Influence of zinc ions addition to different lots of 2-amino-2-methyl-1-propanol (AMP) buffer on the alkaline phosphatase activities.

The influence of Zn2+ on the inactivation of alkaline phosphatase [orthophosphoric monoester phosphohydrolase (alkaline optimum), EC 3.1 3.1] in serum during preincubation with 2-amino-2-methyl-1-propanol (AMP) buffers was investigated. Addition of Zn2+ to the buffer before preincubation increases the enzyme activity. An optimum Zn2+ concentration different for each lot of AMP buffer can be found, at which the enzyme activities are restored to a level equal to activities measured without preincubation. There is a relation between the inactivating properties of the different AMP buffers and the amount of Zn2+ needed to prevent this inactivation. Since Zn2+ chelating substituted diamines are held responsible for the inactivation by removing Zn2+ from the enzyme, we assume that the addition of Zn2+ to the buffer prevents this removal. As Zn2+ itself is an inhibitor of the enzyme, the addition of both too much or too little Zn2+ results in lower enzyme activities after preincubation with AMP buffer.

Functional heterogeneity of transferrin-bound iron: iron uptake by cell suspensions from bone marrow and liver and by cell cultures of fibroblasts and lymphoblasts.

According to the hypothesis of Fletcher and Huehns, functional differences exist between both iron-binding sites of transferrin. The site designated A should mainly be involved in the delivery of iron to erythroid cells, whereas site B should donate its iron preferentially to cells involved in the absorption and storage of iron. In the present study this hypothesis could be confirmed by in vitro experiments with various cell types. Iron transferrin preincubated with rat bone marrow cells donates less iron to rat bone marrow cells, Chinese hamster fibroblasts, human fibroblasts and human lymphoblasts than freshly prepared iron transferrin equal in iron and transferrin concentraion. Rat liver parenchymal cells, however, take up more iron from preincubated than from freshly prepared iron transferrin. Obviously, site A not only donates iron preferentially to erythroid cells but also to (rapidly) dividing nonerythroid cells in culture. From experiments with iron transferrin mixtures in which radioiron was present at low or high iron saturation, it could be concluded that rat bone marrow cells take up iron equally well from monoferric as from diferric transferrin. The observed functional heterogeneity could, therefore, not be ascribed to differences between monoferric and diferric transferrin.

Heme synthetase activity in normal human and rat erythroid cells and in sideroblastic anemia.

The enzyme heme synthetase, involved in the final step of the biosynthesis of heme, has been assessed in rat and human bone marrow and peripheral blood. The pH optimum of the enzyme in bone marrow appeared to be pH 7.6, whereas the Michaelis constant in human and rat bone marrow was found to be 1.6 micrometer and 0.6 micrometer, respectively. Rat reticulocytes showed approximately 100-times higher heme synthetase activities than did rat erythrocytes. By contrast, human reticulocytes did not show significantly higher activities than human erythrocytes. This difference between rat and human reticulocytes could be confirmed by in vitro experiments with intact cells in which iron uptake and heme synthesis of human and rat cells were compared. Finally, heme synthetase activity was assessed in bone marrow cells of two patients with sideroblastic anemia. In both cases the enzyme activities were found to be comparable to those in control bone marrow.