• 제목/요약/키워드: Sulfhydryl reagent

검색결과 17건 처리시간 0.02초

삼화산(三和散)이 심장(心臟) Na-K-ATPase 활성(活性)에 미치는 영향(影響) (Effect of Samhwasan on Na-K-ATPase Activity in Microsomal Fraction of Rabbit Heart Ventricles)

  • 신현철;윤철호;정지천
    • 대한한의학회지
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    • 제17권2호
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    • pp.264-276
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    • 1996
  • This study was carried out to evaluate the effect of Samhwasan on the Na-K-ATPase activity of heart muscle. The Na-K-ATPase activity was prepared from rabbit heart ventricles. Samhwasan markedly inhibited the Na- K - ATPase activity in a dose-dependent manner with an estimated $I_{50}$ of 0.56%. Hill coefficient was 1.70, indicating that the enzyme has more than one binding site for the Samhwasan. Inhibition of enzyme activity by Samhwasan increased as pretreatment time was prolonged. Inhibition by the drug was not affected by a change in enzyme protein concentration. Kinetic studies of substrate activation of the enzyme indicated classical noncompetitive inhibition, showing significant reduction in Vmax without a change in Km value. Inhibitory effect by Samhwasan was not altered by changes in concentration of $Mg^{2+}$, $Na^+$ or $K^+$, dithiothreitol. a sulfhydryl reducing reagent, did not protect the inhibition of Na-K-ATPase activity by Samhwasan combination of Samhwasan and ouabain showed a cumulative inhibition fashion. These results suggest that Samhwasan inhibits Na-K-ATPase activity of heart ventricles with an unique binding site different from that of ATP, $Mg^{2+}$, $Na^+$ or $K^+$ and ouabain.

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Recombinant Interferon-${\alpha}$ Cross-linked with Thymosin ${\alpha}$1 is Biologically Active

  • Jeong, Jee-Yeong;Chung, Hye-Shin
    • BMB Reports
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    • 제29권4호
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    • pp.365-371
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    • 1996
  • Partially reduced interferon-a ($IFN-{\alpha}$) was cross-linked with thymosin ${\alpha}1$ ($T{\alpha}1$) using sulfo-succinimidyl (4-iodoacetyl) amino benzoate (SIAB), a bifunctional cross-linking reagent. The partially reduced $IFN-{\alpha}$ optimal for the cross-linking reaction was obtained by incubating native $IFN-{\alpha}$ with 0.5 mM DTT at $30^{\circ}C$ for 60~100 min. $T{\alpha}1$ was activated by incubating with sulfo-SIAB at $37^{\circ}C$ for 30 min to produce $T{\alpha}1-IAB$. The $T{\alpha}1-IFN-{\alpha}$ cross-linking was achieved by the reaction of the partially reduced $IFN-{\alpha}$ with $T{\alpha}1-IAB$. This cross-linking was between the sulfhydryl group of Cys1 in $IFN-{\alpha}$ and the N-terminal amino group of $T{\alpha}1$ through acetyl amino benzoate as a spacer. The immunological activity of the cross-linked molecule showed the same extent as that of $T{\alpha}1$, and most of the antiviral activity was retained compared to that of the partially reduced $IFN-{\alpha}$.

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X-선(線) 조사(照射)가 Ehrlich 암세포(癌細胞)의 용적(容積), 단백양(蛋白量) 및 수종(數種) Sulfhydryl 기(基)에 미치는 영향(影響)에 관(關)하여 (Effect of X-Irradiation on the Levels of some Sulfhydryl Groups, Protein and Cell Volume of Ehrlich Ascites Tumour Cells)

