Di Girolamo P M, Bradbeer C
J Bacteriol. 1971 Jun;106(3):745-50. doi: 10.1128/jb.106.3.745-750.1971.
The uptake of (60)Co-labeled cyanocobalamin (vitamin B(12)) by cells of Escherichia coli K-12lambda 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 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 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 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 B(12) transport. The cells were also able to take up (60)Co-labeled cobinamide cyanide.
研究表明,大肠杆菌K-12λ细胞对(60)Co标记的氰钴胺素(维生素B(12))的摄取包括一个初始快速阶段(在<1分钟内完成),随后是一个较慢的第二阶段。在研究B(12)摄取的初始速率时,使用了能够在孵育1至2秒后测量(60)Co-B(12)摄取的方法。这个初始过程呈现饱和动力学,每个细胞每秒的V(max)为56个分子,K(m)为5纳米,并且基本上与细胞能量代谢无关。氰化物、氟化物、亚砷酸盐或2,4-二硝基苯酚均未产生抑制作用,根据其在15至35℃之间对温度变化的响应,计算出该摄取初始阶段的活化能为3.8千卡/摩尔。HgCl(2)(0.1毫米时50%抑制)有抑制作用,而1毫米N-乙基马来酰亚胺或1毫米对氯汞苯甲酸则无抑制作用,这与初始B(12)结合位点处一个相对难以接近的巯基残基的作用一致。B(12)摄取的第二阶段明显依赖于细胞的能量代谢,根据其对温度的响应,计算出活化能约为17千卡/摩尔。氰化物(10毫米)、亚砷酸盐(10毫米)和2,4-二硝基苯酚(0.1毫米)对第二阶段的速率至少有70%的抑制作用。其他钴胺素的形成,如5'-脱氧腺苷钴胺素,不是B(12)运输的必然组成部分。这些细胞也能够摄取(60)Co标记的氰钴胺酰胺。