Chemical Modification of Sulfhydryl Groups in Porcine Pancreatic α-Amylase, and Purification and Properties of the Modified Amylase(Biological Chemistry)

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概要

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• The behavior of SH groups of porcine pancreatic α-amylase, called PPA II, was studied by chemical modification with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). Only two SH groups in PPA II reacted, in a pseudo-first-order reaction, and the modification was accompanied with the inactivation of the amylase. The reactivity of SH groups with DTNB was influenced by the ionic strength of the medium. The SH groups were protected against modification by the addition of some substrate analogs; maltopentaitol, maltotetraitol, maltotriitol and cyclomaltohexaose were effective analogs, whereas maltitol, D-glucitol and methyl α-D-glucoside did not protect these groups. The modified enzymes (M_1 and M_2), in which one and two SH groups reacted with DTNB, respectively, were purified in an electrophoretically homogeneous state by chromatography on Bio-Gel P-2 and TSK-Gel DEAE-Toyopearl 650S. The optimum pH of the modified enzyme (M_2) was 6.9〜7.0, which was the same as that of the native PPA II. The isoelectric points of M_1 and M_2 were estimated to be 5.8 and 5.2, respectively, by the method of Catsimpoolas. The CD spectrum of PPA II was altered partially by the modification of SH groups with DTNB. Moreover, a precipitin line with a spur was observed in a double immunodiffusion test of PPA II and M_2 to rabbit antiserum of PPA II. It is concluded that the free SH group(s) in PPA II, located near the substrate binding site, don't participate directly in its catalytic activity, but that the SH group(s) are involved in the antigenicity of PPA II.

• 社団法人日本農芸化学会の論文
• 1986-12-23

著者

• Takahashi Shozo

  Pesticide Research Institute Kyoto University

• Takahashi Shunya

  Riken(the Institute Of Physical And Chemical Research)

• Kobayashi Toshitaka

  Central Research Institute Meiji Milk Products Co. Ltd.

• Kobayashi T

  Nagoya Univ. Nagoya Jpn

• TAKAHASHI Shin-Ichiro

  Laboratory of Biological Chemistry, Department of Agricultural Chemistry, Faculty of Agriculture, To

• SAKANO Yoshiyuki

  Laboratory of Biological Chemistry, Department of Agricultural Chemistry, Faculty of Agriculture, To

• Takahashi S

  Nihon Univ. Chiba Jpn

• ISHIZUKA Yasuyuki

  Laboratory of Biological Chemistry, Department of Agricultural Chemistry, Faculty of Agriculture, To

• KOBAYASHI Tsuneo

  Laboratory of Biological Chemistry, Department of Agricultural Chemistry, Faculty of Agriculture, To

• Ishizuka Yasuyuki

  Laboratory Of Biological Chemistry Department Of Agricultural Chemistry Faculty Of Agriculture Tokyo

• Sakano Y

  Department Of Applied Biological Science Faculty Of Agriculture Tokyo University Of Agriculture And

• Sakano Yoshiyuki

  Laboratory Of Biochemistry Department Of Agricultural Chemistry Tokyo University Of Agriculture And

• Takahashi S

  Riken(the Institute Of Physical And Chemical Research)

• KOBAYASHI Tsuneo

  Laboratory of Biochemistry, Department of Agricultural Chemistry, Faculty of Agriculture, Tokyo University of Agriculture and Technology

• TAKAHASHI Shin-Ichiro

  Laboratory of Animal Cell Regulation, Graduate School of Agricultural and Life Sciences, The University of Tokyo

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