• Title/Summary/Keyword: Gel filtration

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Heterogeneity of Lactate Dehyrogenase Isozymes in tissues of Lampetra japonica (칠성장어(Lampetra japonica) 조직내 젖산수소이탈효소 동위효소들의 이질성)

  • 조성규;염정주
    • The Korean Journal of Zoology
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    • v.36 no.3
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    • pp.319-328
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    • 1993
  • All vertebrates other than lampreys exhibit multiple loci encoding lactate dehydrogenase (EC 1.1.1.27,LDH). From the result shown by cellulose acetate and starch gel electrophoresis, the lampreys were-reported to have only one isozyme. However in our results the LDH of skeletal muscle, heart and kidney in Lampetra japonica were separated into three isozymes and that of liver was separated into two isozymes by polyacrylamide gel electrophoresis. The LDH of skeletal muscle and heart were separated into four isozymes and that of liver was separated into two isozymes by polyacrylamide gel isoelectric focusing (PAGlEF). The LDH of skeletal muscle were separated into four isozymes through the chromatofocusing. The molecular weight of LDH isozymes in skeletal muscle was approximately estimated to be 140,000 by Sephadex G-200 gel filtration. The LDH isozymes of skeletal muscle, heart and liver were inhibited by pyruvate to the nearly similar degree. And the degree of inhibition by pyruvate showed the value between LDH A$_4$and LDH B$_4$isozyme. And the LDH isozymes in heart, liver and skeletal muscle were thermostable. The results mentioned above indicate that the LDH isozyme in lamprey (Lampetra japonica) has not one isozyme but isozymes. And it is also found out that the two structures of their subunits are similar each others.

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Partial Purification and Properties of Non-specific $\beta$ -fructofuranosidase Produced by Bacillus subtilis (Bacillus subtilis가 생산하는 비특이적 $\beta$-fructofuransoidase의 부분정제 및 특성)

  • 송근섭;엄태붕
    • Microbiology and Biotechnology Letters
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    • v.18 no.5
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    • pp.484-489
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    • 1990
  • An intracellular inulase ( fJ-fructofuranoside fructohydrolase, EC 3.2.1.26) from Bacillus subtilis has been partially purified and its mode of action and general properties were studied. The enzyme had an apparent molecular weight of 49,000 as estimated by gel filtration and its pI point was 5.2. Substrate concentration studies showed an apparent Km of 10 mM for sucrose and of 18 mM for raffinose. The enzyme was an acid-labile protein with a pH optimum of 6.6. The optimum temperature was 50$^{\circ}$C. The enzyme acts on straight chain oligo- and poly-fructosides of the inulin series via a exo-wise cleavage mechanism, as well as on sucrose.

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Purification and Characterization of Mouse Liver Rhodanese

  • Lee, Chul-Young;Hwang, Jae-Hoon;Lee, Young-Seek;Cho, Key-Seung
    • BMB Reports
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    • v.28 no.2
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    • pp.170-176
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    • 1995
  • Rhodanese from mouse liver was purified to near homogeneity by ammonium sulfate precipitation, CM-Sephadex ion exchange, hydroxyapatite and Sephacryl S-200-HR gel filtration chromatographies with a purification of 776 folds. The molecular weight was determined by Sephadex G-150 gel filtration and found to be 34.8 KDa. SOS-PAGE showed molecular weight 34 KDa and two identical subunits splitting by aging for 3 weeks at $-70^{\circ}C$ the molecular weight of which was 17 KDa. The optimal pH of enzyme activity was 9.4 and the pI value of the enzyme was 6.6. Rhodanese showed the optimal reaction temperature of $25^{\circ}C$ and near linear increasing pattern until 10 min. incubation. $K_m$ values of rhodanese for KCN and $Na_{2}S_{2}O_{3}$ as substrates were 12.5 mM and 8.3 mM, respectively. Rhodanese activity was inhibited by more than 70% at a concentration of 100 ${\mu}M$ of $Ni^{2+}$, $Zn^{2+}$, $Cd^{2+}$, $Hg^{2+}$ and $Cu^{2+}$. Other metal ions, such as $Mn^{2+}$, $Mg^{2-}$, $Ca^{2+}$, and $Fe^{2+}$ showed no effect on rhodanese activity.

