• 한국미생물·생명공학회지
• /
• 제9권4호
• /
• pp.207-212
• /
• 1981

토양에서 분리된 여러가지 곰팡이류 가운데 $\beta$-galactosidase 분비력이 강하고 중성부근에서 효소안정성이 높은 Penicillium sp. 균주를 $\beta$-galactosidase 생산균주로 선정하였다. 이 균주는 밀기울고체배지에서 3$0^{\circ}C$ 72시간 배양에서 galactosidase 분비력이 가장 높았으며 질소원 첨가배지에서 질소원의 종류에 따라 14%~85%까지 증가되었다. 고체배지에서 얻은 효소추출액을 ammonium sulfate fractionation, SP-Sephadex C-50 chromatography, ultrogel AcA44 filtration, hrdroxrapatite chromatography등에 의하여 비활성도는 101 units/mg protein으로 5050배 정제되었다. 최종적으로 정제된 galactosidase는 analytical ultracentrifuge에서 단일단백으로 Schlieren pattern을 나타냈고 Disc electrophoresis에서는 단일 band로서 전기영동되었으며 영동된 $\beta$-galactosidase 활성 band와 일치하였다.

• Molecules and Cells
• /
• 제37권7호
• /
• pp.526-531
• /
• 2014

Human PARP family consists of 17 members of which PARP-1 is a prominent member and plays a key role in DNA repair pathways. It has an N-terminal DNA-binding domain (DBD) encompassing the nuclear localisation signal (NLS), central automodification domain and C-terminal catalytic domain. PARP-1 accounts for majority of poly-(ADP-ribose) polymer synthesis that upon binding to numerous proteins including PARP itself modulates their activity. Reduced PARP-1 activity in ageing human samples and its deficiency leading to telomere shortening has been reported. Hence for cell survival, maintenance of genomic integrity and longevity presence of intact PARP-1 in the nucleus is paramount. Although localisation of full-length and truncated PARP-1 in PARP-1 proficient cells is well documented, subcellular distribution of PARP-1 fragments in the absence of endogenous PARP-1 is not known. Here we report the differential localisation of PARP-1 Nterminal fragment encompassing NLS in PARP-$1^{+/+}$ and PARP-$1^{-/-}$ mouse embryo fibroblasts by live imaging of cells transiently expressing EGFP tagged fragment. In PARP-$1^{+/+}$ cells the fragment localises to the nuclei presenting a granular pattern. Furthermore, it is densely packaged in the midsections of the nucleus. In contrast, the fragment localises exclusively to the cytoplasm in PARP-$1^{-/-}$ cells. Flourescence intensity analysis further confirmed this observation indicating that the N-terminal fragment requires endogenous PARP-1 for its nuclear transport. Our study illustrates the trafficking role of PARP-1 independently of its enzymatic activity and highlights the possibility that full-length PARP-1 may play a key role in the nuclear transport of its siblings and other molecules.

• 한국임상수의학회지
• /
• 제2권1호
• /
• pp.105-114
• /
• 1985

The optimal conditions for the evaluation of serum ornithine carbamyltransferase activity, based on the do-termination of citrulline formed during the enzymatic reaction, were investigated and the serum ornithine carbamyltransferase activity of cattle were surveyed. Barbital-acetate buffer(70m moles/L, pH 7.0 at $37^{\circ}C$) were usea for the entire experiment. The results were as follows. 1. When the concentration of $H_2SO_4$ in color reagent exceeds 3.0 m1/100m1 the serum protein precipitated and absorbance increased. 2. The concentrations of antipyrine and diacetylmonoxime required for maximal color formation were 1g/L and 5g/L, respectively. 3. The absorbance was maximal when the reaction mixture was boiled for 25 minutes. 4. The chromogen were stable for at least 60 minutes under loon lighting condition, but decolorized rapidly under direct sunlight. 5. The minimal concentration of urease solution(Sigma Chemical Co., Type III) required for elimination of serum urea was 0, 6mg/ml. 6. When the concentration of L-ornithine solution increased up to 22m moles/L, the ornithine carbamyltransferase activity was not inhibited by zwitterion of ornithine. 7. In accordance with the increase of carbamylphosphate concentration the ornithine carbamyltransferase activity increased and the nonenzymatic citrulline production also increased slightly. 8. The standard curve of citrulline revealed linear pattern within the range of this experiment (0.1~4.0m moles/L). 9. The ornithine carbamyltransferase activities of normal cattle investigated in this laboratory were 6.85$\pm$4.38U/L (mean$\pm$SD) in cows and 2.89$\pm$2.50U/L in bulls. The range of the activities were 0.39～29.12U/L in cows and 0.06~17.34U/L in bulls.