  • 유춘식;주영은
    • The Korean Journal of Physiology
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    • 제3권2호
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    • pp.9-16
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    • 1969
  • It is well known that a number of -SH and -SS containing substances afford a certain measure of protection against radiation effects in many biological systems, and it is conceivable that inherent -SH levels in Ehrlich ascites tumour (ELD)cells may be of decisive improtance with respect to the development of cellular radiation injury. So far, little effort has been directed to elucidate the changes in levels of different -SH and -SS groups in ELD cells when the tumour-bearing whole animal was subjected to the sublethal dose of X-irradiation. The present study was designed to bring some lights in the possible changes of and relationship between various sulfhydryl levels, such as P-SH, NP-SH and NP-SS, as well as the content of protein and cell volume of ELD cells, after subjecting the ELD mice to 1,200 r of X-irradiation. The animals used in this experiment were all mixed bred mice of $20{\sim}25\;gm$ in body weight (approximately 2 months old) irrespective of sex. 12 mice in one experiment were inoculated intraperitoneally with 0.2 ml of ascites tumour cells $(2{\times}10^6\;cells)$, and on the 7th day of the tumour growth, they were X-irradiated with 1,200 r, using the conventional X-ray machine under the following conditions: 200 Kv at 15 mA, 0.5 mm Cu filter, target-skin distance: 50 cm. Radiation dose was measured with the the Philip integrating dosimeter. At 24, 36, 48 and 60 hours after the X-irradiation, the mice were killed by cervical dislocation, and the tumours were taken out. Freshly withdrawn ascites tumours were placed in ice, and immediately the cell concentration was measured with the Coulter Cell Counter (Model B), and the hematocrit of the tumour cells were also determined. Cell volume was thus calculated by the cell concentration and hematocrit value. P-SH content of ELD cells was measured potentiometrically according to the method of Calcutt & Doxey, and NP-SH and NP-SS contents were measured spectrophotometrically by the method described by Ellman. Protein content of ELD cells was determined with the Folin phenol reagent by Lowry et al. Altogether, 48 experimental mice were used, and 12 mice with the only exception of X-irradiation were used as the control. Results obtained indicate that the contents of all the cellular sulfhydryl groups as well as cell volume and protein content of the ELD cells increase significantly as time progresses after the sub-lethal X-ray dose of 1,200 r was given and that all the increase is in a lineal fashion. The regression lines of the relative values, (i. e., taking each control value as 1) of all the values obtained, and the regression lines of cell volume, protein and NP-SH are identical, whereas those of NP-SS and P-SH appear to be widely seperated. However, the difference of those two lines (NP-SS & P-SH) were found to be not significant statistically (p>0.05). Therefore, it can be concluded from the above results that all the values examined increase in a lineal fashion with no statistically significant difference among them. Also, with the radiation dose of 1,200 r, the ELD cell becomes enlarged and swollen progressively up to 60 hours post-irradiation and it becomes more than two times of the original normal size at 60 hours after the irradiation, and up to this stage, it seems apparent that the cell division has been slow due to the X-irradiation applied in this experiment. It is well understandable that the contents of NP-SH, NP-SS, P-SH and protein of the ELD cells increase in parallel with the increase of the cell volume by the X-ray does used, but it also seems interesting to note that all the cellular substances tested show no appreciable difference in the pattern of increase.

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Antioxidant Effects of Gamma-oryzanol on Human Prostate Cancer Cells

  • Klongpityapong, Papavadee;Supabphol, Roongtawan;Supabphol, Athikom
    • Asian Pacific Journal of Cancer Prevention
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    • 제14권9호
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    • pp.5421-5425
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    • 2013
  • Background: To assess the antioxidant effects of gamma-oryzanol on human prostate cancer cells. Materials and Methods: Cytotoxic activity of gamma-oryzanol on human DU145 and PC3 prostate cancer cells was determined by proliferation assay using 3-(4, 5-dimethylthiazol, 2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) reagent. mRNA levels of genes involved in the intracellular antioxidant system, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and glutathione reductase (GSR) were determined by reverse transcription-polymerase chain reaction (RT-PCR). Cancer cell lysates were used to measure lipid peroxidation using thiobarbituric acid reactive substance (TBARS). Glutathione contents of the cell lysates were estimated by the reaction between sulfhydryl group of 5, 5'-dithio (bis) nitrobenzoic acid (DTNB) to produce a yellow-color of 5-thio-2-nitrobenzoic acid using colorimetric assay. Catalase activity was also analysed by examining peroxidative function. Protein concentration was estimated by Bradford's assay. Results: All concentrations of gamma-oryzanol, 0.1-2.0mg/ml, significantly inhibited cell growth in a dose- and time-dependent fashion in both prostate cancer cell lines, DU145 and PC3. Gene expression of catalase in DU145 and PC3 exposed to gamma-orizanol at 0.5mg/ml for 14 days was down regulated, while mRNA of GPX was also down regulated in PC3. The MDA and glutathione levels including catalase activity in the cell lysates of DU145 and PC3 treated with gamma-oryzanol 0.1 and 0.5mg/ml were generally decreased. Conclusions: This study highlighted effects of gamma-oryzanol via the down-regulation of antioxidant genes, catalase and GPX, not cytotoxic roles. This might be interesting for adjuvant chemotherapy to make prostate cancer cells more sensitive to free radicals. It might be useful for the reduction of cytotoxic agents and cancer chemoprevention.

파인애플 bromelain의 정제 및 특성 (Purification and characterization of bromelain isolated from pineapple)

  • 서형주;이호;조홍연;양한철
    • Applied Biological Chemistry
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    • 제35권4호
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    • pp.300-307
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    • 1992
  • 파인애플의 과육과 줄기로부터 fruit-bromelain을 정제도 18배와 수율 23%, stem-bromelain을 정제도 46배와 수율 23%로 각각 전기영동적으로 균일하게 정제하였다. 두 효소의 분자량을 Sephadex G-200에 의해 측정한 결과, fruit-와 stem-bromelain의 분자량은 32.5 KDa와 37 KDa이었으며, 이들은 공히 1개의 subunit로 구성된 효소단백질이었다. Fruit-bromelain은 pH 8.0과 $70^{\circ}C$, stem-bromelain은 pH 7.0과 $60^{\circ}C$ 에서 각각 최대 활성을 나타내었다. 동 식물 유래의 천연단백질을 중심으로 기질 특이성을 검토한 결과 동물성 단백질보다는 밀단백 또는 ISP와 같은 식물성 단백질에 높은 기질 특이성을 보였으며, casein에 대한 fruit와 stem-bromelain의 또 $K_m$값은 0.18 mM와 0.34 mM이었다. pCMB는 경쟁적 저해제로서 fruit와 stem-bromelain의 $K_i$값은 0.18 mM과 0.10 mM이었다. pCMB에 의해 저하된 두 효소의 활성은 cysteine에 의해 재활성화 되었으며 또한 과량의 cysteine 첨가에 의해 활성화됨으로써 이 효소들은 전형적인 thiol계 단백분해효소임을 알 수 있었다.

