Biomedical subjects
Publications and source records attributed to E Frieden.
The rate of formation of diferric-transferrin has been studied using various combinations of Fe(II), Tf, Cp and h serum. When the reactants were added in a correct physiological sequence, ceruloplasmin and diluted human serum showed the fastest rate of saturation of transferrin.
Cyclic GMP (cGMP) and cyclic AMP (cAMP) were determined in bullfrog tadpole liver and tail fin using 125I-RIA. cGMP increased approx. 100% 1-6 h after the injection of T3 (3 X 10(-10) mol/g body wt.). Reducing the dose of T3 to 1, 3, and 10 X 10(-11) mol/g body wt. provided increases in cGMP of 50-100% above the control value after 2 h. In contrast, only small increases (less than 20%) in cAMP were observed 2-24 h after T3 injection. We conclude that T3 produces a rapid and significant increase in cGMP in the liver and tail fins of premetamorphic tadpoles. These results suggest that thyroid hormones in amphibia may not be an exclusively nucleus-mediated hormone.
Several features of the catalytic oxidation of cysteine by ceruloplasmin and nonenzymic Cu(II) at pH 7 have been compared. The oxidation of cysteine by ceruloplasmin has several properties in common with the Cu(II) catalyzed oxidation of cysteine: pH maxima, thiol specificity, lack of inhibition by anions, and high sensitivity to inhibition by copper complexing reagents. These two catalysts differed in their molecular activity, in their ability to oxidize penicillamine and thioglycolate, and in that H2O2 was produced as a primary product only during Cu(II) oxidation. The oxidation of cysteine by ceruloplasmin was compared also with the ceruloplasmin catalyzed oxidation of o-dianisidine, a classical pH 5.5 substrate. The mechanism of the oxidation of cysteine by ceruloplasmin at pH 7 differed from that of o-dianisidine oxidation because the latter substrate was inhibited by anions but not by copper complexing agents. Spectral and other data suggest that during the ceruloplasmin reaction with cysteine there is a one electron transfer from cysteine to ceruloplasmin resulting in the specific reduction of type 1b Cu(II).
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Ceruloplasmin is a blue copper protein found in the alpha 2-globulin fraction of vertebrate plasma. It is a single-chain glycoprotein of molecular weight 132 000. It contains six copper atoms per molecule, comprising three or possibly four different types of copper. Its many functions may be related to the heterogeneous nature of these six copper atoms and to the various catalytic activities which they provide. Caeruloplasmin resembles albumin and transferrin in that all three serum proteins are regarded primarily as transport proteins. However, each has numerous other action as important as this transport function. Caeruloplasmin directly mobilizes iron into the serum and provides the major molecular link between copper and iron metabolism; it is the most prominent serum antioxidant, preventing deleterious oxidation of polyenoic acids and other substrates; it scavenges superoxide radicals; it serves as an acute-phase reactant (an endogenous modulator) of the inflammatory response; finally, caeruloplasmin may regulate the serum concentration of the biogenic amines, adrenaline (epinephrine) and serotonin (5-HT).
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The release of iron from transferrin was investigated by incubating the diferric protein in the presence of potential iron-releasing agents. The effective chemical group appears to be pyrophosphate, which is present in blood cells as nucleoside di- and triphosphates, notably adenosine triphosphate (ATP). An alternative structure with comparable activity is represented by 2,3-diphosphoglycerate. Neither 1 mM adenosine monophosphate (AMP) nor 1 mM orthophosphate released iron from transferrin. The ATP-induced iron-releasing activity was dependent on weak acidic conditions and was sensitive to temperature and sodium chloride concentration. The rate of iron release rapidly increased as transferrin was titrated with HCl from pH 6.8 to 6.1 in the presence of 1 mM ATP and 160 mM NaCl at 20 degrees C. Iron release from transferrin without ATP was observed below pH 5.5. Ascorbate (10(-4) M) reduced Fe(III), but only after iron release from transferrin by a physiological concentration of ATP. A proposal for the mechanism of iron release from transferrin by ATP and the utilization of reduced iron by erythroid cells is described.
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NADH-FMN oxidoreductase has been proposed as an enzyme involved in the release of iron from ferritin. The effects of riboflavin and/or iron deficiencies and of dietary allopurinol on the activities of this enzyme and on the iron contents of liver, kidney and duodenum were investigated. Allopurinol, a xanthine oxidase inhibitor, did not affect organ enzyme activities nor iron contents. Riboflavin-deficient rats and iron-deficient rats both had significantly lower organ enzyme activities and iron contrnts than controls. Organ enzyme activities and iron contents of rats fed a diet deficient in both iron and riboflavin were significantly lower than those of controls. After dietary iron and/or riboflavin repletion, organ enzyme activities and iron contents increased. Rats fed an irons-overload diet had enzyme activities similar to that of controls, but organ iron contents were significantly increased over those of controls. Effects of riboflavin and/or deficiencies in rats on NADH-FMN oxidoreductase activities and iron contents of liver, kidney and duodenum appeared to be reversible by riboflavin and/or iron supplementation. The data support the view that NADH-FMN oxidoreductase may be a controlling enxyme in iron release from ferritin.
