Abstract
We describe the characterization, cloning, and genetic analysis of tripeptidyl peptidase II (TPP II) from Drosophila melanogaster. Mammalian TPP II removes N-terminal tripeptides, has wide distribution, and has been identified as the cholecystokinin-degrading peptidase in rat brain. Size exclusion and ion exchange chromatography produced a 70-fold purification of dTPP II activity from Drosophila tissue extracts. The substrate specificity and the inhibitor sensitivity of dTPP II is comparable to that of the human enzyme. In particular, dTPP II is sensitive to butabindide, a specific inhibitor of the rat cholecystokinin-inactivating activity. We isolated a 4309-base pair dTPP II cDNA which predicts a 1354-amino acid protein. The deduced human and Drosophila TPP II proteins display 38% overall identity. The catalytic triad, its spacing, and the sequences that surround it are highly conserved; the C-terminal end of dTPP II contains a 100-amino acid insert not found in the mammalian proteins. Recombinant dTPP II displays the predicted activity following expression in HEK cells. TPP II maps to cytological position 49F4-7; animals deficient for this interval show reduced TPP II activity.
Highlights
Intercellular communication depends critically on both the generation and termination of biological signals
We describe the characterization, cloning, and genetic analysis of tripeptidyl peptidase II (TPP II) from Drosophila melanogaster
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF035351 for Drosophila, M73047 for human, and U23176 for C. elegans
Summary
Materials—Chromogenic substrates were obtained from Bachem (Bubendorf, Switzerland) and Sigma. For the determination of Km, 2–5 units of enzyme prepared from Drosophila according to Table I or purified recombinant dTPP II were incubated in triplicates with AAF-pNA at concentrations of 1.0, 0.5, 0.2, 0.1, 0.05, and 0.025 mM in 0.1 M potassium phosphate buffer, pH 7.5, that contained 15% glycerol and 2.5 mM DTT and 1% Me2SO. HPLC Analysis—For analysis of cleavage products, 6 units of enzyme were incubated with the nonapeptide GVLRRASVA (10 nmoles) in 0.1 M potassium phosphate buffer, pH 7.5, that contained 2 mM DTT and 3% glycerol, in a final volume of 100 l at 37 °C. The same cycling conditions, without the initial denaturing step, were used and produced a distinct band This product was cloned into the T/A vector according to manufacturer’s recommendations (Promega, Madison, WI) with the exception that the Klentaque LA enzyme was used.
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