EPR study of serum ceruloplasmin and iron transferrin in myocardial infarction.

Electron paramagnetic resonance (EPR) analysis of frozen serum from myocardial infarction patients has been conducted. Signal at g=4.3 was found definitively attributable to iron(III)-transferrin complex. Imcrease of serum ceruloplasmin as compared to normal was confirmed, with a concomitant decrease of iron-transferrin content. A mechanism for such correlated variation is hypothesized.     

EPR study of nitric oxide production in rat tissues under hypokinesia

EPR spectroscopy was used to study the intensity of nitric oxide (NO) production upon modeling 60-day progressive hypokinesia (restriction of motor activity) in rats and estimating the content of (DETC)₂-Fe²⁺-NO complexes in heart and liver tissues. In 30 days of hypokinesia, there was a 2–3-fold increase in tissue NO. Administration of a nonspecific inhibitor of NO synthases, L-NAME, to hypokinetic rats prior to measurement decreased their NO level even below the untreated control. Our results show that the intensified NO production in hypokinesia is mainly due to NO synthases, rather than to the nitrite reductase pathway.     

EPR spin-trapping study of nitric oxide formation during bilateral carotid occlusion in the rat

The formation of nitric oxide (NO) radicals was demonstrated by electron paramagnetic resonance spectroscopy in the rat during varying degrees of brain ischemia. Diethyldithiocarbamate and Fe-citrate were used as in vivo spin-trapping reagents. The signal of NO spin adducts increased in accordance with the degree of ischemic insults. The formation of NO radicals was inhibited by N^G-nitro]l-arginine methyl ester.     

Interaction of nitric oxide with ceruloplasmin lacking an EPR-detectable type 2 copper.

Nitric oxide (NO) has previously been reported to modify the EPR spectrum of multicopper blue oxidases, disclosing a pure type 2 copper and inducing half-field transitions at g = 4. In the present work the reactivity of NO was reinvestigated with respect to ceruloplasmins having an apparently EPR-silent type 2 copper in their native state. The optical properties of NO-treated ceruloplasmin were independent of the initial redox state of the metal sites. Addition of NO caused the absorption at 600 nm to decrease in the case of oxidized ceruloplasmin and to increase when starting from the reduced proteins. In this latter case the absorbance at 330 nm was also restored, indicating that NO was able to reoxidize the reduced protein. In all cases the band at 600 nm leveled to ca. 60% of the intensity of the native untreated protein, and new bands below 500 nm appeared in the spectra. While the blue absorption band was restored by removal of NO, the absorbance below 500 nm remained higher even after dialysis. The EPR spectrum resulting from reaction of NO with either oxidized, partially reduced, or fully reduced ceruloplasmin consisted in all cases of a broad, structureless resonance around g = 2. NO caused the reversible disappearance of the type 1 copper EPR spectrum in oxidized ceruloplasmin. Also, the transient novel copper signal that arises during the anaerobic reduction process by ascorbate completely disappeared in the presence of NO and did not reappear upon removal of the gas.(ABSTRACT TRUNCATED AT 250 WORDS)     

EPR spectroscopy of common nitric oxide - spin trap complexes

Two commonly used hydrophobic and hydrophilic spin traps for NO, namely Fe2+(DETC)(2)and Fe2+(MGD)(2), respectively, were analyzed via EPR spectroscopy. EPR spectra of trapped NO, together with field position standards, were recorded both in the frozen state and at room temperature. We present a detailed characterization of the EPR spectra of the above paramagnetic NO complexes, concerning g-value, hyperfine splitting and linewidths. This study also provides spectroscopic data required to develop a quantitative and sensitive detection system for nitric oxide both in hydrophobic and hydrophilic aqueous media.     

Kinetic study of the oxidase reaction of ceruloplasmin

The effects of ionic strength, buffer composition and pH on the oxidase activity of ceruloplasmin isolated from human donor blood were studied. The steady-state kinetics of ceruloplasmin-catalyzed oxidation of organic substrates (pyrocatechine, adrenaline, rho-phenyldiamine) and Fe(II) was analyzed. The relationship between the initial reaction rate and Fe(II) concentration is described by the Michaelis--Menten kinetics, that for organic substrates--by substrate activation or by a scheme which implicates the existence of two catalytic centers in the enzyme molecule. The inhibition of adrenaline oxidation by its reaction product, adrenochrome, is competitive. The reactions studied were shown to occur via the formation of a ternary complex.     

Electron microscope study on human ceruloplasmin.

Electron microscopy of human ceruloplasmin (CP) molecules revealed a few distinctive types of particle images. Analysis of these images allows to propose a tentative model for CP: six "subunits" (which we call domains) not much different in size are arranged with 32 point group pseudosymmetry. The determination of the number of polypeptides arising at the spontaneous specific proteolytic fragmentation of CP and their molecular weights conform with this assumption. The electrophoretic studies of the CP samples prepared both with and without potent proteolytic inhibitor, PMSF, revealed that CP is a single-chain protein with molecular weight of 130 000. Isolated and stored without PMSF the polypeptide chain of CP undergoes specific proteolytic cleavage which results in the appearance of polypeptides with molecular weights of 16 000, 48 000, and 64 000. The latter two polypeptides degradate to about two- and three-fold decreased molecular weights fragments, respectively. Therefore, the single polypeptide chain of CP contains at least five peptide bonds which are particularly susceptible to proteolytic attack and which connect six principal segments of the chain. The hydrolysis of these bonds results in liberation of the six fragments which were integrated in the enzymatically active globule of CP.     

