Isotopic studies of the conversion of oxophlorins and their ferrihaems into bile pigments in the rat

1. Tritiated oxymesoporphyrins and their ferrihaems were tested as possible intermediates in the catabolism of haemoglobin. The tritiated compounds were injected into rats with biliary fistulae and the incorporation of the isotope into bile, bile pigment, urine, faeces, liver, kidney and spleen was measured. 2. alpha-Oxymesoferrihaem was extensively converted into bile pigment and specifically to the expected mesobilirubin. 3. beta-Oxymesoferrihaem was poorly converted into bile pigment and was not converted into mesobiliverdin IXbeta. The latter was independently shown to be excreted rapidly in bile. 4. The free oxyporphyrins were also poor precursors of bile pigment, and alpha-oxymesoporphyrin competed with bilirubin for excretion by the liver. 5. By analogy with the results obtained with alpha-oxymesoferrihaem it is concluded that alpha-oxyprotoferrihaem is an intermediate in the catabolism of haemoglobin, undergoing further oxidation to bile pigment under the catalysis of an enzyme of definite specificity.     

Role of amino acid residue 90 in bioactivity and receptor binding capacity of tumor necrosis factor mutants

We have previously produced two bioactive lysine-deficient mutants of TNF-alpha (mutTNF-K90R,-K90P) and found that these mutants have bioactivity superior to wild-type TNF (wtTNF). Because these mutants contained same amino acid except for amino acid 90, it is unclear which amino acid residue is optimal for showing bioactivity. We speculated that this amino acid position was exchangeable, and this amino acid substitution enabled the creation of lysine-deficient mutants with enhanced bioactivity. Therefore, we produced mutTNF-K90R variants (mutTNF-R90X), in which R90 was replaced with other amino acids, to assay their bioactivities and investigated the importance of amino acid position 90. As a result, mutTNF-R90X that replaced R90 with lysine, arginine and proline were bioactive, while other mutants were not bioactive. Moreover, these three mutants showed bioactivity as good as or better than wtTNF. R90 replaced with lysine or arginine had especially superior binding affinities. These results suggest that the amino acid position 90 in TNF-alpha is important for TNF-alpha bioactivity and could be altered to improve its bioactivity to generate a "super-agonist".