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Post-proline dipeptidyl aminopeptidase (dipeptidyl aminopeptidase IV) from lamb kidney: Purification and some enzymatic properties
Ist Teil von
BBA - Enzymology, 1977-12, Vol.485 (2), p.391-401
Ort / Verlag
Netherlands: Elsevier B.V
Erscheinungsjahr
1977
Link zum Volltext
Quelle
MEDLINE
Beschreibungen/Notizen
Post-proline dipeptidyl aminopeptidase (dipeptidylpeptide hydrolase, EC 3.4.14.1), also known as glycylprolyl β-naphthylamidase or dipeptidyl aminopeptidase IV, was isolated and purified in an overall yield of 20% from autolyzed extracts of lamb kidney by CM-cellulose and column chromatography on DEAE-Sephadex and Sephadex G-200. Purified enzyme was homogenous by disc gel electrophoresis and ultracentrifugal analysis and was most active at pH 7.8 using Gly-Pro β-napthylamide as substrate. The
K
m values for Gly-Pro β-naphthylamide and Ala-Ala β-naphthylamide were 0.63 and 0.77 mM, respectively. The proline-containing peptides were hydrolysed more than 10-fold faster. By isoelectric focusing a pI of 4.9 was determined. The enzyme has a sedimentation coefficient of 10 S. The molecular weight of the enzyme was estimated to be 230 000 ± 15 000 by the sedimentation equilibrium method and sodium dodecyl sulfate polyacrylamide gel electrophoresis indicating that the enzyme is composed of two identical subunits with molecular weights of 115 000. It was inhibited by the active-site directed, irreversible inhibitor diisopropylphosphorofluorofluoridate. Post-proline dipeptidyl aminopeptidase, in contrast to the endopeptidase post-proline cleaving enzyme [9,10] (Walter R. (1976) Biochim. Biophys. Acta 422, 138–158, and Koida, M. and Walter, R. (1976) J. Biol. Chem. 251, 7593–7599) exhibits no endopeptidase activity. Instead it is an exopeptidase with a high specificity for NH
2-terminal-free peptides containing a proline residue in the penultimate position and releases the dipeptide with proline being the COOH-terminal moiety. The name “post-proline dipeptidyl aminopeptidase” is suggested.