Canavanine 이 Lowry 방법에 의한 단백질 정량에 미치는 영향

전방욱,권영명 ( Bang Ook Jun,Young Myung Kwon ) 생화학분자생물학회 1983 BMB Reports Vol.16 No.4

Canavanine, the guanidinooxy structural analog of arginine, significantly treats error in Lowry protein determination by being response to the Lowry reagents. It increases the absorbance of the reagent blank at 750 ㎚ and additively affects the apparent protein value. Two hundred and eighty unit mole of canavanine yields the colorimetric equivalent of 1.0 unit mole of bovine serum albumin. The interference of canavanine can be corrected by the use of appropriate calibration curves or by prior separation of protein from canavanine by trichloroacetic acid precipitation.

Canavanine 에 의한 보리 무배부 (無胚部) 종자의 Amylase 활성과 단백질 함량의 변화

전방욱(Bang Ook Jun),고석찬(Suck Chan Koh),권영명(Young Myung Kwon) 한국식물학회 1983 Journal of Plant Biology Vol.26 No.4

L-canavanine was added to GA_3 treated barley seeds, and induced amylase activity, soluble protein content, and arginine content were measured. Canavanine, added at the beginning of the incubation period, inhibited amylase activity and protein accumulation. Amylase activity decreased markedly by addition of canavanine at 6 hr after incubation, where soluble protein content was not affected. The addition of canavanine after 12 hr incubation did not show serious inhibitory effect on the amylasa activity and protein accumulation. GA_3 incubation caused decrement in arginine content per ㎎ protein, but it was somewhat recovered by canavanine treatment. The longer the time between GA_3 and canavanine addition was, the less the recovery ratio was. Arginine content in the α-amylase fraction (ammonium sulfate 20∼50% saturation) was lower than in 0∼20% fraction, but higher than in 50∼80% fraction. These results and control experiments, using cordycepin and cycloheximide, support the idea that canavanine might incorporate into protein.

Canavanine 존재시 합성된 보리 $\alpha$-Amylase의 정제와 그 생화학적 성질

전방욱,권영명,Jun, Bang-Ook,Kwon, Young-Myung 생화학분자생물학회 1986 한국생화학회지 Vol.19 No.2

$GA_3$에 의하여 유도된 $\alpha$-amylase (대조구의 $\alpha$-amylase)와 $GA_3$ 처리 12시간 경과 후 canavanine을 첨가하여 얻은 $\alpha$-amylase (canavanine 처리구의 $\alpha$-amylase)를 ammonium sulfate분획, CHA-affinity chromatography와 DEAE-cellulose chromatography를 사용하여 정제하였고 이들 두 효소의 성질을 비교 조사하였다. Canavanine 처리구의 $\alpha$-amylase를 아마노산 분석한 결과 $\alpha$-amylase 내에 1.5~1.7 mol의 canavanine이 투입되었음이 확인되었다. 대조구의 $\alpha$-amylase와 canavanine 처리구의 $\alpha$-amylase의 pI값은 각각 5.2와 4.9였다. 그러나 분자량은 50,000 dalton으로 동일하게 나타났으며, 최적온도, 최적 pH, 그리고 반응산물도 동일하였다. 그러나 amylose를 기질로 사용하였을 때의 최대반응속도는 대조구의 $\alpha$-amylase의 경우 canavamne 처리구의 $\alpha$-amylase의 경우보다 1.6배 빨랐다. 그러나 $K_m$값은 동일하였다. 또한 $Ca^{2+}$, $Mg^{2+}$ 및 $Mn^{2+}$과 같은 $\alpha$-amylase의 activator를 첨가하였을 예는 대조구의 $\alpha$-amylase의 경우 $10^{-3}M$보다 $10^{-2}\;M$처리시 그 활성이 높게 나타났으나 canavanine 처리구의 $\alpha$-amylase의 경우 $10^{-3}\;M$ 보다 오히려 그 활성이 감소하였다. 이상의 결과를 종합해 보면 보리종자에 canavanine을 처리하였을 때 canavanine이 $\alpha$-amylase내로 투입되어 효소구조를 변화시킴으로써 효소활성을 감소시키는 것으로 사료된다. The effect of canavanine on the $GA_3$-induced $\alpha$-amylase in barley halfseeds were investigated. $GA_3$-induced $\alpha$-amylase (control $\alpha$-amylase) and the $\alpha$-amylase to which canavanine was added 12 h after $GA_3$ treatment (canavanine treated $\alpha$-amylase) were purified through ammonium sulfate fractionation, CHA-affinity chromatography and DEAE-cellulose chromatography, and the properties of both kinds of enzyme were comparatively studied. The amino acid analysis of canavanine treated $\alpha$-amylase revealed that 1.5-1.7 mol of canavanine was incorporated into $\alpha$-amylase instead of arginine. The pI values of control $\alpha$-amylase and canavanine treated $\alpha$-amylase were 5.2 and 4.9 respectively. However, their molecular weights were determined as the same dalton of 50,000 and their optimum temperature and optimum pH as well as the reaction products were the same in both kinds. While $K_m$ values were the same in both kinds, the maximum enzyme reaction velocity using amylose as substrate were 1.6 times faster for control $\alpha$-amylase than for canavanine treated $\alpha$-amylase. When the enzyme activators such $Ca^{2+}$, $Mg^{2+}$, and $Mn^{2+}$ were treated upon the purified enzymes, the activator concentration of $10^{-3}\;M$ had less effect on the activation of control $\alpha$-amylase then the concentration of $10^{-2}\;M$. However, the situation was reversed in the case of canavanine treated $\alpha$-amylase. In summary, when canavanine was treated upon barley seeds, its incorporation into $\alpha$-amylase is considered to decrease the $\alpha$-amylase activity through. changing the structure of the enzyme.

