1983 Volume 25 Issue 3 Pages 535-544
In order to clarify the pathophysiological role of glycylprolyl dipeptidyaminopeptidase, partial purification and some properties of this enzyme in guinea pig peritoneal exudate fluids by injection of formalintreated Bacteroides gingivalis cells were studied. The supernatant fluids of guinea pig peritoneal exudates after injection of Bacteroides gingivalis 381 cells were collected by washing with phosphate-buffered saline (PBS). Glycylprolyl dipeptidylaminopeptidase was partial purified by ammoniumsulfate fractionation, DEAE sephadex column chromatography, and Sephadex G-200 gel filtration. Final enzyme sample was separated from collagenase-like peptidase, elastase-like peptidase, trypsin-like peptidase and leucine aminopeptidase, and the purification ratio was 31-fold as specific activity per protein.
The enzyme properities were as follows;
1) The optimum pH for the enzyme activity was 8.6 using Tris-maleate buffer.
2) The Km value was 0.1mM using glycylproline p-nitroanilide tosylate as a substrate.
3) The enzyme activity was not affected by EDTA, but strongly inhibited by HgCl2.
4) The enzyme activity was slightly inhibited by PCMB but activated by dithiothreitol.
5) Protease inhibitors, diisopropylfluorophosphate and bestatin inhibited the enzyme activity.
In order to clarify the origin of glycylprolyl dipeptidylaminopeptidase in peritoneal exudates, lymphocytes and macrophages were isolated from peritoneal exudates using Percoll density gradient centrifugation. The homogenates of lymphocytes or macrophages were fractionated by linear sucrose density gradient centrifugation. The activity of glycylprolyl dipeptidylaminopeptidase was associated with acid phosphatase activity. These data suggest that the origin of glycylprolyl dipeptidylaminopeptidase is confined in lysosomes in lymphocytes and macrophages.
