Monoubiquitinated alpha globin is an intermediate in the ATP-dependent proteolysis of alpha globin.
A dialyzed whole human reticulocyte (beta-thalassemic) lysate was incubated with human 125I-alpha globin at 37 degrees C for 15 min in a medium supporting proteolysis with ATP and added ubiquitin (Ub). Analysis of this reaction mixture by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that approximately 0.5% of the initial 125I-alpha globin substrate radioactivity (125I cpm) was in the conjugate (Ub1-alpha), of one Ub monomer with one 125I-alpha globin monomer and substantially less in the Ub2-alpha conjugate. When the reaction mixture contained 0.5 microM ubiquitin aldehyde (Ubal) to inhibit endogenous isopeptidases, there was a 7-9-fold increase in the Ub1-alpha and Ub2-alpha conjugates and an appearance of higher molecular weight species including polyubiquitinated 125I-alpha globin (Ub > 10-alpha). Purified fractions of Ub1-alpha, Ub2-alpha, Ub3,4-alpha, 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 fractions with Ubal-treated lysate in a proteolysis reaction mixture showed a progressive increase in the initial rate of degradation (conversion to acid-soluble 125I cpm) with increase in molar amount of Ub from approximately 3 times that of the unconjugated 125I-alpha globin precursor for Ub1-alpha to approximately 6 times for Ub > 10-alpha. Analytical SDS-PAGE of the Ub1-alpha mixture also showed a rapid conversion to higher M(r) conjugates. These results suggest that monoubiquitinated alpha globin, in addition to higher M(r) conjugates, is an intermediate in the ATP-dependent proteolysis of alpha globin. The finding that addition of Ubal enhances the amount of these conjugates suggests that isopeptidase activity contributes to the poor turnover of hemoglobin alpha chains observed in intact beta-thalassemic reticulocytes.
Shaeffer
J R
JR
Center for Blood Research, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115.
D000255:Adenosine Triphosphate / Q000378:metabolism*; D005914:Globins / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008970:Molecular Weight; D014452:Ubiquitins / Q000378:metabolism*
D014452:Ubiquitins; D000255:Adenosine Triphosphate; D005914:Globins