Purification and characterization of a cathepsin D protease from bovine chromaffin granules.
Purification and potential tachykinin and enkephalin precursor cleaving enzymes from bovine chromaffin granules was undertaken using as substrates the model precursors 35S-(Met)-beta-preprotachykinin [35S-(Met)-beta-PPT] and 35S-(Met)-preproenkephalin [35S-(Met)-PPE]. Purification by concanavalin A-Sepharose, Sephacryl S200, and chromatofocusing resulted in a chromaffin granule aspartyl protease (CGAP) that preferred the tachykinin over the enkephalin precursor. CGAP was composed of 47-, 30-, and 16.5-kDa polypeptides migrating as a single band in a nondenaturing electrophoretic gel system, and coeluting with an apparent molecular mass of 45-55 kDa by size-exclusion chromatography. These results suggest that two forms exist: a single 47-kDa polypeptide and a complex of 30 + 16.5-kDa-associated subunits. CGAP was optimally active at pH 5.0-5.5, indicating that it would be active within the acidic intragranular environment. Cleavage at basic residues was suggested by HPLC and HVE identification of 35S-(Met)-NKA-Gly-Lys as the major acid-soluble product generated from 35S-(Met)-beta-PPT. Neuropeptide K was cleaved at a Lys-Arg basic residue site, as determined by identification of proteolytic products by microsequencing and amino acid composition analyses. Structural studies showed that the three CGAP polypeptides were similar to bovine cathepsin D in NH2-terminal sequences and amino acid compositions, indicating that CGAP appears to be a cathepsin D-related protease or cathepsin D itself. The 47- and 16.5-kDa polypeptides of CGAP possessed identical NH2-terminal sequences, suggesting that the 16.5-kDa polypeptide may be derived from the 47-kDa form by proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)
Krieger
T J
TJ
Department of Biochemistry, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814.
Hook
V Y
VY
D000595:Amino Acid Sequence; D000818:Animals; D002402:Cathepsin D / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002417:Cattle; D002837:Chromaffin Granules / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009479:Neuropeptides / Q000737:chemistry / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D015320:Tachykinins
D009479:Neuropeptides; D011480:Protease Inhibitors; D015320:Tachykinins; C045206:neuropeptide K; D002402:Cathepsin D