sodium-dodecyl-sulfate and sezolamide

Many functions of polymorphonuclear leukocytes (PMNs) appear to alter and be affected by changes in the intracellular and/or extracellular acid-base milieu, suggesting that carbonic anhydrase (CA) may be important. Although small amounts of CA activity in PMNs have been reported, it has not been characterized fully. We therefore studied isolated mature circulating human PMNs and cultured HL-60 cells, an undifferentiated myelopoietic cell line, and compared these to human red cells (RBCs) for CA activity. Activity and sulfonamide inhibition were measured by a modified micromethod assay. Isoenzyme and total CA concentrations were determined by radioimmunoassay for human isozyme CA I, differential inhibition by MK-927, inhibition by 0.2% sodium dodecyl sulfate (SDS), and quantitative sulfonamide binding. Total CA activity (units/10(6) cells) was 0.04 in PMNs, 0.06 in HL-60 cells, and 0.62 in RBCs. Human PMNs have a total CA concentration of 1.3 microM, of which 0.9 microM is CA I and the remainder is CA II. Total loss of CA activity with 100 microM ethoxzolamide and 0.2% SDS ruled out significant CA III or CA IV activity. Subcellular fractionation of PMNs revealed that all CA activity was cytosolic. The absence of CA activity in mitochondrial and microsomal membrane fractions argues against any mitochondrial CA V or membrane-bound CA IV contribution to total CA activity. Neutrophils contain both CA I and II isozymes in roughly the same proportion as RBCs but at much lower concentrations, suggesting that in the course of maturation the CA content of neutrophils is regulated differently from that in erythrocytes.

Topics: Carbonic Anhydrase Inhibitors; Carbonic Anhydrases; Chromatography, High Pressure Liquid; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Ethoxzolamide; Humans; Immunoblotting; Isoenzymes; Leukemia, Myeloid; Neutrophils; Radioimmunoassay; Sodium Dodecyl Sulfate; Sulfonamides; Thiophenes; Tumor Cells, Cultured