RESUMEN

Carbonic anhydrase (CA) expression was examined in the red cells of two mammals that have adapted to low oxygen stress: the llama, which has adapted to high altitudes, and the beluga (or white) whale, which routinely dives for extended periods. Immunodiffusion analyses of their Hb-free hemolysates and partial amino acid sequencing of their HPLC-separated nonheme proteins indicate that the low-activity CA I isozyme is the major nonheme protein in erythrocytes of both the beluga whale and the llama. The high-activity CA II isozyme was not detected in the whale red cells but was present at low levels in erythrocytes of the llama. These results suggest that the absence or decrease in the expression of the high-activity CA II isozyme may be advantageous under hypoxic conditions.

Asunto(s)

Camélidos del Nuevo Mundo/sangre , Anhidrasas Carbónicas/sangre , Eritrocitos/enzimología , Ballenas/sangre , Secuencia de Aminoácidos , Animales , Camelus , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Masculino , Datos de Secuencia Molecular , Oxígeno , Alineación de Secuencia

Asunto(s)

Anhidrasas Carbónicas/genética , Anhidrasas Carbónicas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Secuencia de Aminoácidos , Animales , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Mamíferos , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Familia de Multigenes , Proteínas del Tejido Nervioso/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido

Asunto(s)

Anhidrasas Carbónicas/genética , Anhidrasas Carbónicas/metabolismo , Investigación/tendencias , Animales , Anhidrasas Carbónicas/química , Eritrocitos/enzimología , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo

RESUMEN

Carbonic anhydrase VII (CA VII) appears to be the most highly conserved of the active mammalian carbonic anhydrases. We have characterized the catalytic activity and inhibition properties of a recombinant murine CA VII. CA VII has steady-state constants similar to two of the most active isozymes of carbonic anhydrase, CA II and IV; also, it is very strongly inhibited by the sulfonamides ethoxzolamide and acetazolamide, yielding the lowest Ki values measured by the exchange of 18O between CO2 and water for any of the mammalian isozymes of carbonic anhydrase. The catalytic measurements of the hydration of CO2 and the dehydration of HCO3- were made by stopped-flow spectrophotometry and the exchange of 18O using mass spectrometry. Unlike the other isozymes of this class of CA, for which Kcat/K(m) is described by the single ionization of zinc-bound water, CA VII exhibits a pH profile for Kcat/K(m) for CO2 hydration described by two ionizations at pKa 6.2 and 7.5, with a maximum approaching 8 x 10(7) M-1 s-1. The pH dependence of kcat/K(m) for the hydrolysis of 4-nitrophenyl acetate could also be described by these two ionizations, yielding a maximum of 71 M-1 s-1 at pH > 9. Using a novel method that compares rates of 18O exchange and dehydration of HCO3-, we assigned values for the apparent pKa at 6.2 to the zinc-bound water and the pKa of 7.5 to His 64. The magnitude of Kcat, its pH profile, 18O-exchange data for both wild-type and a H64A mutant, and inhibition by CuSO4 and acrolein suggest that the histidine at position 64 is functioning as a proton-transfer group and is responsible for one of the observed ionizations. A truncation mutant of CA VII, in which 23 residues from the amino-terminal end were deleted, has its rate constant for intramolecular proton transfer decreased by an order of magnitude with no change in Kcat/K(m). This suggests a role for the amino-terminal end in enhancing proton transfer in catalysis by carbonic anhydrase.

Asunto(s)

Anhidrasas Carbónicas/metabolismo , Isoenzimas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión/genética , Anhidrasas Carbónicas/química , Anhidrasas Carbónicas/genética , Catálisis , Transporte de Electrón/genética , Humanos , Concentración de Iones de Hidrógeno , Isoenzimas/química , Isoenzimas/genética , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Protones , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo

RESUMEN

Chromatographic separation of the non-heme proteins from the erythrocytes of the subterranean mole rat belonging to the superspecies Spalax ehrenbergi from Israel revealed two major peaks. On sequence analyses, the larger peak corresponded to a 56 kDa selenium-binding protein (SeBP) previously characterized from mouse and human liver, and the second peak to the low-activity carbonic anhydrase (CA) isozyme, CA I. There was no evidence of the high-activity CA II isozyme normally found in the red cells of all amniotes tested to date. Thus, the mole rat appears to be the first mammalian species to express both a SeBP and the low-activity CA I isozyme, as the major non-heme proteins in its red blood cells. It is possible that the absence of the high-activity CA II isozyme may be advantageous to the mole rat in adapting to the low O2 and high CO2 environment of its underground burrows. It is also likely that the 56 kDa SeBP may play an important adaptive role in the physiology of the red cell.

