Biochemical Genetics, Vol. 17, Nos. 7/8, 1979

Evidence for 15 Genetically Determined Electrophoretic Variants of Transcobalamin II in Rabbit Serum Piero Masina, 1 Luigi Ramunno, 1 and Domenico Iannelli 1 Received 5 Jan. 1979--Final 9 Feb. 1979

By starch gel electrophoresis and autoradiography two classes of vitamin Bj2 binding proteins were detected in rabbit serum. By analogy to the nomenclature used in man, the two classes of proteins were named "transcobalamin I" ( TCI) and "transcobalamin H" ( TCII). Fifteen TCH phenotypes were observed, and family data indicated that they are controlled by five allelic codominant genes. The possibility that the five genes arise from the action of at least two polymorphic and closely linked structural loci is discussed. KEY WORDS: rabbits; vitamin B12 binding proteins; transcobalamins; genetic variants; starch gel electrophoresis; autoradiography.

INTRODUCTION Vitamin B12 is present in nature in very small amounts, and many animals have developed complex mechanisms for its absorption, transport, and storage. The stomach synthesizes a glycoprotein, known as intrinsic factor, which mediates the absorption of vitamin B12 from the gastrointestinal tract. In plasma, serum, and other biological fluids, vitamin B~2 also binds to various proteins which facilitate its storage and transport. In human plasma two classes of vitamin B~2 binding proteins have been identified: transcobalamin I (TCI) and transcobalamin II (TCII), which on electrophoresis migrate in the e- and ]%globulin regions, respectively. TCI is probably involved in the storage of vitamin B12 and TCH in its transport (for This work was supported by a grant from the Consiglio Nazionale delle Ricerche (Contract No. 78.00223.80). I Institute of Animal Production, University of Naples, 80055 Portici, Italy. 757

0006-2928/79/0800-0757503.00/0© 1979PlenumPublishingCorporation

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review, see Glass, 1974). A third transcobalamin (TCIII), also present in human serum, migrates in the fl region and probably is an isoprotein of TCI (Stenman, 1974). In man, Daiger et al. (1978) have identified electrophoretic variants of TCII controlled by four alleles at an autosomic locus. This report describes the identification in rabbit serum of two major classes of proteins which bind vitamin B12 (TCI and TCII). The latter class (TCII) displays 15 phenotypes which appear to be under genetic control. Three of these phenotypes, the most common in our material, have been reported previously (Masina et al., 1978). MATERIALS AND METHODS Individual serum samples (30 #1) obtained from about 1700 rabbits (1300 White New Zealand, 50 Fawn Burgundy, and 350 crosses between these two breeds), varying in age from 1 month to 3 years, were incubated with 0.1 ng of vitamin B I2 [57Co] (specific activity 180 #Ci/#g, Radiochemical Centre, Amersham, England) at 37 C for 30 min. Starch gel electrophoresis and autoradiography were performed as described by Geldermann (1970) and Daiger et al. (1975), respectively. Ammonium sulfate separation was made as described by Daiger et al. (1978). RESULTS Rabbit serum samples incubated in vitro with [57Co] vitamin B12 and subsequently examined by electrophoresis and autoradiography showed two major classes of vitamin Bl2 binding proteins. By analogy to the nomenclature used in man, the one with mobility of a fl-globulin was named "transcobalamin II" (TCII); the other, with mobility of an a-globulin, was "transcobalamin I" (TCI). On ammonium sulfate separation, rabbit TCII and TCI were found, respectively, in the precipitate and in the supernatant, in analogy to that already observed for human TCII and TCI (Carmel, 1974). Under the conditions used in the present investigation, the serum of all the females close to parturition so far examined and the serum of a few individuals (about 20) of both sexes displayed the presence of TCI alone or of both TCI and TCII. All the remaining serum samples displayed only TCII (Fig. 1). None of the serum samples examined was shown to lack both TCI and TCII. No appreciable difference was noticed in the electrophoretic pattern of TCII between serum and plasma samples collected with EDTA. On the other hand, heparinized plasma samples gave unclear electrophoretic patterns and their phenotype could not be established. This phenomenon, reported also by Daiger et al. (1978), may be due to the combination of heparin with TCII (Cooper, 1970).

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Fig. 1. Autoradiographyof [57Co]vitamin BI2 binding proteins in rabbit scrum after separation by starch gel electrophoresis. 1, Serum sample showingTCI and TCII (AIC2phenotype);2, serum samplc showingTCI; 3, serum sampleshowingTCII (A1AIphenotype).

Fifteen different TCII phenotypes (shown diagrammatically in Fig. 2) were detected in the serum of the rabbits tested. Family data (Table I) indicated that they are controlled by five genes, named TCII A1, TCII A2, TCII ~1, TCII cl, and TCII c2. (For brevity, the prefix TCII will be omitted wherever such an omission does not cause ambiguity.) These genes, on transmission, behave like autosomal codominant alleles at one locus, but probably arise from the action of at least two polymorphic and closely linked loci (see Discussion). Phenotypes Al, A2, B1, Cl, and C2 correspond to the homozygous genotypes (A1/A1, Ae/A2, B~/B1, C1/CI, and C2/C2, respectively) and phenotypes

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Evidence for 15 genetically determined electrophoretic variants of transcobalamin II in rabbit serum.

Biochemical Genetics, Vol. 17, Nos. 7/8, 1979 Evidence for 15 Genetically Determined Electrophoretic Variants of Transcobalamin II in Rabbit Serum Pi...
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