Error in Lowry Protein Determination by Canavanine

전방욱,권영명,Jun, Bang-Ook,Kwon, Young-Myung 생화학분자생물학회 1983 한국생화학회지 Vol.16 No.4

Canavanine은 arginine의 guanidinooxy structural analog로서 Lowry시약과 반응하여 Lowry 방법에 의한 단백질 정량시 심각한 오차를 일으킨다. Canavanine은 750 nm에서 reagent blank의 흡광도를 증가시키며 또한 단백질의 측정치를 증가시킨다. 280단위몰의 canavanine은 1단위몰의 소혈청단백질과 동일한 흡광도를 나타낸다. 단백질 정량에 미치는 canavanine의 이와 같은 간섭은 적절한 calibration curve를 사용하거나 trichloroacetic acid 첨전법에 의해 단백질로부터 canavanine을 분리하는 방법을 사용하여 제거할 수 있다. Canavanine, the guanidinooxy structural analog of arginine, significantly creats error in Lowry protein determination by being response to the Lowry reagents. It increases the absorbance of the reagent blank at 750nm and additively affects the apparent protein value. Two hundred and eighty unit mole of canavanine yields the colorimetric equivalent of 1.0 unit mole of bovine serum albumin. The interference of canavanine can be corrected by the use of appropriate calibration curves or by prior separation of protein from canavanine by trichloroacetic acid precipitation.

열처리가 보리 종자에서 추출한 Amylase 활성에 미치는 영향

최재은,전방욱,권영명,Choi, Jae-Eun,Jun, Bang-Ook,Kwon, Young-Myung 생화학분자생물학회 1984 한국생화학회지 Vol.17 No.2

