FREE ADP-RIBOSE IN HUMAN ERYTHROCYTES - PATHWAYS OF INTRAERYTHROCYTICCONVERSION AND NONENZYMATIC BINDING TO MEMBRANE-PROTEINS

We have previously identified free ADP-ribose (ADPR) as a normal metab olite in mature human erythrocytes. In this study the metabolic transf ormations of ADPR were investigated in both supernatants from erythroc yte lysates and intact erythrocytes, loaded with ADPR by means of a pr ocedure involving hypotonic haemolysis and isotonic resealing. In both experimental systems, the main pathway was a dinucleotide pyrophospha tase-catalysed hydrolysis to yield AMP, which was readily converted in to the adenylic and inosinic nucleotide pools. To a lesser extent, ADP R underwent conversion into a compound that was identified as ADP-ribu lose (ADPRu), on the basis of m.s., n.m.r. spectroscopy and enzymic an alysis. 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. Isomeriz ation of ADPR to ADPRu was markedly enhanced by ATP. Incubation of uns ealed haemoglobin-free crythrocyte membranes with labelled ADPR did no t cause any transformation of this nucleotide and resulted in its tric hloroacetic acid- and formic acid-resistant binding to a number of mem brane cytoskeletal proteins. These proteins include spectrin, glyceral dehyde 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 masse s 58 and 41 kDa. Acid-resistant binding of ADPR, as well as of NAD+, t o Ga3PDH was confirmed for the enzyme purified from human erythrocytes .