Nonpigmenting Serratia sp . 에서 균체의 단백질 분해효소의 생성과 정제에 관한 연구

김승수 ( Soung Soo Kim ) 한국미생물생명공학회 1985 한국미생물·생명공학회지 Vol.13 No.4

Nonpigmenting Serratia sp . 에서 생성되는 균체 외 alkaline Protease 의 특성규명

김승수 ( Soung Soo Kim ) 생화학분자생물학회 1985 BMB Reports Vol.18 No.4

Some chemicial and physical properties of the extracellular alkaline protease from a nonpigmented Serratia sp. are described. Enzyme purification was achieved by ammonium sulfate fractionation and DEAE-cellulose chromatography. The purified enzyme has a broad pH optimum in the alkaline range with a maximum around pH 9. The enzyme was stable at 40℃ over the pH range from 6 to 8, wherease it was unstable at 30℃ in alkaline condition and inactivated completely by heating at 55℃ for 15 min. The purified enzyme moved as a single symmetrical peak with a S^0_(20,w) of 4.18 in ultracentrifugation and the molecular weight was determined to be 40,000 by Yphantis sedimentation equilibrium. Amino acid analysis of the enzyme indicated no sulfur-containing amino acids in the purified enzyme preparation. Specificity studies using carboxymethyl β-insulin and trypsin digested insulin as substrates reveal that the specificity of the purified protease on these substrates is very similar to the protease of pigmented Serriatia marcescens ATCC 25419.

한국산 무우로부터의 isoperoxidase 에 관한 연구

유원일,김승수 ( Won Il Yoo,Soung Soo Kim ) 생화학분자생물학회 1988 BMB Reports Vol.21 No.3

An anionic isoperoxidase, named A₂, was isolated from Korean-radish (Raphanus Sativas L.) root. Purification of the enzyme was accomplished by using CM-Sephadex chromatography, DEAE-Sephacel chromatography and Sephadex G-100 gel filtration. The enzyme was glycosylated and its molecular weight was approximately 43,000 as determined by SDS-PAGE and 43,700 by Sephadex G-150 gel filtration. Isoperoxidase A₂ was a hemoprotein that had RZ value (A_(439)/A_(275)) of 1.5. The Km values for guaiacol and H₂O₂ were 6.7 and 1.38 mM, respectively. The Km value against o-dianisidine was 0.63 mM. Optimal pHs and Km values were also determined for various phenolic compounds such as scopoletin, caffeic acid, esculetin, chlorogenic acid and ferulic acid.

담배 조직배양에서 6 - Phosphogluconater Dehydrogenase 의 정제와 성질에 관한 연구

최현민,김승수 ( Hyun Min Choi,Soung Soo Kim ) 생화학분자생물학회 1984 BMB Reports Vol.17 No.4

One 6-phosphogluconate dehydrogenase isoenzyme, designated 6PG DH II, was purified from tobacco callus culture line (Nicotiana tobacum L., var. Virginia 115) utilizing ammonium sulfate precipitation, DEAE-cellulose chromatography, Red Sepharose CL-6 B affinity chromatography followed by Sephadex G-100 gel filtration. The purified enzyme was homogeneous according to the criteria of polyacrylamide gel electrophoresis. 6 PG DH II appears to have a dimeric structure with subunit molecular weight of 44,000 and native molecular weight of approximately 83,000. Double reciprocal plot of reaction velocity vs. 6-phosphogluconate concentrations indicated Km values of 17 μM with a marked inhibition of initial velocity at high 6-phosphogluconate concentrations.The NADP^+ double reciprocal plot showed an abrupt increase in the apparent Km and Vmax with increasing NADP^+. The two Km`s for low and high NADP^+ concentrations were 6.5 μM and 70 μM, respectively. The pH optimum of the enzyme was 8.5 in Tris-HCl and 8.0 in phosphate buffer. MgCl₂ was not required for the enzymatic activity. Furthermore, MgCl₂ inhibited the activity at high concentration.

