Transport of Vitamin B 12 in Escherichia coli

Abstract

The uptake of 60 Co-labeled cyanocobalamin (vitamin B 12 ) 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 60 Co-B 12 uptake after incubation times of 1 to 2 sec were used in studies on the initial rate of B 12 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 HgCl 2 (50% at 0.1 m m ) but not by 1 m m N -ethylmaleimide or 1 m m p -chloromercuribenzoate was consistent with a role for a relatively inaccessible sulfhydryl residue at the initial B 12 binding site. The secondary phase of B 12 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 m m ), arsenite (10 m m ), and 2, 4-dinitrophenol (0.1 m m ) 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 B 12 transport. The cells were also able to take up 60 Co-labeled cobinamide cyanide.