Effect of lactoferrin on the ferroxidase activity of ceruloplasmin.

The effects of various forms of lactoferrin (Lf) interacting with ceruloplasmin (Cp, ferro-O2-oxidoreductase, EC 1.16.3.1) on oxidase activity of the latter were studied. Comparing the incorporation of Fe3+ oxidized by Cp into Lf and serum transferrin (Tf) showed that at pH 5.5 apo-Lf binds the oxidized iron seven times and at pH 7.4 four times faster than apo-Tf under the same conditions. Apo-Lf increased the oxidation rate of Fe2+ by Cp 1.25 times when Cp/Lf ratio was 1 : 1. Lf saturated with Fe3+ or Cu2+ increased the oxidation rate of iron 1.6 and 2 times when Cp to holo-Lf ratios were 1 : 1 and 1 : 2, respectively. Upon adding to Cp the excess amounts of apo-Lf (Cp/apo-Lf < 1 : 1) or of holo-Lf (Cp/holo-Lf < 1 : 2) the oxidation rate of iron no longer changed. Complex Cp-Lf demonstrating ferroxidase activity was discovered in breast milk.

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.

Embryotoxicity of silver ions is diminished by ceruloplasmin--further evidence for its role in the transport of copper.

The effect of alimentary administration of silver salts upon embryogenesis in rats was studied. Feeding of female rats throughout the term on a regular diet supplemented with AgCl did not cause alterations of their physiological functions, despite the fact that enzymatically active copper-containing ceruloplasmin (CP) was eliminated from the blood plasma. However, developmental abnormalities of embryos, their prenatal death or the 100% mortality of the newborns in the first 24 h of life was seen. Copper content in placenta and fetal tissues was strongly diminished. Cu, Zn-superoxide dismutase (SOD) activity decreased in cytoplasm of embryonic cells along with a drop, though less pronounced, in the tissues of the pregnant females. Embryotoxicity of AgCl was seriously diminished by repetitive injections of native CP to the pregnant rats. Such treatment resulted in an increase of SOD activity in placenta and embryonic tissues. The mortality of the newborns also became less. It is suggested that the embryotoxic effect of AgCl is caused by its ability to interfere with copper metabolism, in particular by altering the copper-transporting function of CP.

Evidence that human ceruloplasmin molecule consists of homologous parts.

The products of spontaneous and induced proteolysis of human ceruloplasmin (Cp) were studied. Some physico-chemical properties of the six fragments with electrophoretically determined Mr 130,000 (F1), 110,000 (F2), 66,000 (F3), 48,000 (F4) 22,000 (F5) and 18,000 (F6) were compared. The amino acid compositions and N-terminal amino acid sequences coincide in F1-F5, but differ from those of F6. Limited tryptic proteolysis of Cp causes the accumulation of polypeptide fragment with Mr 22,000, the N-terminal primary structure of which is identical to that of F5 produced by spontaneous proteolysis. Electrophoretic fragments of Cp were extracted from polyacrylamide gel, treated with 125I and then studied by peptide mapping with subsequent radioautography. The comparison of the "finger prints" showed the identity of F1 to F2 and F3 and gross similarity between F4 and F1-F3. It also revealed similar peptides in F5 and F6 hydrolyzates and almost perfect matching of the F4 map to the map of F5 + F6 mixture. On the basis of the obtained data general principles of Cp molecular organization are discussed and intramolecular homology is suggested to be a feature of the protein.

Isolation and partial purification of ceruloplasmin messenger RNA from rat liver.

Partially purified ceruloplasmin mRNA was isolated using indirect immunoprecipitation of rat liver polysomes and poly(U)-Sepharose chromatography of polysomal RNA. This RNA programmed the synthesis of ceruloplasmin polypeptides in a cell-free system from mitochondria. Immunochemical analysis of the translation products revealed a 40-fold enrichment of the ceruloplasmin mRNA activity. The purified ceruloplasmin mRNA migrated as a major homogeneous component with an apparent molecular weight about 1 X 10(6) daltons in polyacrylamide gels containing sodium dodecyl sulfate. The immunoprecipitated products of the cell-free translation had molecular weights in the range 4.5--5.4 X 10(4) daltons as estimated by gel-electrophoresis under denaturating conditions. These values approach the weight of the half-molecule of native ceruloplasmin.

Preliminary X-ray crystallographic and physico-chemical investigations of human ceruloplasmin.

Single crystals of the plasma protein ceruloplasmin (CP) and its two modified forms: neuraminidase-treated CP (asialoCP) and NaN3-inhibited CP (NaN3-CP) suitable for X-ray studies have been grown. The native CP crystallizes as described previously by Magdoff-Fairchield et al. (1969) in the tetragonal space group 14 (a = b = 268.2 A, c = 129.1 A) with two protein molecules in the asymmetric part of a unit cell. AsialoCP crystals belong to the trigonal space group P 3(1)21 or P321 (a = b = 215.0 A, c = 84.5 A) and have one protein molecule in the asymmetric part of a unit cell. NaN3-CP crystals are isomorphous to crystals of native CP. Despite some differences in electrophoretic mobility and optical properties, the conformations of the native CP molecule and its modified forms are similar, as can be concluded from a study of ORD and CD spectra.

On the defect of synthesis ceruloplasmin in the liver polyribosomes in Wilson's disease.

Comparative immunochemical analysis of ceruloplasmin-synthesizing polyribosomes in liver biopsies from control subjects and homozygous carriers of the Wilson's mutation was performed. According to I125-antibody binding data, the amount of ceruloplasmin-forming liver polysomes in patients with Wilson's disease was 10--20 times lower than that in non-Wilson patients. Correspondingly, the pulse labeling of ceruloplasmin polypeptides was decreased several-fold in the cell-free liver preparations from patients with Wilson's disease.