Is the increased biotin uptake into proliferating PBMCs due to an increased number of biotin transporters on the cell surface or to an increased affinity of transporters for biotin? Kinetic data suggest that the increased uptake of biotin is caused by an increased number of transporters. The maximal transport rate (Fmax) of biotin uptake provides an estimate for the number of biotin transporters on the PBMC surface. Theoretically, increased synthesis of biotin transporters in proliferating PBMCs is paralleled by increased Vmax for biotin. Vmax of biotin uptake in proliferating PBMCs was approximately four times greater compared with non-proliferating PBMCs: 9.1 ± 6.6 versus 2.3 ± 1.6fmol/(106 cells X 30 min) (Zempleni and Mock, 1999c). Transporter affinity for biotin (as judged by the Michaelis—Menten constant) was not significantly different in proliferating and quiescent PBMCs. In summary, these findings suggest that the increased biotin uptake into proliferating PBMCs is mediated by increased synthesis of transporters rather than by increased affinity for substrate.
Analysis of SMVT gene expression as judged by mRNA levels is consistent with increased synthesis of SMVT in proliferating PBMCs. SMVT gene expression was determined by reverse transcriptase—polymerase chain reaction (RT—PCR) of total RNA isolated from quiescent and proliferating PBMCs; certain housekeeping genes were quantified for comparison (Zempleni et al., 2001). The concentration of RNA encoding SMVT in proliferating PBMCs was 10.6 ± 1.6 times the control value in quiescent cells (Fig. 5.6). The increased expression of SMVT in proliferating PBMCs exceeded the increased expression of the housekeeping genes encoding transferrin receptor (5.6 ± 6.4 times control), glyceraldehyde-3-phosphate dehydrogenase (1.0 ± 0.1 times control), histone H1.3 (1.8 ± 0.1 times control) and histone H4 (1.1 ± 0.1 times control); typical examples are depicted in Fig. 5.6.
Theoretically, the increased biotin uptake in proliferating PBMCs could also be caused (or supplemented) by increased synthesis of a transporter other than SMVT. However, the following lines of evidence suggest that proliferating and quiescent PBMCs express the same biotin transporter. (i) The transporter affinity for biotin (as judged by the Michaelis-Menten constant) is the same in proliferating and quiescent PBMCs (Zempleni and Mock, 1999c). (ii) The substrate specificity of the biotin transporter is the same in proliferating and quiescent PBMCs (Zempleni and Mock, 1999c). (iii) mRNA encoding SMVT has been identified in both proliferating and quiescent PBMCs (Zempleni et al, 2001).
Notwithstanding the important role ofSMVT in biotin transport, preliminary evidence has been provided that other transporters also might account for some biotin uptake into PBMCs. For example, certain organic acids such as hexanoate, pyruvate and acetate compete with biotin for cellular uptake (J. Zempleni, unpublished observation).
These organic acids are substrates for mono-carboxylate transporters in mammalian cells (Halestrap and Price, 1999). Moreover, two mono-carboxylate transporters (MCT4 and MCT7) from human blood cells have been cloned and sequenced (Halestrap and Price, 1999). In summary, these preliminary findings are consistent with the hypothesis that monocarboxylate transporters might account for some biotin transport in PBMCs.
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