Rat brain enolase isozymes. Purification of three forms of enolase

Three forms of rat brain enolase (Fletcher, L., Rider, C.C., & Taylor, C.B. (1976) Biochim. Biophys. Acta 452, 245-252), which were separable by DEAE-cellulose chromatography (referred as enolase I, enolase II, and enolase III in order of the elution), were purified with a high yield by use of column chromatographies of Sephadex G-150, Blue Sepharose CL-6B, and hydroxylapatite. About ten mg of each isozyme was obtained from 220 g of the brain with a yield of 30-50%. Sodium dodecyl sulfate gel electrophoresis of purified enolase I and enolase III showed single bands with relative mobilities corresponding to molecular weights of 49,000 (enolase I) or 46,000 (enolase III), and that of purified enolase II showed two bands corresponding to the above molecular sizes. Amino acid analysis of three forms of enolase revealed that the amount of each amino acid enolase II was midway between those of enolase I and enolase III. Antiserum to enolase I or enolase III was raised in New Zealand white rabbits. Results of immunochemical neutralization studies and immunolectrophoresis indicated that anti-enolase III was specific to the subunit of enolase III and reacted with enolase II and enolase III. However, anti-enolase I reacted not only with enolase I and enolase II, but also with enolase in various other tissues. These results support previous reports on crude preparations that enolase II was a hybrid molecule consisting of one enolase I subunit and one enolase III subunit.