단백질합성 억제물질이 효모 mitochondria SDH 및 NADH-DH 효소합성에 미치는 영향에 관한 연구

Abstract

[영문]

[한글]

Eukaryotic cell은 핵 DNA로부터 온 mRMA를 가지고 cytoplasmic ribosome에서, 그리고

mitochondrial DNA로부터 온 mRNA를 가지고 mitochondrial ribosome에서 서로 독립적으로

단백질을 합성할 수 있다. 따라서 mitochondria내막에 존재하는 일부 효소들은 이 두 장

소에서 합성된 단백질이 서로 통합되어 그 기능을 발휘한다.

본 연구는 이러한 단백질의 합성을 ribosome 선상에서 선택적으로 억제하는 cyclohexim

ide와 chloramphenicol을 사용하여 효모세포내의 succinate genase(NADH·DH)의 합성장소

를 밝히고 어떤 요소들이 이들 효소합성에 영향을 미치는가를 구명코저 본 연구를 실시하

여 다음과 같은 결론을 얻었다.

효모(Saccharomyces cerevisiae KFCC-35115)세포를 normal medium에서 30℃의 shaking

water bath에서 배양하면서 1시간 간격으로 세포수와 세포흡광도를 동시에 측정한 결과

세포수의 증가와 흡광도의 증가는 일치하였으며 세포증식이 나타나지 않는 lag time은 1

시간, 그후 8시간까지는 exponential phase로 관찰되었다. 또한 세포증식의 doubling tim

e은 약 2시간으로 측정되었다.

Normal medium에서 배양한 후 chloramphenicol을 첨가하여 다시 배양한 효모세포의 mit

ochondria내의 SDH 및 NADH·DH의 활성은 정상으로 배양한 효모세포의 효소활성과 하등의

차이가 없었다. 이러한 결과는 이들 두 효소들은 mitochondria내에서 합성되지 않음을

시사한다.

효모세포를 혐기성상태로 배양하면 효모세포내의 SDH의 활성을 관찰할 수 없었다. 이와

같이 효소활성이 없어진 효모세포를 호기성상태로 배양하면 SDH의 활성은 정상으로 회복

되었으나 이때 cycloheximide를 첨가한 군의 효소활성이 회복되지 아니하였다. 그러나 NA

DH·DH 활성은 혐기성 및 호기성상태에서 배양한 효모세포에서 차이가 없이 정상으로 유

지되었다. 이 결과로 보아 SDH는 cytoplasmic 80S ribosome에서 합성되며 산소와 밀접한

관계를 가진 효소라 생각된다.

혐기성상태로 배양한 효모세포를 호기성상태로 배양하면 효모성장 및 SDH활성은 10시간

까지는 서로 같은 양상으로 회복되고 그후 평행을 유지하였다. 그러나 NADH·DH활성은 배

양과정의 산소의 유무에 관계없이 효소활성에 하등의 변화가 없는 것으로 보아 SDH의 합

성은 O^^2 -dependent, NADH·DH의 합성은 O^^2 -independent한 것으로써 사료되었다.

효모세포를 포도당 농도를 달리한 repression 배지내에서 배양하면 SDH 및 NADH·DH의

활성은 포도당 농도가 감소함에 따라 급격히 감소하고 SDH활성은 NADH·DH보다 그 영향을

강하게 받는 것으로 나타났는데, 이처럼 배지내 포도당의 농도가 이 두 효소활성에 미치

는 영향에 대한 기전은 아직 밝혀지지 않았다.

효모세포를 repression시키면 SDH 및 N4DH·DH활성은 급격히 감소하고 이를 다시 derep

ression medium에서 배양하면 효소활성이 정상으로 회복되었다. Repression된 효모세포를

derepression medium에서 배양할때 chloramphenicol을 첨가하면 두 효소의 활성은 하등

의 영향를 받지 아니하고 정상으로 회복되었으나, derepression medium에 cycloheximide

를 처리하고 배양하면 이 두 효소활성이 회복되지 않았다. 또한 derepression시 혐기성배

양을하면 SDH의 활성은 회복되지 못하였으나 NADH·DH의 활성은 정상으로 회복되었다.

