A. Stratil

Partial characterization of transferrins of some species of the family Cyprinidae

1. Transferrins of five cyprinid fishes (Tinca tinca, Ctenopharyngodon idella, Chondrostoma nasus. Hypophthalmichthys molitrix and Aristichthys nobilis) were isolated in a pure state using Sephadex G-100, DEAE-Sephadex and SP-Sephadex. 2. All the transferrins are heterogeneous. In H. molitrix and A. nobilis the heterogeneity is caused partly, but not exclusively, by the presence of sialic acid; in the other species it is independent of sialic acid. 3. Molecular weights ranging from 69,900 (T. tinca) to 78,800 (H. molitrix) were obtained by sedimentation equilibrium ultracentrifugation. SDS-polyacrylamide gel electrophoresis with beta-mercaptoethanol indicated that the transferrins are composed of a single polypeptide chain. 4. Amino acid compositions of all species were similar, the greatest similarity being in the transferrins of H. molitrix and A. nobilis. 5. Carbohydrate was not found in transferrins of T. tinca and C. idella, but was present in A. nobilis (2.63%) and H. molitrix (5.01%). 6. Alanine was N-terminal in C. idella. No N-terminal amino acid was found in T. tinca, H. molitrix and A. nobilis.

Phylogenetically more conservative epitopes among monoclonal antibody-defined antigenic sites of human transferrin are involved in receptor binding.

Summary Of eight monoclonal antibodies raised against human transferrin, one (H.TF‐14) cross reacted with pig and rabbit transferrins and one (H.TF‐1) showed cross‐reactivity with horse and dog transferrins. While rabbit and pig transferrins exhibited the same patterns of binding to MOLT‐3 cell receptors as human and horse transferrins, binding of mouse and dog transferrins was weaker and bovine and carp transferrins gave entirely negative results. The results of these competitive binding experiments were confirmed by a biological test in which bovine transferrin had no effect on the growth of MOLT‐3 cells when added to a serum‐free medium. The observed correlation between cross‐reactivity of anti‐transferrin monoclonal antibodies and the binding abilities of transferrins to the MOLT‐3 cell receptors may be associated with the conservatism of the part of the transferrin molecule recognized by the cell receptor.

Comparison of biochemical polymorphisms in mouflon and sheep: Isoelectric differences in haemoglobins and quantitative variation of mouflon haemopexin

1. Of ten protein systems studied in mouflon (Ovis musimon), five were polymorphic (Tf, Hpx, EsA, X-protein, Cat). Electrophoretic mobilities of mouflon proteins did not differ from those of sheep. 2. Mouflon haemoglobin B and sheep haemoglobin B differed in isoelectric focusing. 3. Haemopexin levels in mouflon were determined by rocket immunoelectrophoresis. A trimodal distribution was apparent, with no haemopexin, low and high levels of the protein. The results are indicative of genetic control of haemopexin levels, one of the alleles being inactive (Hpx0).

Evidence for the presence of α1 B-glycoprotein in mammalian sera: immunoblotting studies

1. 1. A monospecific antiserum to pig α1 B-glycoprotein (PO2) was produced in rabbits and was used to search for homologues of α1 B in sera of 41 mammalian species belonging to seven orders. 2. 2. Specific reactions were detected in the sera of representatives of Insectivora, Primates, Carnivora, Proboscidea, Perissodactyla and Artiodactyla. No cross-reactions were observed in the sera of two species of Rodentia (mouse, rat). 3. 3. Cross-reactions in the sera of Erinaceus europaeus, Homo sapiens and Macaca mulatta were rather weak; this indicates a greater structural difference between the α1 B of Insectivora and Primates and that of the other mammalian orders. 4. 4. Electrophoretic patterns of α1 B were, in most cases, heterogeneous, the most heterogeneous being in ruminants. 5. 5. Evidence was obtained that the α1 B of sheep is identical with the earlier described (Juneja and Gahne (1980) Anim. Blood Grps Biochem. Genet. 11, 81–92.) polymorphic post-transferrin (Ptf).

