Characterization of a cholecystokinin 8-generating endoprotease purified from rat brain synaptosomes.
An endoproteolytic activity that specifically cleaves CCK 33, producing CCK 8, has been purified from a rat brain synaptosome preparation. The purification, which included anion exchange, chromatofocusing, hydroxyapatite, and gel filtration chromatography, resulted in a greater than 3000-fold increase in specific activity. This neutral endoprotease (pH optimum 8) exists as a 90-kDa species, which can be dissociated into active 40-kDa species. The enzyme is a non-trypsin serine protease, which is inhibited by diisopropyl-fluorophosphate and p-aminobenzamidine but not by soybean trypsin inhibitor, phenylmethylsulfonyl fluoride, aprotinin, or a number of thiol or metalloprotease inhibitors. It is highly substrate-specific and cleaves neither trypsin, enteropeptidase, kallikrein substrates, nor analogues of mono- or dibasic cleavage sites of prohormones other than pro-CCK. The endoprotease will not cleave CCK 12 desulfate or CCK (20-29), although these peptides contain common sequences with CCK-33. The protease does cleave [Glu27]CCK (20-29), a peptide in which the glutamate mimics the negative charge normally present on tyrosine sulfate. This suggests that the negative charge at position 27 is important in substrate recognition. The enzyme will also cleave CCK 33 and CCK (1-21) on the carboxyl-terminal side of a single lysine residue in position 11. The subcellular location and specificity of this endoprotease make it a good candidate for a CCK-processing protease.
Viereck
J C
JC
Department of Pharmacological and Physiological Science, St. Louis University Medical School, Missouri 63104.
Beinfeld
M C
MC
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002766:Cholecystokinin / Q000378:metabolism*; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D051381:Rats; D013379:Substrate Specificity; D013574:Synaptosomes / Q000201:enzymology*
D011480:Protease Inhibitors; D002766:Cholecystokinin; D010450:Endopeptidases