Asunto(s)

Anhidrasas Carbónicas/sangre , Proteínas Portadoras/sangre , Eritrocitos/química , Ratas Topo/sangre , Secuencia de Aminoácidos , Animales , Anhidrasas Carbónicas/química , Proteínas Portadoras/química , Eritrocitos/enzimología , Isoenzimas/sangre , Datos de Secuencia Molecular , Filogenia , Selenio/sangre , Proteínas de Unión al Selenio , Análisis de Secuencia , Homología de Secuencia de Aminoácido

RESUMEN

Carbonic anhydrase II (CAII) plays an important role in the acid-base homeostasis of the body and its deficiency results in renal tubular acidosis. In order to identify the regulatory regions in the CAII gene for the future development of kidney-targeted gene therapy, we investigated the 5' region of the gene for its promoter activity. Deletion constructs with various lengths of the 5' flanking region of the human CAII promoter were ligated to the CAT reporter gene and lipofected in primary cultures of mouse proximal renal tubular cells and in cells of the established porcine proximal tubular cell line, LLC-PK1. The CAT activity was measured 48 hours after gene transfection. The -12000/CAT and -1300/CAT constructs expressed the highest CAT activity in both types of renal tubular cells (143- and 180-fold increase, respectively, in mouse proximal tubular cells; 50- and 70-fold increase, respectively, in LLC-PK1 cells) but not the -420/CAT, -270/CAT, or -180/CAT constructs (9, 12, and 9% of that of -1300/CAT construct, respectively, in mouse proximal tubular cells and, 23, 9, and 8%, respectively, in LLC-PK1 cells, all p <0.01 vs. -1300/CAT construct). No cytotoxicity was detected in the transfected cells. A computer search identified multiple putative transcription factor binding elements including Ap1 and Ap2 binding elements, which are present in the -1300/CAT construct but not in the shorter constructs. In conclusion, we demonstrate that the human CAII 5' sequence of proximal 1.3 kb contains strong promoter sequence(s) for renal tubular cells.

Asunto(s)

Anhidrasas Carbónicas/genética , Túbulos Renales Proximales/enzimología , Regiones Promotoras Genéticas/fisiología , Secuencias Reguladoras de Ácidos Nucleicos , Animales , Secuencia de Bases , Supervivencia Celular/fisiología , Células Cultivadas , Cloranfenicol O-Acetiltransferasa/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , Regulación Enzimológica de la Expresión Génica , Genes Reporteros , Vectores Genéticos , Humanos , Túbulos Renales Proximales/fisiología , Ratones , Porcinos , Transfección

RESUMEN

Conserved amino acid motifs are found in numerous expressed genes. Proteins and peptides with functional relationships may be identified using probes designed to hybridize with these motifs. An oligonucleotide probe was prepared to match the sequence of the expected active region of a frog corticotropin-releasing factor-like peptide sauvagine and used to screen a sheep brain cDNA library. A novel 1331-bp cDNA encoding a putative 328-residue protein with a theoretical mass of 36 kDa was identified. The presence of a strong signal sequence indicates that it is a secreted protein. The amino- and carboxy-terminal regions are characterized by several potential phosphorylation sites and binding motifs, suggesting a role in intracellular signal transduction. Although the protein possesses a 7-residue sequence identical to that found in sauvagine, its overall primary structure most closely resembles those of the alpha-carbonic anhydrases (alpha-CAs). Moreover, the detection of the human and mouse orthologues in the EST databases, together with an evolutionary analysis, indicates that the protein represents a new member of the alpha-CA gene family, which we designate carbonic anhydrase-related protein XI (CA-RP XI), encoded by CA11 (human) and Car11 (mouse, rat). The human CA11 gene appears to be located between the secretor type alpha(1,2)-fucosyltransferase gene cluster (FUT1-FUT2-FUT2P) and the D-site binding protein gene (DBP) on chromosome 19q13.3. Despite potentially inactivating changes in the active-site residues, CA-RP XI is evolving very slowly in mammals, a property indicative of an important function, which has also been observed in the two other "acatalytic" CA isoforms, CA-RP VIII and CA-RP X, whose functions are unknown.

