ABSTRACT
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.
Subject(s)
Aspartic Acid Proteases/genetics , Gastric Mucosa/metabolism , Salamandridae/metabolism , Amino Acid Sequence , Animals , Aspartic Acid Proteases/classification , Aspartic Acid Proteases/isolation & purification , Cathepsin E/classification , Cathepsin E/genetics , Cathepsin E/isolation & purification , Cloning, Molecular , DNA, Complementary/genetics , Electrophoresis, Polyacrylamide Gel , Enzyme Assays , Enzyme Precursors/classification , Enzyme Precursors/genetics , Enzyme Precursors/isolation & purification , Hydrogen-Ion Concentration , Molecular Sequence Data , Molecular Weight , Pepsin A/classification , Pepsin A/genetics , Pepsin A/isolation & purification , Pepsinogens/classification , Pepsinogens/genetics , Pepsinogens/isolation & purification , Pepstatins/pharmacology , Phylogeny , Protease Inhibitors/pharmacologyABSTRACT
Pepsinogen levels in ape stomachs were comparable to those in macaques and significantly higher than those in the stomachs of other mammals, including carnivores and ruminants. The occurrence of multiple forms of pepsinogens was remarkable. Nine, sixteen, eight, and fourteen pepsinogens were purified or partially purified from the gastric mucosa of a gibbon, orang-utan, gorilla, and chimpanzee, respectively. Most of these were type-A pepsinogens, and only one type-C pepsinogen was identified in each ape. The two types could be readily distinguished by staining for proteolytic activity on polyacrylamide gel electrophoresis (PAGE) in the presence/absence of pepstatin. Type-A pepsinogens were further divided into two subtypes. One subtype, constituting a major group of pepsinogens in apes, exhibited high hemoglobin-digestive activity. The other subtype was specified by a relatively high content of Lys and low hemoglobin-digestive activity. It is likely that pepsinogen-A genes have been duplicated several times as hominoids, including humans, evolved in the primate lineage. The presence of multiple pepsinogens in apes might be advantageous in the efficient digestion of a wide variety of foods.
Subject(s)
Hominidae/metabolism , Pepsin A/isolation & purification , Pepsinogens/isolation & purification , Stomach/enzymology , Amino Acids/analysis , Animals , Digestion , Electrophoresis , Electrophoresis, Polyacrylamide Gel , Hemoglobins/metabolism , Hydrogen-Ion Concentration , Molecular Weight , Pepsin A/chemistry , Pepsin A/classification , Pepsinogens/chemistry , Pepsinogens/classificationABSTRACT
Twenty years after B.S. Hartley's 1960 review, on which the present scheme for classification of the proteinases is based, most of the new information that has been obtained appears fully consistent with Hartley's views. A slight amendment is proposed of the name of the four groups of these enzymes to 'serine', 'cysteine', 'aspartic' and 'metallo'-proteinases.
