37] Human peptidyl dipeptidase (converting enzyme, kininase II)

Abstract

This chapter presents the procedure for the large-scale purification of membrane-bound human lung and kidney converting enzyme (CE). 13 mg of homogeneous enzyme is obtained from 15.8 kg of lung tissue, and 29 mg of enzyme from 5.8 kg of kidney. About half the enzyme content of the cadaver lung goes into solution after homogenization; the kidney enzyme is less soluble. The yield after the first three steps of purification reflects the recovery of the bound enzyme. The decrease in activity after tryptic extraction (step 4) is probably because of the release of peptides that inhibit the enzyme. The inhibitory activity was removed during the subsequent chromatography steps. CE is assayed by peptidase activity of the enzyme with a recording UV spectrophotometer by following the hydrolysis of Bz-Gly-Gly-Gly, Bz-Gly-His-Leu, or Bz-Gly-Phe-Arg. The latter two substrates represent the C-terminal dipeptide of angiotensin I and bradykinin. The enzyme in tissue homogenates or biological fluids with low specific activities are assayed by extracting 3H-Bz-Gly from the reaction mixture containing 3H-Bz-GIy-Gly-Gly substrate. High ionic strength in the incubation mixture increases the rate of hydrolysis 3- to 6-fold. To correct for interference by contaminating peptidases, it is advised to use a low concentration of a specific inhibitor such as 10 –7 M captopril.