tested by spectrophotometric measurements, determining hydrogen peroxide production following oxidative deamination of different substrates, such as polyamines (spermine, spermidine, putrescine and cadaverine) and monoamines (dopamine and benzylamine). The activity, observed on polyamines and not on monoamines, was inhibited by semicarbazide and azide, but not by pargyline, clorgyline and l-deprenil. Enzyme specificity was tested on several diamines characterized by different carbon atom chain length in the range 2-6 carbon atoms. The highest activity was found with 1,2-diamino-ethane and the highest affinity with 1,5-diamino-pentane. The above reported results suggest the presence of a novel copper-dependent amine oxidase in liver mitochondria matrix.
Abstract: In order to identify the protein responsible for a dopamine peroxidizing activity, previously described in human normal and parkinsonian substantia nigra by our group, we developed non-denaturing polyacrylamide gel electrophoresis conditions, mimicking the characteristic colour in vitro reaction, resulting from cyclic oxidation of dopamine (DA). After separating protein mixtures from human normal midbrain homogenates on two sets of identical native gels, one gel set was subjected to specific activity staining by using DA and hydrogen peroxide. An activity red/orange band appeared in midbrain tissue lanes, similarly to the lane where commercial horseradish peroxidase (HRP) was present as control of peroxidative activity. The second set of gels, stained with Coomassie Blue, showed other, not enzymatically active protein bands. Mass spectrometry analysis of the bands containing the activity and the corresponding Coomassie Blue bands revealed the presence of proteins that may play a role in neurodegenerative disease, highlighting a possible functional link among dopamine/dopaminochrome redox cycle and protein metabolism.
Abstract: A biosensor to detect hydrogen peroxide, by coulometry, down to submicromolar concentration using a monomolecular layer of horseradish peroxidase was developed. In this device 0.3 pmol of the enzyme were covalently immobilized on the glass surface of the biosensor and the enzyme layer was characterized by atomic force microscopy and activity measurements. The glass surface bearing the peroxidase was faced to a carbon electrode in a cell of 1 microl of active volume. The polarization of the working electrode at -100 mV versus Ag/AgCl, in the presence of 1,4-hydroquinone as mediator, allowed the fast reduction of the injected hydrogen peroxide via the hydroquinone-peroxidase system. This device permitted to measure the total number of H(2)O(2) molecules present in the cell in the concentration range of 0.3-100 microM H(2)O(2), with a sensitivity of 196 nC/microM H(2)O(2), which is close to the theoretical value (193 nC/microM).
Abstract: Atmospheric formaldehyde (CH(2)O) was detected under continuous flow conditions by an on-line system comprising of a wet scrubber for a continuous transfer of the pollutant to an aqueous solution, a micro-reactor containing immobilized formaldehyde dehydrogenase (FDH) and a conductometric transducer. By this system atmospheric formaldehyde concentrations in the range 0.05-2 ppm were detected with a sensitivity of 20 microS/ppm. In this concentration range the immobilized enzyme oxidized all the sampled formaldehyde molecules to formic acid, avoiding cumbersome calibration procedures. The operational stability of the biosensor was at least 3 months, working continuously 10 h/day at room temperature.
Abstract: The effect of calcium ions has been studied on three different isoforms of thioredoxin reductase. The cytosolic (TrxR1), mitochondrial (TrxR2), and the Escherichia coli enzymes were examined and compared. In our condition, TrxR1 appears extremely sensitive to Ca2+ showing an IC50 of about 160 nM, while Ca2+ exerts only a weak inhibitory effect on the mitochondrial isoform. The thioredoxin reductase purified from E. coli is almost completely insensitive to calcium ions. Circular dichroism analysis of highly purified mitochondrial and cytosolic thioredoxin reductases reveals that Ca2+ induces conformational alterations that are particularly relevant only in the cytosolic isoform. These observations are discussed with reference to the physiological role and, in particular, to the regulatory functions of the thioredoxin system.