보리(Hordeum vulgare L. cv. Baecdong) 종자에서 추출한 $\alpha$-amylase와 $\beta$-amylase의 활성에 미치는 열처리($70^{\circ}C$)의 영향을 조사하였다. 20분 열처리 후 $\beta$-amylase 활성은 완전히 소실되었으며 대부분의 $\alpha$-amylase 활성 역시 소실되었으나 남아 있는 $\alpha$-amylase의 활성을 측정할 수는 있었다. amylase를 전기영동한 결과 20분간 열처리 후 $\alpha$-amylase band는 남아 있었으나 $\beta$-amylase band는 완전히 소설되었다. 이 결과로 미루어 보아 열처리에 의한 $\beta$-amylase의 선택적 불활성화 방법을 $\alpha$-amylase 활성 측청시에나 $\alpha$-amylase 단백질 정제시에 사용할 때는 충분한 검토가 필요함을 알 수 있다. The effect of heat treatment ($70^{\circ}C$) on the activities of $\alpha$-amylase and $\beta$-amylase extracted from barley (Hordeum vulgare L.cv. Baecdong) seeds was investigated. After 20 min-heat treatment, $\beta$-amylase activity was completely lost and most of $\alpha$-amylase activity was lost, but somewhat detectable $\alpha$-amylase activity remained. Electrophoretic study of amylases revealed that $\alpha$-amylase bands remained but $\beta$-amylase bands were completely lost after 20 min-heat treatment. These results suggest that precautions must be taken when selective $\beta$-amylase inactivation by heat treatment is to be employed to assay $\alpha$-amylase activity or to purify $\alpha$-amylase protein.

해녀콩(Canavalia lineata) 자엽 Arginase 의 정제와 효소학적 특성

유경희,전방욱,홍영남,권영명,Yu, Gyung-Hee,Jun, Bang-Ook,Hong, Young-Nam,Kwon, Young-Myung 생화학분자생물학회 1988 한국생화학회지 Vol.21 No.4

해녀콩 (Canavalia lineata ) 자엽의 arginase (L-arginine amidino hydrolase, EC3.5.3.1)를 정제하여서 그 특성을 조사하였다. 이 효소는 황산스트렙토마이신 침전, $AmSO_4$ 분획, DEAE-Sephacel, canavanine-Sepharose 4B affinity gel 및 Sephadex G-200 gel 크로마토그래피를 한 결과 4%의 회수율로 fold는 217배 증가되었다. 이상 3 종류의 컬럼크로마토그래피에서 ADA (arginine-dependent activity)와 CDA (canavanine-dependentactivity)의 활성은 항상 같은 fraction에서 peak를 보였다. 그리고 최적 pH는 ADA의 경우9.0이었고 CDA의 경우 8.0과 8.5이었다. 또한 arginine에 대한 $K_m$은 pH 9.0에서 30 mM, pH 8.0에서 82 mM이였다. 이에 비하여 canavanine에 대한 효소의 $K_m$은 pH 9.0에서 62 mM, pH 8.0에서 43 mM이었다. 이러한 결과에서 생리적인 pH에서는 canavanme에 대한 효소의 친화성이 arginine에 대한 친화성 만큼 높은 것으로 추정할 수 있었다. 1 mM $Mn^{2+}$일 때 효소의 활성이 최대로 나타났으며 최적온도는 $40^{\circ}C$이었다. Sephadex G-200 gel 컬럼에서 arginase의 분자량은 180,000이었으며, SDS-PAGE에서 얻은 subunit의 분자량은 44,000임을 알 수 있었다. Arginase (L-arginine amidino hydrolase, EC 3.5.3.1) from cotyledones of Canavalia lineata was purified and characterized. The purification steps involved streptomycin sulfate precipitation, ammonium sulfate fractionation, DEAE-Sephacel ion-exchange chromatography, canavanine-Sepharose 4B affinity chromatography, and Sephadex G-200 gel filtration chromatography. Arginase was purified 217-fold with 4% recovery. These column chromatography revealed that ADA (arginine-dependent activity) and CDA (canavanine-dependent activity) eluted the same peak. The pH optimum was 9 for ADA and 8 / or 8.5 for CDA. Kinetic analyses of purified arginase for arginine revealed an apparent $K_m$, of 30 mM at pH 9 and 82 mM at pH 8. Comparable determinations with canavanine revealed an apparent $K_m$, of 62 mM at pH 9 and 43 mM at pH 8. It is suggested that the affinity for canavanine with this enzyme at physiological pH be as high as that for arginine. 1 mM $Mn^{2+}$ was required for the optimal enzyme activity and maximum activity was exerted at $40^{\circ}C$. The molecular weight was estimated as 180,000 by Sephadex G-200 gel filtration chromatography and subunit molecular weight was obtained as 44,000 by SDS-PAGE.

