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A possible new enzyme, a cytosolic neutral PTH-degrading enzyme previously described by us, was purified by ammonium sulfate fractionation, Sephadex G-200 column chromatography and DEAE cellulose column chromatography from the 100,000 x g supernatant of rat kidney. Three active peaks (designated P-I, P-II and P-III) were obtained through a DEAE cellulose chromatographic column. These three activity peaks were interchangeable on rechromatography. Further characterization of the major P-II fraction was attempted. When PTH was used as the substrate, this enzyme preparation (P-II) was inhibited by Ca2+, ATP and glutathione (GSH), but not by trypsin inhibitor, PMSF, E-64, leupeptin, chymostatin or pepstatin. This enzyme preparation could hardly hydrolyze several synthetic analogs containing MCA but not Leu-MCA and Z-Phe-Arg-MCA. When Z-Phe-Arg-MCA was used as the substrate, this enzyme preparation was activated by ATP and GSH, while no activation was induced when PTH was used. These results indicate that the neutral PTH-degrading enzyme seems to represent a new PTHase capable of specifically splitting PTH at certain locations.

Using unlabeled bovine parathyroid hormone (b-PTH) as the substrate, the PTH-degrading activity in the 100,000 x g supernatant of rat renal cortex was examined. The PTH-degrading activity showed the highest peak at pH 7.25, along with 3 minor peaks at pH 4.5, 6.0 and 8.5. The neutral PTH-degrading activity of the 100,000 x g supernatant (pH 7.25) was eluted at V0 in Sephadex G-200 gel filtration corresponding to a high molecular weight. The neutral PTH-degrading activity was inhibited by ATP and calcium, but the acid PTH-degrading activity (pH 4.5) was slightly activated by ATP and was uninfluenced by calcium. The cytosolic neutral PTH-degrading activity was not inhibited by PMSF, trypsin inhibitor, E-64, chymostatin, leupeptin or pepstatin, whereas the acid PTH-degrading activity was inhibited by pepstatin, leupeptin, trypsin inhibitor and chymostatin. The neutral and acid PTH-degrading activities most probably depend on different enzymes. The neutral PTH-degrading enzyme is unlike any of the PTH-degrading enzymes so far reported.