MONOUBIQUITINATED ALPHA-GLOBIN IS AN INTERMEDIATE IN THE ATP-DEPENDENT PROTEOLYSIS OF ALPHA-GLOBIN

A dialyzed whole human reticulocyte (beta-thalassemic) lysate was incu bated with human I-125-alpha globin at 37 degrees C for 15 min in a me dium supporting proteolysis with ATP and added ubiquitin (Ub). Analysi s of this reaction mixture by sodium dodecyl sulfate-polyacrylamide ge l electrophoresis (SDS-PAGE) showed that similar to 0.5% of the initia l I-125-alpha globin substrate radioactivity (I-125 cpm) was in the co njugate (Ub(1)-alpha) of one Ub monomer with one I-125-alpha globin mo nomer and substantially less in the Ub(2)-alpha conjugate. When the re action mixture contained 0.5 mu M ubiquitin aldehyde (Ubal) to inhibit endogenous isopeptidases, there was a 7-9-fold increase in the Ub(1)- alpha and Ub(2)-alpha conjugates and an appearance of higher molecular weight species including polyubiquitinated I-125-alpha globin (Ub(>10 )-alpha). Purified fractions of Ub(1)-alpha, Ub(2)-alpha, Ub(3,4)-alph a, and Ub(>10)-alpha conjugates were isolated by preparative SDS PAGE, dialyzed and treated with triethylamine to remove SDS, and dissolved in 10 mM HCOO-(Na+), pH 4.0. Incubation of each of the conjugate fract ions with Ubal-treated lysate in a proteolysis reaction mixture showed a progressive increase in the initial rate of degradation (conversion to acid-soluble I-125 cpm) with increase in molar amount of Ub from s imilar to 3 times that of the unconjugated I-125-alpha globin precurso r for Ub(1)-alpha to similar to 6 times for Ub(>10)-alpha. Analytical SDS-PAGE of the Ub(1)-alpha mixture also showed a rapid conversion to higher M(r) conjugates. These results suggest that monoubiquitinated a lpha globin, in addition to higher M(r) conjugates, is an intermediate in the ATP-dependent proteolysis of alpha globin. The finding that ad dition of Ubal enhances the amount of these conjugates suggests that i sopeptidase activity contributes to the poor turnover of hemoglobin al pha chains observed in intact beta-thalassemic reticulocytes.