Degradation of Muscle Proteins by Lysosomal Hydrolases

조무제,Cho, Moo-Je 생화학분자생물학회 1982 한국생화학회지 Vol.15 No.1

백혈구 lysosome으로부터 n-butanol로 추출한 효소를 투석 냉동건조 한 후 0.05 M 인산완충액 (pH 7.0)에 1 mg/ml의 농도로 용해시킨후 우육에다 처리한후 $37^{\circ}C$ 및 $4^{\circ}C$ 에서 각각 12시간 및 7일간 보존하면서 lysosome 효소에 의한 육조직 및 섬유단백질에의 변화를 전자현미경 및 전기영동법으로 관찰한 결과 Z-line, M-line 및 transverse ridges등이 lysosome 효소에 의하여 심한 분해를 받은것이 전자현미경적으로 관찰되었으며 섬유단백질 중에서는 myosin heavy chain, 및 ${\alpha}$-actinin은 lysosome 효소에 아주 민감한 반면 actin 및 troponin T는 비교적 안정함이 SDS-polyacrylamide gel 전기영동으로 관찰되었다. Ultrastructural and biochemical changes of bovine psoas muscle produced by leukocyte lysosomal hydrolases in vitro were studied at two different temperature, 37 and $4^{\circ}C$ by SDS-polyacrylamide gel electrophoresis, scanning and transmission electron microscopy. Severe degradation of endomysial and sacolemmal connective tissue, transverse ridges, Z-lines and M-lines were observed in lysosomal enzyme treated muscle by electron microscopy. Myosin heavy chain and ${\alpha}$-actinin were observed to be susceptible but actin and troponin-T was very stable to lysosomal hydrolases by SDS-polyacrylamide gel electrophoresis.

알파파 근류로부터 분리한 Poly ( A+ ) - mRNA 에 의하여 합성된 Leghemoglobin 유전자의 cDNA Cloning 에 관한연구

조무제,윤한대,최용락,최영주,이경상,Winstion J . Brill ( Moo Je Cho,Min Suk Yang,Han Dae Yun,Yong Lark Choi,Young Ju Choi,Kyung Sang Lee ) 생화학분자생물학회 1985 BMB Reports Vol.18 No.1

Complementary DNAs were synthesized by the leghemoglobin message rich 9S, 18S and 28S-mRNA fractions prepared from the poly(A)^+-mRNA of alfalfa root nodules. The synthetic Sal I linkered double stranded cDNA was inserted into the SalI site of the pBR322, and transformed in E. coli HB101. The leghemoglobin cDNA clones were screened by colony hybridization and Southern blot hybridization with the soybean leghemoglobin cDNA as a probe, and further screened by hybrid-released and hybridarrested translation. One alfalfa leghemoglobin cDNA clone was selected, and restriction map and fingerprints of the cDNA from the selected clone were analyzed.

Lysosomal Enzyme에 의(依)한 근육조직(筋肉組織)의 변화(變化) -제(第)2보(報) Scanning Electron Microscopy에 의(依)한 고찰(考察)-

조무제,윤태규,베일리밀톤이,Cho, Moo-Je,Yoon, Tae-Gyu,Bailey, Milton E. Korean Society of Food Science and Technology 1978 한국식품과학회지 Vol.10 No.1

맥백혈구(脈白血球) Lysosomal 효소를 pH(7.0, 4.0), 온도($37^{\circ}C$, $4^{\circ}C$) 및 처리기간($37^{\circ}C$에서 12 24시간, $4^{\circ}C$에서 36 168시간)을 달리하여 처리(處理)한 우(牛)의 요근조직(腰筋組織)의 변화(變化)에 대(對)하여 endomysial connective tissue, sarcolemma 및 transverse ridge등 근(筋)섬유 표면조직(表面組織)의 변화(變化)를 SEM으로 관찰한 바, 처리온도(溫度)에 관계(關係)없이 pH 7.0에서는 endomysial connective tissue, sarcolenna 및 transverse ridge의 분해(分解)를 나타내어 이 효소의 높은 역가(力價)를 보였으나, pH4.0에서는 이들 표면조직(表面組織)에 변화(變化)가 없었으며 특(特)히 transverse ridge는 $37^{\circ}C$에서 24시간, 그리고 $4^{\circ}C$에서 7일간 처리(處理)하여도 변화(變化)를 나타내지 않아 안정(安定)됨을 보였다. Surface ultrastructural changes in endomysial connective tissue, sarcolemma and transverse ridges of bovine psoas muscle produced by leukocyte lysosomal enzymes in vitro at different pH (pH 7.0 and 4.0), temperature (37 and $4^{\circ}C$) and time interval (12, 24 hours at $37^{\circ}C$ and 36, 168 hours at $4^{\circ}C$ were studied by scanning electron microscope. Muscle incubated with leukocyte lysosomal enzymes at pH 7.0 produced severe degradation of endomysial and sarcolemmal connective tissue and transverse ridges but at pH 4.0 endomysial and sarcolemmal structures remain moderately stable and tranverse ridges are very stable even after 24 hours incubation at $37^{\circ}C$ and 7 days incubation at $4^{\circ}C$.

