Purification and Properties of Malic Enzyme from the Uropygial Gland of Peking Duck.

국문 초록 (Abstract)

Uropygial gland에 있는 malic enzyme을 2',5'-ADP agarose affinity chromatography, DEAE-Sephacel ion exchange chromatography에 의해서 183배 정제하였으며 specific activity는 174 unit/mg protein이었다. 이렇게 정제된 효소는 SDS-polyacrylamide gel electrophoresis를 하였을 때 95% 이상의 순도를 나타냈으며, subunit의 분자크기는 약 60,000dalton이었다. 최적 pH는 7.5이었으며 이 효소의 활성을 나타내는데는 $Mn^{++}$이나 $Mg^{++}$ 이온이 필요하였다. Uropygial gland malic enzyme의 경우에 있어서 malate와 $NADP^+$에 대한 Km은 각각 $370{\mu}M$, $10.6{\mu}M$ 이었으며, liver malic enzyme의 경우는 $606{\mu}M$, $20.8{\mu}M$이었다. Uropygial gland와 liver 효소에 대한 product inhibition pattern은 서로 같은 양상을 나타나는데 즉, bicarbonate는 malate와 $NADP^+$에 대하여 모두 uncompetitive inhibitor이었고, pyruvate에 대하여서는 두 기질 모두 uncompetitive inhibitor이었으며, NADPH는 malate에 대하여 noncompetitive inhibitor이었으나 $NADP^+$에 대해서는 competitive inhibitor이었다. 그러나 uropygial gland 효소의 product (bicarbonate, pyruvate, NADPH)에 대한 Ki 값은 liver 효소의 Ki 값보다 2-3배정도의 높은 값을 나타냈다. Initial velocity plot과 produt inhibition pattern을 조사해본 결과 효소에 $NADP^+$가 먼저 결합한 후 다음에 malate가 들어와서 결합하게 되면 생성물이 $CO_2$, pyruvate, NADPH 순서로 유리되는 ordered kinetic mechanism을 갖는다는 사실을 알아 내었다. Uropygial gland에서 분리한 malic enzyme은 간에서 분리한 같은 효소에 대하여 이상과 같은 차이점이 있음은 물론 전기이동에 의하여서도 이동거리가 서로 다른 것으로 보아 하나의 isozyme인 것으로 예측되며 uropygial gland의 특별한 역할에 잘 맞도록 구성된 것으로 믿어진다.

다국어 초록 (Multilingual Abstract)

Malic enzyme from the uropygial gland of duck was purified by the combination of 2',5'-ADP agarose and DEAE-Sephacel in the electrophoretically homogeneous form. Purification fold and specific activity were 183 and 174 unit/mg protein, respectively. Molecular size of subunit determined by SDS-polyacrylamide gel electrophoresis was 60,000 dalton. Optimal pH was 7.5 and $Mn^{++}$ or $Mg^{++}$ was required for activity. For the enzyme in uropygial gland the apparent Km for malate and $NADP^+$ were $370{\mu}M$ and $10.6{\mu}M$ respectively. In case of liver enzyme apparent Km for malate and $NADP^+$ were $606{\mu}M$ and $20.8{\mu}M$, respectively. In uropygial gland and liver, the following product inhibition patterns were observed with malate and $NADP^+$ as variable substrates: NADPH, noncompetitive ($Ki_G$, $166{\mu}M$; $Ki_L$, $97{\mu}M$) and competitive($Ki_G$, $43{\mu}M$; $Ki_L$, $22{\mu}M$): pyruvate, uncompetitive($Ki_G$, $26{\mu}M$; $Ki_L$, $11{\mu}M$) and uncompetitive ($Ki_G$, $32{\mu}M$; $Ki_L$, $20{\mu}M$): bicarbonate, noncompetitive ($Ki_G$, $112{\mu}M$; $Ki_L$, $36{\mu}M$) and noncompetitive ($Ki_G$, $183{\mu}M$; $Ki_L$, $63{\mu}M$). In addition to this kinetic difference between the enzymes from the gland and liver, electrophoretic mobility of the gland enzyme was also different from that of the liver enzyme, suggesting that these enzymes are isozymes each other and the gland enzymes are specially designed to meet the spcialized function of the unopygial gland.