• Proceedings of the Korean Society of Food Hygiene and Safety Conference
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• 2002.05a
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• pp.215-215
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• 2002

Catalytic subunit of pyruvate dehydrogenase phosphatase (PDPc) has been suggested to have three major funational domains such as dihydrplipoamide adetyltransferase(E2)-binding domain, regulatory subunit of PDP(PDP)r-binding domain, and calcium-binding domain. In order to identify functional domains, recombinant catalytic subunit of pyruvate dehydrogenase phosphatase(rPDPc) was expressed in E. coli JM101 and purified to near homogeneity using the unique property of PDPc: PDPc binds to the inner lipoyl domain (L2) of E2 of ppyruvate dehydrogenase complex (PDC) in the presence of Ca+2, not under EGTA. PDPc was limited-proteolysed by typsin, chymotypsin, Arg-C, and elastase at pH 7.0 and 30C and N-terminal analysis of the fragments was done. Chymotrypsin, trypsin, and elastase made two major fragments: N-terminal large fragment, approx. 50kD and C-terminal small fragment, approx.10 kDa. Arg-C made three major fragments: N-terminal fragment, approx. 35kD, and central fragment, approx. 15 kD, and C-terminal fragment, approx. 10 kD. This study strongly suggest that PDPc consists of three major functional domains. However, further study should be necessary to identify the functional role.

• Environmental Analysis Health and Toxicology
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• v.17 no.1
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• pp.73-80
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• 2002

Pyruvate dehydrogenase phosphatase (PDP)와 kinase는 당대사시 해당과정에서의 대사 산물인 pyruvate를 acetyl CoA로 만들어 구연산 회로로 진입시켜 주는 효소인 pyruvate dehydrogenase complex (PDC)의 활성을 조절하는 중요한 효소이다. PDP의 catalytic subunit는 PDC의 dihydrolipoamide acetyltransferase (E2), PDP regulatory subunit (PDPr), 그리고 칼슘 결합 도메인 등으로 구성되어 있는 것으로 추측되어지고 있다. 본 연구에서는 그 구조와 기능과의 상관관계를 알아보기 위해 PDPc를 E. coli JM101에서 발현시켜 순수 정제 후 단백분해 효소를 이용한 제한적 가수분해 방법을 이용해 그 구조와 기능과의 상관관계에 대해 연구하고자 하였다 정제된 PDPc는 trypsin, chymotrypsin, Arg-C 그리고 elastase를 이용하여 3$0^{\circ}C$ 그리고 pH 7.0에서 제한적으로 분해시켰으며 각 분해산물의 아미노 말단의 아미노산 배열을 분석하였다. 그 결과 PDPc는 trypsin, chymotrypsin, elastase에 의해 N-terminal의 50 kD과 C-terminal의 10 kD의 두개의 분해산물을 만들었으며, Arg-C에 의해 50kD의 분해산물은 약 35kD와 15kD으로 더 가수분해가 되었다. 이러한 결과로 볼 때 PDPc는 앞에서 추측한데로 세개의 주요한 기능적 도메인으로 이루어져 있음을 알 수 있었다 또한 C-terminal의 10kD은 PDPc의 활성에는 영향을 주지 않는 것으로 밝혀졌으나 다른 도메인의 기능은 더 연구가 되어져야 할 것으로 생각된다.

• Proceedings of the Korean Society of Applied Pharmacology
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• 2002.07a
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• pp.236-236
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• 2002

Pyruvate dehydrogenase phosphatase (PDP) is a mitochondrial protein serine/threonine phosphatase that catalyzes the dephosphorylation and concomitant reactivation of the pyruvate dehydrogenase componant of the pyruvate dehydrogenase complex (PDC). PDP consists of a Mg$\^$+2/ -dependent and Ca$\^$+2)-stimulated catalytic subunit (PDPc) of Mr 52,600 and a FAD-containing regulatory subunit (PDPr) of Mr 95.600. Catalytic subunit of pyruvate dehydrogenase phosphatase (PDPc) has been suggested to have three major functional domains such as dihydrolipoamide acetyltransferase(E$_2$)-binding domain, regulatory subunit of PDP(PDPr)-binding domain, and calcium-binding domain. In order to identify functional domains, recombinant catalytic subunit of pyruvate dehydrogenase phosphatase (rPDPc) was expressed in E. coli JM101 and purified to near homogeneity using the unique property of PDPc: PDPm binds to the inner lipoyl domain (L$_2$) of E$_2$ of pyruvate dehydrogenase complex (PDC) in the presence of Ca$\^$+2/, not under EGTA. PDPc was limited-proteolysed by trypsin, chymotrypsin, Arg-C, and elastase at pH7.0 and 30$^{\circ}C$ and N-terminal analysis of the fragment was done. Chymotrypsin, trypsin, and elastase made two major framents: N-terminal large fragment, approx. 50kD and C-terminal small fragment, approx. 0 kDa. Arg-C made three major fragments: N-terminal fragment, approx. 35 kD, and central fragment, approx. 15 kD, and C-terminal fragment, approx. 10 kD. This study strongly suggest that PDPc consists of three major functional domains. However, further study should be necessary to identify the functional role.