In Vitro Translation and Characterization of Peroxidase mRNAs from Tobacco Callus

김재종,김승수,Kim, Jae-Jong,Kim, Soung-Soo 생화학분자생물학회 1987 한국생화학회지 Vol.20 No.3

담배 callus (Nicotiana tabacum L., var Virginia 115)로 부터 guanidium thiocyanate 및 guanidine HCl 방법을 이용 total cellular RNA를 분리 하였으며 oligo (dT)-cellulose 친화성 크로마토그라피 방법에 의해 $poly(A)^+$ RNA를 분리하였다. Protein-synthesizing cell-free system을 이용한 peroxidase mRNA의 translation을 위 해 rabbit reticulocyte lysate의 peroxidase mRNA에 대한 최적 translation 조건을 조사한 결과 2mM $Mg^{2+}$, 80 mM $K^+$, $15\;{\mu}M$ hemine 및 $1\;{\mu}g$의 $poly(A)^+$ RNA 일때 최대의 방사능 유입과 peroxidase 활성을 나타냈다. 그러나 어떤 peroxidase population에 있어서는 1 mM $Mg^{2+}$을 최적 조건으로 갖는 경우도 있었다. 6M urea agarose electrophoresis에 의해 total cellular RNA를 분석해 본 결과 16S 및 23S 부근에서 peroxidase mRNA를 확인할 수 있었다. 또한 Anti isoperoxidase $C_3$ 및 $A_4$antiserum을 이용한 immunoprecipitation에 의해 in vitro translation products 내에서 anti $C_3$와 crossreactivity를 갖는 isoperoxidase $C_3$, $C_4$, $C_1/C_2$ 및 anti $A_4$와 crossreactivity를 갖는 isoperoxidase $A_4$, $A_1$을 확인할 수 있었다. Total celluar RNA was isolated from tobacco callus (Nicotiana tabacum L., var Virginia 115) with guanidium thiocynate and guanidine HCl method. The total RNA was further fractionated by oligo (dT)-cellulose chromatography. For translation of peroxidase mRNAs in cell-free protein-synthesizing system, rabbit reticulocyte lysate was tested and the optimum translational conditions of peroxidase mRNAs were established. The lysate containing 2 mM $Mg^{2+}$, 80 mM $ K^+$ and $15\;{\mu}M$ hemine was found to be the optimum condition for the translation of one population of peroxidase mRNAs. On the other hand, 1 mM $Mg^{2+}$ was the optimum for the other population of peroxidase mRNAs. The incorporation of [$^{35}S$]-Met into peroxidase was increased up to $1\;{\mu}g$ of $poly(A)^+$ RNA and then it was decreased. Size fractionation of total cellular RNA was carried out by 6M urea agarose electrophoresis. Peroxidase mRNAs were distributed at slightly above 16S and 23S regions. As a result of immunoprecipitation of in vitro translation products with anti isoperoxidase $C_3$ and $A_4$ antiserum, we found $C_3$, $C_1$ or/and $C_2$, $C_4$ crossreactive to anti $C_3$ antiserum and $A_4$, $A_1$ to anti $A_4$antiserum.

Shoot 을 형성시키는 두 호르몬 levels 에 있어서 peroxidase 의 활성과 isoenzyme pattern 변화에 관한 연구

김은성,김승수 ( Eun Sung Kim,Soung Soo Kim ) 생화학분자생물학회 1985 BMB Reports Vol.18 No.2

The effects of various ratios of kinetin and IAA in media on shoot initiation, peroxidase activity and isoperoxidase pattern changes of tobacco callus were examined. Tissues grown on 5×10^(-7) M kinetin/10^(-8) M IAA or 10^(-5) M kinetin/10^(-5) M IAA began to form masses of cells in nodular form for shoot initiation after 15 days. The peroxidase activities of shoot intiating calli were higher than the normal non-shoot forming calli at the initial stage of shoot formation. It was also noted that the activity of peroxidase in 10^(-8) M IAA cultures was about twice higher than that of 10^(-5)M kinetin cultures. The increased enzyme activity in 10^(-8) M IAA cultures was due to the increased amount of cathodic isoperoxidases while anodic isoperoxidases were responsible for the increase of peroxidase activity in 10^(-5)M kinetin cultures. The results indicate that kinds or groups of isoperoxidases not over-all concentration of enzymes are important in shoot initiating process.

Serratia marcescens 로 부터 두 가지 형태의 acetolactate Synthease 분리와 성질

양정희,김승수 ( Jeong Hee Yang,Soung Soo Kim ) 생화학분자생물학회 1992 BMB Reports Vol.25 No.1

Two forms of Serratia marcescens acetolactate synthase (designated ALS I and ALS II) have been separated by utilizing DEAE-Sephacel column chromatography with the combination of stepwise and a linear gradient elution of increasing ionic strength. The native molecular weights of the two forms of ALS as determined by Sephacryl S-400 gel filtration method appears to be 572,000 and 531,400 for ALS I and ALS II, respectively. Feedback inhibition studies indicate that ALS I is far more sensitive to inhibition by valine, leucine and isoleucine than ALS II. On the other hand, ALS II is far more sensitive to α-ketobutyrate inhibition than ALS I. The two forms of ALS also have differences in sensitivity against two different classes of herbicides, sulfonylurea and imidazolinone. These data suggest that the two forms of ALS are physically and functionally distinct.