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Purification and Characterization of Protein Methylase II from Porcine Testis

  • Jung, Ki-Kyung;Kwon, Myung-Hee;Lee, Hoi-Young;Lee, Hyang-Woo;Hong, Sung-Youl
    • BMB Reports
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    • v.28 no.2
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    • pp.149-154
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    • 1995
  • Protein methylase II (S-adenosyl-L-methionine : protein O-methyl-transferase, EC 2.1.1.24; PM II) was purified approximately 1250-fold from porcine testis by fractional precipitation and DEAE-cellulose chromatography, followed by gel filtration on a Sephadex G-75 column and HPLC on a Protein Pak 125 column. The molecular weight of the enzyme was estimated to be 33,000 daltons by SDS-PAGE, which agreed with the value determined by gel filtration. Isoelectric focusing of purified PM II showed a single protein species with an isoelectric point of 6.2. The optimum pH for the reaction was 6.0. The $K_m$ value of the enzyme was $1{\times}10^{-5}M$ with a $V_{max}$ value of 769 pmol/min/mg of enzyme. S-adenosyl-L-homocysteine is a competitive inhibitor of PM II with a $K_i$ value of $1.38{\times}10^{-6}M$.

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Purification and Characterization of S-adenosylmethionine Synthetase from Soybean (Glycine max) Axes

  • Kim, Dae-Gun;Park, Tae-Jin;Kim, Jong-Yeol;Cho, Young-Dong
    • BMB Reports
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    • v.28 no.2
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    • pp.100-106
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    • 1995
  • S-adenosylmethionine (SAM) synthetase was purified to homogeneity from soybean (Glycine max) axes. The enzyme was purified 216-fold with a 1.5% yield by ammonium sulfate fractionation, acetone fractionation, ion exchange chromatography with DEAE-sephacel, gel filtration with Sephacryl S-300, and afffinity chromatography with ATP-agarose. The enzyme activity reached a maximum 3 days after germination. SAM synthetase had a subunit molecular weight of 57,000 daltons from a silver stained single band on SDS-PAGE. The molecular weight of the enzyme was 110,000 daltons from Sephacryl S-300 gel filtration. The enzyme was composed of two identical subunits. The $K_m$ values of the enzyme for L-methionine and ATP were 1.81 and 1.53 mM, respectively. The enzymatic activity was not affected by polyamines, agmatine, or SAM analogues, but was inhibited by SAM. The inhibition pattern was showed non-competitive for L-methionine and uncompetitive for ATP. The activity of SAM synthetase was inhibited by thiol-blocking reagents. The enzyme was induced by treatment with $10^{-3}$ M putrescine at germination. Experimental data revealed a possible novel regulation mechanism of polyamine biosynthesis through several endogenous intermediates.

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Purification and Properties of Trehalase from Rhizoctonia solani (Rhizoctonia solani가 생산하는 Trehalase의 정제 및 특성)

  • 오태광;서영배;고영희
    • Microbiology and Biotechnology Letters
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    • v.20 no.1
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    • pp.53-60
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    • 1992
  • Nonregulatory trehalase has been purified from mycelia of Rhztoctonia solani. a pathogen of rice sheath blight. Purification procedures involved sonification, gel filtration and fast protein liquid chromatography. Purity was confirmed by isoelectric focusing with silver staining. The purified trehalase was estimated to have a molecular weight of 54,000 and pI point of 5.1. Maximal activity was observed at pH 5.4 and temperature $45^{\circ}C$. The purified trehalase exhibited on apparent Km for trehalose of 3.11 mM and a Vmax of 105.3 $\mu mol min^{-1}\times mg^{-1}$. Validamycin, a commercial antibiotics of rice sheath blight, was a non-competitive inhibitor of Rhzzoctoniu solani trehalase.

