Active site of tripeptidyl peptidase II from human erythrocytes is of the subtilisin type.

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.

Download Full-text

Exploring the active site of tripeptidyl-peptidase II through studies of pH dependence of reaction kinetics

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics ◽

10.1016/j.bbapap.2012.01.004 ◽

2012 ◽

Vol 1824 (4) ◽

pp. 561-570 ◽

Cited By ~ 1

Author(s):

Sandra Eklund ◽

Ann-Christin Lindås ◽

Emil Hamnevik ◽

Mikael Widersten ◽

Birgitta Tomkinson

Keyword(s):

Reaction Kinetics ◽

Active Site ◽

Ph Dependence ◽

Tripeptidyl Peptidase ◽

Tripeptidyl Peptidase Ii

Download Full-text

Supramolecular structure of tripeptidyl peptidase II from human erythrocytes as studied by electron microscopy, and its correlation to enzyme activity

Biochemical Journal ◽

10.1042/bj2480259 ◽

1987 ◽

Vol 248 (1) ◽

pp. 259-263 ◽

Cited By ~ 38

Author(s):

E Macpherson ◽

B Tomkinson ◽

R M Bålöw ◽

S Höglund ◽

O Zetterqvist

Keyword(s):

Electron Microscopy ◽

Enzyme Activity ◽

Supramolecular Structure ◽

Ammonium Molybdate ◽

Human Erythrocytes ◽

Native Enzyme ◽

Polymeric Form ◽

Large Size ◽

Tripeptidyl Peptidase ◽

Tripeptidyl Peptidase Ii

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.

Download Full-text

Immunological cross-reactivity between human tripeptidyl peptidase II and fibronectin

Biochemical Journal ◽

10.1042/bj2670149 ◽

1990 ◽

Vol 267 (1) ◽

pp. 149-154 ◽

Cited By ~ 18

Author(s):

B Tomkinson ◽

O Zetterqvist

Keyword(s):

Active Site ◽

Immunoblot Analysis ◽

Cross Reactivity ◽

Binding Domain ◽

Heparin Binding ◽

Cell Binding ◽

Collagen Binding ◽

Tripeptidyl Peptidase ◽

Heparin Binding Domain ◽

Tripeptidyl Peptidase Ii

Tripeptidyl peptidase II (TPP II) is a large intracellular exopeptidase with an active site of the subtilisin type. Affinity-purified hen antibodies against human erythrocyte TPP II cross-reacted with fibronectin in an immunoblot analysis. Furthermore, antibodies against human fibronectin cross-reacted with TPP II. Antibodies against a 65 kDa cell-binding fragment of fibronectin specifically reacted with TPP II, whereas antibodies against the collagen-binding domain, the main heparin-binding domain or the N-terminal fibrin-binding domain did not react. Moreover, the affinity-purified antibodies against TPP II reacted with a 105 kDa cell-binding fragment of fibronectin but not with the fibrin-binding domain or the collagen-binding domain. When native TPP II was dissociated into smaller units through dialysis against a dilute Tris buffer, it could be digested by chymotrypsin into three stable fragments of 70 kDa, 42 kDa and 20 kDa. It could be demonstrated that the 42 kDa fragment was specifically recognized by antibodies against the 65 kDa cell-binding fragment of fibronectin. Furthermore, labelling with di-[3H]isopropyl phosphorofluoridate and N-terminal sequence determination showed that the 70 kDa fragment contained the active-site serine residue. In conclusion, our findings suggest that one domain of the TPP II molecule bears structural resemblance to a cell-binding fragment of fibronectin.

Download Full-text