• Applied Biological Chemistry
• /
• 제43권1호
• /
• pp.18-23
• /
• 2000

찹쌀 14 품종의 전분 미세구조는 효소처리에 의해서 debranching시킨 전분의 glucose chain length 분포 비교에 의해서 수행되었고, 찹쌀 품종별로 미세구조상에 차이가 있었다. 짧은 쇄장을 다량 함유하고 있는 찰품종은 병곡이었으며, 상대적으로 긴 쇄장을 다량 함유하고 있는 품종은 TP2579Al이었다. 15% $H_2SO_4$에 대한 가수분해도는 샤레벼-152-1-B가 가장 높았으며, 산동 47이 가장 낮았다. X선 회절도는 14 품종 찹쌀 모두 전형적인 A형이었다. Glucoamylase에 의한 가수분해도의 양상은 품종에 따라 차이가 있었으며, 거창 1 및 병곡은 $37^{\circ}C$에서 3시간 작용시킴에 의해 100% 가수분해 되었다.

• 한국식품영양학회지
• /
• 제10권1호
• /
• pp.102-109
• /
• 1997

효소반응과 부분 산가수분해 결과를 해석하여 Leuconostoc mesenteriodes M-12 덱스트란수크라제 억셉터 반응 산물인 새로운 분지올리고당의 구조를 확인하였다. 분지올리고당 B4의 구조는 62-O-$\alpha$-D-kojibiosylmaltose인 것으로 확인되었으며, 분지올리고당 B5의 구조는 63-O-$\alpha$-D-kojibiosylpanose였다. 억셉터 반응산물을 덱스트라나제로 분해한 결과 새로운 올리고당인 D4를 확인할 수 있었다. 억셉터 반응산물을 억셉터로 이용한 두 번째 억셉터 반응의 생성물을 덱스트라나제 처리하여 D4를 얻었는데 덱스트라나제와 글\ulcorner아밀라제에 의해 분해되지 않았다. 그 구조는 62-O-$\alpha$-D-kojibiosylisomaltose로 확인되었다. 직선상 또는 분지 결합을 가진 d.p. 6 이하의 억셉터 반응산물의 생성 패턴도 확인하였다.

• 생명과학회지
• /
• 제14권2호
• /
• pp.252-254
• /
• 2004

$\alpha$소단위체 56번 잔기가 치환된 돌연변이 (D56E/G/N) 트립토판 합성효소의 효소활성도는 매우 낮다. 이러한 돌연변이 효소에 $\alpha$와 $\beta$소단위체 특이 리간드를 처리하여 그 영향을 조사하였다. 양이온은 야생종과 잔기치환체에 다른 흡광도를 보여주었다. 반면, glycerophosphate는 모두 비슷한 양상의 흡광도를 보여주고 있다. glycerophosphate는 $\alpha$소단위체의 활성부위에 결합함으로 $\alpha$소단위체에 기질이 결합된 반응 단계에서는 56번 잔기가 $\alpha$와 $\beta$소단위체간의 이소조절에 관여하지 않고 있음을 시사한다. 따라서, 잔기 56번 치환 효소는 $\alpha$소단위체로부터 기질이 떨어진 이후에 일어나는 반응 단계에 결함이 있는 것으로 추정된다.

• 한국식품영양과학회지
• /
• 제18권3호
• /
• pp.348-355
• /
• 1989

Alkaline protease 생성능이 강한 Aspergillus fumigatus 균주를 토양에서 분리하고, 생성효소를 정제하여 특성을 조사한 결과 최적 pH는 9.0, pH안정성은 $pH\;8.0{\sim}10.0$, 최적온도는 $50^{\circ}C$였으며, $50^{\circ}C$이하의 온도에서 안정하나 그 이상의 온도에서는 급격한 효소 불활성화를 보였고, 금속염 $Mn^{++},\;Cu^{++},\;Ba^{++},\;Mg^{++}$ 등에 의해서 활성이 다소 증대되나 $K^+,\;Fe^{+++},\;Ag^{++},\;Pb^{++},\;Na^+,\;Ca^{++},\;Hg^+,\;Zn^{++}$에 의해 저해를 받았다. 활성저해제인 EDTA, 2,4-DNP, ${\varepsilon}-amino$ caproic acid에는 큰 저해를 받지 않으나, PCMB에 많은 저해를 받는 것으로 미루어 활성 부위가 SH기인 cystein protease로 추정되었다. Km값은 $8.33{\times}10^{-4}mole/{\ell}$, Vmax는 $47.62{\mu}g/min$였으며, casein과 hemoglobin을 trypsin보다 더 잘 분해하고, casein을 hemoglobin보다 잘 분해하였다.

