Purification and characterization of human salivary carbonic anhydrase.
A novel carbonic anhydrase was purified from human saliva with inhibitor affinity chromatography followed by ion-exchange chromatography. The molecular weight was determined to be 42,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis, indicating that the human salivary enzyme is larger than the cytosolic isoenzymes CA I, CA II, and CA III (Mr 29,000) from human tissue sources. Each molecule of the salivary enzyme had two N-linked oligosaccharide chains which were cleaved by endo-beta-N-acetylglucosaminidase F but not by endo-beta-N-acetylglucosaminidase H, indicating that the oligosaccharides are complex type. The isoelectric point was determined to be 6.4, but significant charge heterogeneity was found in different preparations. The human salivary isozyme has lower specific activity than the rat salivary isozyme and the human red blood cell isozyme II in the CO2 hydratase reaction. The inhibitory properties of the salivary isozyme resemble those of CA II with iodide, sulfanilamide, and bromopyruvic acid, but the salivary enzyme is less sensitive to acetazolamide and methazolamide than CA II. Antiserum raised in a rabbit against the salivary enzyme cross-reacted with CA II from human erythrocytes, indicating that human salivary carbonic anhydrase and CA II must share at least one antigenic site. CA I and CA III did not crossreact with this antiserum. The amount of salivary carbonic anhydrase in the saliva of the CA II-deficient patients was greatly reduced, indicating that the CA II deficiency mutation directly or indirectly affects the expression of the salivary carbonic anhydrase isozyme. From these results we conclude that the salivary carbonic anhydrase is immunologically and genetically related to CA II, but that it is a novel and distinct isozyme which we tentatively designate CA VI.
D000596:Amino Acids / Q000032:analysis; D002257:Carbonic Anhydrase Inhibitors / Q000494:pharmacology; D002256:Carbonic Anhydrases / Q000172:deficiency / Q000276:immunology / Q000302:isolation & purification*; D002845:Chromatography; D000939:Epitopes / Q000276:immunology; D006026:Glycoside Hydrolases / Q000378:metabolism; D006801:Humans; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000172:deficiency / Q000276:immunology / Q000302:isolation & purification*; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism; D012463:Saliva / Q000201:enzymology*
D000596:Amino Acids; D002257:Carbonic Anhydrase Inhibitors; D000939:Epitopes; D007527:Isoenzymes; D009844:Oligosaccharides; D006026:Glycoside Hydrolases; D002256:Carbonic Anhydrases