Studies on the in vivo elution of 51Cr from baboon red blood cells.

Four anticoagulant solutions were added to baboon red blood cells prior to labeling with 51Cr to determine how each would influence the distribution of 51Cr within the red blood cells, the loss of 51Cr from the red blood cells after transfusion, and the calculated red cell survival value. The 51Cr label was detected in the hemoglobin and in the low molecular weight compounds within the red blood cells. The elution of 51Cr from labeled baboon red blood cells following transfusion could not be explained by the distribution of 51Cr between hemoglobin and low molecular weight compounds within the red blood cells.

In vitro interactions of 51Cr in human red blood cells and hemolysates.

The intracellular distribution of radioactivity was studied in normal and sickle erythrocytes labeled with sodium 51Cr chromate. Both types of cells had a higher fraction of 51Cr bound to hemoglobin when labeled in the presence of ACD at a pH of 7.09 than when labeled in the presence of CPD at a pH of 5.96. Citrate at a pH of 5.96 or less entered the red blood cells and decreased the 51Cr binding hemoglobin. Binding of 51Cr to hemoglobin within the red cells was also reduced when the ATP and DPG levels in the red blood cells were elevated. Studies with hemoglobin solution showed that 51Cr binding to hemoglobin was influenced by 2,3-DPG, ATP and citrate.

Tryptophanyl transfer ribonucleic acid synthetase of Escherichia coli. Character of required thiol group and structure of thiol peptides.

Native tryptophanyl-tRNA synthetase purified from Escherichia coli B has on each identical subunit a single thiol group which rapidly forms a mixed disulfide with a thionitrobenzoate moiety of 5,5'-dithiobis(2-nitrobenzoic acid). The reaction and the concomitant inactivation of the enzyme are both reversible by reductive removal of the thionitrobenzoate with dithiothreitol. Iodoacetamide and N-ethylmaleimide also react with the thiol group required for enzyme activity, but iodoacetic acid inactivates the enzyme through another mechanism. Three or 4 half-cystine residues/subunit were detected by amino acid analysis and by titration of the denatured enzyme with 5,5'-dithiobis(2-nitrobenzoic acid); no disulfide bonds were detected by borohydride reduction. Cleavage of the subunit (molecular weight 37,000) with 2-nitro-5-thiocyanobenzoic acid gave fragments of molecular weights 32,000, 27,000, and 9,500. Five carboxymethylated peptides were isolated from the trypsin products of the denatured enzyme after treatment with iodo[14C]lacetate. Three of these peptides represented unique sequences surrounding thiol groups in the enzyme. One cysteine-containing nonapeptide has a heptapeptide sequence homologous to a heptapeptide sequence in a cysteine containing decapeptide from the tryptophanyl-tRNA synthetase of human placenta. The nonapeptide appears to bear the thiol group required for enzyme activity.