Monocyte‐specific esterase isoenzyme demonstrated by isoelectric focusing

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Abstract
Using horizontal thin‐layer isoelectric focusing in polyacrylamide gels, we separated the isoenzymes of carboxylic esterase (EC 3.1.1.1) of cell extracts prepared from human hematopoietic cells. Isoenzyme bands were visualized by staining with naphthol ester as substrate and coupling to an azo dye. Staining intensities of isoenzymes were quantified by densitometric scanning. On isoelectric focusing in a pH 2–11 gradient, distinct esterase isoenzyme profiles could be discerned and correlated to various types of normal hematopoietic cells and their leukemic counterparts. One unique isoenzyme, termed monoband, could be clearly identified on the basis of its isoelectric point (pI 6.0), its strong expression by normal and malignant monocytes and its complete and selective inhibition by sodium fluoride. This band was only found in monocytes of either normal or leukemic origin, but not in lymphoid or myeloid cells. The monocyte esterase could be inhibited by sodium fluoride whereas other isoenzyme bands were resistant to this inhibition. However, the specificity of this inhibitory reaction was relative, depending on the concentration of sodium fluoride. Compared with normal monocytes, leukemic monocytes often showed an overexpression of the mono‐bands. Dilution experiments established the distinct prominence of the mono‐band which could be detected among the other isoenzymes even when only 1% of the total cell population consisted of monocytes. Immature myeloid, but mono‐band negative leukemic cells whose arrest of differentiation can be overcome by in vitro 12‐O‐tetradecanoylphorbol 13‐acetate‐promoted differentiation to more mature cells, could be induced to express the mono‐band which paralleled their maturation to monocytes. Isoelectric focusing of esterase isoenzymes represents a powerful tool for the isoenzymatic analysis of hematopoietic cells and an enzymological classification of leukemias.