Reaction of human ceruloplasmin and anion treated ceruloplasmin with diethyldithiocarbamate

The reaction of human ceruloplasmin and anion treated ceruloplasmin with diethyldithiocarbamate was studied at pH 5.5. The analysis of optical and EPR spectra at 9 GHz showed that ceruloplasmin contains five paramagnetic copper ions, two of which, X and Y, not involved in enzymatic activity, are chelated by diethyldithiocarbamate; the complex thus formed is easily removed by high-speed centrifugation. However, the enzyme depleted of these two X and Y copper ions is able to compete with the Cu(II)-diethyldithiocarbamate complex, as time elapses, recovering both Cu(II) atoms. In addition diethyldithiocarbamate acts as a reducing agent for the two type-I copper atoms when added in large excess to the enzyme or the anion treated enzyme.     

Molecular pathology of ceruloplasmin

Individual preparations of CP isolated from one normal donor and two homozygotes for the Wilson's disease gene were subjected to a comparative structural and analytical examination. Analysis of CP tryptic hydrolysate by the peptide map technique revealed 64–66 peptides in each sample. Patient P.'s peptide map lacked one acidic, one neutral, and two basic peptides, but had one basic peptide not found in the normal control. Patient R. lacked one acidic and one basic peptide present in the normal. These changes in the peptides of both patients did not affect the capacity of the protein to bind copper but were associated with diminished oxidase activity.     

Dolphin ceruloplasmin: the first proteolytically stable mammalian ceruloplasmin.

1. Ceruloplasmin, the blue protein of the plasma of vertebrates, was isolated from dolphin, a marine mammal. The protein showed overall physico-chemical parameters very similar to those of all other mammalian ceruloplasmins. The spectroscopic properties indicated a conservation of the copper binding sites. 2. Non-denaturing electrophoresis revealed a conformation similar to that of other mammalian ceruloplasmins. EPR spectroscopy and calorimetric analyses indicated a three-domain arrangement of the protein typical of "aged" ceruloplasmin. 3. Dolphin ceruloplasmin is the only mammalian ceruloplasmin insensitive to trypsin, plasmin or chymotrypsin. This property, however, does not result in a higher conformational stability of the molecule. Thus, susceptibility of ceruloplasmin to aging is not directly related to the lability to proteases, which is typical of all other mammalian ceruloplasmins so far studied.     

The immunoenzyme analysis of ceruloplasmin

A highly specific and sensitive enzyme immunoassay (EIA) system, suitable for the qualitative analysis of ceruloplasmin, has been developed. The possibility of its use for the examination of children with mononucleosis and pseudotuberculosis has been studied. An increase in the concentration of ceruloplasmin has been more pronounced in infectious mononucleosis (0.506 +/- 0.026 g/l) and pseudotuberculosis (0.421 +/- 0.157 g/l). The results of EIA coincided with the data obtained by radial immunodiffusion.     

Properties of silver-containing rat ceruloplasmin

Ceruloplasmin (Cp) was isolated from the sera of albino rats fed with silver nitrate (60 mg/kg of body weight). The oxidase activity of the enzyme was sharply decreased, while its concentration in the blood (as assayed immunologically) was slightly lower than in controls. The drop in the oxidase activity was caused by the replacement of several coppers by silver ions in the Cp molecule. Ag-Cp contained about four silver atoms per 1 mole of protein, its spectrum lacking maxima at 450 and 610 nm that are typical of normal Cp. When subjected to PAAG electrophoresis, Ag-Cp displayed two bands, one of which (Ag-Cp2) had the anodic mobility of normal Cp. The other band (Ag-CpI) migrated at a slower rate. Both bands were separately subjected to SDS-PAAG electrophoresis which revealed the dissimilarities among the proteolytic fragment patterns of Ag-CpI, Ag-Cp2 and normal Cp. Both Ag-CpI and Ag-Cp2 contained peculiar fragments produced by spontaneous limited proteolysis of the native molecule. The binding of silver ions by Cp seems to alter significantly the molecule conformation, which may cause the exposure of new peptide bonds susceptible to proteolytic attack. Cp seems to participate in the binding and detoxication of heavy metals in mammals.     

Interaction of lactoferrin with ceruloplasmin

When added to human blood serum, the iron-binding protein lactoferrin (LF) purified from breast milk interacts with ceruloplasmin (CP), a copper-containing oxidase. Selective binding of LF to CP is evidenced by the results of polyacrylamide gel electrophoresis, immunodiffusion, gel filtration, and affinity chromatography. The molar stoichiometry of CP:LF in the complex is 1:2. Near-uv circular dichroism spectra of the complex showed that neither of the two proteins undergoes major structural perturbations when interacting with its counterpart. K(d) for the CP/LF complex was estimated from Scatchard plot as 1.8 x 10(-6) M. The CP/LF complex is found in various fluids of the human body. Upon injection into rat of human LF, the latter is soon revealed within the CP/LF complex of the blood plasma, from where the human protein is substantially cleared within 5 h.     

Hereditary ceruloplasmin deficiency with hemosiderosis

Hereditary ceruloplasmin deficiency with hemosiderosis (aceruloplasminemia) is a new disease characterized by systemic hemosiderosis, diabetes mellitus, neurological abnormalities and pigment degeneration of the retina. Loss of the ferroxidase activity of ceruloplasmin results in systemic iron deposition and tissue damage. Neuroimaging studies reveal iron deposition in basal ganglia and in the red and dentate nuclei. Cerebellar ataxia, extrapyramidal signs and dementia develop after middle age. We report a patient with undetectable serum ceruloplasmin levels and the above clinical manifestations. Sequence analysis of the cDNA of ceruloplasmin from this patient revealed an insertion of adenine in exon 3; this produced a premature stop codon. Received: 10 November 1995