알파파 Leghemoglobin mRNA 의 분리 정제 및 in vitro Translation 에 관하여

조무제,양민식,윤한대,최용락,최영주,이경상 ( Moo Je Cho,Min Suk Yang,Han Dae Yun,Yong Lark Choi,Young Ju Choi,Kyung Sang Lee ) 생화학분자생물학회 1985 BMB Reports Vol.18 No.1

The five components of leghemoglobin from alfalfa root nodules were purified by DEAE-cellulose(DE52) and Sephadex G-100 chromatography and antiserum against the purified alfalfa leghemoglobin was prepared. RNA was extracted from alfalfa root nodules and poly(A)-mRNA was purified by oligo(dT)-cellulose affinity chromatography. The leghemolgobin messages were observed to be 23 % of the poly(A)-mRNA. The poly(A)-mRNA was fractionated by sucrose density gradient centrifugation and the mRNA had 9, 18 and 28S value populations with 58, 21 and 36 % of the leghemoglobin messages.

Purification of Leghemoglobin mRNA from Alfalfa(Medicago Sativa L. Vernal) Root Nodules and In Vitro Translation

조무제,양민석,윤한대,최용락,최영주,이경상,Cho, Moo-Je,Yang, Min-Suk,Yun, Han-Dae,Choi, Yong-Lark,Choi, Young-Ju,Lee, Kyung-Sang 생화학분자생물학회 1985 한국생화학회지 Vol.18 No.1

알파파 근류로부터 DEAE-Cellulose (DE52) 및 Sephadex G-100 크로마토그라피에 의하여 5종의 leghemoglobin component를 분리 정제한 후 이것을 이용하여 antlserum을 만들었다. 한편 알팔파 근류에서 분리한 RNA로 부터 oligo (dT)-cellulose 크로마토그라피에 의하여 poly $(A)^+$-mRNA를 분리 정제하고 이들을 sucrose density gradient에 의하여 분획하여 in vitro translation시킨 후 면역 침전법에 의하여 leghemoglobin message 함유비가 높은 9S, 18S 및 28S mRNA를 정제하였는데 각 fraction의 leghemoglobin message 함유비는 각각 58%, 27% 및 36% 였다. The five components of leghemoglobin from alfalfa root nodules were purified by DEAE-cellulose(DE52) and Sephadex G-100 chromatography and antiserum against the purified alfalfa leghemoglobin was prepared. RNA was extracted from alfalfa root nodules and poly(A)-mRNA was purified by oligo(dT)-cellulose affinity chromatography. The leghemolgobin messages were observed to be 23% of the poly(A)-mRNA. The poly(A)-mRNA was fractionated by sucrose density gradient centrifugation and the mRNA had 9, 18 and 28S value populations with 58, 21 and 36% of the leghemoglobin messages.

Cloning of nitA Gene from Klebsiella pneumoniae and Expression in Azospirillum

조무제,정명철,강규영,갈상완,Cho, Moo-Je,Chung, Myung-Chul,Kang, Kyu-Young,Gal, Sang-Wan 생화학분자생물학회 1989 한국생화학회지 Vol.22 No.3

Klebsiella pneumoniae로부터 nifA 유전자를 크로닝하여 Azospirillum에 도입 발현시킴으로서 암모니아 비제어 질소고정을 유도하고자 하였다. Azospirillum 숙주에서 안정하게 복제 유지될 수 있는 vector로서 RK2 (IncP-1 group plasmid) 변형 vector인 pRK290과 pVK100를 선별하였으며 pKT230 (RSF1010 변형 vector, IncQ group) 및 pSa151(pSa 변형 vector, IncW group)는 불안정하였다. nifA 유전자 크로닝은 nifA 3.0 kb Sal1 절편을 pRK290의 Bgl II 위치에 그리고 pVK100의 Xho1 위치에 각각 크로닝하고 pMK23 및 pMK26으로 명명하였다. pMK23에서 nifA의 발현은 $Tc^r$ 유전자 promoter pMK 26에서는 $Km^r$ 유전자 promoter에 의하여 constitutive expression을 유도하였다. A lipoferum KY 6 에서는 16mM의 암모니아 첨가로 질소고정효소의 발현이 완전히 억제되었으나 A.lipoferum KY6 (pMK23) 및 A.lipoferum KY6 (pMK26)에서는 암모니아를 첨가하지 않았을 경우의 질소고정력의 18% 및 10%를 나타내므로써 K. pneumoniae nifA 유전자의 Azospirillum에서 발현이 확인되었다. 한편 암모니아 존재하에서의 질소고정 효소 합성도 전기영동 결과 확인되었다. The nifA gene in the 3.0 kb SalI fragment from the nifA gene cluster of Klebsiella pneumoniae was cloned into BglII site of pRK290 and XhoI site of pVK100, a derivative of a wide host range plasmid RK2(Inc P-1 group) and named pMK23 and pMK26, respectively. The constructed pMK23 and pMK26 carring nifA gene could be introduced into Azospirillum by transformation and conjugation with the helper plasmid pRK2013 and maintained stably in the host in constrast to some instability of RSF1010 (Inc Q group) or pSa (Inc W group)-derived vector. The nifA gene in the former recombinant nifA plasmid was expressed under the $Tc^r$ gene promoter of pRK290 and the latter under the $Km^r$ gene promoter of pVK100 in the Azospirillum host. The degree of derepression of the nitrogenase activity by the nifA expression from K. pneumoniae in the presence of 16mM ammonia was about 18% in A. lipoferum (pMK23) and 10% in A. lipoferum (pMK26) compared to the nitrogenase activity in the absence of fixed nitrogen. Different level of MoFe-protein (nitrogenase component I) and Fe-protein (nitrogenase component II) synthesis between nifA plasmid introduced Azospirillum transconjugant and wild type were observed in the cells grown with the fixed nitrogen.