Abstract: An amperometric flow biosensor, using laccase from Rigidoporus lignosus as bioelement was developed. The laccase was kinetically characterized towards various phenolics both in solution and immobilized to a hydrophilic matrix by carbodiimide chemistry. A bioreactor connected to an amperometric flow cell by a FIA system was filled with the immobilized enzyme and the operational conditions of this biosensor were optimized as regards pH. Under the adopted experimental conditions, the immobilized enzyme oxidizes all the substrate molecules avoiding the need of cumbersome calibration procedures. The biosensor sensitivity, which was found to be 100 nA/microM for some of the tested substrates, resulted to be constant for more than 100 working days. This biosensor permits the detection of phenolics in aqueous solutions at concentrations in the nanomolar range and was successfully used to detect phenolics in wastewaters from olive oil mill without sample preparation.
Abstract: Pregnancy is associated with changes in circulating red blood cells, mainly involving band 3 protein and membrane lipid peroxidation. Membrane band 3 is a multifunctional protein containing four Tyr-phosphorylatable residues which modulate the physiological status of erythrocytes by regulating glycolysis, cell shape and membrane transport. Erythrocytes from nine pregnant and 12 age-matched non-pregnant healthy women were subjected to oxidative and hyperosmotic stress conditions and the extent of band 3 Tyr-phosphorylation and membrane Syk recruitment as a membrane marker were evaluated. Results indicated that, in pregnancy, red blood cells show a decrease in band 3 Tyr-phosphorylation and a clear-cut rearrangement of band 3 protein within the membrane. In fact, band 3 shows a decrease in high molecular weight aggregates (HMWA), with different subdivision between Triton-soluble and -insoluble compartments, and an increase in proteolytic fragments. In conclusion, it is demonstrated that pregnancy is associated with membrane adjustments which reduce the sensitivity of erythrocytes to both oxidative and osmotic stress. Band 3 Tyr-phosphorylation is proposed as a new parameter in the evaluation of erythrocyte membrane arrangement.
Abstract: Laccases from various sources were tested, and laccase from Rigidoporus lignosus was found to be the most active towards syringaldazine and ABTS, which are typical substrates of this class of enzymes, and towards the phenols found in olive oil mill wastewaters. This laccase was covalently immobilised by carbodiimide chemistry, on a self-assembled monolayer of 3-mercaptopropionic acid deposited on a gold surface. A flow biosensor, using the monolayer of laccase as bioelement and a glassy carbon electrode as amperometric transduction system, was developed. Although the amount of the immobilised enzyme (about 140 ng/cm2 effective surface area) was tiny, the biosensor showed a sensitivity of 3 nA/microM when 1,4-hydroquinone was used as substrate, and a half-life of 35 days. The proposed device permits detection of phenols in aqueous solutions at concentrations in the low micromolar range, i.e. below European Community limits. The biosensor was successfully used to detect phenols in wastewaters from an olive oil mill after minimal sample preparation (incubation of the aqueous sample with sodium borohydride for a few minutes) to suppress the current due to oxidised compounds present in the wastewaters.
Abstract: A steady-state kinetic study of bovine serum amine oxidase activity was performed, over a wide range of pH values (5.4-10.2) and ionic strength (10-200 mM), using various (physiological and analogue) substrates as specific probes of the active-site binding region. Relatively small changes in k (cat) values (approx. one order of magnitude) accompanied by marked changes in K(m) and k(cat)/K(m) values (approx. six orders of magnitude) were observed. This behaviour was correlated with the presence of positively charged groups or apolar chains in the substrates. In particular, it was found that the docking of the physiological polyamines, i.e. spermidine and spermine, appears to be modulated by three amino acid residues of the active site, which we have named L(-)H(+), G(-)H(+) and IH(+), characterized by p K (a) values of 6.2+/-0.2 [Di Paolo, Scarpa, Corazza, Stevanato and Rigo (2002) Biophys. J. 83, 2231-2239], 8.2+/-0.3 and 7.8+/-0.4 respectively. The electrostatic interaction between the protonated substrates and the enzyme containing the residues L(-)H(+), G(-)H(+) and IH(+) in the deprotonated form, the on/off role of the IH(+) residue and the role of hydrophobic interactions with substrates characterized by apolar chains are discussed.