Peroxidase mRNA 의 In vitro Translation 및 특성에 관한 연구

김재종,김승수 ( Jae Jong Kim,Soung Soo Kim ) 생화학분자생물학회 1987 BMB Reports Vol.20 No.3

Total celluar RNA was isolated from tobacco callus (Nicotiana tabacum L., var Virginia 115) with guanidium thiocynate and guanidine HCl method. The total RNA was further fractionated by oligo (dT)-cellulose chromatography. For translation of peroxidase mRNAs in cell-free protein-synthesizing system, rabbit reticulocyte lysate was tested and the optimum translational conditions of peroxidase mRNAs were established. The lysate containing 2 mM Mg^(2+), 80 mM K^+ and 15 μM hemine was found to be the optimum condition for the translation of one population of peroxidase mRNAs. On the other hand, 1 mM Mg^(2+) was the optimum for the other population of peroxidase mRNAs. The incorporation of [^(35)S]-Met into peroxidase was increased up to 1 ㎍ of poly(A)^+ RNA and then it was decreased. Size fractionation of total cellular RNA was carried out by 6M urea agarose electrophoresis. Peroxidase mRNAs were distributed at slightly above 16S and 23S regions. As a result of immunoprecipitation of in vitro translation products with anti isoperoxidase C₃ and A₄ antiserum, we found C₃, C₁ or/and C₂, C₄ crossreactive to anti C₃ antiserum and A₄, A₁ to anti A₄ antiserum.

Diethylpyrocarbonate 에 의한 토끼 헤모글로빈의 Peroxidase 활성의 불활성화

주한승,김승수 ( Han Seung Joo,Soung Soo Kim ) 생화학분자생물학회 1991 BMB Reports Vol.24 No.6

Diethylpyrocarbonate inhibits the peroxidase activity of rabbit hemoglobin with a second order rate constant of 220 M^(-1)min^(-1) at pH 6.0 and 30℃. The increase in absorbance at 242 nm due to formation of carbethoxyhistidyl derivatives and other modification studies suggest that the modification of histidine residues is responsible for the loss of activity. However, treatment of inactivated hemoglobin with hydroxylamine does not restore catalytic activity due to the inhibitory effect of hydroxylamine itself on the peroxidase activity of hemoglobin. The substrate guaiacol have partially reduced the diethylpyrocarbonate dependent inactivation, and a reconstitution experiment using diethylpyrocarbonate treated apohemoglobin shows that it is not reconstituted to hemoglobin having full activity. The decrease of absorption at Soret region (406 nm) upon diethylpyrocarbonate modification of native hemoglobin and the acceleration of inactivation by preincubation of the diethylpyrocarbonate inactivated hemoglobin with H₂O₂ (5 mM) implies that the essential residue is distal histidine as previously proposed in case of cytochrome C peroxidase.

담배 Callus 와 Shoots 에서 Isoperoxidase 와 Translatable Isoperoxidase mRNAs 의 비교 연구

김은성,김승수 ( Eun Sung Kim,Soung Soo Kim ) 생화학분자생물학회 1985 BMB Reports Vol.18 No.3

Tobacco callus grown on 5×10^(-7) M kinetin/10^(-8) M IAA or 10^(-5) M kinetin/10^(-5)IAA formed shoots after 15-20 days of culture. The total peroxidase activities in shoots were lower than those of non-shoot-forming callus. However, cathodic isoperoxidases were increased significantly in shoots formed on 10^(-8) M IAA media while anodic isoperoxidases were increased in shoots formed on 10^(-5) M kinetin media as is the case of shoot initiating callus. The gene expression patterns of isoperoxidases in shoot and non-shoot-forming tissue were also examined by rabbit reticulocyte lysate in vitro translation system. These studies indicate that fraction of translatable poly(A)isoperoxidase mRNA was increased considerably in shoots formed on 10^(-5) M kinetin, whereas poly(A)^+ isoperoxidase mRNA contents were twice higher than poly(A)isoperoxidase mRNAs in non-shoot-forming callus. At the present time, at least five isoperoxidases could be detected from the translation mixture of total cellular RNA. Preliminary translation data indicate that less cathodic isoperoxidases and more anodic isoperoxidases were expressed from poly(A)^- RNAs than from poly(A)^+ RNAs.