Characterization and biosynthesis of soluble and membrane-bound carbonic anhydrase in brain.
Carbonic anhydrase from both the cytoplasmic and membrane fractions of the forebrains of rats was characterized with respect to enzymatic activity, immunoreactivity, and in vitro biosynthesis. A procedure for the rapid purification of both membrane-bound and soluble brain carbonic anhydrase is presented that permits retention of full enzymatic activity. Both forms of the enzyme were found to show specific activities of approximately 5500 Units/mg protein when CO2 hydrating activity was determined. In addition, they exhibited similar esterase activity when assayed with p-nitrophenyl acetate. The membrane-bound form, although requiring detergent for extraction from membranes, was freely soluble in aqueous buffers after purification. The molecular weights of both soluble and membrane-bound carbonic anhydrase are 30,000 daltons, and mixing experiments failed to show any significant differences with respect to size. The two forms also exhibit isoelectric points of 7.2. However, the two proteins were found to differ in two respects. Complement fixation indicated that antibodies to soluble carbonic anhydrase had a higher affinity for the soluble form than for the membrane-bound form. The failure to observe any precursor-product relationship between these two proteins with pulse chase studies and the establishment that carbonic anhydrase-like proteins are synthesized on both free polysomes and the rough endoplasmic reticulum indicated that these proteins are synthesized by two separate mechanisms. In vitro synthesis on both free and bound polysomes was determined by two independent methods using different antibodies and different analytical procedures. The basis for these findings and their physiologic importance are discussed.
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002256:Carbonic Anhydrases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D007700:Kinetics; D008970:Molecular Weight; D011132:Polyribosomes / Q000201:enzymology; D014176:Protein Biosynthesis; D051381:Rats; D012995:Solubility
D002256:Carbonic Anhydrases