Transport of Vitamin B12 in Escherichia coli1

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RESUMO

The uptake of 60Co-labeled cyanocobalamin (vitamin B12) by cells of Escherichia coli K-12λ 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 Vmax of 56 molecules per sec per cell and a Km 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.

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