• ALGAE
• /
• 제21권2호
• /
• pp.261-266
• /
• 2006

The innate soluble polysaccharides and phenolic compounds of marine macroalgae are serious contaminants which interfere with experimental procedures such as restriction enzyme digestion, polymerase chain reaction (PCR) and other enzymatic reactions using extracted DNA samples. The viscous polysaccharides are co-precipitated with DNA samples by isopropanol or ethanol precipitation in conventional experiment. To overcome the problem, a method for the isolation of high molecular weight DNA from Porphyra tenera is developed with the application of diatomaceous earth column. The isolated DNAs by this method were about 50-100 kb in size and could be digested well with restriction enzymes. The nuclease activity seemed to be minimal, and high reproducibility in the arbitrary primed PCR for RAPD analyses was a distinctive feature. These results suggest that this method is very efficient in isolating nucleic acid from macroalgae including Porphyra.

• Preventive Nutrition and Food Science
• /
• 제1권1호
• /
• pp.111-116
• /
• 1996

Polyphenol oxidase(PPO) isolated from the crude extract of ascidian, Halocynthia roretzi, showed higher affinity for catechol than tyrosine or DL-DOPA. Successful enzyme assay could be performed at $25^{\circ}C$, 10min. by mixing 0.2ml of crude enzyme extract with 2.8ml of 0.13M catechol in 0.1M sodium phosphate buffer(pH 6.4). The specific activity of PPO which had been purified with a combination of ammonium sulfate treatment, ion exchange chromatography on DEAE-cellulose, and gel filtration on Sepharose 6B was 13-fold disc gel electrophoresis. The activity of PPO was stable from pH 5.0 to 8.0 and showed the peak activity at pH 6.4 .The optimum reaction temperature for PPO oxidation on catechol was 35$^{\circ}C$ and those enzyme were heat stable up to 4$0^{\circ}C$. Molecular weigth of the enzyme was estimated about 170kDa. One molecule was found to be composed of gour subunits. Two of them had molecular weigh of 55kDa and the others 30kDa. The {TEX}$K_{m}${/TEX} values, {TEX}$V_{max}${/TEX} and catalytic efficiency({TEX}$V_{max}${/TEX}/{TEX}$K_{m}${/TEX}) for catechol were 0.12mM, 2.5mM/liter/min. and {TEX}$0.18min^{-1}${/TEX} respectively. The substrate affinity and electrophorectic pattern suggested that the enzyme of ascidian was considered to be not tyosine but catechol oxidase.

• Journal of Nutrition and Health
• /
• 제53권6호
• /
• pp.563-569
• /
• 2020

Purpose: The vascular smooth muscle cells (VSMCs) in mature animals have implicated to play a major role in the progression of cardiovascular diseases such as atherosclerosis. This study aimed at optimizing the protocol in culturing primary VSMCs (pVSMCs) from rat thoracic aorta and investigating the effect of cellular zinc (Zn) deficiency on cell proliferation of the isolated pVSMCs. Methods: The thoracic aorta from 7-month-old Sprague Dawley rats was isolated, minced and digested by the enzymatic process of collagenase I and elastase, and then inoculated with the culture Dulbecco Modified Eagle Medium (DMEM) at 37℃ in an incubator. The primary cell culture morphology was observed using phase-contrast microscopy and cellular Zn was depleted using Chelex-100 resin (extracellular zinc depletion only) or 3 µM N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) (extracellular and intracellular zinc depletion). Western blot analysis was used for the detection of SM22α and calponin as smooth muscle cell marker proteins and von Willebrand factor as endothelial cell marker protein to detect the culture purity. Cell proliferation by Zn depletion (1 day) was measured by MTT assay. Results: A primary culture protocol for pVSMCs from rat thoracic aorta was developed and optimized. Isolated cultures exhibited hill and valley morphology as the major characteristics of pVSMCs and expressed the smooth muscle cell protein markers, SM22α and calponin, while the endothelial marker von Willebrand factor was hardly detected. Zn deprivation for 1 day culture decreased rat primary vascular smooth muscle cell proliferation and this pattern was more prominent under severe Zn depletion (3 µM TPEN), while less prominent under mild Zn depletion (Chelexing). Conclusion: Our results suggest that cellular Zn deprivation decreased pVSMC proliferation and this may be involved in phenotypic modulation of pVSMC in the aorta.