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Extraction and Separation of Protein-bound Polysaccharide by Lentinus edodes (표고버섯 배양액으로부터 단백다당류의 추출 및 정제 방법)

  • 박경숙;이별나
    • The Korean Journal of Food And Nutrition
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    • v.10 no.4
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    • pp.503-508
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    • 1997
  • The extraction and separation methods of protein-bound polysaccharides from the mycelium and culture broth of L. edodes were investigated. The use 2% solution of surface active agent, Triton X-100 was effective for extraction of the protein-bound polysaccharide from the mycelium. The extraction of the protein-bound polysaccharides from mycelium with hot water was achieved by 4 hours extraction at 10$0^{\circ}C$. For the separation and partial purification of the protein bound polysaccharides the column chromatography using DEAE-Cellulose, DEAE-Sephadex and Sephadex proved to be effective.

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Purification and Properties of an Extracellular Acid Phytase from Pseudomonas fragi Y9451

  • In, Man-Jin;Jang, Eun-Seok;Kim, Young-Jin;Oh, Nam-Soon
    • Journal of Microbiology and Biotechnology
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    • v.14 no.5
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    • pp.1004-1008
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    • 2004
  • An extracellular acid phytase from Pseudomonas fragi Y9451 was purified to homogeneity from the culture supernatant by salting-out, DEAE-Sepharose column chromatography, CM-Sepharose column chromatography, and Sephacryl S-300 gel filtration. The molecular weight of the purified enzyme was estimated to be 74 kDa on gel filtration and 54 kDa and 25 kDa on SDS-PAGE, suggesting that the native enzyme was a heterodimeric protein. The purified enzyme was most active at pH 4.5 and $70^{\circ}C$ and fairly stable from pH 4.0- 6.0. It was specific for phytate and exhibited a $K_{m}$ value of 27 mM (sodium phytate, pH 4.5, $50^{\circ}C$). The phytase activity was strongly inhibited (at maximum by 87%) by $Fe^{3+},\;Cu^{2+},\;Fe^{2+}$, and $Zn^{2+}$ at 5 mM concentration, and greatly inhibited by $Ca^{2+}$ at 10 mM concentration. However, EDTA notably stimulated the phytase activity at 10 mM concentration. With optimum pH and stability, Pseudomonas fragi phytase could be a potential candidate for animal feed applications.

Identification and purification of Wx protein involved in biosynthesis of amylose in Rice (벼에서의 아밀로즈 생합성 관련 Wx 단백질의 동정 및 분리)

  • Nahm, Baek-Hie;Kim, Jin-Ku;Choi, Hae-Choon
    • Applied Biological Chemistry
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    • v.36 no.6
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    • pp.533-538
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    • 1993
  • The Wx protein, known as starch synthase or starch glucosyl transferase (E.C. 2.4.1.11), is responsible for the amylose synthesis. In an effort to explain the mechanism of amylose biosynthesis, the starch synthase known as Wx protein was identified by analyzing the various wx rice mutants with SDS-PAGE of proteins extracted from rice starch. Finally, the 66 kDa protein was purified by extracting the starch-bound protein fractions followed by Suprose 12 gel filtration chromatography.

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New Antimicrobial Activity from Korean Radish Seeds (Raphanus sativus L.)

  • Park, Jong-Heum;Shin, Keuyn-Kil;Hwang, Cher-Won
    • Journal of Microbiology and Biotechnology
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    • v.11 no.2
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    • pp.337-341
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    • 2001
  • To isolate antifungal substances from Korean radish (Raphanus Sativus L.) seeds, various purification techniques such as DE52 cellulose anion exchange, SP-Sephadex C-25 cation exchange, and Sephadex G-50 gel filtration chromatographies were used. The molecular masses of two purified R. sativus antifungal proteins (RAPs) were estimated to be about 6.1 kDa (RAP-1) and 6.2 kDa (RAP-2) by SDS-PAGE, and 5.8 kDa(RAP-1) and 6.2 kDa (RAP-2 by a gel filtration chromatography, respectively. Purified proteins RAP-1 and 2 clearly exhibited different growth inhibitory activities against other microorganisms like Candida albicans and Saccharomyces cerevisiae. Although they have similar molecular masses, both RAP-1 and 2 proteins are not identical because their microbial inhibitory actions were different. Therefore, RAP-1 could be a new antifungal protein when compared with the antifungal activities of 2S albumins, Rs-AFPs, Mj-AMPs, chitinase, glucanase, permatin, and ribosome inactivating proteins, all of which are anifungal proteins of plants.

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