Abstract: The assessment of kidney viability before transplantation (with a view of discarding nonviable organs) remains an obstacle to confidently extending organ harvesting to marginal donors. In the present study phosphorus magnetic resonance spectroscopy was used to monitor metabolic changes in (31)P-containing metabolites in isolated porcine kidneys. After various warm ischemia times, the organs were stored at 0 degrees C. Time-dependent changes in the phosphomonoester/inorganic-phosphate ratio were recorded at 0 degrees C were shown to follow a biexponential decay. The first-order kinetic rate constant of the short-time decay was strongly dependent on the warm ischemia time, a result that was discreted in terms of the underlying biochemistry. The metabolic events responsible for the dramatic decrease in phosphomonoester/inorganic phosphate ratio that occur immediately after organ perfusion and storage, suggest that any procedure to minimize organ damage must occur immediately after harvesting.
Abstract: An assay to estimate the amount of glucose oxidase immobilised as a monolayer onto a flat surface is reported. This method is based on the electrochemical detection of the flavin adenine dinucleotide (FAD) cofactor released by the immobilised enzyme in acid solutions. FAD concentration in the acid solution was measured by amperometry, using a flow injection analysis (FIA) system equipped with a wall-jet electrode, and with a sensitivity of (9.2+/-2.0)x10(-2) nA/nM. By this method, the amount of glucose oxidase molecules present in a monolayer deposited on a silanised glass slide was easily detected, in which the detection limit is more than one order of magnitude lower than the maximum loading of the surface with an ordered monolayer of glucose oxidase.
Abstract: The development of reliable methods for assessing the viability of currently available livers is expected to increase the number of successful transplantations.
Abstract: A stable ESR signal, centred at g = 2.0037 +/- 0.0002, characterised by a single resonance and assignable to a free radical, was found in all the bottled red wines, both commercial and experimental, that we have examined. The radical concentration was calculated to be in the range of 5-82 nM. After exposure of the wines to air for a few minutes a two fold increase of the ESR signal, followed by a slow decrease with time, was observed. The intensity of ESR signal in experimental red wines, was found to increase with the ageing of the wines and was strictly correlated to the total content of polyphenols. The formation of semiquinone radicals of polyphenols is suggested as one possible mechanism leading to the presence of stable free radicals in red wines.
Abstract: Active uniform films of horseradish peroxidase (HRP) have been prepared by covalent binding on Si/SiO(2) or glass supports previously activated by silanization and succinylation. Labeling by fluorescent or by Electron Spin Resonance (ESR) probes was used to quantify the surface density of active groups and of horseradish peroxidase. Atomic Force Microscopy (AFM) imaging was used to characterize the surface morphology. We observed that a non-uniform protein adsorption due to physical interactions was present when the supports were not activated for covalent binding and was, in large part, removed by washing. The enzyme deposited by covalent binding formed homogeneous layers with a height in the range 60-90 A. By using a fluorescent label, we calculated a protein density of 3.6 x 10(12) molecules cm(-2) on Si/SiO(2), corresponding to an estimated area per molecule of 2800 A(2) which is in agreement with the value expected on the basis of the crystallographic data considering the formation of a monomolecular layer. The protein density of the layer immobilized on glass was similar (1.9 x10(12) molecules cm(-2)). The enzyme immobilized on both supports showed a k(cat)/K(M) being of the order of 3-5x10(5) M(-1)s(-1) that is 1/20th of free HRP. The half-life time of the activity of the enzyme immobilized by covalent binding was longer than 40 days at 6 degrees C.
Abstract: Highly reactive radicals, ROO(*), were generated from 2, 2'-azobis[2-(2-imidazolin-2-yl)propane] and linoleic acid. The ROO(*) scavenging capacity of some Italian red wines was evaluated following the changes in oxygen consumption. Under the experimental conditions the time course of oxygen consumption shows two typical behaviors: trolox-like (class I) and gallic acid-like (class II). Usually the time course of wine was similar to that of gallic acid. The rate of oxygen consumption was found to decrease exponentially with the amount of wine or gallic acid added to the test solution. On this basis the capacity of red wines to scavenge peroxy radicals was expressed as content of gallic acid (S(GA)). The S(GA) values were found to be correlated to the amount of total proanthocyanidins and total polyphenols of some Italian red wines (p < 0.01). The proanthocyanidins extracted from seeds were shown to make a major contribution to the peroxy radical scavenging capacity of red wines, whereas, interestingly, the chemical class of the low molecular weight tannins reactive to vanillin did not correlate with the S(GA) values.
