• 유기물자원화
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• 제1권1호
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• pp.115-125
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• 1993

Euglena gracilis 에서 arginine deiminas는 mitochondrial matrix 내에 존재한다. 고도로 정제된 효소가 0.23 nM의 $K_m$ 값을 갖고 효소반응을 하기 위해서는 $Co^{2+}$가 필요하며, 이때 최적 pH는 9.3~10.3이었다. Gel filtration에 의해서 얻어진 조효소 단백질의 분자량은 87,000이었으며, SDS-acrylamide gel electrophoresis에 의해 효소는 48,000의 분자량을 갖는 2개의 동일한 subunit로 구성되어 있음이 밝혀졌다. Euglena의 arginine deiminas는 sulfhydryl inhibitors에 의해서 활성이 저지되었는데, 이는 sulfhydryl group이 효소의 활성부위에 관여함을 나타낸다. 이 sulfhydryl group은 arginine이 효소와 결합하는데 있어서 negative cooperativity를 나타내었다. ${\beta}-guanidinopropionate$, ${\gamma}-guanidinobutyrate$와 guanidinosuccinate는 효소의 활성을 저지시키지 않는데 반하여, $L-^{\alpha}-amino-{\beta}-guanidino-propionate$, D-arginine, 그리고 L-homoarginine은 효소의 활성을 강력하게 저지시켰다. Citrulline과 ornithine에 의해서도 상당한 정도의 효소활성저지가 관찰되었다. 우리는 Euglena의 arginine deiminase의 독특한 성질이 Euglena 라는 원생동물 내에서 arginine 대사의 조절에 어떻게 영향을 미치는지를 토의하고자 한다.

• 한국식품저장유통학회지
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• 제9권4호
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• pp.429-433
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• 2002

Candida sp. L-16 균주가 생산하는 D-xylulokinase는 배양균체를 초음파 파쇄한 조효소액으로 하여 황산암모늄 염석, DEAE-cellulose, chromatography, Sephadex G-100과 Sephadex G-200 gel filtration 과정으로 정제하여 최종 수율 11.2%로 약 23.2배 정제하였다. 정제 효소의 분자량은 SDS-PAGE로 분석한 결과 분자량은 75,000 dalton으로, Sephadex G-200겔 여과에 의해 150,000 dalton으로 나타나 dimer로 확인되었다. 효소 활성에 미치는 최적 반응 온도는 4$0^{\circ}C$로 나타났고, 온도안정성은 비교적 불안정하여 3$0^{\circ}C$이상에서는 빠르게 실활되었다. 정제 효소의 최적 반응 pH는 pH 8.0이었고, pH 7.0에서 pH 9.0 사이에서 비교적 효소활성이 높았다. 본 효소는 D-xylulose, D-arabinose, D-ribose등에서는 높은 기질특이성을 가지고 있었으나 D-xylose, D-glucose, L-arabinose 등은 기질로서 작용하지 못하였다. 정제 효소의 활성화 에너지값(Ea)은 $25^{\circ}C$ 내지 4$0^{\circ}C$$^{\circ}C$의 온도 범위에서 4.75kcal/mol이었다. 효소의 활성화제로는 EDTA, cysteine-HCl, DTT, glutathione 등이 존재하며 억제제로는 6-phosphogluconic acid, 2-koeto-gluconic acid 등으로 나타났다.

• Mass Spectrometry Letters
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• 제12권4호
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• pp.179-185
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• 2021

Collision-induced dissociation (CID) combined with electrospray ionization mass spectrometry (ESI-MS) was used to obtain structural information on rat islet amyloid polypeptide (rIAPP) monomers (M) and dimers (D) observed in the multiply charged state in the MS spectrum. MS/MS analysis indicated that the rIAPP monomers adopt distinct structures depending on the molecular ion charge state. Peptide bond dissociation between L₂₇ and P₂₈ was observed in the MS/MS spectra of rIAPP monomers, regardless of the monomer molecular ion charge state. MS/MS analysis of the dimers indicated that D⁵⁺ comprised M²⁺ and M³⁺ subunits, and that the peptide bond dissociation process between the L₂₇ and P₂₈ residues of the monomer subunit was also maintained. The observation of (M+ b₂₇)⁴⁺ and (M+ y₁₀)³⁺ fragment ions were deduced to originate from the two different D⁵⁺ complex geometries, the N-terminal and C-terminal interaction geometries, respectively. The fragmentation pattern of the [M_rIAPP + M_hIAPP]⁵⁺ MS/MS spectrum showed that the interaction occurred between the two N-terminal regions of M_rIAPP and M_hIAPP in the heterogeneous dimer (hetero-dimer) D⁵⁺ structure.

