|
pubmed:abstractText |
We have previously identified free ADP-ribose (ADPR) as a normal metabolite in mature human erythrocytes. In this study the metabolic transformations of ADPR were investigated in both supernatants from erythrocyte lysates and intact erythrocytes, loaded with ADPR by means of a procedure involving hypotonic haemolysis and isotonic resealing. In both experimental systems, the main pathway was a dinucleotide pyrophosphatase-catalysed hydrolysis to yield AMP, which was readily converted into the adenylic and inosinic nucleotide pools. To a lesser extent, ADPR underwent conversion into a compound that was identified as ADP-ribulose (ADPRu), on the basis of m.s., n.m.r. spectroscopy and enzymic analysis. ADPRu was also susceptible to degradation by the dinucleotide pyrophosphatase, which was partially purified from erythrocyte lysates and characterized with respect to its substrate specificity. Isomerization of ADPR to ADPRu was markedly enhanced by ATP. Incubation of unsealed haemoglobin-free erythrocyte membranes with labelled ADPR did not cause any transformation of this nucleotide and resulted in its trichloroacetic acid- and formic acid-resistant binding to a number of membrane cytoskeletal proteins. These proteins include spectrin, glyceraldehyde 3-phosphate dehydrogenase (Ga3PDH), three proteins of molecular masses 98, 79 and 72 kDa, which apparently comigrate with bands 3, 4.1 and 4.2 respectively, and two additional proteins of molecular masses 58 and 41 kDa. Acid-resistant binding of ADPR, as well as of NAD+, to Ga3PDH was confirmed for the enzyme purified from human erythrocytes.
|
