Red blood cell loading with hexokinase and hexokinase-inactivating antibodies. A new strategy for studying the role of enzymes in red cell metabolism and removal.
Human erythrocytes were loaded with homogeneous hexokinase purified from human placenta or with inactivating anti hexokinase IgG, using a procedure of encapsulation based on hypotonic hemolysis, isotonic resealing and reannealing. As a result of these procedures we were able to obtain human erythrocytes with hexokinase levels up to 15-times higher than controls and red blood cells (RBC) with only 10-20% of normal hexokinase activity. RBC with increased hexokinase activity were able to metabolize 1.8-time more glucose than controls, while anti-hexokinase loaded RBC have glycolytic abilities that are only 30% of controls. In both cases the amount of glucose metabolized in the hexose monophosphate pathway was unmodified under resting conditions, but strongly dependent on hexokinase levels in the presence of an oxidative stress. RBC overloaded with hexokinase have a steady-state concentration of glycolytic intermediates that is higher than controls, while 2, 3-diphosphoglycerate and adenine nucleotide levels were almost unchanged. In contrast, RBC with reduced hexokinase activity have a reduced 2, 3-diphosphoglycerate concentration and are not able to maintain their ATP concentration. Inactivation of endogenous RBC hexokinase promotes autologous IgG binding on the RBC membrane. Since the phenomenon is known to be associated with red cell phagocytosis, it could be speculated that in hexokinase deficiency red blood cells are mainly removed by phagocytosis. These results are consistent with suggestions by several investigators that glucose metabolism in human erythrocytes is regulated by hexokinase.
Magnani
M
M
Istituto di Chimica Biologica G. Fornaini, Università degli Studi, Urbino, Italy.
Rossi
L
L
Bianchi
M
M
Serafini
G
G
Stocchi
V
V
D000906:Antibodies / Q000276:immunology; D004789:Enzyme Activation; D004912:Erythrocytes / Q000201:enzymology* / Q000276:immunology; D006593:Hexokinase / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D007773:Lactates / Q000096:biosynthesis; D010587:Phagocytosis
D000906:Antibodies; D007074:Immunoglobulin G; D007773:Lactates; D006593:Hexokinase