The localization of peptidases in microvilli from the brush border of the proximal renal tubule of the rabbit.

We have previously reported that a neutral peptidase capable of hydrolysing [1251]iodinated insulin B chain (pH7-5 peptidase) is, like arylamidase and alkaline phosphatase, localized in the microvilli of the proximal renal tubule. Moreover, electron micrographs of negatively stained preparations have revealed that the surface of the microvillus has a covering of discrete granules 80-1001 in diameter (Wong-Leung, George, Aparicio & Kenny, 1968). It is also clear that the pH7.5 peptidase is readily distinguishable from arylamidase in properties and substrate specificity (Kenny, Wong-Leung & Ingram, 1968). We now report some further experiments concerned with the localization of these enzymes in the microvillus and with the purification of the arylamidase. Deoxycholate treatment of a light-microsomal fraction of the kidney cortex disrupted all the membranous elements and solubilized both the pH7*5 peptidase and arylamidase to about the same extent. On the other hand, incubation of the fraction with papain brought about the selective release of arylamidase in a soluble form, while the pH7-5 peptidase and alkaline phosphatase remained in the microsomal pellet. Electron micrographs of the pellets, negatively stained with phosphotungstate, were examined. After papain treatment the surface of many microvilli appeared smooth, in contrast with the control (which had been incubated without papain) in which the surface covering of particles was preserved. The supernatant fraction obtained after papain treatment provided a useful starting point for the purification of arylamidase. Ammonium sulphate precipitation, DEAE-cellulose chromatography and column electrophoresis in a sucrose density gradient yielded a preparation with a specific activity of 57 units/mg. (1 unit1 ,umole of L-leucine pnitroanilide hydrolysed/min. at 370 at pH7.0) and which appeared to be homogeneous on disc electrophoresis. This preparation also hydrolysed L-aamino acid P-naphthylamides, the relative rates for leucine, arginine and glutamic acid derivatives being 100: 70:28. lodinated insulin B chain was attacked about 1000 times more slowly than was L-leucine fl-naphthylamide. Studies in the analytical ultracentrifuge using the sedimentation-equilibrium method of Yphantis (1964) gave a mol.wt. of 136 000 + 13 000. Our results indicate that alkaline phosphatase and the pH7*5 peptidase are situated within the microvillus and that arylamidase is located superficially. The results obtained from the electron microscope and the ultracentrifuge are consistent with the hypothesis that arylamidase is associated with the surface particles.