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RISS 인기검색어
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- 저자 : 양철학 ( Jong Hwa Kim (검색결과 2 건)
- 무료
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Three ${\beta}$-N-Acetyl-D-glucosaminidase from Human Semen : Their Purifications and Properties
김종화,양철학,Kim, Jong-Hwa,Yang, Chul-Hak 생화학분자생물학회 1982 한국생화학회지 Vol.15 No.4
하나의 A형태와 두개의 B형태의 ${\beta}$-N-acetyl-D-glucosaminidase가 사람의 정액으로부터 분리되었다. 사람의 seminal plasma를 희석시킨 다음 $(NH_4)_2SO_4$로 분획 분리시켰다. DEAE-cellulose column으로부터 ${\beta}$-N-acetyl-D-glucosaminidase B는 완충 용액만으로 용출되어졌으며 $A_1$과 $A_2$는 각각 0.1M NaCl과 0.25M NaCl에서 용출되어졌다. 각 효소는 P11 cellulose phosphate와 Sephadex G-200 크로마토그래피법에 의하여 더욱 정제되어졌다. 고순도로 정제된 ${\beta}$-N-acetyl-D-glucosaminidase B는 pH 8.3의 disc gel electrophoresis에서 하나의 단백질 띠로 나타나는 반면, $A_1$과 $B_2$는 하나의 주된 띠와 다른 하나의 가는 띠로 나타났다. 이 세효소는 모두 pH 4.5에 서 가장 높은 활성도를 나타내고 있으며 온도에 대한 가장 높은 활성도는 B 형태가 $50-54^{\circ}C$에서 나타났고 $A_1$과 $A_2$ $43^{\circ}-47^{\circ}C$에서 나타났다. 세 효소는 기질로서 p-nitrophenyl-${\beta}$-N-acetyl-D-glucosaminide를 사용하였을 때 모두 Km값이 1.33 mM로 동일하였다. Sulfite와 acetate 및 N-acetyl-D-glucosamine은 세 효소의 활동도를 모두 방해하였으며 특히 N-acetyl-D-glucosarnine은 ${\beta}$-N-acetyl-D-glucosaminidase들을 경쟁적으로 방해하였으며 Dixon의 그래프법에 의하여 KI값을 구한 결과 B형태는 1.64 mM이었고 A형태는 1.96 mM이었다. 베타-엔-아세틸-디-글루코 사미니다아제의 분자량은 젤 여과법에 의하여 구한 결과 170,000이었다. DEAEcellulose column용출액 중에서의 ${\beta}$-N-acetyl-D-glucosaminidase B는 $60^{\circ}C$에서 4시간 반응한 후에도 70%의 활성도를 유지하였으나 $A_1$과 $A_2$형태의 효소는 대단히 불안정하였다. 그러나 고순도로 정제된 세 효소는 모두 $60^{\circ}C$에서 30분이내에 완전히 활성도를 잃었다. Three ${\beta}$-N-acetyl-D-glucosaminidase (E.C. 3.2.1.30) of one B form and two A forms were isolated from human semen. The enzyme from human seminal plasma was fractionated by $(NH_4)_2SO_4$. By DEAE-cellulose chromatography, ${\beta}$-N-acetyl-D-glucosaminidase B was eluted with buffer alone and $A_1$ form and $A_2$ form of ${\beta}$-N-acetyl-D-glucosaminidase were eluted with 0.1 M NaCl and 0.25 M NaCl respectively. Each enzyme was further purified by P11 cellulose phosphate and Sephadex G-200 chromatography. The highly purified ${\beta}$-N-acetyl-D-glucosaminidase B showed one major protein band and $A_1$ form and $A_2$ form showed one major and one minor bands on disc gel electrophoresis at pH 8.3. Three of the enzyme had maximum activities at pH 4.5, but the temperature optimum was $50-54^{\circ}C$ for B, $43-47^{\circ}C$ for $A_1$ and $A_2$. The three enzymes had identical Km values of 1.33mM with p-nitrophenyl-${\beta}$-N-acetyl-D-glucosaminide as substrate. Sulfite, acetate and ${\beta}$-N-acetyl-D-glucosaminidase the ${\beta}$-N-acetyl-D-glucosaminidase B, $A_1$ and $A_2$ activities. N-acetyl-n-glucosmine inhibited ${\beta}$-N-acetyl-D-glucosaminidase competitively and the $K_I$ values were 1.64 mM for ${\beta}$-N-acetyl-D-glucosaminidase B and 1.96 mM for ${\beta}$-N-acetyl-D-glucosaminidase A by Dixon plot. The molecular weight of human semen ${\beta}$-N-acetyl-D-glucosaminidase was 170,000 by gel filtration. The DEAE-cellulose fractions which showed ${\beta}$-N-acetyl-D-glucosaminidase B activity retained 70% of its activity at $60^{\circ}C$ for 4 hrs, whereas $A_1$ and $A_2$ were heatlabile. However incubation at $60^{\circ}C$ for 30 min completely inactivated the three purified enzymes.
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사람의 정액에서 세 종류의 β - N - Acetyl - D - glucosaminidase 의 분리 및 성질에 관한 연구
김종화,양철학 ( Jong Hwa Kim,Chul Hak Yang ) 생화학분자생물학회 1982 BMB Reports Vol.15 No.4
Three β-N-acetyl-D-glucosaminidases (E. C. 3. 2. 1. 30) of one B form an d two A forms were isolated from human semen. The enzyme from human seminal plasma was fractionated by (NH₄)₂SO₄. By DEAE-cellulose chromatography, β-N-acetyl-D-glucosaminidase B was eluted with buffer alone and A₁ form and A₂ form of β-N-acetyl-D-glucosaminidase were eluted with 0.1 M NaCl and 0.25 M NaCl respectively. Each enzyme was further purified by Pll cellulose phosphate and Sephadex G-200 chromatography. The highly purified β-N-acetyl-D-glucosaminidase B showed one major protein band and A₁ form and A₂ form showed one major and one minor bands on disc gel electrophoresis at pH 8.3. Three of the enzyme had maximum activities at pH 4.5, but the temperature optimum was 50-54 ℃ for B, 43-47 ℃ for A1 and A2. The three enzymes had identical Km values of 1. 33 mM with p-nitrophenyl-β-N-acetyl-D-glucosaminide as substrate. Sulfite, acetate and β-N-acetyl-D-glucosaminidaseinhibited the β-N-acetyl-D-glucosaminidase B, A₁ and A₂ activities. N-acetyl-n-glucosmine inhibited β-N-acetyl-D-glucosaminidase competitively and the KI values were 1. 64 mM for β-N-acetyl-D-glucosaminidase B and 1. 96 mM for β-N-acetyl-D-glucosaminidase A by Dixon plot. The molecular weight of human semen β-N-acetyl-D-glucosaminidase was 170,000 by gel filtration. The DEAE-cellulose fractions which showed β-N-acetyl-D-glucosaminidase B activity retained 70% of its activity at 60 ℃ for 4 hrs, whereas A₁ and A₂ were heatlabile. However incubation at 60 ℃ for 30 ruin completely inactivated the three purified enzymes.
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