RESUMEN
Six aspartic proteinase precursors, a pro-cathepsin E (ProCatE) and five pepsinogens (Pgs), were purified from the stomach of adult newts (Cynops pyrrhogaster). On sodium dodecylsulfate-polyacrylamide gel electrophoresis, the molecular weights of the Pgs and active enzymes were 37-38 kDa and 31-34 kDa, respectively. The purified ProCatE was a dimer whose subunits were connected by a disulphide bond. cDNA cloning by polymerase chain reaction and subsequent phylogenetic analysis revealed that three of the purified Pgs were classified as PgA and the remaining two were classified as PgBC belonging to C-type Pg. Our results suggest that PgBC is one of the major constituents of acid protease in the urodele stomach. We hypothesize that PgBC is an amphibian-specific Pg that diverged during its evolutional lineage. PgBC was purified and characterized for the first time. The purified urodele pepsin A was completely inhibited by equal molar units of pepstatin A. Conversely, the urodele pepsin BC had low sensitivity to pepstatin A. In acidic condition, the activation rates of newt pepsin A and BC were similar to those of mammalian pepsin A and C1, respectively. Our results suggest that the enzymological characters that distinguish A- and C-type pepsins appear to be conserved in mammals and amphibians.
Asunto(s)
Proteasas de Ácido Aspártico/genética , Mucosa Gástrica/metabolismo , Salamandridae/metabolismo , Secuencia de Aminoácidos , Animales , Proteasas de Ácido Aspártico/clasificación , Proteasas de Ácido Aspártico/aislamiento & purificación , Catepsina E/clasificación , Catepsina E/genética , Catepsina E/aislamiento & purificación , Clonación Molecular , ADN Complementario/genética , Electroforesis en Gel de Poliacrilamida , Pruebas de Enzimas , Precursores Enzimáticos/clasificación , Precursores Enzimáticos/genética , Precursores Enzimáticos/aislamiento & purificación , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Peso Molecular , Pepsina A/clasificación , Pepsina A/genética , Pepsina A/aislamiento & purificación , Pepsinógenos/clasificación , Pepsinógenos/genética , Pepsinógenos/aislamiento & purificación , Pepstatinas/farmacología , Filogenia , Inhibidores de Proteasas/farmacologíaRESUMEN
A cDNA library was constructed from a poly(A)(+) RNA fraction of the gastric mucosa of bullfrog Rana catesbeiana. We cloned a cDNA encoding preprocathepsin E (Pre-Pro-CE) from the library. The present study is the first demonstration of the Pre-Pro-CE cDNA of lower vertebrate such as amphibian. Amino acid sequence deduced from the cDNA was compared with partial amino acid sequence determined by Edman degradation, suggesting that the cDNA comprises an open reading frame encoding a signal peptide (16 amino acids), a pro-sequence (33 amino acids) and a mature protein region (348 amino acids). Two consensus tri-peptide sequences (FDT and VDT) as active site and positions of seven cysteine residues were conserved in this amphibian CE. Although the bullfrog CE was deduced to contain one potential N-linked glycosylation site, its position (Asn(139)-Leu(140)-Thr(141)) was different from that of mammalian CEs. Molecular phylogenetic analysis showed that the bullfrog Pro-CE belongs to the typical Pro-CE group among various aspartic proteinases.