Purification and characterization of a detergent-requiring membrane-bound metalloendopeptidase from porcine brain.
A detergent-requiring metalloendopeptidase cleaving a progastrin-C-terminal peptide (progastrin-(88-101)) mainly at the Arg95-Gly96 bond was solubilized from porcine cerebral vesicular membranes and purified to homogeneity as examined by PAGE. The purified enzyme had a molecular mass of approximately 76 kDa as estimated by both SDS/PAGE and Sephacryl S-300 gel filtration. It hydrolyzed progastrin-(88-101) peptide, BAM-12P, and bradykinin fairly specifically, and more efficiently than various other neuropeptides and related oligopeptides examined as substrates. It was inactive in the absence of detergents, and required certain detergents such as Triton X-100 or Lubrol PX for activity. Its optimum pH was about 6.5 and was strongly inhibited by metal-chelating agents such as EDTA, EGTA, and o-phenanthroline. It was extremely sensitive to EDTA and was completely inhibited even by 0.3 microM EDTA; the activity was fully restored by addition of a 10-fold higher concentration of Zn2+, CO2+, or Mn2+ ions over EDTA. On the other hand, dynorphin A-(1-13) peptide, a strong inhibitor of neurolysin, failed to inhibit the enzyme. The various characteristics indicated that the present enzyme is a unique membrane-bound metalloendopeptidase.
Jeohn
G H
GH
Laboratory of Molecular Biochemistry, School of Life Science, Tokyo University of Pharmacy and Life Science, Japan.
Matsuzaki
H
H
Takahashi
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002499:Centrifugation, Density Gradient; D003035:Cobalt / Q000378:metabolism; D003374:Coumarins / Q000378:metabolism*; D003902:Detergents; D004399:Dynorphins / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D007425:Intracellular Membranes / Q000201:enzymology; D008345:Manganese / Q000378:metabolism; D008666:Metalloendopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D012995:Solubility; D013379:Substrate Specificity; D013552:Swine; D015032:Zinc / Q000378:metabolism
C111961:Boc-Gly-Arg-Arg-MCA; D003374:Coumarins; D003902:Detergents; D009842:Oligopeptides; D011480:Protease Inhibitors; D003035:Cobalt; D008345:Manganese; D004399:Dynorphins; D008666:Metalloendopeptidases; D015032:Zinc