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Streptomyces sp. YSA-130이 생산하는 Alkaline Protease의 정제 및 특성 (Purification and Properties of Alkaline Protease from Streptomyce sp. YSA-130)

  • 윤성우;이강표;유주현;신철수;오두환
    • 한국미생물·생명공학회지
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    • 제17권4호
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    • pp.358-364
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    • 1989
  • 토양으로부터 분리한 Streptomyces sp. YSA-130으로부터 활성이 좋은 결정화된 alkaline protease를 분리하였다. Alkaline protease 생산의 최적 배양조건은 2.0% soluble starch, 1.0% soytone, 0.3% $K_2$HPO$_4$, 0.02% MgSO$_4$.7$H_2O$, 0.8% $Na_2$CO$_3$ 3$0^{\circ}C$, pH 10.5에서 72 시간 배양하였을 때 였다. Alkaline protease이 정제는 (NH$_4$)$_2$SO$_4$. 분별침전, 투석, DEAE cellulose column chromatography, Sephadex G-75 gel filtration, crystallization으로 하였으며, 그 결과 비활성도 14,290unit/mg, 정제도 23.8 배였고, 수율은 20.0% 이었다. Alkaline protease의 반응 최적온도와 pH는 6$0^{\circ}C$ 와 11.5이었으며, 효소의 pH 안정성은 5.5-12.0에서 안정하였고, 온도 안정성은 5$0^{\circ}C$까지 안정하였으며, $Ca^{++}$ ion 첨가시 6$0^{\circ}C$까지 안정성이 증가하였다. Alkaline protease의 분자량은 30,000이었으며 금속이온, EDTA, 환원제는 활성에 영향이 없었고 DFP에 의해 저해되었다. 계면활성제에 저항성이 크고 $H_2O$$_2$에 대한 잔존활성은 60% 을 유지하였다.

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Aspergillus oryzae의 세포내 효소인 Adenosine Deaminase의 기질 유사체와 억제물질에 대한 반응속도론적 분석 (Kinetics of Intracellular Adenosine Deaminase to Substrate Analogs and Inhibitors in Aspergillus oryzae)

  • 최혜선
    • 미생물학회지
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    • 제32권1호
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    • pp.84-90
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    • 1994
  • Adenosine deaminase (ADA)의 여러 기질과 억제물질의 반응 속도론적 상수가 Aspergillusoryzae의 세포내 효소인 ADA의 활성자리에 어떻게 부착하고 어떤 요인을 필요로 하는지를 알기위해 측적되어졌다. kcat/$K_m$값에 의하면 조사된 기질로 작용하는 화합물 중에 3'-deoxyadenosine이 가장 좋은 기질로 작용하는 것으로 밝혀졌다. 몇 개의 유사체가 억제물질로 조사되었는데 purine riboside가 $3.7{\times}10^{-5}$M의 값 $K_i$값을 가지고 가장 강한 억베물질로 나타났다. Adenine은 기질로도 억제물질로도 작용을 못하므로 adenine의 N-9 위치의 ribose가 효소에 부착하는데 중요하다는 것을 시사하고 있다. 또 ADA는 6-chloropurine riboside(6-CPR)의 dechlorination을 촉매화하여 inosine과 Cl이온을 생성한다. 6-CPR의 ADA에 대한 기질 특이성은 정상 기질인 adenine의 0.86%로 측정되었다. ADA의 경쟁적 억제물질인 purine riboside는 비슷한 $K_i$값을 가지고 dechlorination도 억제하므로 deamination과 dechlorination 반응은 효소의 부착자리를 공유하고 있다고 생각되어진다. SH기에 작용하는 화합물중 수은제인 p-chloromercuribenzoate(PCMB), mersalyl acid, $HgCl_2$는 효소의 deamination 반응을 억제했다. Mersalyl acid에 의해 활성이 억제된 ADA는 thiol reagent인 dithiothreitol이나 2-mercaptoethanol에 의해 활성이 회복되지만 PCMB에 의해 억제된 효소는 회복되지 않았다. 각 수은제들이 억제물질로 작용할 때 $K_i$값과 억제양상이 측정되었는데 모두 경쟁적 방해를 보였다. $K_i$값은 10mM 인산완충용액에서 측정한 것이 100 mM 인산완충용액에서 측정한 것보다 훨씬 낮아 인산기가 기질이 아니어도 효소의 부착에 큰 영향을 미치는 것을 보여주고 있다.

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