Abstract: The esterification of the three polysaccharides, starch, amylose and amylopectin was carried out in pyridine-DMSO by succinic anhydride. The carboxylic groups in the succinylated polysaccharides were measured by FT-IR spectroscopy. The succinic derivatives were tested as alpha-amylase (1,4-alpha-D-glucan glucano hydrolase, E.C. 3.2.1.1) substrates. A colorimetric assay of the alpha-amylase activity indicated that this enzyme is active on succinic esters of starch and amylose and that the activity shows a linear decrease with the number of succinic units introduced into the polysaccharide. Since the colorimetric test was not suitable for the detection of the alpha-amylase activity when succinylated amylopectin was the substrate, we set-up an assay based on the labeling by a paramagnetic probe of the free carboxylic groups of succinylated polysaccharides. The kinetics of the alpha-amylase reaction were monitored by ESR spectroscopy through the increase of the mobility of the paramagnetic probe. The spin label used was the commercially available 4-amino-tempo. By this method we demonstrated that alpha-amylase is active on succinylated amylopectin. The utility of the assay for monitoring alpha-amylase activity when other methods (i.e. colorimetric tests) fail, is discussed.
Abstract: A novel, simple, and rapid procedure for the purification of pea seedling amine oxidase is reported. The crude enzyme, obtained by ammonium sulfate fractionation, was purified in two steps: the first one by anion-exchange chromatography and the second one by affinity chromatography. The first chromatography step was carried out on a diethylaminoethyl-cellulose column. By lowering the amount of protein loaded on the column and the buffer concentration it was possible to obtain an enzyme pure at 95% (sp act 1.2 microkat/mg). To achieve a higher degree of purification various affinity resins were prepared and tested. The resins were obtained by covalent immobilization of polyamines on Sepharose according to three different procedures. The best results were obtained with 6-aminohexyl-Sepharose 2B, prepared using CNBr as coupling agent, and eluting the enzyme by a solution containing 1, 4-diaminocyclohexane. This last compound was found to be a relatively strong competitive inhibitor of the oxidative deamination of cadaverine catalyzed by pea seedling amine oxidase (Ki = 32 microM). According to this procedure an electrophoretically homogeneous enzyme, characterized by a specific activity of 1.63 microkat/mg, was obtained.
Abstract: Five peptides containing (His-X2)-His or (His-X3)-His motifs have been designed and synthesized to coordinate Cu(II). Structural information was obtained by various spectroscopic techniques and was used as constraint to search for local conformational energy minima by molecular mechanics. Thermodynamic stability constants of the Cu(II) chelates was obtained by 19F-NMR. The synthesized Cu(II)-peptide chelates were tested as catalysts of some important red-ox processes occuring in biological systems, in particular oxidation of ascorbate and dismutation of superoxide ion. The catalytic efficiency of the five chelates was much lower than that of ascorbate oxidase. On the contrary, two of them showed kinetic constants for superoxide dismutation about one order of magnitude lower than that of the enzyme Cu,Zn superoxide dismutase. In both cases, the catalytic properties were dependent on the peptide sequence. The relationships between structure and activity are discussed to find the structural parameters crucial for catalytic activity that can be modulated by appropriate design and synthesis of the peptides.
Abstract: Glucose Oxidase (GOD) has been covalently bound to functionalized glass cover slips. The surface density of immobilized GOD molecules was measured by a method based on the amperometric determination of Flavin Adenine Dinucleotide (FAD). Atomic Force Microscopy (AFM) images, obtained in aqueous solution for the covalently bound enzyme, show a monomolecular layer of the enzyme on a functionalized glass surface. The catalytic constants were measured for the immobilized GOD and compared with those of the free enzyme.