Serum proteins of rhinoceroses: inter- and intra-specific variation

Abstract 1. 1. Serum proteins of Ceratotherium simum cottoni Lydekker (C.s. cottoni), Diceros bicornis L. (D. bicornis) and Rhinoceros unicornis L. (R. unicornis) were studied by ID PAGE, 2D agarose-PAGE, immunoblotting and inhibitions of trypsin and chymotrypsin. 2. 2. In all species studied albumin, transferrin, α1β glycoprotein, vitamin D binding protein (GC), α2HS glycoprotein, haptoglobin, haemopexin, ceruloplasmin, esterase and protease inhibitors were found. 3. 3. ID PAGE and 2D agarose-PAGE patterns of serum proteins of rhinoceroses were found to be species-specific. 4. 4. In C. s. cottoni intra-specific variation was observed in vitamin D binding protein (GC), protease inhibitors AC and ATC2 and haptoglobin. Less well defined variation was also detected in protease inhibitor ATCl, a postalbumin (PSA) and an esterase (ES3).

Transferrins of Barbus barbus, barbus meridionalis petenyi and their hybrids. Genetic polymorphism, heterogeneity and partial characterization

1. 1. The transferrins of Barbus barbus and B. meridionalis petenyi are highly polymorphic. Thirteen variants were detected in B. barbus and 6 variants in B. meridionalis petenyi. The electrophoretic patterns of transferrin from B. meridionalis petenyi are distinct from those of B. barbus. 2. 2. The distribution of phenotypes indicates a simple genetic determination from one locus by a set of codominant alleles in each species. 3. 3. Transferrin variants of both species are heterogeneous. In B. barbus differences in heterogeneity occur that have no analogy in other vertebrates. If electrophoretic mobility is not taken into account, five patterns can be differentiated. The variants also differ in the response to neuraminidase treatment. 4. 4. Amino acid composition of transferrins of the two species are remarkably similar, and mol. wts are 74,000 and 76,000 for B. barbus and B. meridionalis petenyi, respectively. 5. 5. Assumed hybrids B. barbus × B. meridionalis petenyi have transferrin variants of both species.

A comparison of molecular weights of transferrins of various vertebrates

Abstract 1. 1. SDS-polyacrylamide gel electrophoresis has been used to study molecular weights of transferrins of 27 fish, 6 avian and 14 mammalian species. 2. 2. Mol. wts of fish transferrins ranged from 61,000 to 87,000. 3. 3. The values for avian transferrins were in the range from 70,000 to 72,000. 4. 4. For mammalian transferrins mol. wts were estimated to range from 69,000 to 77,000.

Partial characterization of transferrins of catfish (Silurus glanis L.) and pike (Esox lucius L.).

Basic composition and properties of isolated transferrins of Silurus glanis and Esox lucius have been compared. In transferrin of S. glanis carbohydrate is absent, but it is present in transferrin of E. lucius (2.5%). The N-terminal amino acid is alanine in both species. Mol. wts are 68,400 (S. glanis) and 86,800 (E. lucius). Transferrins of the two species are heterogeneous, but genetic polymorphism was not observed.

Inter- and intra-specific differences in serum proteins of different species and subspecies of zebras

1. Serum proteins of Equus grevyi, E. zebra hartmannae, E. burchelli boehmi, E. b. chapmanni and E. b. antiquorum were studied using starch-gel electrophoresis, 1-D polyacrylamide-gel electrophoresis, inhibitions of trypsin and chymotrypsin, immunoblotting, and specific staining for esterase. 2. Clear species-specific patterns were observed in albumin, transferrin, and for E. grevyi in protease inhibitor-1. Specific esterase was detected only in E. z. hartmannae. 3. Protein polymorphism was found in all studied species: E. grevyi--transferrin; E. z. hartmannae--protease inhibitor-1; E. b. boehmi--albumin, GC, transferrin, protease inhibitor-1, protease inhibitor-T; E. b. chapmanni--albumin, GC, transferrin, protease inhibitor-1; E. b. antiquorum--GC, transferrin, protease inhibitor-1. 4. Phenotype patterns of the polymorphic proteins were indicative of simple codominant inheritance. Further studies of polymorphism of protease inhibitor-2 and variability of protease inhibitor-X are needed. 5. alpha 1B glycoprotein in all zebra species was monomorphic. 6. The main transferrin components and alpha 1B glycoprotein of zebra (E. b. boehmi) were characterized for terminal sialic acid content.

Immunological Comparison of Transferrins of Some European Cypirinid Fish

Abstract Double immunodiffusion in agar gel with antiserum to carp transferrin was used to prove immunological relatedness between the transferrins of some European cyprinid fish. Quantitative differences between transferrins of nine species of cyprinid fish were studied by micro-complement fixation with the use of entisera to transferrins of four species. Immunological distances were conspicuously great and amounted on average to about 125 immunological distance units (IDU). A high rate of evolution of transferrins wes obtained. namely 4.2 IDU/million years, which represents about 0.84% sequence differences per million years.