RESUMO
Human skin fibroblasts and bone marrow cells were tested for their ability to synthesize the cobalamin-binding protein transcobalamin II. Cobalamin binders secreted in the media of cultured fibroblasts and of dextran-sedimented bone marrow cells in liquid culture could be identified as transcobalamin II on the basis of immunological, electrophoretical and chromatographical identity with serum transcobalamin II. The net secretion of transcobalamin II increased linearly with time of culture, up to 30 days after confluence. The reversible inhibition of transcobalamin II secretion by cycloheximide demonstrated that human fibroblasts are capable of de novo transcobalamin II synthesis. Addition of cyanocobalamin to the fibroblast culture medium induced a reduction of transcobalamin II net secretion, most likely due to preferred uptake of transcobalamin II saturated with cobalamin, as opposed to unsaturated protein. Addition of lysozymal enzyme inhibitors, ammonium chloride and chloroquine, resulted in a markedly increased secretion of transcobalamin II. In the culture medium of fibroblasts, obtained from two transcobalamin II-deficient patients, functionally deficient transcobalamin II was demonstrated on the basis of strongly reduced secretion of immunoreactive transcobalamin II, and the absence of apotranscobalamin II. Individual phenotypes in the culture media of the fibroblasts and bone marrow cells were identical to the corresponding serum transcobalamin II types.
Assuntos
Medula Óssea/metabolismo , Pele/metabolismo , Transcobalaminas/biossíntese , Células Cultivadas , Fibroblastos/metabolismo , Humanos , Transcobalaminas/deficiência , Transcobalaminas/metabolismoRESUMO
Transcobalamin II ( TC2 ) phenotyping was performed in Nigerian black males from several ethnic groups, using polyacrylamide gel electrophoresis in combination with autoradiography. The TC2 gene frequencies in the total Nigerian sample amounted to .064 (X), .124 (S), .498 (M), and .313 (F). Compared to white populations, the frequency of TC2 *X is low while the frequencies of TC2 *S and TC2 *F are high, which confirms previous studies on blacks. In some of the rare phenotypes we found, in addition to common TC2 variants, TC2 gene products with an electrophoretic mobility intermediary to X and S, and to M and F, indicative of further microheterogeneity in the genetic system of TC2 . The TC2 gene frequency data indicate interpopulation heterogeneity for this marker. Major differences were found between the Sudan and Guinea Savanna Group ( SGSG ) and the Middle Belt Group, and between the SGSG and the Ibos . The results of our comparative study confirm to some extent similar surveys, which have been based on the distribution of the ABO blood groups.
Assuntos
População Negra , Etnicidade , Variação Genética , Transcobalaminas/genética , Autorradiografia , Eletroforese em Gel de Poliacrilamida , Frequência do Gene , Humanos , Masculino , Nigéria , FenótipoRESUMO
Electrophoresis and subsequent autoradiography of 57Co-cobalamin (57 Co-Cbl)-labeled serum show intensity differences between the genetic variants of human transcobalamin II (TC2), suggesting differences in the unsaturated (apo-) TC2 concentration. In order to distinguish between variant-specific differences in the Cbl binding affinity and those in the total-TC2 concentration, techniques were developed to determine total, apo-, and holo-TC2. Prolonged incubation at 37 degrees C with a 20-fold excess of 57Co-Cbl resulted in an almost complete exchange of endogenously bound Cbl, which allowed determination of the total TC2. The holo-TC2 concentration of both gene products in TC2 heterozygotes could be estimated by comparison of the labeling levels of apo- and total TC2, using densitometric quantification of the autoradiographs. By means of ion-exchange chromatography, TC2 could be separated from other Cbl-binding proteins, permitting a simple quantitative assay of apo- and total TC2, the results of which correlate fairly well with those measured by an immunoadsorption assay. The results obtained in the present investigation indicate that the variant-specific variation in the apo-TC2 concentration is caused by differences in the total-TC2 concentration rather than in the Cbl binding affinity.