이상과 같은 결과를 종합하여 보면 NADH·DH 및 SDH는 cytoplasmic ribosome에서 합성

되어 mitochondria막으로 유입되며, repression시킨 효모세포의 SDH 및 NADH·DH의 활성

이 감소되는 현상은 효소합성 감소와 밀접한 관계가 있는 것으로 사료되며, SDH합성은 O^

^2 -dependent, NADH·DH 합성은 O^^2 -independent 하다는 사실을 입증하였다.

Effect of Antibiotics on the Synthesis of SDH and NADH-DH in Yeast Mitochondria

Beon Sook Choi

Department of Medical Science The Graduate School, Yonsei University

(Directed by Professor Yoon Soo Kim M.D., Ph.D.)

Protein synthesis in eukaryotic cell can occur independently at cytoplasmic

ribosome utilizing mRNA from nuclear DNA or at mitochondrial ribosome utilizing

mRNA from mitochondrial DNA, and some functional enzymes localized in mitochondrial

inner membrane are composed of protein subunits synthesized from these two systems.

In an attempt to study the synthetic sites of succinate dehydrogenase and NADH

dehydrogenase in yeast cell and the factors involved in the protein synthesis,

experiments were carried out using cyoloheximide and chloramphenicol which are

known as selective inhibitors of ribosomal protein synthesis level, and the

following results were obtained.

Yeast cells (Saccharomyces cerevisiae KFCC-35115) were cultured at 30℃ in normal

medium, and the cells were counted and optical density was measured at one hour

intervals. The result showed that the increase in cell count was corresponded to

the increase in optical density of cell. Cells were proliferated in exponential

phase after one hour of lag time, and the doubling time of cell growth was about

two hours.

When yeast cells were cultured in the presence of chloramphenicol after culturing

in normal medium, enzyme activities of yeast cell mitochondrial succinate

dehydrogenase and NADH dehydrogenase remained same as in cells cultured in normal

medium, indicating that these two enzymes are not synthesized in mitochondria.

No succinate dehydrogenase activity was detected in the mitochondria of yeast

cells cultured under anaerobic condition for 48 hours. When these cells were then

cultured under aerobic condition, the activity of succinate dehydrogenase was

recovered to its normal value, whereas the enyzme activity was not recovered when

the yeast cells were treated with cycloheximide. However. specific activity of NADH

dehydrogenase remained same level whether the yeast cells were cultured under

anaerobic or aerobic condition.

These results suggest that succinate dehydrogenase is synthesized on cytoplasmic

80 S ribosome and oxygen is an important factor for its synthesis.

Cell growth and succinate dehydrogenase activity were recovered in the same

manner upto 10 hours and reached plateau when yeast cells were cultured under

aerobic condition after culturing under anaerobic condition. However, specific

activity of NADH dehydrogenase showed no change whether the cells were cultured

under aerobic or anaerobic condition, indicating that the synthesis of succinate

dehydrogenase is O^^2 -dependent and the synthesis of NADH-dehydrogenase is O^^2

-independent.

When yeast cells were cultured in repression medium in the presence of varying

concentration of glucose, the specific activities of succinate dehydrogenase and

NADH dehydrogenase were decreased markedly as the concentration of glucose was

decreased. This effect of glucose concentration on the enzyme activity was stronger

far succinate dehydrogenase than NADH dehydrogenase. The mechanism by which the

glucose concentration affects the activities of these two enzymes is not yet known.

The specific activities of succinate dehydrogenase and NADH dehydrogenase

decreased rapidly when cells were cultured in repression medium, and the activities

were recovered when the cell growth was derepressed.

However, the cell cultured in repression medium were then cultured in

derepression medium including chloramphenicol showed no effect on the activities of

the two enzymes, whereas the enzyme activities were not recovered when

cycloheximide was added to the derepression medium. Anaerobic condition of the cell

growth in derepression medium had an inhibitory effect on the recovery of enzyme

activity of succinate dehydrogenase but not that of NADH dehydrogenase.

These results indicate that yeast mitochondrial succinate dehydrogenase and NADH

dehydrogenase are synthesized in the cytoplasmic ribosome and imported into

mitochondrial membrane, and that the decrease in enzyme activities when the cell

growth was repressed is closely related to the decrease in protein synthesis. The

results also provided the evidences that the synthesis of succinate dehydrogenase

is 0^^2 -dependent and that of NADH-dehydrogenase is O^^2 -independent.