ATP-dependent proteolysis of hemoglobin alpha chains in beta-thalassemic hemolysates is ubiquitin-dependent

• Published in: The Journal of biological chemistry Vol. 263; no. 27; pp. 13663 - 13669
• Main Author: Shaeffer, J R
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 25-09-1988; American Society for Biochemistry and Molecular Biology
• Subjects: Adenosine Triphosphate - pharmacology; Antibodies - physiology; Cell-Free System; Ethylmaleimide - pharmacology; Globins - metabolism; Hemoglobins - metabolism; Humans; Macromolecular Substances; Peptide Hydrolases - blood; Phenanthrolines - pharmacology; Phenylmethylsulfonyl Fluoride - pharmacology; Protease Inhibitors - pharmacology; Reticulocytes - metabolism; Thalassemia - blood; Ubiquitins - immunology; Ubiquitins - pharmacology; Vanadates - pharmacology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)68292-4

beta-Thalassemia is an inherited human disorder which is characterized by a deficient production of hemoglobin beta chains and an attendant accumulation of structurally normal alpha chains in the erythropoietic cells. The objective of this work is to understand the mechanism of intracellular proteolysis of these excess alpha chains. Dialyzed stroma-free hemolysates (32 mg/ml hemoglobin) of blood reticulocytes from four individuals with beta-thalassemia intermedia were incubated with human hemoglobin 3H-alpha chains (0.13 mg/ml) at 37 degrees C in a reaction mixture supporting protein degradation. In the presence of ATP and an ATP-generating system, the fraction of alpha chain 3H radioactivity made acid-soluble after 4 h ranged from 4 to 12% among the different hemolysates; in the absence of ATP or when hemolysates of normal human erythrocytes were used, only 1 to 2% of the 3H-alpha chains were degraded. It is likely that the ATP-dependent proteolysis of 3H-alpha chains in the beta-thalassemic hemolysates corresponds to the ATP-dependent turnover of newly synthesized soluble alpha chains in intact beta-thalassemic reticulocytes observed previously (Shaeffer, J. (1983) J. Biol. Chem. 258, 13172-13177) because of the following similarities between the two systems: (a) free 3H-alpha chains, but not 3H-labeled tetrameric hemoglobins, were readily degraded; (b) the rate of 3H-alpha chain proteolysis in the cell-free system was at least one-half of that observed for the turnover of newly synthesized alpha chains (t1/2 approximately 6 h) in intact cells; and (c) the ATP-dependent proteolytic activity of both systems was inhibited substantially by certain chemical agents (orthovanadate, N-ethylmaleimide, o-phenanthroline, and phenylmethylsulfonyl fluoride) but only slightly, if at all, by others (epsilon-aminocaproic acid and leupeptin). When excess human erythrocyte ubiquitin was added to the beta-thalassemic cell-free systems, a stimulation in ATP-dependent proteolysis of 3H-alpha chains ranging from 30 to 58% was observed. Conversely, addition of from 1.25 to 2.50 mg/ml affinity-purified rabbit antiubiquitin inhibited almost all (greater than 90%) of the ATP-dependent 3H-alpha chain proteolysis; in control experiments, antiubiquitin neutralized with excess ubiquitin inhibited only 13 to 30% of the total (including ubiquitin-stimulated) ATP-dependent proteolysis.