Abstract
Lysosome-free preparations of rat liver mitochondria take up hydroxo[57Co]cobalamin by a process which is dependent on mitochondrial swelling, rather than on energy or ion fluxes. The uptake system is saturable and unidirectional, leading to inside/outside concentration ratios of 17. The process also exhibits specificity: cyano[57Co]cobalamin is taken up less rapidly and to a lesser extent than hydroxocobalamin; methylcobalamin and adenoslcobalamin inhibit hydroxocobalamin uptake markedly, while cyanocobalamin does not. The [57Co]cobalamin ([57Co]Cbl) taken up is bound to a mitochondrial protein whose apparent molecular weight is 120,000 by Sephadex G-150 chromatography. Double reciprocal plots of bound [57Co]Cbl versus medium [57Co]Cbl concentration yield estimates for bound Cblmax of 29 pmol/mg of protein and for Kd is 8.2 muM. We conclude that mitochondrial uptake of cobalamins occurs via the diffusion of free cobalamins into the mitochondria and their subsequent binding to a high affinity mitochondrial protein(s) which we propose to be the source of the unidirectional character, the saturability, and the specificity of the uptake system.
Highlights
Lysosome-free preparations of rat liver mitochondria take up hydroxo[57Co]cobalamin by a process which is dependent on mitochondrial swelling, rather than on energy or ion fluxes
This paper reports experiments which define the characteristics of the system by which intact rat liver mitochondria take up cobalamins, including the ion and respiratory state dependence, the cobalamin specificity, and the role of cobalaminprotein interaction
Mitochondrial Preparation-As a result of several recent studies [7, 22], it is apparent that lysosomes are involved in the initial uptake of cobalamins by tissues
Summary
Lysosome-free preparations of rat liver mitochondria take up hydroxo[57Co]cobalamin by a process which is dependent on mitochondrial swelling, rather than on energy or ion fluxes. The process exhibits specificity: cyano[“Co]cobalamin is taken up less rapidly and to a lesser extent than hydroxocobalamin; methylcobalamin and adenosylcobalamin inhibit hydroxocobalamin uptake markedly, while cyanocobalamin does not. Double reciprocal plots of bound [57Co]Cbl versus medium [57Co]Cbl concentration yield estimates for bound Cbl max of 29 pmol/mg of protein and for Kd of 0.75 FM for OH-Cbl. For CN-Cbl, bound Cbl,,, is uptake of cobalamins occurs via the diffusion of free cobalamins into the mitochondria and their subsequent binding to a high affinity mitochondrial. This paper reports experiments which define the characteristics of the system by which intact rat liver mitochondria take up cobalamins, including the ion and respiratory state dependence, the cobalamin specificity, and the role of cobalaminprotein interaction
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