CATHEPSIN-D FROM HUMAN-BRAIN - PURIFICATION AND MULTIPLE FORMS

Abstract

Cathepsin D was purified .apprx. 1000-fold from human brain cortex by a procedure involving ammonium sulfate fractionation (30-70%), Sephadex G-75 chromatography, affinity chromatography on pepstatin-Sepharose and isoelectric focusing. The enzyme was assayed fluorometrically at pH 3.2, the substrates used were globin or Hb modified with pyridoxal-5''-phosphate. Six multiple forms of cathepsin D were resolved in the isoelectric focusing step with pI [isoelectric point] values 4.4, 4.8, 5.3, 6.2, 6.5 and 6.8 Km of pyridoxal-globin, and pyridoxal Hb for all 6 multiple forms is 1.8-2.0 .RTM. 10-5 M and 1.3-4 .RTM. 10-6 M, respectively, and Ki of pepstatin is 2-4 .RTM. 10-9 M. Gel filtration of the multiple forms on Sephadex G-100 column showed that each has a MW of .apprx. 50,000. Human brain cathepsin D has a pH optimum of 3.2 with a smaller 2nd optimum at pH 4.0 (pyridoxal-Hb being used as substrate). All the multiple forms have the same pH-dependence curve. On SDS [sodium dodecyl sulfate]-polyacrylamide gel electrophoresis the purified enzyme produced 3 bands corresponding to MW .apprx. 50,000, .apprx. 35,000 and .apprx. 15,000. Study of the breakdown of substance P and its C-terminal heptapeptide by cathepsin D shows that cleavage occurs at the Phe-Phe linkages of both substrates tested.