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SMALL-SCALE ISOLATION OF FERRITIN FOR THE ASSAY OF THE INCORPORATION OF 14C-LABELLED AMINO ACIDS.

ABSTRACT: 1. A new procedure is described for the isolation of pure ferritin from small amounts of tissue. After the removal of most of the tissue proteins by heat coagulation, the ferritin fraction was chromatographed successively on CM-cellulose and Sephadex G-200. 2. The isolated ferritin appeared to be free from other proteins, as judged by its sedimentation pattern in the ultracentrifuge, by electrophoresis on polyacrylamide gel and by immunoelectrophoresis. 3. After the injection of [(14)C]leucine into a series of rats, the specific activity of liver ferritin isolated by the new procedure bore a constant relationship to that of liver ferritin separated by antigen-antibody precipitation. The procedure can thus be used to obtain ferritin of suitable purity for studies of amino acid incorporation. 4. The new procedure can be used to measure the total ferritin protein content of a tissue. It is not possible to use ultraviolet absorption for the measurement of ferritin protein because of considerable interference from the iron that it contains.

SUBMITTER: DRYSDALE JW

PROVIDER: S-EPMC1206816 | biostudies-other | 1965 Jun

REPOSITORIES: biostudies-other

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Project description:In confirmation of the findings of Gaitonde et al. (1974), a decrease in the brain concentration of threonine and serine, and an increase in glycine, were observed in rats maintained on a thiamin-deficient diet. Similar changes were found in the blood, and the concentration of several other amino acids in the blood decreased significantly. There was a correlation between the concentrations of threonine, serine, aspartate and asparagine in the brain and blood. In experiments in which [U-14C]threonine was injected into rats most of the radioactivity in the brain and blood of control rats was, as expected, in threonine in the acid soluble metabolites. In contrast, a considerable proportion of radioactivity was also found in other amino acids, namely glutamate, glutamine, aspartate, gamma-aminobutyrate and alanine, in the brain of thiamin-deficient rats. [U-14C]Threonine was also converted into 14C-labelled lactate and glucose, but the extent of this conversion was severalfold higher in thiamin-deficient than in control rats. This finding gave evidence of the stimulation in thiamin-deficient rats of the catabolism of [U-14C]threonine to [14C]lactate by the aminoacetone pathway catalysed by threonine dehydrogenase, and into succinate via propionate by the alpha-oxobutyrate pathway catalysed by threonine dehydratase (deaminase). The measurement of specific radioactivities of glutamate, aspartate and glutamine after injection of [U-14C]threonine, indicated a stimulation of the activities of threonine dehydrogenase and threonine dehydratase (deaminase) in the brain of thiamin-deficient rats. The specific radioactivities of glutamate, asparatate and glutamine int he brain were consistent with an alteration in the metabolism of threonine, mainly in the 'large' compartment of the brain of thiamin-deficient rats. The measurement of relative specific radioactivity of proteins after injection of [U-14C]threonine indicated a marked decrease in the synthesis of proteins, mainly in the liver of thiamin-deficient rats.

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SMALL-SCALE ISOLATION OF FERRITIN FOR THE ASSAY OF THE INCORPORATION OF 14C-LABELLED AMINO ACIDS.

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