Asunto(s)

Anhidrasas Carbónicas/genética , Cromosomas Humanos Par 19 , Proteínas de Unión al ADN , Proteínas del Tejido Nervioso/genética , Péptidos , Factores de Transcripción/genética , Secuencia de Aminoácidos , Proteínas Anfibias , Animales , Secuencia de Bases , Biomarcadores de Tumor , Encéfalo/enzimología , Mapeo Cromosómico , Secuencia Conservada , Etiquetas de Secuencia Expresada , Humanos , Ratones , Datos de Secuencia Molecular , Familia de Multigenes , Hormonas Peptídicas , Filogenia , Biosíntesis de Proteínas , Ratas , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Ovinos

RESUMEN

The spatial expression patterns of the two alpha-carbonic anhydrase genes, CA VII and CA-RP VIII (called Car7 and Car8 in the mouse) were examined in the mouse brain by in situ hybridization. These two genes are the most highly conserved evolutionarily among the mammalian alpha-CAs. Both genes showed a similarly wide expression pattern in the brain. In the cerebrum, mRNA expression was detected in the pia, choroid plexus, and neurons of the cortical layer, thalamus, and medial habenulae. A high level of expression appeared in the pyramidal and granular cells of the hippocampus. In the cerebellum, both Car7 and Car8 were transcribed to different degrees in the Purkinje cells, and a lower expression level occurred in the molecular and granular cell layers. Transcription signals for both genes were excluded from the white matter regions.

Asunto(s)

Encéfalo/enzimología , Anhidrasas Carbónicas/genética , Isoenzimas/genética , Animales , Encéfalo/metabolismo , Cerebelo/metabolismo , Femenino , Expresión Génica , Hibridación in Situ , Ratones , Ratones Endogámicos C57BL , ARN Mensajero

Asunto(s)

Anhidrasas Carbónicas/genética , Mapeo Cromosómico , Mitocondrias/enzimología , Animales , Humanos , Ratones , Ratones Endogámicos C57BL

RESUMEN

The carbonic anhydrase (CA)-like protein, CA VIII, lacks the typical carbon dioxide hydrase activity of the CA isozymes. However, the high degree of amino acid sequence similarity between the products of the mouse and the human CA VIII genes suggests an important biological function. We have attempted to investigate the function of this gene in mammalian development by conducting an in situ hybridization study on sagittal sections of mouse embryos at gestation days of 9.5-16.5 using a 35S-labelled riboprobe. Results indicate that this gene (called Car8 in mice) is expressed as early as day 9.5 in a variety of organs including liver, branchial arches, neuroepithelium and developing myocardium. Between days 10.5 and 12.5, it showed a widespread distribution of mRNA expression that became more restricted as development progressed. The level of expression of Car8 mRNA was relatively high in the brain, liver, lung, heart, gut, thymus and epithelium covering the head and the oronasal cavity.

Asunto(s)

Desarrollo Embrionario y Fetal , Regulación del Desarrollo de la Expresión Génica , Proteínas del Tejido Nervioso/genética , Animales , Biomarcadores de Tumor , Anhidrasas Carbónicas/genética , Hibridación in Situ , Ratones , ARN Mensajero/biosíntesis , Distribución Tisular

RESUMEN

The alpha-carbonic anhydrase (alpha-CA) gene family in mammals encodes 10 CA or CA-like proteins (CA I-CA X). Although the gene for human CA VII has been cloned and characterized, the corresponding protein has not previously been purified, and hence, the CO2 hydrase activity of its product has not as yet been demonstrated. In this study, we have cloned the mouse CA VII cDNA in an E. coli, glutathione-S-transferase (GST) expression vector. The CO2 hydrase activity of the expressed protein is about 4% that of the high-activity CAII isozyme, demonstrating that this evolutionarily highly conserved protein is a catalytically active member of this CA gene family.

Asunto(s)

Anhidrasas Carbónicas/genética , Isoenzimas/genética , Secuencia de Aminoácidos , Animales , Anhidrasas Carbónicas/metabolismo , Catálisis , Clonación Molecular , ADN Complementario , Escherichia coli/genética , Humanos , Isoenzimas/metabolismo , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Plásmidos

RESUMEN

The carbonic anhydrases (CA) catalyze with high efficiency the reversible hydration of carbon dioxide, a reaction underlying many diverse physiological processes in animals, plants, archaebacteria, and eubacteria. We examined the evolutionary history and functional convergence of the CAs encoded by members of three independent CA gene families (alpha-CA, beta-CA and gamma-CA). Surprisingly, the six mammalian alpha-CA isozymes of defined function and tissue expression are evolving more rapidly than four mammalian alpha-CA-related proteins of unknown function. We have identified and included several previously unrecognized CA homologues present in the sequence databases, many of which are the fruits of genome project sequencing and expressed cDNA studies. We examined alpha-CA active site evolution and the putative beta-CA and gamma-CA active sites. We found support for the "introns late" hypothesis by analysis of alpha-CA intron locations. The view that alpha-CAs would be restricted to the animal kingdom and plant green algae (Chlamydomonas), the beta-CAs to plants and eubacteria, and the gamma-CAs to archaebacteria and eubacteria is breaking down. The plant Arabidopsis has homologues of all three families.