해녀콩 ( Canavalia lineata ) 자엽 Arginase 의 정제와 효소학적 특성

유경희,전방욱,홍영남,권영명 ( Gyung Hee Yu,Bang Ook Jun,Young Nam Hong,Young Myung Kwon ) 생화학분자생물학회 1988 BMB Reports Vol.21 No.4

Arginase (L-arginine amidino hydrolase, EC 3.5.3.1) from cotyledones of Canavalia lineata was purified and characterized. The purification steps involved streptomycin sulfate precipitation, ammonium sulfate fractionation, DEAE-Sephacel ion-exchange chromatography, canavanine-Sepharose 4B affinity chromatography, and Sephadex G200 gel filtration chromatography. Arginase was purified 217-fold with 4% recovery. These column chromatography revealed that ADA (arginine-dependent activity) and CDA (canavanine-dependent activity) eluted the same peak. The pH optimum was 9 for ADA and 8 / or 8.5 for CDA. Kinetic analyses of purified arginase for arginine revealed an apparent K_m of 30 mM at pH 9 and 82 mM at pH 8. Comparable determinations with canavanine revealed an apparent K_m of 62 mM at pH 9 and 43 mM at pH 8. It is suggested that the affinity for canavanine with this enzyme at physiological pH be as high as that for arginine. 1 mM Mn^(2+) was required for the optimal enzyme activity and maximum activity was exerted at 40℃. The molecular weight was estimated as 180,000 by Sephadex G200 gel filtration chromatography and subunit molecular weight was obtained as 44,000 by SDS-PAGE.

Canavanine 존재시 합성된 보리 α - Amylase 의 정제와 그 생화학적 성질

권영명,전방욱 생화학분자생물학회 1986 BMB Reports Vol.19 No.2

The effect of canavanine on the GA₃-induced α-amylase in barley halfseeds were investigated. GA3-induced α-amylase (control α-amylase) and the α-amylase to which canavanine was added 12 h after GA₃ treatment (canavanine treated α-amylase) were purified through ammonium sulfate fractionation, CHA-affinity chromatography and DEAE-cellulose chromatography, and the properties of both kinds of enzyme were comparatively studied. The amino acid analysis of canavanine treated α-amylase revealed that 1.5-1.7 ㏖ of canavanine was incorporated into α-amylase instead of arginine. The pI values of control α-amylase and canavanine treated α-amylase were 5.2 and 4.9 respectively. However, their molecular weights were determined as the same dalton of 50,000 and their optimum temperature and optimum pH as well as the reaction products were the same in both kinds. While K_m values were the same in both kinds, the maximum enzyme reaction velocity using amylose as substrate were 1.6 times faster for control α-amylase than for canavanine treated α-amylase. When the enzyme activators such Ca^(2+), Mg^(2+), and Mn^(2+) were treated upon the purified enzymes, the activator concentration of 10^(-3) M had less effect on the activation of control α-amylase then the concentration of 10^(-2) M. However, the situation was reversed in the case of canavanine treated α-amylase. In summary, when canavanine was treated upon barley seeds, its incorporation into α-amylase is considered to decrease the α-amylase activity through changing the structure of the enzyme.

열처리가 보리 종자에서 추출한 Amylase 활성에 미치는 영향

권영명,전방욱,최재은 생화학분자생물학회 1990 BMB Reports Vol.17 No.2

The effect of heat treatment (70℃) on the activities of α-amylase and α-amylase extracted from barley (Hordeum vudgare L.cv. Baecdong) seeds was investigated. After 20 min-heat treatment, α-amylase activity was completely lost and most of α-amylase activity was lost, but somewhat detectable α-amylase activity remained. Electrophoretic study of amylases revealed that α-amylase bands remained but α-amylase bands were completely lost after 20 min-heat treatment. These results suggest that precautions must be taken when selective α-amylase inactivation by heat treatment is to be employed to assay α-amylase activity or to purify α-amylase protein.