On the mechanism of citrate inhibition of ceruloplasmin ferroxidase activity

Ceruloplasmin is a plasma protein, which oxidizes ferrous ions in a catalytic manner. It is considered to function as a ferroxidasein vivo. Citrate was found to inhibit the reaction. The ceruloplasmin catalyzed oxidation ofp-phenylenediamines, however, was not affected by citrate. The inhibitory effect is proposed to be due to formation of Fe²⁺-citrate, which does not react with ceruloplasmin. The stability constant for the Fe²⁺-citrate complex estimated from the present inhibition study is in good agreement with previously published data.     

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.     

An X-ray structural study of human ceruloplasmin in relation to ferroxidase activity

The role of ceruloplasmin as a ferroxidase in the blood, mediating the release of iron from cells and its subsequent incorporation into serum transferrin, has long been the subject of speculation and debate. However, a recent X-ray crystal structure determination of human ceruloplasmin at a resolution of around 3.0?Å, in conjunction with studies associating mutations in the ceruloplasmin gene with systemic haemosiderosis in humans, has added considerable weight to the argument in favour of a ferroxidase role for this enzyme. Further X-ray studies have now been undertaken involving the binding of the cations Co(II), Fe(II), Fe(III), and Cu(II) to ceruloplasmin. These results give insights into a mechanism for ferroxidase activity in ceruloplasmin. The residues and sites involved in ferroxidation are similar to those proposed for the heavy chains of human ferritin. The nature of the ferroxidase activity of human ceruloplasmin is described in terms of its three-dimensional molecular structure.     

Pre-steady state kinetics of the ferroxidase reaction catalyzed by ceruloplasmin

Analysis of the presteady-state step of the ferroxidase reaction catalyzed by ceruloplasmin revealed that the kinetic curve for the reaction product accumulation versus time is characterized by an induction period, tau, whose value does not change within the pH range of 5.0-7.5. The value of tau decreases with a rise in the enzyme concentration and increases with a rise in the substrate concentration at constant Fe(II) and E0 concentrations. A reaction scheme is proposed which points to the existence of an induction period in the ceruloplasmin-catalyzed ferroxidase reaction. It was shown that the calculated values of tau = f([Fe(II)]0) and tau = f([E]0) obtained through the use of differential equations qualitatively coincide with the corresponding experimental values.     

Purification and characterization of the serum ferroxidase inhibitor

The inhibitor of the serum ferroxidases, recently detected in rabbit serum, has been purified to homogeneity from human serum by a combination of gel-filtration and ion-exchange chromatography. The molecular weight, chromatographic behavior, electrophoretic mobility, electrofocusing pH, carbohydrate content, and reactivity with anti-human albumin during immunodiffusion indicate that the ferroxidase inhibitor is serum albumin. Copper-binding studies, proteolytic fragmentation studies, and a comparison of the inhibitory potencies of several albumin species which differ in their affinity for copper strongly indicate that albumin elicits its inhibitory effect on the serum ferroxidases by interacting with the functional copper of these enzymes. Kinetic analyses further suggest that albumin competes with substrate (ferrous iron) for binding to the functional copper of the serum ferroxidases.     

Thrombin inhibits the anti-myeloperoxidase and ferroxidase functions of ceruloplasmin: relevance in rheumatoid arthritis

