Chicken pepsinogen

We also have crystals of chymosin (6), the acid proteinase from Mucor pusillus (7), chicken pepsin (8), and chicken pepsinogen (8), the first two of which are large and very suitable for x-ray analysis. Dr. C. W. Bunn and his co-workers made preliminary x-ray studies of chymosin but the method of isomorphous replacement was unsuccessful, as simple heavy atom derivatives proved impossible to prepare. Both we and Professor B. Foltmann and Dr. S. Larsen of Copenhagen have continued x-ray studies on chymosin, but have also met difficulties. More recently, we have began to use the structural information from the Endothia parastica enzyme to solve the structure of chymosin by using the method of molecular replacement. 

Pepsinogen from porcine stomachs was purified to homogeneity (10) with a final step of chromatography on polylysine-Sepharose (11). Polylysine-Sepharose was made from polylysine (12) obtained from Cambrian Chemicals, Croydon, U. K. Bovine and canine pepsinogens, chicken pepsinogen, calf prochymosin, and pepstatin were very... 

Activation of Bovine, Canine, and Chicken Pepsinogens and Calf Prochymosin... 

The supernatants from bovine and canine pepsinogens were fractionated on polylysine-Sepharose at pH 3.5 as already described whereas, since neither prochymosin nor chicken pepsinogen has affinity for this resin, these preparations were chromatographed on DEAE-cellulose in 0.02 M phosphate buffer, pH 5.4, which separated the unretarded peptide fraction from the adsorbed protein. ... 

The peptide fractions from activation of canine and chicken pepsinogens are shown in Table VII. The composition of canine pepsinogen is known (26) but that of the pepsin is not, so it is not possible to calculate a difference for the activation segment. Assuming a modest homology with the proteins in Figure 1, however, it seems reasonable to assume that about 50 residues should be released. Only about 22 amino acids were released in the presence of pepstatin. 

The compositions of chicken pepsinogen (27) and pepsin (27,28) are known and the difference calculated for the activation segment is given in Table VII Activation in the absence of pepstatin gave a peptide composition (Table VII) in reasonable agreement with these predicted values. In contrast, inclusion of pepstatin during the activation produced significantly smaller numbers of residues. 

The composition of the protein generated on activation of chicken pepsinogen in the absence of pepstatin (Table VIII) bears a marked resemblance to chicken pepsin (the low Met and Tyr values were due to poor hydrolysis conditions) whereas the protein obtained in the presence of pepstatin has a composition intermediate to those of the zymogen and pepsin and quite similar to the calculated values obtained by subtraction of the peptide composition (Table VII) from that of pepsinogen. 

Amino Acid Compositions (mol of residue/mol) of the Peptide Fractions from Canine and Chicken Pepsinogens Samples were hydrolyzed in vacuo for 48 hr in 6M HCl in 105 ... 

Peptide(s) from chicken pepsinogen in presence of pepstatin... 

By incubation of porcine, bovine, canine, or chicken pepsinogens and calf prochymosin with pepstatin at pH 2.5, the first active protein generated on activation is trapped in an inactive complex. The first activation peptide liberated from porcine pepsinogen has been identified as residues 1-16 whereas that from prochymosin is derived from residues 1-27. This suggests that pepsin and chymosin are not formed by one-step conversions from their zymogens, but by (different) sequential, activation mechanisms. 

In chicken pepsinogen, a protein that contains six tryptophan residues, the various indole side chains have different environments and thus showed somewhat different emission spectra and different optical activity 381). 

Several pepsinogens and pepsins have been reported in dogfish and chickens, but we do not find the information sufficient to classify these enzymes in the general scheme suggested above. 

Thus, in the cases of bovine pepsinogen and calf prochymosin where the activation segment sequences are known, peptides were released corresponding to the NH2-terminal parts of the sequence only. For the canine and chicken proteins where no sequence data is available, it appears that inclusion of pepstatin stops the activations before all of the activation segment is released. Consequently, a sequential activation mechanism and not a one-step transformation must be operative with all five of these zymogens. 

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