Human red cell protein kinase in normal subjects and patients with hereditary spherocytosis, sickle cell disease, and autoimmune hemolytic anemia

Abstract

Protein kinase-catalyzed phosphorylation of normal and HS red cell membranes by ATP proceeds at a rate linear with time and membrane concentration for only short periods. In the case of HS membranes, some loss in radioactivity is noted after 20 to 40 min. After 60min incubation, phosphorylation of HS membranes is less than that observed in normal membranes. Findings similar to those in HS membranes were observed in patients with other hemolytic disorders, particularly in sickle cell disease. The K(m) of normal and HS protein kinase for ATP is approximately 10-5M. Phosphate binding sites on band 3 are not saturated in either normal or HS membranes after 1 h of incubation with ATP. There is some loss of binding sites of bands 1 + 2 after such incubation, but no difference is observed between normal and HS membrane. GTP is a very inefficient phosphate donor. Under the conditions of measurement employed, slight stimulation of enzyme activity by 1 μM cAMP is noted, but no increase is produced by 1 μM cGMP. Dephosphorylation of red cell membranes after labeling occurs at similar rates in HS and normal membranes. The calcium chelating agent [ethylenebis (oxyethelenenitrilo)]-tetraacetic acid (EGTA) has little effect on phosphorylation of band 1 + 2, but strongly stimulated phosphorylation of band 3. Stimulation was similar, however, in membranes from normal subjects and those with HS or other hemolytic disorders.