Human ceruloplasmin (CP) is a multifunctional copper-binding protein produced in the liver. CP oxidizes Fe²⁺ to Fe³⁺, decreasing the concentration of Fe²⁺ available for generating harmful oxidant species. CP is also a potent inhibitor of leukocyte myeloperoxidase (MPO) (K_d=130 nM), a major source of oxidants in vivo. Rheumatoid arthritis (RA) is an inflammatory autoimmune disease affecting flexible joints and characterized by activation of both inflammatory and coagulation processes. Indeed, the levels of CP, MPO, and thrombin are markedly increased in the synovial fluid of RA patients. Here we show that thrombin cleaves CP in vitro at ⁴⁸¹Arg–Ser⁴⁸² and ⁸⁸⁷Lys–Val⁸⁸⁸ bonds, generating a nicked species that retains the native-like fold and the ferroxidase activity of the intact protein, whereas the MPO inhibitory function of CP is abrogated. Analysis of the synovial fluid of 24 RA patients reveals that CP is proteolytically degraded to a variable extent, with a fragmentation pattern similar to that observed with thrombin in vitro, and that proteolysis is blocked by hirudin, a highly potent and specific thrombin inhibitor. Using independent biophysical techniques, we show that thrombin has intrinsic affinity for CP (K_d=60–270 nM), independent of proteolysis, and inhibits CP ferroxidase activity (K_I=220±20 nM). Mapping of thrombin binding sites with specific exosite-directed ligands (i.e., hirugen, fibrinogen γ′-peptide) and thrombin analogues having the exosites variably compromised (i.e., prothrombin, prethrombin-2, β_T-thrombin) reveals that the positively charged exosite-II of thrombin binds to the negatively charged upper region of CP, while the protease active site and exosite-I remain accessible. These results suggest that thrombin can exacerbate inflammation in RA by impairing the MPO inhibitory function of CP via proteolysis and by competitively inhibiting CP ferroxidase activity.Notably, local administration of hirudin, a highly potent and specifc thrombin inhibitor, reduces the concentration of active MPO in the synovial fluid of RA patients and has a beneficial effect on the clinical symptoms of the disease.     

The ferroxidase activity of yeast frataxin

Frataxin is required for maintenance of normal mitochondrial iron levels and respiration. The mature form of yeast frataxin (mYfh1p) assembles stepwise into a multimer of 840 kDa (alpha(48)) that accumulates iron in a water-soluble form. Here, two distinct iron oxidation reactions are shown to take place during the initial assembly step (alpha --> alpha(3)). A ferroxidase reaction with a stoichiometry of 2 Fe(II)/O(2) is detected at Fe(II)/mYfh1p ratios of < or = 0.5. Ferroxidation is progressively overcome by autoxidation at Fe(II)/mYfh1p ratios of >0.5. Gel filtration analysis indicates that an oligomer of mYfh1p, alpha(3), is responsible for both reactions. The observed 2 Fe(II)/O(2) stoichiometry implies production of H(2)O(2) during the ferroxidase reaction. However, only a fraction of the expected total H(2)O(2) is detected in solution. Oxidative degradation of mYfh1p during the ferroxidase reaction suggests that most H(2)O(2) reacts with the protein. Accordingly, the addition of mYfh1p to a mixture of Fe(II) and H(2)O(2) results in significant attenuation of Fenton chemistry. Multimer assembly is fully inhibited under anaerobic conditions, indicating that mYfh1p is activated by Fe(II) in the presence of O(2). This combination induces oligomerization and mYfh1p-catalyzed Fe(II) oxidation, starting a process that ultimately leads to the sequestration of as many as 50 Fe(II)/subunit inside the multimer.     

Functional link between ferroxidase activity of ceruloplasmin and protective effect of apo-lactoferrin: studying rats kept on a silver chloride diet

Strongly pronounced argyrosis caused by adding AgCl to the feed of laboratory rats efficiently mimics the deficiency of ceruloplasmin (CP) ferroxidase activity. Bringing the concentration of AgCl in the feedstuff of lactating rats to 250 mg % and keeping their progeny (Ag-rats) for 3 months on the same silver-containing feed provided the serum iron content 1.4 times lower than that in the control group. Besides, the ferroxidase activity of CP dropped to zero. In CP purified from sera of Ag-rats two copper ions were substituted with two silver ions. Using rat models of both post-hemorrhagic and hemolytic anemia we showed that the deficiency of CP ferroxidase activity in Ag-rats affects the iron content in serum, though does not prevent the recovery of hemoglobin level accompanied by exhaustion of iron caches in liver and spleen. When apo-lactoferrin (apo-LF) was administered to Ag-rats suffering from either post-hemorrhagic or hemolytic anemia, both hemoglobin and serum iron were restored more rapidly than in the control animals. In independent experiments Ag-rats were compared with those fed on regular diet and the former displayed a prolonged 3-day stabilization of hypoxia-inducible factors 1 and 2 alpha (HIF-1a and HIF-2a) along with an increased serum concentration of erythropoietin. Introduction to Ag-rats of active CP separately or together with apo-LF reduced that effect to 1 day only. It is concluded that saturation of apo-LF with iron, provided by active CP, can strongly affect its protective capacity.     

