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 <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 mm EDTA; the activity was fully restored by addition of a 10-fold higher concentration of Zn 2+ , Co 2+ , or Mn 2+ 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.
Abstract:Proteinase A from Aspergillus niger var. macrosporus is a non-pepsintype acid proteinase, whose catalytic site and enzymatic mechanism remain to be clarified. As the first step toward elucidating its three-dimensional structure by X-ray crystallography, proteinase A was crystallized in ammonium sulfate solutions by the hanging-drop vapor diffusion method. In the absence of an organic solvent, only the aggregates of long, thin needles were formed. On the other hand, thicker rodlike single crystals were grown at around pH 1.75 when dimethylsulfoxide (DMSO) was added to a concentration of around 5%. The single crystals of proteinase A thus obtained were suitable for X-ray diffraction measurements with a precession camera.
The serine proteinase previously isolated and partially characterized from the microsomal membrane fraction of rat liver was shown to be composed of two peptide chains of molecular masses of approximately 31 kDa and 19 kDa cross-linked by disulfide bond(s). Partial sequence analysis of peptides obtained by enzymatic hydrolysis indicated that the enzyme shares the same amino acid sequence with rat hepsin. These results provide evidence for the identity of these two enzymes.
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