Assuntos
Transcobalaminas/metabolismo , Vitamina B 12/metabolismo , Apoproteínas/metabolismo , Eletroforese , Humanos , Neuraminidase/metabolismo , Polimorfismo Genético , Ligação Proteica , Transcobalaminas/genéticaRESUMO
Phenotypes of transcobalamin II (TC2) were determined in 95 maternal-cord serum pairs in order to identify the origin of TC2 in human cord blood. Unsaturated (apo) TC2 in serum was labeled with radioactive (57Co) cobalamin (CbI) and separated into isoproteins by polyacrylamide gel electrophoresis and autoradiography. Discordancy between the maternal and the cord serum type was observed in 45% of the pairs. The results demonstrated that, at the end of pregnancy, the fetus is capable of TC2 synthesis and that there is no detectable transplacental passage of maternal apo-TC2. Presence of maternal saturated (holo) TC2 in cord serum could be excluded in 9 informative discordant pairs by exchanging endogenously bound CbI with 57Co-CbI. Our finding that TC2 in human cord serum is of fetal rather than maternal origin suggests an essential role for fetal TC2 in CbI utilization and appears to contradict the hypothesis that transplacental passage of maternal TC2 may explain the normal fetal development in cases of congenital TC2 deficiency. The total immunoreactive TC2 content in 23 maternal serum samples collected at the end of pregnancy (812 +/- 175 pM CbI equivalent) was significantly higher than in the corresponding cord sera (605 +/- 148 pM; p less than 0.001) and did not significantly differ from the value in a control group of healthy male and female adults (841 +/- 192 pM). At the end of pregnancy, the apo-TC2 content in 12 maternal serum samples (760 +/- 347 pM) was significantly higher than in the corresponding cord sera (501 +/- 254 pM; p less than 0.05) and did not significantly differ from the value in the control group (747 +/- 137 pM).
Assuntos
Proteínas Sanguíneas/genética , Transcobalaminas/genética , Cromatografia em Gel , Feminino , Sangue Fetal , Humanos , Recém-Nascido , Troca Materno-Fetal , Fenótipo , GravidezRESUMO
Somatic cell hybrids, recombinant inbred (RI) mouse strains, and backcross breeding experiments were used to locate the gene of transcobalamin II (Tcn-2), the vitamin B12 binding protein in mouse serum. TCN-2 was found to be useful genetic marker in the somatic cell hybrids. Selected hybrid clones were derived from fusions between GR mouse cells and the Chinese hamster cell line E36. Analysis of mouse specific chromosomal enzyme markers in relationship to TCN-2 secretion, in the hybrid clones, provided provisional evidence for assignment of the Tcn-2 locus to chromosome 11. The strain distribution pattern of the TCN-2 variants S and F in the RI series CXS, constructed from the cross of BALB/cHeA (TCN-2S) with STS/A (TCN-2F), implied a close linkage with the hemoglobin alpha-chain locus (Hba) on chromosome 11. Backcross breeding using inbred strains confirmed these findings and located the Tcn-2 gene closest to the centromere, linked with waved 2 (wa-2) and Hba with recombination frequencies of 6.9 and 19.2% each. The linkage group Tcn-2/wa-2/Hba was established.
Assuntos
Mapeamento Cromossômico , Ligação Genética , Marcadores Genéticos , Hemoglobinas/genética , Transcobalaminas/genética , Animais , Linhagem Celular , Cricetinae , Cricetulus , Cruzamentos Genéticos , Globinas/genética , Células Híbridas , Camundongos , Camundongos Endogâmicos , Fenótipo , Recombinação GenéticaRESUMO
Human transcobalamin II (TC2), a vitamin B12 binding serum protein, is synthesized and secreted into the medium by cells growing in vitro. Mouse-man somatic cell hybrids were analyzed in order to map the locus of TC2. The presence of human TC2 in the culture media was correlated with the results of genetic marker and chromosome analysis of the hybrid cells. Chromosome 22 showed 100% concordancy. However, chromosome 6 (90% concordancy) and chromosome 7 (96% concordancy) were not completely excluded. Meningioma cells obtained from patients heterozygous for TC2 showed a concomitant loss of one chromosome 22 and one of the TC2 alleles, strongly supporting the assignment to chromosome 22.