Alternative pathways of nitric oxide formation in lactobacilli: EPR evidence for nitric oxide synthase activity

The study of the ability of Lactobacillus plantarum 8P-A3 to synthesize nitric oxide (NO) showed that this strain lacks nitrite reductase. However, analysis by the EPR method revealed the presence of nitric oxide synthase activity in this strain. Like mammalian nitric oxide synthase, lactobacillar NO synthase is involved in the formation of nitric oxide from L-arginine. L. plantarum 8P-A3 does not produce NO in the course of denitrification process. The regulatory role of NO in symbiotic bacteria is discussed.     

Presence of reduced type 1 copper in ceruloplasmin as revealed by reaction with hydrogen peroxide

The reaction of hydrogen peroxide with ox or sheep ceruloplasmin leads to approximately 10% increase of the optical absorption band at 610 nm and of the Type 1 EPR signal. No inactivation or denaturation of the protein is apparent up to 15 H2O2 molar excess. Oxygen is able to restore about 50% of the Type 1 copper absorption in ascorbate-reduced ceruloplasmin, while the other half is recovered after addition of H2O2. It appears that H2O2 undergoes a specific redox reaction with ceruloplasmin, which reveals a fraction of the total copper to be present in the native protein as reduced copper. This fraction is apparently Type 1 copper, while Type 2 is not affected by H2O2.     

Alternative pathways of nitric oxide formation in Lactobacilli: Evidence for nitric oxide synthase activity by EPR

The study of the ability of Lactobacillus plantarum 8P-A3 to synthesize nitric oxide (NO) showed that this strain lacks nitrite reductase. However, analysis by the EPR method revealed the presence of nitric oxide synthase activity in this strain. Like mammalian nitric oxide synthase, lactobacillar NO synthase is involved in the formation of nitric oxide from L-arginine. L. plantarum 8P-A3 does not produce NO in the denitrification process. The regulatory role of NO in symbiotic bacteria is emphasixed.     

Immunochemical and enzymatic study of ceruloplasmin in rheumatoid arthritis

Forty adult patients (30 women and 10 men) with rheumatoid arthritis (RA), treated with nonsteroidal anti-inflammatory drugs, were studied. Serum levels of immunoreactive ceruloplasmin, oxidase activity of the ceruloplasmin and total copper, as well as the specific oxidase activity (enzyme activity per unit of mass) and the copper/immunoreactive ceruloplasmin relationship were significantly higher in the group of patients than in the healthy control group (p < 0.001). However, no significant difference was found for the concentration of non-ceruloplasmin copper between both groups. A statistically significant negative correlation was obtained for the concentration of serum thiobarbituric acid-reacting substances with the immunoreactive ceruloplasmin and its oxidase activity in the group of patients (p < 0.005). These results suggest that in RA increases of serum copper are produced at the expense of the fraction linked to the ceruloplasmin, diminishing the proportion of apoceruloplasmin and other forms poor in copper. Although the increase in the serum concentration of ceruloplasmin might offer an additional safeguard against oxidative stress. it does not appear to have a beneficial effect upon the activity of the illness as evaluated by means the biological inflammation markers C-reactive protein, erythrocyte sedimentation rate and sialic acid.     

A study on ascorbate inhibition of ceruloplasmin ferroxidase activity

The ferroxidase activity of ceruloplasmin is often determined according to the method of Johnson et al. (1967), using apotransferrin for trapping ferric ions generated by the enzyme; spectrophotometrically monitoring the Fe–transferrin formation at pH6.0. Reports have shown that ascorbate inhibits this reaction, and it is hypothesized that the effect could be of physiological significance in individuals with a high ascorbate to ceruloplasmin ratio in plasma (e.g. premature babies).The present study shows that the inhibitory effect of ascorbate rapidly decreases with increasing pH. At pH7.4 no significant effect was observed, the result suggesting that ascorbate is not a physiological inhibitor of ceruloplasmin. Furthermore, experiments demonstrate that at acidic pH the inhibitory effect of ascorbate on the rate of Fe–transferrin formation is not primarily due to an interaction with ceruloplasmin, but to a reduction of enzymically generated ferric ions before they are bound to apotransferrin.     

Comparative study of ceruloplasmin biosynthesis in various cell-free systems

Some peculiarities of mRNA translation of ceruloplasmin (CP) from rat liver were investigated, using three cell-free protein biosynthesis systems (wheat embryo extracts, rabbit reticulocyte lysates and Zajdela ascite hepatoma extracts). It was shown that reticulocyte lysates and tumour cell extracts synthesize full-size CP mRNA translation products, whose molecular mass is close to that of mature CP molecules, i. e., 122-132 kD. Wheat embryo extracts synthesize the NH2-terminal fragment of the CP molecule (Mr = 84 kD). Addition of liver membrane fractions to wheat embryo extracts translating CP mRNA results in the reconstitution of proteolytic steps of CP maturation.