Carbonic Anhydrase in the Membrane of the Endoplasmic Reticulum of Male Rat Liver

We have prepared subcellular fractions of male rat liver homogenate by the method of Lewis and Tata [Lewis, J. A. \& Tata, J. R. (1973) J. Cell Sci. 23, 447-459], further purifying the membranes of the microsomal fraction by exposure to 0.01% Triton X-100 and centrifugation. We determined the pu...

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Published in:	Proceedings of the National Academy of Sciences - PNAS Vol. 89; no. 24; pp. 11721 - 11725
Main Authors:	Ono, Yoshiaki, Ridderstrale, Y., Forster, R. E., Chu, Zhi Gou, Dodgson, S. J.
Format:	Journal Article
Language:	English
Published:	Washington, DC National Academy of Sciences of the United States of America 15-12-1992 National Acad Sciences National Academy of Sciences
Subjects:	Analytical, structural and metabolic biochemistry Animals Biological and medical sciences carbonate dehydratase Carbonic Anhydrase Inhibitors - pharmacology Carbonic Anhydrases - metabolism Cell membranes Cellular biology Cytosol Endoplasmic reticulum Endoplasmic Reticulum - enzymology enzymatic activity Enzymes Enzymes and enzyme inhibitors Fundamental and applied biological sciences. Psychology Hepatocytes Histocytochemistry Intracellular Membranes - enzymology Isoenzymes - metabolism kinetics Liver Lyases Male Microsomes Microsomes, Liver - enzymology P branes purification Rats Rats, Sprague-Dawley Rodents Subcellular fractions Subcellular Fractions - enzymology Sulfonamides Intracytoplasmic membrane Rat Enzyme Isozyme Liver Rodentia Male Microsome Vertebrata Mammalia Enzymatic activity Carbonic anhydrase Localization Endoplasmic reticulum Histochemistry
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Summary:	We have prepared subcellular fractions of male rat liver homogenate by the method of Lewis and Tata [Lewis, J. A. \& Tata, J. R. (1973) J. Cell Sci. 23, 447-459], further purifying the membranes of the microsomal fraction by exposure to 0.01% Triton X-100 and centrifugation. We determined the purity of the fractions with marker enzymes and measured carbonic anhydrase (CA; EC 4.2.1.1) activity in intact and solubilized particulates with18O exchange between CO2/HCO- 3and water. We measured the concentration of CA by titration with a sulfonamide inhibitor, ethoxzolamide, obtaining an average value of 3.8 μmol/mg of microsomal membrane protein. The equilibrium constant for binding ethoxzolamide was 0.49 x 10-9M. The Kmfor CO2was 1.7 mM and the turnover number was 560,000 sec-1, characterizing this as a membrane-bound, high-activity isozyme of type IV. By electron microscopy of tissue sections after staining with a cobalt precipitation technique, CA was seen in small cytoplasmic vesicles in hepatocytes and in microsomal particles and membranes. There was a sulfonamide-resistant (isozyme type III) and a sulfonamide-sensitive (isozyme type II) CA in the cytosol but none in the rapidly sedimenting endoplasmic reticulum. We conclude that there is no CA normally within the matrix of the cell endoplasmic reticulum but that the CA type III found in the microsome may have been captured from the cytosol during resealing. Thus the adult male rat hepatocyte contains CA type IV in the membrane of the endoplasmic reticulum and CA type II and CA type III in the cytoplasm.
Bibliography:	ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2
ISSN:	0027-8424 1091-6490
DOI:	10.1073/pnas.89.24.11721