Anomalies in the electrophoretic resolution of Na+/K(+)-ATPase catalytic subunit isoforms reveal unusual protein--detergent interactions.
Three different isozymes of the Na+/K(+)-ATPase have slightly different different electrophoretic mobilities in sodium dodecyl sulfate (SDS). Certain procedures (reduction and alkylation, heating, and the use of sodium tetradecyl sulfate) have been reported either to improve the electrophoretic separation of isoforms or to reveal the presence of new isoforms. The variables affecting gel electrophoretic mobility were investigated here. Reduction and alkylation decreased the mobility of all three isozymes, and slightly improved the separation of alpha 1 from alpha 2 and alpha 3 without causing a qualitative change in the alpha isoforms detected. Heating the enzyme in SDS caused splitting into two bands. Both bands were intact polypeptides but migrated differently in 5% and 15% polyacrylamide, disclosing an anomalous conformation in detergent. The use of sodium tetradecyl or decyl sulfate instead of dodecyl sulfate altered the relative mobilities of the isozymes, revealing differences in detergent affinity, but no new isoforms were found. In conclusion, Na+/K(+)-ATPase alpha-subunit mobility reflects complex detergent-protein interaction that can be affected by experimental conditions. The existence of more than one band on gels may reflect different conformations in detergent, but should not be accepted alone as evidence for subunit structural heterogeneity.
Sweadner
K J
KJ
Massachusetts General Hospital, Boston 02214.
D000477:Alkylating Agents / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology*; D004586:Electrophoresis; D006358:Hot Temperature; D007527:Isoenzymes / Q000737:chemistry*; D008623:Mercaptoethanol; D008861:Microsomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D051381:Rats; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*; D013379:Substrate Specificity
D000477:Alkylating Agents; D007527:Isoenzymes; D012967:Sodium Dodecyl Sulfate; D008623:Mercaptoethanol; D000254:Sodium-Potassium-Exchanging ATPase