Mitochondrial carbonic anhydrase (isozyme V) in mouse and rat: cDNA cloning, expression, subcellular localization, processing, and tissue distribution.

Abstract

When the human cDNA, isolated on the basis of homology to the murine carbonic anhydrase (CA) "Y" was expressed in COS cells, the human CA was targeted to and processed in mitochondria, as expected for CA-V. However, tissue distribution reported for the corresponding mouse CA Y mRNA was much more limited than that reported for the distribution of CA-V immunostaining in rat tissues. To determine whether the murine cDNA actually encodes a mitochondrial CA activity and to compare the tissue distribution of the homologous murine and rat gene products, we used reverse transcription-PCR to reisolate the murine CA-V candidate cDNA and used the murine cDNA probe to isolate the homologous rat cDNA. We compared the two cDNA sequences, the activities they expressed after transfection of COS cells, and the sites of N-terminal processing of expressed products. In addition, we used antibodies to the C-terminal peptides predicted from each cDNA to compare distribution of CA-V in mouse and rat tissues and to identify CA-Vs in mitochondria isolated from mouse and rat liver. From these studies, we conclude that both mouse and rat CA-V candidate cDNAs encode active CAs that are targeted to and processed in mitochondria and that there are real differences in tissue distribution of CA-V between mouse and rat. However, the findings that are M(r) of CA-V in rat tissues is smaller than that previously reported and that the tissue distribution also differs lead us to conclude that the antibody used in prior reports most likely misidentified another antigen in rat tissues as CA-V.

Structure determination of murine mitochondrial carbonic anhydrase V at 2.45-A resolution: implications for catalytic proton transfer and inhibitor design.

“…Located in the alkaline environment of the mitochondrial matrix (4)(5)(6)(7)(8), CA-V is unique among the family of mammalian carbonic anhydrases because it is the only isozyme known to be compartmentalized in a cellular organelle. Since the inner mitochondrial membrane is impermeable to bicarbonate (9), CA-V ensures a constant supply of bicarbonate in the mitochondrial matrix for the generation of carbamoyl phosphate in the urea cycle and the conversion of pyruvate into oxaloacetate in gluconeogenesis (10,11).…”

Targeted mutagenesis of mitochondrial carbonic anhydrases VA and VB implicates both enzymes in ammonia detoxification and glucose metabolism

“…Affinity-pure first antibodies against mouse CA VA C-tail were purified using a peptide containing the last 8-aa C-tail peptide-Affigel 10 column. First antibodies against the carboxyl terminal 17-aa CA VB peptide were produced by injecting the C-terminal peptide of mouse CA VB conjugated to thyroglobulin into rabbits in complete Freund's adjuvant (21). The titer and specificity of the antiserum were checked by Western blot.…”

“…Rabbit antibodies against mouse CA VA C-tail peptides were the same as described (21). Affinity-pure first antibodies against mouse CA VA C-tail were purified using a peptide containing the last 8-aa C-tail peptide-Affigel 10 column.…”

“…The murine Car5A transcript was identified by Northern blots only in mRNA from mouse liver (20). Western blot data (21) and immunohistochemical results (23) suggested a wider distribution in rat tissues. A possible solution to the apparent discrepancies in tissue distribution in different studies was suggested by GenBank expressed sequence tag (EST) data indicating that there are at least two mitochondrial CAs with differing tissue distributions.…”

“…Subsequently, mouse, rat, and human cDNAs for CA V were cloned (20)(21)(22). The murine CA genes are called Car5A and Car5B.…”