Abstract
Adenylate kinase exists in the human erythrocyte in a number of molecular forms with two allels at a single polymorphic locus coding for most of the enzyme forms. The predominant enzyme form, AK a, was purified to constant specific activity in excess of 3000 and appeared homogeneous by chromatography, electrophoresis, and ultracentrifugation. Sedimentation velocity and partial specific volume measurments of AK a yielded values of s20,w = 2.1 S and 0.722 cm-3per g. The molecular weight of the native enzyme was estimated to be 22,500 by sedimentation equilibrium and gel filtration analyses. The molecular weight of the denatured enzyme did not differ, indicating an absence of subunit structure in confirmation of genetic evidence of a single locus coding for the enzyme. The isolated enzyme demonstrated remarkable stability to denaturants (heat, guanidine HCl, urea) in the presence of appropriate stabilizing agents and could not be distinguished from rabbit muscle enzyme on this basis (as well as by a number of other kinetic and physicochemical parameters). The erythrocyte adenylate kinases have a common molecular size but differ in their charge properties. They demonstrate anomalous electrophoretic behavior, migrating anionic to hemoglobin in starch gel, yet exhibit isoelectric points considerably alkaline to hemoglobin (e.g. AK a, pI = 9.0) by isoelectric focusing.
Highlights
Specificactivity in excessof 3000 and appearedhomogeneous exhibited at frequencies of around 90 and 10% within a large by chromatography, electrophoresis, and ultracentrifugation
Procedures employed for the isolation of Adenylate Kinase a (AK a), the predominant molecular form of the human erythrocyte enzyme, are detailed
Adenylate kinase exists in the human erythrocyte in multiple molecular form and comprises about 0.005% of the cell protein
Summary
Adenylate kinase exists in a number of molecular forms in Adenylate kinase exists in the human erythrocyte in a human tissues (6). The most common phenotype, AK 1, exhibits a major and several minor electrophoretically distinguishable enzyme forms. The molecular weight of the denatured enzyme did not differ, indicating an absence of subunit structure in confirmation of genetic evidence of a singlelocus. The erythrocyte adenylate kinaseshave a commonmolecular size but differ in their charge properties. They demonstrate anomalouselectrophoretic behavior, migrating anionic to hemoglobin in starch gel, yet exhibit isoelectric points ture consistent with genetic evidence (7, 8) for the involvement of a single locus in its production. Contrary to an apparent near neutral isoelectric point, indicated by electrophoretic patterns exhibited in starch gel (7-9), the isolated enzyme showed strongly basic properties with ion exchange celluloses and a p1 of 9.0 by isoelectric focusing technique.
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