Tripeptidyl peptidase II is an extralysosomal serine peptidase of an unusually large size, i.e. Mr greater than 10(6) for the native enzyme and Mr 135000 for the subunit. The enzyme from human erythrocytes was studied by electron microscopy on samples negatively stained by ammonium molybdate. Two different structural representations of the purified enzyme were obtained, both with a length of about 50 nm, and consisting of repetitive substructures. Upon dialysis of the enzyme against a Tris/HCl buffer, the activity was gradually decreased. This decrease was shown to parallel the dissociation of the large enzyme structures into smaller ones, the smallest measuring 3 nm by 10 nm and apparently corresponding to the repetitive substructures. The results indicate that a large polymeric form of the enzyme is a prerequisite for full activity.

Rabbit antibodies against purified tripeptidyl peptidase II (TPP II) of human erythrocytes have been prepared and used in immunoblot analysis. Antibodies affinity-purified against the denatured 135,000-Mr subunit of the human peptidase were shown to cross-react with purified rat liver TPP II, as well as a polypeptide of Mr 135,000 in haemolysates and liver homogenates from several other species. TPP II activity in haemolysates from monkey, hog, horse and rabbit corresponded to the levels found in human erythrocytes, whereas this activity was absent or low in erythrocytes from calf and hen. On immunoblot analysis of the calf haemolysates, the 135,000-Mr polypeptide could not be detected. In contrast, analysis of liver homogenates from these species revealed both TPP activity and immunoreactive polypeptides. Immunoreactive polypeptides with Mr about 105,000 and 84,000 of variable occurrence were also detected. In addition, extracts of Escherichia coli showed no TPP II activity under the conditions of the standard assay, although polypeptides of Mr 135,000 and 40,000 were revealed on immunoblot analysis of this species.

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