Endopeptidase 24:11 activity in cerebral microvessels.

The cndothelial cells of cerebral microvessels constitute a major site o f the blood-brain barrier. This barrier has physical and 'enzymic' components which together maintain the metabolic environment of the central nervous system. Major peptidase activities in cerebral microvessels are thought to be due to angiotensin-converting cnzymc (ACE) [ I ] and aminopeptidase(s) [2]. Although these enzymes may inactivate a range of biologically active peptides, it is important to determine if other peptidases contribute to the 'enzymic' blood-brain barrier. Bovine cerebral microvessels were assayed for endopeptidase 24:11 (EP 24:l 1 ) activity using thc fluorogenic substrate succinyl-alanyl-alanylphenylalanyl-7-amido-4-methylcoumarin (Suc-AAF-AMC) in a coupled assay procedure 131. EP 24: 1 1 cleaves this substrate to release phenylalanyl-7-amido-4-mcthylcoumarin (F-A MC) which, in the presence of cxccss aminopeptidasc, is broken down to phenylalanine and 7-amino-4-methylcoumarin (AMC). The increase in fluorescence caused by the production of AMC is directly proportional to the activity o f EP 24: 11 if aminopeptidase activity is not ratclimiting. Bovine cerebral microvessels were prepared using techniques similar to those already published 14. 51. All procedures were carried out at 4°C. After removal o f pial vessels and white matter, the cortex was homogenized using a Potter-Elvehjem homogenizer driven at 400 rev./min. The homogenate was centrifuged at 1000 g for 10 min. The pellet was resuspended in 4 vol. of 15%) (w/v) dextran solution and centrifuged at 5000 g for 20 min. This step was then repeated. The pellet was suspended in buffer and sequentially passed through 355 pm and I18 pm pore nylon meshes. Microvessels were collected by passing the filtrate over a 50 p m mesh. The preparation consisted only of capillary networks and smaller segments as seen by phase contrast microscopy. The endothelial cell marker, yglutamyltranspeptidase. was enriched 1 8-fold in the preparation. The microvessels were stored in 50 mM Tris-HCI, pH 7.4, at 20°C. Because Suc-AAF-AMC is a potential substrate for both EP 24: 1 1 and ACE, rates of cleavage of the substrate by pure preparations of these two enzymes were first compared. Assays were carried out in 50 mM-Tris-HCI, pH 7.4, containing 0.1% (w/v) Triton X-100, 30 p~ substrate, 0.25 pg of enzyme and 57 pg of leucine aminopeptidase (LAP) in a final volume of 240 p l . Development of fluorescence was monitored continuously for up to 30 min at 37°C. It was found that the commercially supplied LAP itself turned over the substrate at a rate of 6.03 x 10l ' mol of AMC/h per 57 pg of LAP. This activity was completely inhibited by the aminopeptidase inhibitor bestatin (100 p M ) and was reduced to 50% by the endopeptidase inhibitor phosphoramidon (2.5 p ~ ) , indicating contamination of the LAP by endopeptidase( s). This background hydrolysis was subtracted from subsequent rate determinations. The rate of production of AMC from Suc-AAF-AMC with ACE, after the lag phase, was 3.14x 10-I'' mol of AMC h-I 0.25 pg-l of ACE 57 pg lo fLAP.ForEP24 : l l itwas 1 . 4 3 ~ lO-'rnolAMCh-'