Features detect the activity of transmembrane enzymes in erythrocytes and membrane preparations of mammalian erythrocytes

About the author:

Alexander D. Shalabodov, Dr. Biol. Sci., Professor, Director at the Institute of Mathematics, Natural Sciences and Information Technologies, Tyumen State University

Abstract:

The article analyzes the methods of detecting the activity of transmembrane enzymes in erythrocytes and in membrane preparations of erythrocytes of mammals (including humans). A widespread method of detecting the activity of transport ATPases in whole erythrocytes with the use of Tween-20 can detect the activity of the enzyme without changing the cooperative interaction of the subunits of the Na,K-ATPase during the reaction cycle. Upon receipt of membrane preparations of erythrocytes using hypoosmotic hemolysis by the method of Dodge et.al. about 80% of the membrane preparations at their premises on environ of the enzyme activity determination are «closed», forming a latent form of the enzyme, to identify which is not possible without a mild increase in the permeability of the membrane to open access of substrate and cofactors to the active sites. The introduction of 0.5-1 mm EDTA in the environment of the hemolysis prevents the process that allows a 2-4 fold increase in detectable enzyme activity in the membrane preparations of erythrocytes. It is assumed that the membrane preparations (shadows) of erythrocytes when they are received (hemolysis of erythrocytes) in the presence of chelators of divalent ions lose the ability to «closure» as a result of loss of peripheral membrane proteins of the skeleton (most likely the speсtrin and actin).

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