Molecular Cloning of Alfalfa(Medicago Sativa L. Vernal) Leghemoglobin Structural Genes Synthesized by $Poly(A)^+$-mRNA from Alfalfa Root Nodules

조무제,윤한대,최용락,최영주,이경상,Cho, Moo-Je,Yun, Han-Dae,Choi, Yong-Lark,Choi, Young-Ju,Lee, Kyung-Sang,Brill, Winston J. 생화학분자생물학회 1985 한국생화학회지 Vol.18 No.1

알팔파 근류에서 분리 정제한 $poly(A)^+$-mRNA로부터 leghemoglobin message 함유비율이 높은 9S, 18S 및 28S mRNA를 이용하여 cDNA를 합성하였다. 이들 cDNA를 Sal I linker에 연결 pBR322의 Sal I site에 크로닝하여 cDNA library를 만든 후 이들 library로부터 colony hybridization, Southern blot hybridization, hybrid-released 및 hybrid-arrestes translation에 의하여 leghemoglobin cDNA clone을 동정하였으며, 동정된 clone으로부터 alfalfa leghemoglobin cDNA의 제한효소 지도를 작성하였다. Complementary DNAs were synthesized by the leghemoglobin message rich 9S, 18S and 28S-mRNA fractions prepared from the $poly(A)^+$-mRNA of alfalfa root nodules. The synthetic Sal I linkered double stranded cDNA was inserted into the Sal I site of the pBR322, and transformed in E. coli HB101. The leghemoglobin cDNA clones were screened by colony hybridization and Southern blot hybridization with the soybean leghemoglobin cDNA as a probe, and further screened by hybrid-released and hybrid-arrested translation. One alfalfa leghemoglobin cDNA clone was selected, and restriction map and fingerprints of the cDNA from the selected clone were analyzed.

Klebsiella pneumoniae 로 부터 nif A Gene 의 크로닝 및 Azospirillum 에서의 발현

조무제,정명철,강규영,갈상완 ( Moo Je Cho,Myung Chul Chung,Kyu Young Kang,Sang Wan Gal ) 생화학분자생물학회 1989 BMB Reports Vol.22 No.3

The nifA gene in the 3.0 kb SalI fragment from the nifA gene cluster of Klebsiella pneumoniae was cloned into BglII site of pRK290 and XhoI site of pVK100, a derivative of a wide host range plasmid RK2 (Inc P-1 group) and named pMK23 and pMK26, respectively. The constructed pMK23 and pMK26 carring nifA gene could be introduced into Azospirillum by transformation and conjugation with the helper plasmid pRK2013 and maintained stably in the host in constrast to some instability of RSF1010 (Inc Q group) or pSa (Inc W group)-derived vector. The nifA gene in the former recombinant nifA plasmid was expressed under the Tc^r gene promoter of pRK290 and the latter under the Km^r gene promoter of pVK100 in the Azospirillum host. The degree of derepression of the nitrogenase activity by the nifA expression from K. pneumoniae in the presence of 16 mM ammonia was about 18% in A. lipoferum (pMK23) and 10% in A. lipoferum (pMK26) compared to the nitrogenase activity in the absence of fixed nitrogen. Different level of MoFe-protein (nitrogenase component I) and Fe-protein (nitrogenase component II) synthesis between nifA plasmid introduced Azospirillum transconjugant and wild type were observed in the cells grown with the fixed nitrogen.

Legume - Rhizobium Symbiosis 와 관련된 기주식물유전자의 구조 및 발현

조무제 ( Moo Je Cho ) 한국생화학분자생물학회 (구 한국생화학회) 1986 생화학총설집 Vol.1 No.-

Lysosomal Hydrolase 에 의한 육단백질의 분해

조무제 ( Moo Je Cho ) 생화학분자생물학회 1982 BMB Reports Vol.15 No.1

Ultrastructural and biochemical changes of bovine psoas muscle produced by leukocyte lysosomal hydrolases in vitro were studied at two different temperature, 37 and 4℃ by SDS-polyacrylamide gel electrophoresis, scanning and transmission electron microscopy. Severe degradation of endomysial and sacolemmal connective tissue, transverse ridges, Z-lines and M-lines were observed in lysosomal enzyme treated muscle by electron microscopy. Myosin heavy chain and α-actinin were observed to be susceptible but actin and troponin-T was very stable to lysosomal hydrolases by SDS-polyacrylamide gel electrophoresis.