The relative strength of binding of several triiodothyronine (T3) analogs by cytosol prepared from the tail fin of the bullfrog tadpole has been tested and compared with their thyromimetic activity in tail tissue. In competitive binding experiments, four tri-substituted analogs were bound much more strongly than two tetra-derivatives. Correlation between binding and relative thyromimetic activity was observed only for three triiodo-substituted compounds, triiodothyronine, its methylene bridge analog and the acetic acid analog. The affinity for T3 was 250 times that of T4, making it unlikely that the T3 binding sites in tadpole tail fin cytosol bind T4 under physiological conditions. The difference in binding by tadpole tail fin cytosol constitutes one of the largest differences observed between T3 and T4 in a potentially significant biological system.
125I-labeled deoxyuridine (IdUrd) has been used to estimate the turnover of DNA in liver, tail, and hind limb during spontaneous and triiodothyronine-induced metamorphosis. It was found that the total amount of liver DNA remained constant and there was no significant loss of the label from the liver DNA, which would be expected if there was an increase in DNA turnover during metamorphosis. Also, the change in specific activity of liver DNA parallels that of tail DNA during spontaneous metamorphosis. These data suggest that metamorphic transitions in the tadpole liver do not involve significant changes in DNA turnover. It was observed that the incorporation of label into hind limb DNA showed a high variability among individual animals as compared to liver and tail tissue. The data presented suggest that the observed variability is not a random phenomenon but related directly to the rate at which animals will metamorphose.
Ceruloplasmin, the blue copper-protein of vertebrate plasma, has been reviewed mainly from a functional point of view. However we have surveyed the chemistry and state copper in the molecule because of the implications of the recent data of Ryden (13,28). His observations suggest that unless special precautions are taken in the isolation of ceruloplasmin degradation, probably proteolytic, produces fragments of various sizes. When isolated, these fragments appear to be held together by noncovalent interactions. Comparison of their catalytic and spectral properties reveals no significant differences from a single homogeneous species of molecular weight of 134,000 isolated by Ryden's methods. On the other hand, the homogeneous molecule may differ in properties highly sensitive to conformation and three-dimensional parameters. Three types of copper atoms have been identified in ceruloplasmin, but their amino acid environment is still unknown. Ceruloplasmin possesses significant oxidase activity towards Fe(II) and numerous aromatic amines and phenols. Its ferroxidase activity has led to the discovery that it is a molecular link between copper and iron metabolism. Ceruloplasmin mobilizes iron into the plasma from iron storage cells in the liver. An equally important duty is that ceruloplasmin, after its rapid biosynthesis in the liver, serves as a major copper transport vehicle, comparable to transferrin. Evidence is accumulating that the copper atoms of ceruloplasmin are a prerequisite for copper utilization in the biosynthesis of cytochrome oxidase and other copper proteins. The ability of ceruloplasmin to release copper at specific cellular sites may be related to its broad substrate spectrum of biological reducing agents. A possible third role of ceruloplasmin is as a contributor to the regulation of the balance of biogenic amines through its oxidase action on the epinephrine and the hydroxyindole series. Thus ceruloplasmin is a copper-protein with several important functions, all of which are directly related to its oxidase activity.
Ceruloplasmin (ferroxidase) the blue Cu-protein of vertebrate plasma, possesses significant oxidase activity towards Fe(II) and numerous aromatic amines and phenols. Its ferroxidase activity has led to the discovery that it is a molecular link between copper and iron metabolism. Ceruloplasmin mobilizes iron into the plasma from iron storage cells in the liver. An additional role of Cp may be as a contributor to the regulation of the balance of biogenic amines through its oxidase action on the epinephrine and the hydroxyindole series. Ceruloplasmin also serves as a major copper transport vehicle, comparable to transferrin for iron. Evidence is presented that the copper atoms of Cp are a prerequisite for copper utilization in the biosynthesis of cytochrome oxidase. The ability of Cp to release copper at specific cellular sites is believed to be related to its broad substrate spectrum of biological reducing agents. Thus Cp is a serum protein with several important functions, all of which are directly related to its oxidase activity.
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