Purification and properties of two active components from crystalline preparation of porcine pancreatic .ALPHA.-amylase.

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Two active components (PPA Iand PPA II) of porcine pancreatic α-amylase [EC 3.2.1.1] from the crystalline preparation were highly purified by a novel ion-exchanger, TSK-Gel DEAE-Toyopearl® 650S (DEAE-Fractogel® TSK 6505) chromatography. The PPA I and II purified showed a single band in polyacrylamide gel electrophoreses with or without sodium dodecylsulfate. PPA II migrated to the anode more rapidly than PPA Iin the polyacrylamide gel electrophoresis (pH 9.0). Isoelectric points of PPA I and II were estimated to be 6.5 and 6.1, respectively. Activation by chloride in concentration above 50 mM was more marked on PPA I than on PPA II. Except the differences described above, the enzymatic properties of PPA I and II examined were almost same as shown below: 1) optimum pH and temperature for enzyme action on starch were 6.9 and 53°C, respectively, 2) thetwo components were more stable at alkaline pH in the presence of Ca2+ than in the absence of Ca2+, 3) both activities of PPA I and II were markedly inhibited by Hg2+, Cu2, Ni2+, Zn2+, N-bromosuccinimide, 5, 5′-dithiobis-(2-nitrobenzoic acid) and some microbial α-amylase inhibitors to nearly the same extent, and 4) Km, and k0 values of PPA I and II for short chain amylose (DP= 17), and Ki values for α-amylase inhibitors calculated from Lineweaver-Burk plots were coincident with each other, respectively.
日本応用糖質科学会の論文