Identification of an endothelial cell product as an inhibitor of tissue factor activity

Summary A endothelial cell glycoprotein that inhibits the initiation of the coagulation process promoted by tissue factor has been isolated by heparin-sepharose, hydroxyapatite and gel filtration chromatography. As is the case for several other species synthesized by this cell type, the N-linked carbohydrate moiety is sulfated. The identification of this inhibitor hints at the possible existence of a functional balance, between the actvvator of the coagulation cascade and its inhibitor, which may become perturbed in various pathophysiological conditions. This work was supported by grants from NHLB Institute, RJR Nabisco and the Council for Tobacco Research. Editor's statement This paper describes a heparin-binding factor capable of inhibiting coagulation. The work contributes toward understanding the processes of cellular hemostasis, and also may represent the first report of a protein that could find widespread therapeutic use.     

Identification of albumin-bound fatty acids as the major factor in serum-induced lipid accumulation by cultured cells

Factors responsible for the high lipogenic activity of rabbit serum were investigated using an assay procedure based on the gravimetric determination of the 24 hr increase in cell lipid. Cellular synthesis of fatty acids was inhibited by the presence of serum in the assay medium. Approximately 90% of the increase in cell lipid produced by serum fractions was due to triglyceride accumulation. Fractionation of rabbit serum by precipitation with ammonium sulfate or by ultracentrifugation in high density medium, both indicated that three-quarters of its lipogenic activity was associated with albumin. The lipoproteins prepared by ultracentrifugation also exhibited about one-half the activity of whole serum. The lipogenic activity of albumin was confirmed by the high potency of the albumin isolated in a nearly pure form from proteins of d>1.21 by precipitation with trichloroacetic acid and extraction with ethanol. As judged from chemical and isotopic analysis, neither the lipid content nor the lipid composition of the albumin was appreciably altered during its isolation. Of the albumin-bound lipids, only the free fatty acids, as determined by DEAE column chromatography, were present in an amount sufficient to account for the observed increase in cell triglycerides. In control experiments with horse serum of low lipogenic activity, the proteins of d>1.21 also possessed low activity in conjunction with a low content of free fatty acid. However, the albumin isolated from the latter preparation exhibited the high lipogenic activity of rabbit serum albumin. Chemical and isotopic analysis of the recovered horse serum albumin revealed that its free fatty acid content was the same as that of rabbit serum albumin. These results indicated that the isolation of horse serum albumin was attended by a substantial increase in its free fatty acid content. When the rabbit serum and horse serum content of media were adjusted to provide equivalent concentrations of albumin-bound fatty acids, the rabbit liver cells grown on the former media accumulated more lipid than cells grown on the latter media. This difference was shown to be due to the higher concentration of albumin per micro mole of fatty acid in horse serum as compared with rabbit serum. Consequently, the albumin to fatty acid ratio also controls the lipogenic activity of a serum. A linear relationship is presented which relates the cell lipid content to the molar ratio of albumin to free fatty acids and to the absolute concentration of free fatty acids in the medium.     

Identification of the thiol groups in human ceruloplasmin

Human ceruloplasmin was attached to activated thiol-Sepharose via its thiol groups and was then digested with pepsin. After appropriate washings the thiol peptides were eluted by reduction and were carboxymethylated and purified by column chromatography and electrophoresis. Amino acid sequencing showed that the peptides were derived from five different areas in the molecule and together accounted for 92 residues, six of which were cysteines. Since one of the peptides contained two cysteines it seemed evident that, prior to the reductive elution of the peptides, one of these had been paired in a disulfide bridge with one of the four remaining thiol peptides present in the mixture. The disulfide was isolated and identified by digesting the immobilized protein with pepsin followed by trypsin. The second (tryptic) digestion released the disulfide peptide. Three of the true thiol peptides obtained occur in regions of sequence that have already been reported and which account for 564 of the approximately 1050 residues present in the protein. Three of them also show about 40% identity with each other, whereas no relatedness is observed with the fourth. The three related peptides are, moreover, clearly homologous to the copper-binding areas in the small blue plant and bacterial proteins plastocyanin and azurin. Homologous regions are also evident when the peptides are compared to the two sequences reported for the blue oxidase, fungal laccase, one of which contains a disulfide bridge.