An amphiphilic form of dipeptidyl peptidase IV from pig small-intestinal brush-border membrane. Purification by immunoadsorbent chromatography and some properties

Birte Svensson, M. Danielsen, M. Staun, L. Jeppesen, O. Norén, H. Sjöström

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Dipeptidyl peptidase IV (EC 3.4.14.-) of pig small intestinal brush border membrane was solubilized by Triton X-100 and purified by immunoadsorption on Sepharose conjugated to antibodies against kidney dipeptidyl peptidase IV. The recovery was around 20% and the purification factor around 500. The enzyme appeared homogeneous in crossed immunoelectrophoresis and migrated in polyacrylamide gel electrophoresis in dodecyl sulphate as a single polypeptide chain corresponding to a molecular weight of 137000. Gel filtration under non-denaturing conditions indicated a molecular weight around 230000, which suggested a dimeric structure for the isolated form of dipeptidyl peptidase IV.

The enzyme showed a strong tendency to aggregate and its hydrophobic properties were further characterized in studies on detergent binding. Interaction with sodium deoxycholate or cetyltrimethylammonium bromide in charge-shift crossed immunoelectrophoresis thus resulted in an anodic or cathodic shift in mobility, respectively. Furthermore, by crossed immunoelectrophoresis in the presence of 14C-labelled Triton X-100 the detergent binding could be directly visualized by autoradiography. After trypsin treatment the enzyme no longer formed complexes with the detergents.

The intestinal dipeptidyl peptidase IV released the N-terminal dipeptides from glycyl-l-proline-4-nitroanilide, glycyl-l-proline-2-naphthylamide, glycyl-l-prolyl-l-alanine, L-alanyl-l-alanyl-l-alanyl-l-alanine, and l-leucyl-l-leucyl-l-valyl-l-tyrosyl-l-serine. It had a very low activity on l-alanine-4-nitroanilide and showed no endopeptidase activity. The activity on all hydrolyzed substrates was sensitive to the serine protease inhibitor diisopropylfuorophosphate. The Km for the hydrolysis of glycyl-l-proline-4-nitroanilide was 0.24 mM. The dipeptides glycyl-l-proline and glycyl-l-leucine competitively inhibited the hydrolysis of glycyl-l-proline-4-nitroanilide with Ki values of 3.7 mM and 1.7 mM, respectively.
Original languageEnglish
JournalEuropean Journal of Biochemistry
Volume90
Issue number3
Pages (from-to)489-498
ISSN0014-2956
DOIs
Publication statusPublished - 1978
Externally publishedYes

Fingerprint

Dive into the research topics of 'An amphiphilic form of dipeptidyl peptidase IV from pig small-intestinal brush-border membrane. Purification by immunoadsorbent chromatography and some properties'. Together they form a unique fingerprint.

Cite this