Abstract: The hapten atrazine was detected under continuous flow conditions using a micro-column which contained immobilized monoclonal antibodies (Ab) against atrazine and atrazine labeled with alkaline phosphatase (An*). The equilibrium of the antibody-hapten system, was achieved by a continuous flow of the tracer An* through the micro-column containing the immobilized antibodies. The activity of the tracer was monitored continuously, after the micro-column, by an amperometric detector using p-hydroquinone phosphate as substrate. When pulses of unlabeled atrazine (An) were added to the An* flowing continuously through the micro-column, An* bound to the antibody was displaced, with a consequent change of the detector signal. By this method atrazine concentrations in the range 9-180 micrograms/l were monitored under conditions of continuous operation. Since the equilibrium condition for the system Ab-An* was continuously restored by the flow of An* through the micro-column the regeneration of the antibody was not required.
Abstract: 2H-NMR spectroscopy has been used to study the distribution of 2H atoms in brain lactate, after intraperitoneal injection of 2H2O. Information on brain metabolism was obtained from the ratio [(3-2H) lactate]/[(2-2H)-lactate] which was found very sensitive to the PO2 in the breathing air. The dependence of this ratio on the rates of metabolic pathways is an example of the possibility to use the distribution of 2H atoms in a molecule to probe metabolic flow through different pathways.
Abstract: A systematic study on the disappearance of the electron spin resonance (ESR) signal of nitroxides based on six-or five-membered ring and bearing various charges was carried out in vitro and in vivo. The second-order kinetic rate constants of the reaction of spin probes with ascorbate were measured in vitro at various temperatures in phosphate buffered saline, and the relative activation energies were calculated. Clearance rates of the nitroxide radicals in rat brain homogenates and in blood indicate that the ascorbate contribution to nitroxide removal is about 50-70% in brain and 50-90% in blood. These rates can be easily calculated on the basis of the ascorbate concentration and of the second-order kinetic rate constants measured in phosphate buffered saline. ESR spectra acquired in vivo in rat head and tail, by an L-band resonator, indicated that the nitroxide decay rate is a first-order kinetic process in both domains and that the positively charged nitroxides are not retained in the brain, whereas the anionic and uncharged nitroxides are. Once nitroxides with piperidine ring enter the brain, their decay appears controlled mainly by ascorbate, while the ascorbate has a negligible influence on disappearance in brain of five-membered ring proxyl nitroxides.
Abstract: The effect of incubation temperature on the activity of bovine serum amine oxidase was studied using natural substrates of this enzyme, namely spermine and spermidine. The activity behavior was found to be rather complex and different from that observed using benzylamine as substrate. The enzyme is fully active after 3 min incubation at temperatures up to 60 degrees C, while at higher temperatures it shows an S-shaped irreversible decrease of the activity with a T50 of about 70 degrees C. The dependence of the kinetic parameters Kcat and Km on temperature was also studied in the range 5-66 degrees C, measuring the initial rates of oxidation of spermidine. The Arrhenius plot of kcat shows a continuous bend in the thermal stability range of the enzyme. This nonlinearity is due to the dramatic change of kcat activation entropy (from +11 eu at 8 degrees C, to -31 eu at 50 degrees C) and indicates that kcat is a composite constant, involving two or more successive steps characterized by different kinetic parameters. In the case of the ratio kcat/Km, which for natural substrates of bovine serum amine oxidase was shown that its dependence on ionic strength gives information on the influence of electrostatic field on k1 (Stevanato et al. (1994) Biochem. J. 299, 317-320), where k1 is the kinetic constant of interaction between the polyamine and the enzyme, the Arrhenius plot is linear in the range 12-45 degrees C, and an activation entropy of 18.8 eu was calculated at 37 degrees C. This value is in accord with a mechanism involving the formation of a polyamine-enzyme complex which is accompanied by neutralization of charges between the polycationic substrate and negatively charged groups of the enzyme.