Asunto(s)

Anhidrasas Carbónicas/genética , Evolución Molecular , Isoenzimas/genética , Familia de Multigenes , Secuencia de Aminoácidos , Animales , Bacterias/enzimología , Bacterias/genética , Secuencia de Bases , Sitios de Unión/genética , Secuencia Conservada , ADN/genética , Bases de Datos Factuales , Variación Genética , Humanos , Intrones , Modelos Genéticos , Datos de Secuencia Molecular , Filogenia , Plantas/enzimología , Plantas/genética , Homología de Secuencia de Aminoácido

RESUMEN

Although the proximal, 5' 115 bp of the human carbonic anhydrase II (CA II) gene was sufficient for expression of a reporter gene in some transfected cell lines, we found previously that 1100 bp of this promoter (or 500 bp of the mouse CA II promoter) was not sufficient for expression in transgenic mice. We have now studied the expression of linked reporter genes in mice transgenic for either (1) 11 kb of the human 5' promoter or (2) 8 kb of the human 5' promoter with mouse sequences from the first exon, part of the first intron (since a CpG island spans this region), and the 3' sequences of the gene. Expression was found in both cases, but the tissue specificity was not appropriate for CA II. Although there was a difference in the sensitivity of the assays used, the first construct led to expression in many tissues, while the second construct was expressed only in spleen. These findings indicate considerable complexity of DNA control regions for in vivo CA II expression.

Asunto(s)

Anhidrasas Carbónicas/genética , Regulación Enzimológica de la Expresión Génica/genética , Animales , Secuencia de Bases , Cartilla de ADN , Genes Reporteros , Humanos , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes de Fusión/genética

RESUMEN

The human carbonic anhydrase (CA) VIII gene (CA8) has been mapped to chromosome 8 at q11-->q12 by human-mouse hybrid mapping and by fluorescence in situ hybridization. The closely-linked human CA isozyme genes, CA1, CA2 and CA3, are also located on chromosome 8, but at q22, and therefore not closely linked to the CA8 locus.

Asunto(s)

Anhidrasas Carbónicas/genética , Cromosomas Humanos Par 8 , Animales , Mapeo Cromosómico , Humanos , Células Híbridas , Hibridación Fluorescente in Situ , Ratones

RESUMEN

The structure of the gene encoding carbonic anhydrase I (CA I) was determined for the pigtail macaque Macaca nemestrina. When the deduced amino-acid sequence was compared with those of five other primates, four non-primate mammals and a turtle, seven residues were found to be unique and invariant to all of the CA I sequences. A scheme is presented for the probable evolutionary order of the six polymorphic nucleotide changes found in the coding regions of the CA I locus of pigtail macaques.

Asunto(s)

Anhidrasas Carbónicas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Evolución Biológica , ADN , Humanos , Macaca nemestrina , Ratones , Datos de Secuencia Molecular , Homología de Secuencia de Ácido Nucleico , Tortugas

RESUMEN

To date, three different structural gene mutations have been identified in patients with carbonic anhydrase II deficiency (osteopetrosis with renal tubular acidosis and cerebral calcification). These include a missense mutation (H107Y) in two families, a splice junction mutation in intron 5 in one of these families, and a splice junction mutation in intron 2 for which many Arabic patients are homozygous. We report here a novel mutation for which carbonic anhydrase II-deficient patients from seven unrelated Hispanic families were found to be homozygous. The proband was a 2 1/2-year-old Hispanic girl of Puerto Rican ancestry who was unique clinically, in that she had no evidence of renal tubular acidosis, even though she did have osteopetrosis, developmental delay, and cerebral calcification. She proved to be homozygous for a single-base deletion in the coding region of exon 7 that produces a frameshift that changes the next 12 amino acids before leading to chain termination and that also introduces a new MaeIII restriction site. The 27-kD truncated enzyme produced when the mutant cDNA was expressed in COS cells was enzymatically inactive, present mainly in insoluble aggregates, and detectable immunologically at only 5% the level of the 29-kD normal carbonic anhydrase II expressed from the wild-type cDNA. Metabolic labeling revealed that this 27-kD mutant protein has an accelerated rate of degradation. Six subsequent Hispanic patients of Caribbean ancestry, all of whom had osteopetrosis and renal tubular acidosis but who varied widely in clinical severity, were found to be homozygous for the same mutation.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Acidosis Tubular Renal/genética , Anhidrasas Carbónicas/deficiencia , Mutación del Sistema de Lectura , Hispánicos o Latinos/genética , Osteopetrosis/genética , Adulto , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encefalopatías/genética , Calcinosis/genética , Anhidrasas Carbónicas/genética , Región del Caribe/etnología , Línea Celular Transformada , Niño , Preescolar , Chlorocebus aethiops , Desoxirribonucleasas de Localización Especificada Tipo II , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Masculino , Datos de Secuencia Molecular , Linaje , Mutación Puntual , Reacción en Cadena de la Polimerasa , Mapeo Restrictivo , Estados Unidos

