Cyanocobalamin (vitamin B12) uptake by Ochromonas malhamensis

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Abstract
Uptake of cyanocobalamin-Co60 was studied in Ochromonas malhamensis, a phytoflagellate requiring the vitamin for growth, at 25 C in washed cells suspended in iNaCl, O.I [image]; OH-butyrate buffer, pH 5.0, .010 [image]; NaCN, 7 x 10-5 [image]; vitamin B12, 30 m[mu]g/ml. Uptake occurred in a rapid (1 min) phase of .65 [mu]g/ml packed cells and a slower phase reaching a steady state of 2-3 [mu]g/ml in 4 hr. Both phases showed saturation kinetics (half max. concn for rapid phase,< 5 m[mu]g/ml; slow phase, 7.1 m[mu]g/ml); only slower phase was sensitive to metabolic inhibitors. B12 uptake was pH dependent, falling off rapidly above pH 6.5; at higher pH, calcium ion (half max. concn 2 x 10-5 [image]) completely restored uptake. Subcellular localization of Co60-labeled B12 on sonicated cells showed 55% in the 30,000-g pellet and 24% in the 195,000-g pellet; only 12% was found in the 195,000-g supernatant material. Findings suggest that B12 entered cytoplasm by carrier mechanism cr pinocytosis against little or no concentration gradient and that binding or concentration by intracellular organelles accounted for large observed uptakes.

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