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Transport of Vitamin B₁₂ in Escherichia coli¹

^a Department of Biochemistry, University of Virginia School of Medicine, Charlottesville, Virginia 22901

ABSTRACT

The uptake of ⁶⁰Co-labeled cyanocobalamin (vitamin B₁₂) by cells of Escherichia coli K-12 was shown to consist of an initial rapid phase (complete in <1 min), followed by a slower secondary phase. Methods enabling the measurement of ⁶⁰Co-B₁₂ uptake after incubation times of 1 to 2 sec were used in studies on the initial rate of B₁₂ uptake. This initial process showed saturation kinetics, with a V_max of 56 molecules per sec per cell and a K_m of 5 nm, and was essentially independent of cellular energy metabolism. No inhibition was obtained with cyanide, fluoride, arsenite, or 2, 4-dinitrophenol, and an energy of activation of 3.8 kcal/mole for this initial phase of uptake was calculated from its response to temperature changes between 15 and 35 C. The inhibition by HgCl₂ (50% at 0.1 mM) but not by 1 mMN-ethylmaleimide or 1 mMp-chloromercuribenzoate was consistent with a role for a relatively inaccessible sulfhydryl residue at the initial B₁₂ binding site. The secondary phase of B₁₂ uptake was clearly dependent on the energy metabolism of the cell, and, from its response to temperature, an energy of activation of about 17 kcal/mole was calculated. Cyanide (10 mM), arsenite (10 mM), and 2, 4-dinitrophenol (0.1 mM) gave at least 70% inhibition of the rate of the secondary phase. The formation of other cobalamins, such as 5'-deoxyadenosyl cobalamin, was not an obligate part of B₁₂ transport. The cells were also able to take up ⁶⁰Co-labeled cobinamide cyanide.

¹ A preliminary report of this work was presented at the 70th Annual Meeting of the American Society for Microbiology in Boston, Mass., 26 April-1 May 1970.

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