RESUMEN

Bone marrow transplantation (BMT) in mice was utilized to determine the relative importance of carbonic anhydrase II (CA II) deficiency in blood compared to kidney in the pathogenesis of the ammonium chloride intolerance observed in CA II-deficient mice. "Normal" BMT experiments utilized normal donors and CA II-deficient recipients (NL-->DEF), "reverse" BMT experiments utilized CA II-deficient donors and normal recipients, and control BMT experiments utilized normal mice with a hemoglobin polymorphism (Hbb d/s). Unstressed urinary pH was not significantly altered by normal or reverse BMT, nor was any change induced by control BMT. However, DEF-->NL mice showed markedly altered weight changes when placed on oral ammonium chloride, an effect apparently secondary to dehydration due to decreased water intake. In addition, some CA II-deficient mice have a urinary concentrating defect. Red blood cell and kidney CA II deficiency contribute additively to these effects.

Asunto(s)

Cloruro de Amonio/farmacología , Trasplante de Médula Ósea/fisiología , Médula Ósea/metabolismo , Anhidrasas Carbónicas/deficiencia , Animales , Células de la Médula Ósea , Anhidrasas Carbónicas/metabolismo , Tolerancia a Medicamentos , Concentración de Iones de Hidrógeno , Riñón/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Fenotipo , Orina/química

Asunto(s)

Anhidrasas Carbónicas/genética , Análisis Mutacional de ADN , Zinc/metabolismo , Anhidrasas Carbónicas/metabolismo , Activación Enzimática/genética , Humanos , Hidrólisis , Mutación Puntual , Reacción en Cadena de la Polimerasa

RESUMEN

The genes encoding carbonic anhydrase I (CA I) have been characterized for chimpanzee (Pan troglodytes) and gorilla (Gorilla gorilla). In addition, 44 nucleotides (nt) at the 5' end of the noncoding first exon (exon 1a), which is unique to the erythroid CA I mRNA, together with 188 nt of the adjacent 5' flanking regions, were sequenced for the corresponding positions of the CA I of orangutan, pigtail macaque, and squirrel monkey. When these 5' flanking regions are compared, along with those published for human and mouse CA I, they were found to contain several conserved sequences that may bind factors involved in the erythroid-specific expression of CA I. Comparisons of the human, chimpanzee, and gorilla coding and noncoding CA I sequences do not significantly deviate from a pattern of trichotomy for the evolutionary origins of these three hominoid species.

Asunto(s)

Anhidrasas Carbónicas/genética , Eritrocitos , Gorilla gorilla/genética , Pan troglodytes/genética , Regiones Promotoras Genéticas , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Evolución Biológica , ADN , Exones , Humanos , Macaca nemestrina , Datos de Secuencia Molecular , Pongo pygmaeus , Saimiri , Homología de Secuencia de Ácido Nucleico

RESUMEN

A recently reported mRNA, encoding 'carbonic anhydrase-related polypeptide' (CARP) from the Purkinje cells of mouse cerebellum, was shown to have a 30-40% deduced amino acid sequence identity with the carbonic anhydrases (CA) of mammals. In order to compare the mouse and human CARP sequences, we used the polymerase chain reaction (PCR) to amplify human CARP sequences from several cDNA libraries (salivary gland, testis and placenta). The sequence has an 89.3% sequence identity with mouse CARP at the nucleotide level and 97.9% at the amino acid level. This extremely high evolutionary conservation suggests an important function for the CARP gene product.

Asunto(s)

Anhidrasas Carbónicas , Proteínas del Tejido Nervioso/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN , Humanos , Ratones , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico