RESUMEN
The effect of hormone treatment on the abundance of Na+-K+-ATPase alpha- and beta-subunit mRNA in Sparus sarba branchial tissue was investigated. Groups of seawater (33/1000) and hypo-osmotic (6/1000) acclimated fish were injected daily, with either saline, cortisol, recombinant bream growth hormone (rbGH) or ovine prolactin (oPRL). Total RNA from branchial tissue was analyzed by Northern blotting using PCR amplified Na+-K+-ATPase alpha- and beta-subunit cDNA clones. Na+-K+-ATPase alpha- and beta- subunit transcripts of 3.3kb and 2.4kb respectively, were detected and their abundance, after hormone treatment was assessed using RNA dot blots. The abundance of subunit mRNAs increased 1.4-1.9 fold, relative to controls, after cortisol treatment. The alpha:beta mRNA ratio also increased in cortisol treated seawater acclimated fish. Growth hormone treatment did not cause any significant changes in Na+-K+-ATPase subunit mRNA, whereas prolactin significantly reduced alpha-subunit mRNA levels by approximately 0.5 fold in both seawater and hypo-osmotic conditions. The data from this study add further support to the generally accepted roles that cortisol and prolactin have in the modulation of Na+-K+-ATPase activity. It can be concluded from this study that S. sarba branchial Na+-K+-ATPase subunit expression is multihormonally regulated.
Asunto(s)
Región Branquial/efectos de los fármacos , Hormona del Crecimiento/farmacología , Hidrocortisona/farmacología , Perciformes , Prolactina/farmacología , ARN Mensajero/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Región Branquial/enzimología , Cartilla de ADN/química , ADN Complementario/metabolismo , Isoenzimas , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ATPasa Intercambiadora de Sodio-Potasio/genéticaRESUMEN
We have isolated the gene encoding the beta subunit of mitochondrial processing peptidase (beta-MPP) from the shiitake mushroom Lentinula edodes. It is a nuclear gene with two small introns. Comparison with known beta-MPP genes revealed that the L. edodes gene is most closely related with that from Neurospora crassa, with 60.8% identities and 87% similarity in the amino-acid sequences. The deduced L. edodes beta-MPP peptide sequence contains the inverse zinc-binding motif (H-X-E-H) that has been found in a large family of zinc-binding metalloproteinases including bacterial proteinases, insulin degrading enzymes and beta-MPPs. The two histidines are thought to contribute two of the three residues for zinc binding. The expression of L. edodes beta-MPP is higher during the development of the fruiting bodies, suggesting that higher mitochondrial activities may be required to meet the energy demand in the rapid growth of the fruiting bodies.