Tadeusz Krajewski

Purification and partial characterization of goose ceruloplasmin

The preparation and properties of ceruloplasmin from goose blood plasma are described. Ammonium sulfate was used to precipitate the crude protein followed by adsorption and elution from DEAE-Sephadex A-50. Further treatment with an ethanol-chloroform mixture and Sephadex G-200 yielded an intensely blue protein possessing a high degree of chemical purity and biological activity. Goose ceruloplasmin, existing in two forms, appears to be a single polypeptide, apparent Mr121,300, with an A610/A280 ratio of 0.07. Copper represented 0.32%, which corresponded to six atoms of copper per protein molecule. Although the amount of EPR-detectable copper was the same as in mammalian ceruloplasmins there were some differences in EPR parameters, mainly in A parallel. Goose ceruloplasmins amino acid composition, although similar in many residues to human ceruloplasmin, was lower in tyrosine, cystine/cysteine, and acidic amino acids. Valine was found as the N-terminal amino acid. Hexose, hexosamine, sialic acid, and fucose accounted for 6.65% of the weight. Goose protein contained only half the sialic acid of human ceruloplasmin. Two values for Km using either p-phenylenediamine (0.64 and 0.053 mM) or o-dianisidine (0.76 and 0.15 mM) were evaluated from Lineweaver-Burk plots. EPR studies on reactions with water radiolysis products at cryogenic temperatures allowed us to discover that goose ceruloplasmin, like human and bovine ceruloplasmins, possesses superoxide dismutase activity.

Binding of fibrinogen molecules to pig platelets and their membranes

Following addition of ADP, 125I-labelled fibrinogen binds specifically to pig platelets. This binding is completely inhibited by the unlabelled fibrinogen. Quantitative analysis indicates the presence of 12,400-25,000 molecules of fibrinogen which can be bound with an association constant of 5 . 10(8) M-1 to platelets. Fibrinogen receptors were found to be active in the isolated platelet membranes as well. Quantitative analysis of the saturable binding of fibrinogen to the platelet membranes showed that these receptors react with the same affinity with fibrinogen molecules. In contrast to the intact platelets, the platelet membranes can specifically bind fibrinogen in the absence of ADP. We conclude that a specific receptor for fibrinogen is exposed on the surface as a result of cell damage which is the first step of the platelet membrane isolation.

Phosphorus-rich Aα polypeptide chains of pig and sheep fibrinogen

Abstract Pig and sheep fibrinogen isolated from citrated plasma were sulphitolysed and afterwards separated by column chromatography into polypeptide chains Aα, Bβ and γ. By means of quantitative analysis and chromatography studies it was established that the whole phosphorus of pig and sheep fibrinogens was present in the Aα chains and existed there as a phosphoserine (sheep fibrinogen) and phosphoserine as well as phosphothreonine (pig fibrinogen).

Comparative studies of fibrin polypeptide composition of two avian families: Sylvidae and Hirundinidae

Abstract The polypeptide subunits of crosslinked and noncrosslinked fibrin of two bird families /Sylvidae, Hirundinidae/ of the same order /Passeriformes/ were examined by polyacrylamide gel electrophoresis. It was found that α, β and γ polypeptide chains were usually visible on gel as single bands if fibrin was obtained from plasma of young birds. Fibrin polypeptide subunits of all Acrocephalus species had almost the same molecular weight: α 57000–57900, β 52200–56000 and γ 46200–50400 daltons. The corresponding values of Hirundinidae were 61500–61700, 56500–58200 and 50000–51000 daltons, respectively. The crosslinked fibrin apart from single polypeptide chains would always give additional band on polyacrylamide gel electrophoresis patterns /γ-γ dimer/ in the range of molecular weight 86100–91900 daltons. Our investigations indicated that polypeptide subunit structure of fibrin of the examined avian groups was similar to mammalian ones i.e. fibrin was composed of α, β and γ polypeptide chains.

The presence of phosphoserine and phosphothreonine in tryptic digest degradation products of pig fibrinogen.

Abstract Pig fibrinogen isolated from citrated plasma was subjected to the action of trypsin. Phosphoserine and phosphothreonine were isolated from the hydrolyzate by column ion-exchange chromatography and low voltage electrophoresis chromatography.

Comparative effects of selenite and selenite on the glutathione-related enzymes activity in pig blood platelets

The effects of inorganic selenium (Se) compounds (sodium selenite and selenate) on the activities of glutathione-related enzymes (glutathione peroxidase, glutathione-S-transferase [GST] and glutathione reductase [GR]) in pig blood platelets were investigated in vitro. GST activity in blood platelets treated with 10−4M of selenite was reduced to 50%, whereas no decrease GST activity was observed after the treatment of platelets with the same dose of selenate. In platelets incubated with physiological doses (10−7, and 10−6M) of Se compounds, the activity of glutathione peroxidase (GSH-Px) was enhanced (about 20%). GR activity after the exposure of platelets to tested Se compounds was unaffected.

Effects of gamma radiation on pig platelet function

The effects of gamma radiation (60Co source) on pig platelet aggregation, protein secretion, and malonyldialdehyde (MDA) formation were studied. It was observed that gamma radiation (0.1-1 kGy ) modified platelet function, i.e., inhibited platelet aggregation and secretion with concomitant lipid peroxidation expressed by increased amounts of malonyldialdehyde. MDA production in irradiated platelets was dependent on the dose of gamma radiation.

Chemical structure and properties of duck and goose fibrinogen.

Duck and goose fibrinogen were isolated from fresh pooled plasma by three different methods. To minimize proteolytic activity, epsilon-aminocaproic acid and trasylol were used throughout the preparation procedures. Amino acid composition of fibrinogens and carbohydrate content (hexose, hexosamine, sialic acid) as well as phosphorus were analysed. Intact preparations showed single band on SDS-polyacrylamide gel electrophoresis. After reduction and modification of the thiol groups, the material could be separated by SDS-polyacrylamide gel electrophoresis into four bands corresponding to the gamma, partially degraded A alpha, B beta and intact A alpha chain. Intact polypeptide subunits were separated by ion-exchange chromatography or preparative SDS-polyacrylamide gel electrophoresis and their amino acid compositions were determined. Evidences supporting the view that bird fibrinogen is very sensitive to proteolytic degradation and that a partial degradation of the A alpha chain takes place even when inhibitors are used in all steps of the purification procedures are presented.

Homology of polymerization domains in vertebrate fibrinogens

Abstract Pig fibrinogen and fibrin monomers were coupled to CNBr-activated Sepharose 4B and used to test the homology of polymerization sites of some vertebrate fibrinogens. It was found that fibrinogens and fibrin monomers of analysed mammalia, aves, amphibia and pisces bound specifically to pig fibrin and fibrinogen, respectively. This finding suggests that, despite structural evolution of vertebrate fibrinogen molecules, regions providing polymerization sites remain unchanged.