Abstract: The kinetic characterization of soybean seedling amine oxidase (SSAO) and the effect of ionic strength and pH on the enzyme activity have been studied. A strong dependence of the activity on the carbon chain length of alpha-omega diamines, peaking at C8 and at C5, for Km and kc, respectively, was found. The analysis of ionic strength effects on activity showed a high sensitivity of Km and an insensitivity of kc to this parameter. This behavior and the different dependence of Km and kc on the carbon chain length suggest a two-stage equilibrium model for the oxidative deamination of polyamines by SSAO. The formation of the first intermediate (complex amine-enzyme) is controlled mainly by ionic interactions and by the structure of the polyamine, while the formation of the second intermediate depends only on the length of the carbon chain of the diamine. The molecular dissociation constants of the system soybean seedling amine oxidase-cadaverine were obtained from the dependence of kc, Km, and kc/Km on pH. A mechanism of the initial steps of reaction, involving a two-stage equilibrium model, is proposed.
Abstract: 19F nuclear magnetic resonance (NMR) was utilized to obtain information on the uptake and half-life time of fluoride ion in rats. Changes in tissue fluoride level after acute loading were monitored over time in blood and tissue homogenates obtained from liver and brain. The rate of fluoride elimination from various tissues was roughly similar, following in all cases a first-order kinetic rate law. The F- concentration in brain was about 20% of that found in liver, indicating a reduced fluoride diffusion across the blood-brain barrier. In vivo F- spectra were obtained in rat brain in few minutes with a good signal-to-noise ratio; this confirms the possibility of extending the use of F- as a probe of biomolecules to in vivo applications.
Abstract: A simple and rapid procedure for purification of soybean seedling amine oxidase is reported. The crude enzyme, obtained by ammonium sulfate fractionation was purified by ion-exchange chromatography on a cellulose phosphate column and batch affinity chromatography on 6-aminohexyl-Sepharose. Cyclohexylamine, a competitive inhibitor, was utilized to elute the enzyme. A homogeneous enzyme was obtained with a yield higher than 25%, the content of minor components being < or = 2%. The M(r) estimated by gel filtration is 113,000 and 77,000 by sodium lauryl sulfate-polyacrylamide gel electrophoresis. The enzyme is a dimer and contains two Cu2+ ion per molecule. Its EPR spectrum is typical of Cu2+ in a tetragonal symmetry. The enzyme oxidizes cadaverine at high rate, the specific activity being 4.3 mukat/mg. Molecular, spectroscopic, and kinetic properties of this enzyme are reported.
Abstract: A novel method for isolation of amine oxidase from bovine plasma is reported; it involves a two-step procedure, namely ammonium sulfate fractionation and affinity chromatography with elution by aniline, which is a competitive inhibitor of the enzyme. A homogeneous enzyme, characterized by a specific activity of 0.44 U/mg, was obtained with a yield higher than 50%.
Abstract: Two protein kinases active on casein and phosvitin were partially purified from the soluble fraction of ejaculated bovine spermatozoa. They were operationally termed casein kinase A and B based on the order of their elution from a phosphocellulose column. CK-A showed an approximate molecular mass of 38 kDa, and it phosphorylated serine residues of casein and phosvitin utilizing ATP as a phosphate donor (Km 19 microM). Enzyme activity was maximal in the presence of 10 mM MgCl2, whereas it decreased in the presence of spermine, polylysine, quercetin, and NaCl (20-250 mM). CK-B seemed to have a monomeric structure of about 41 kDa; it underwent autophosphorylation and cross-reacted with polyclonal antibodies raised against recombinant alpha, but not beta, subunit of human type 2 casein kinase. It phosphorylated both serine and threonine residues of casein and phosvitin, utilizing ATP (Km 12 microM) but not GTP as a phosphate donor. Threonine was more affected in the phosphorylated phosvitin than in the partially dephosphorylated substrate. CK-B was active toward the synthetic peptide Ser-(Glu)5 and calmodulin (in the latter case, in the presence of polylysine), and it was activated by spermine, polylysine, MgCl2 (30 mM), and NaCl (20-400 mM). The activity of the enzymes was not affected by cAMP, or the heat-stable inhibitor of the cAMP-dependent protein kinase, or calcium.
Abstract: F- added to an erythrocyte suspension shows two separated resonances arisen from intra and extracellular compartments. Cu, Zn superoxide dismutase dominates the longitudinal relaxation rate of the intracellular F- resonance, while diamagnetic interactions with hemoglobin contribute mainly to the transversal relaxation rate.
