Protein carboxyl methyltransferase (PCM)에 의한 메칠화 반응의 생리적 기능을 알아보기 위해 percoll/hypaque density gradient centrifugation 방법으로 사람 적혈구를 노화된 정도에 따라 분류하고, 각 층에서 세포질내의 PCM 활성도와 적혈구 막의 methylatability를 관찰하였다. 그 결과 노화된 정도에 따라 적혈구 막의 methylatability는 증가하였고, 세포질내의 PCM 활성도는 거의 변화가 없었다. 또한 적혈구 막을 열 $(80^{circ}C)$, 염기 (0.1 N $NH_4OH$) 또는 자외선으로 손상을 입힌 후 methylatability를 측정한 결과 시간의 함수로써 증가하였다. 당뇨병 환자 적혈구 막의 methylatability를 조사한 결과 Hb $A_{1c}$/total blood (%)가 커짐에 따라 methylatability가 증가하였다. 메칠화 반응에 관여하는 적혈구 막 단백질의 종류를 알아보기 위해 CPC/PAGE로 분리해 본 결과 band 4.5가 PCM에 대한 좋은 기질임이 확인되었고, 이들 단백질의 methylatability가 노화되어지는 정도에 따라 증가하였다. 당뇨병 환자의 경우 band 3와 band 4.5의 methylatability가 정상인에 비해 증가하였다. 이러한 결과로 볼 때, 이 효소에 대한 기질인 비정상 단백질이 적혈구 노화에 수반되는 deamidation이나 산화적 손상에 의해 증가됨을 제시해 볼 수 있다.
For the purpose of showing the physiological role of methyl esterification by protein carboxyl methyltransferase (PCM) as a post-translational modification, the intact human erythrocyte populations were separated by percoll/hypaque density gradient centrifugation according to erythrocyte aging. PCM activity in cytosolic fraction and methylatability of erythrocyte membrane was measured. The level of cytosolic PCM activity has shown insignificant change according to aging, but a twofold increase of methylation was observed in the oldest cells compared with the youngest ones. From this result, we could presume that abnormal protein as a substrate for the enzyme are formed during deamidation or oxidative damage events which accompany the aging process of protein molecules in cells. We observed the level of methylatability of membrane protein by chemical treatment. After heat treatment $(80^{circ}C)$, base treatment (0.1N $NH_4OH$ at $37^{circ}C$) or UV radiation, the extent of the methylation of the treated membrane also increased as a function of time. We also studied for the diabetic blood. The more increased Hb $A_{1c}$/total blood(%), the more increased methylatability of diabetic erythrocyte membrane. From these results, we could suggest that the ability of methyl esterification by PCM converts damaged protein into normal protein. Erythrocyte membrane protein was separated by CPC/PAGE in order to show protein participated in methylation. Band 4.5 was good substrate, methylatability of these proteins increased according to cell aging. Methylatabilities of band 3 and 4.5 were higher in diabetic blood.
