Multicatalytic, High-Mr Endopeptidase from Postmortem Human Brain

Autor: John R. McDermott, A. M. Gibson, J. A. Biggins, A. E. Oakley
Rok vydání: 1991
Předmět:
Zdroj: Journal of Neurochemistry. 56:1509-1517
ISSN: 1471-4159
0022-3042
DOI: 10.1111/j.1471-4159.1991.tb02045.x
Popis: The main high molecular weight (650K) multica-talytic endopeptidase has been purified from postmortem human cerebral cortex. As in other tissues and species, this enzyme is composed of several subunits of 24-3 IK and has three distinct catalytic activities, as shown by the hydrolysis of the fluorogenic tripeptide substrates glutaryl-Gly-GIy-Phe-7-amido-4-methylcoumarin, benzyloxycarboxyl - Gly - Gly -Arg-7-amido-4-methylcoumarin, and benzyloxycarboxyl-Leu-Leu-Glu-2-naphthylamide with hydrophobic (Phe), basic (Arg), and acidic (Glu) residues in the P, position, respectively. These activities are distinguishable by their differential sensitivity to peptidase inhibitors. The enzyme hydrolysed neu-ropeptides at pH 7.4 at multiple sites with widely differing rates, ranging from 113 nmol/min/mg for substance-P, down to 2 nmol/min/mg for bradykinin. The enzyme also had pro-teinase activity as shown by the hydrolysis of casein. For the hydrolysis of the Tyr5-Gly6 bond in luteinizing hormone-releasing hormone, the Km was 0.95 mM and the specificity constant (kcat/Km) was 4.7 ± 103 M-l s-1. The bond specificity of the enzyme at neutral pH was determined by identifying the degradation products of 15 naturally occurring peptide sequences. The bonds most susceptible to hydrolysis had a hydrophobic residue at P1, and either a small (e.g., -Gly or -NH2) or hydrophobic residue at P1. Hydrolysis of -Glu-X bonds (most notably in neuropeptide Y) and the Arg6-Arg7 bond in dynorphin peptides was also seen. Thus the three activities identified with fluorogenic substrates appear to be expressed against oligopeptides.
Databáze: OpenAIRE
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