I. The amino acid sequences of the cytochromes c from elephant seal and emu were determined.
The chymotryptic haemopeptide from seal cytochrome c was separated from the other chymotryptic peptides by gel filtration on Sephadex G-50 followed by chromatography on phosphocellulose. The remaining peptides were separated on Dowex-50 X 2.
The sequence of seal cytochrome c was deduced from the sequences of 27 chymotryptic peptides which were aligned by comparison with the sequences of other cytochromes c. It differs in 1 and 7 positions from the dog and horse proteins respectively. The single substitution between the dog and seal proteins was found at position 100, which is occupied by isoleucine in the seal.
This finding is incompatible with the view that the lack of an hydrophobic residue is a structural requirement of the C-terminus.
Partial chymotryptic digestion of emu cytochrome c yielded 21 peptides which could be resolved on phosphocellulose.
Gel filtration on Sephadex G-50 of the CNBr fragments of emu cytochrome c revealed at least 4 components. These were partly purified by several rechromatographies on Sephadex G-50. The peptides corresponding to residue 1-65 and 66-80 were further purified by paper electrophoresis and chromatography, but the peptide corresponding to 81-104 could not be separated from a contaminating haemopeptide.
The complete sequence of emu cytochrome c was deduced from the CNBr,chymotryptic peptides and by comparison with the sequencesof other cytochromes c.
It differs in 2 positions from both penguin and chicken cytochromes c. The most notable feature of the sequence is the absence of the His residue found in position 33 in most cytochromes c. This His is also absent from the proteins from the turtle, bullfrog, kangaroo and tuna. This might be a characteristic of these c l a s s e s , which appeared early in the evolutionary process.
II. The amino acid sequences around the reactive serine residues in the active sites of pig, ox, sheep and chicken liver carboxylesterases were determined to be:
Pig and Sheep Gly-Glu-Ser- (Ala, Gly. Gly)-Glu-Ser
III. Pig carboxypeptidase A was found to possess endopeptidase activity. Studies with the oxidized insulin A and B chains and oxidized ribonuclease suggest that the endopeptidase cleaves bonds involving the amino groups of certain residues of aspartic, glutamic and cysteic acids.
IV Leucine aminopeptidase was purified 560-fold from an acetone/chloroform powder of pig kidney; by chromatography on DEAEcellulose and gel filtration on Sephadex G-200.
However, the enzyme was not pure. Comparison with a similar procedure published recently suggests that the lack of an ammonium sulphate fractionation step might explain the failure to obtain a highly purified enzyme.