Differential molecular sieving permits visualization of hexokinase isoenzymes following electrophoretic separation in high resolution acrylamide gels.
1. Differential molecular sieving is the concept applied to bring together isoenzymes of ATP:D-hexose-6-phosphotransferase (hexokinases) with glucose 6-phosphate dehydrogenase in acrylamide gels by utilization of their dissimilar electrophoretic mobilities. 2. The hexokinase isoenzymes migrate and separate in gels with pore sizes selected to entrap glucose 6-phosphate dehydrogenase in their interstices. The locations of the bands of specific activity are visualized by fluorescence of NADPH under long wave, ultraviolet radiation. 3. A new discontinuous electrochemical system has been devised to deliver protective thiol groups into the gel. Cysteine (trailing ion) and SO4(2-) (leading ion) form a sharp moving boundary. 4. The high resolution of the system has permitted visualization of a rapidly migrating, high Km hexokinase in murine spleen, fat, kidney and lymph nodes. Hexokinase Types I and II, were observed in all tissues tested, but Type IV was seen only in the liver. 5. The importance of glucose concentration effects on hexokinase activity is emphasized by inactivation of slowly migrating low Km hexokinase Types I and II following exposure to 200 mM glucose during preparation of extracts.
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Virginia Commonwealth University, Medical College of Virginia Health Sciences Center, Department of Pathology, Richmond, Va. 23219, USA.
D000818:Animals; D004563:Electrochemistry / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation / Q000379:methods; D006593:Hexokinase / Q000032:analysis / Q000302:isolation & purification*; D007527:Isoenzymes / Q000032:analysis / Q000302:isolation & purification; D051379:Mice; D008809:Mice, Inbred C3H; D008815:Mice, Inbred Strains; D008820:Mice, Obese; D013045:Species Specificity
D007527:Isoenzymes; D006593:Hexokinase