• Journal of Microbiology and Biotechnology
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• 제26권1호
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• pp.20-27
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• 2016

Lactobacillus kefiranofaciens ZW3 was obtained from kefir grains, which have high lactose hydrolytic activity. In this study, a heterodimeric LacLM-type β-galactosidase gene (lacLM) from ZW3 was isolated, which was composed of two overlapping genes, lacL (1,884 bp) and lacM (960 bp) encoding large and small subunits with calculated molecular masses of 73,620 and 35,682 Da, respectively. LacLM, LacL, and LacM were expressed in Escherichia coli BL21(DE3) and these recombinant proteins were purified and characterized. The results showed that, compared with the recombinant holoenzyme, the recombinant large subunit exhibits obviously lower thermostability and hydrolytic activity. Moreover, the optimal temperature and pH of the holoenzyme and large subunit are 60℃ and 7.0, and 50℃ and 8.0, respectively. However, the recombinant small subunit alone has no activity. Interestingly, the activity and thermostability of the large subunit were greatly improved after mixing it with the recombinant small subunit. Therefore, the results suggest that the small subunit might play an important role in maintaining the stability of the structure of the catalytic center located in the large subunit.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2000년도 국제심포지움
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• pp.10-12
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• 2000

Members of the glycoprotein family, which includes CG, LH, FSH and TSH, comprise two noncovalently linked $\alpha$- and $\beta$-subunits. Equine chorionic gonadotropin (eCG), known as PMSG, has a number of interesting and unique characteristics since it appears to be a single molecule that possesses both LH- and FSH-like activities in other species than the horse. This dual activity of eCG in heterologous species is of fundamental interest to the study of the structure-function relationships of gonadotropins and their receptors. CG and LH $\beta$ genes are different in primates. In horse, however, a single gene encodes both eCG and eLH $\beta$-subunits. The subunit mRNA levels seem to be independently regulated and their imbalance may account for differences in the quantities of $\alpha$ - and $\beta$ -subunits in the placenta and pituitary. The dual activities of eCG could be separated by removal of the N-linked oligosaccharide on the $\alpha$-subunit Asn 56 or CTP-associated O-linked oligosaccharides. The tethered-eCG was. efficiently secreted and showed similar LH-like activity to the dimeric eCG. Interestingly, the FSH-like activity of the tethered-eCG was increased markedly in comparison with the native and wild type eCG. These results also suggest that this molecular can implay particular models of FSH-like activity not LH-like activity in the eCG/indicate that the constructs of tethered molecule will be useful in the study of mutants that affect subunit association and/or secretion. A single-chain analog can also be constructed to include additional hormone-specific bioactive generating potentially efficacious compounds that have only FSH-like activity. The LH/CG receptor (LH/CGR), a membrane glycoprotein that is present on testicular Leydig cells and ovarian theca, granulosa, luteal, and interstitial cells, plays a pivotal role in the regulation of gonadal development and function in males as well as in nonpregnant and pregnant females. The LH/CGR is a member of the family of G protein-coupled receptors and its structure is predicted to consist of a large extracellular domain connected to a bundle of seven membrane-spanning a-helices. The LH/CGR phosphorylation can be induced with a phorbol ester, but not with a calcium ionophore. The truncated form of LHR also was down-regulated normally in response to hCG stimulation. In contrast, the cell lines expressing LHR-t63I or LHR-628, the two phosphorylation-negative receptor mutant, showed a delay in the early phase of hCG-induced desensitization, a complete loss of PMA-induced desensitization, and an increase in the rate of hCG-induced receptor down-regulation. These results clearly show that residues 632-653 in the C-terminal tail of the LHR are involved in PMA-induced desensitization, hCG-induced desensitization, and hCG-induced down-regulation. Recently, constitutively activating mutations of the receptor have been identified that are associated with familial male-precocious puberty. Cells expressing LHR-D556Y bind hCG with normal affinity, exhibit a 25-fold increase in basal cAMP and respond to hCG with a normal increase in cAMP accumulation. This mutation enhances the internalization of the free and agonist-occupied receptors ~2- and ~17-fold, respectively. We conclude that the state of activation of the LHR can modulate its basal and/or agonist-stimulated internalization. Since the internalization of hCG is involved in the termination of hCG actions, we suggest that the lack of responsiveness detected in cells expressing LHR-L435R is due to the fast rate of internalization of the bound hCG. This statement is supported by the finding that hCG responsiveness is restored when the cells are lysed and signal transduction is measured in a subcellular fraction (membranes) that cannot internalize the bound hormone.

• 한국가축번식학회지
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• 제24권4호
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• pp.357-364
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• 2000

Members of the glycoprotein family, which includes CG, LH, FSH and TSH, comprise two noncovalently linked $\alpha$- and $\beta$-subunits. Equine chorionic gonadotropin (eCG), known as PMSG, has a number of interesting and unique characteristics since it appears to be a single molecule that possesses both LH- and FSH-like activities in other species than the horse. This dual activity of eCG in heterologous species is of fundamental interest to the study of the structure-function relationships of gonadotropins and their receptors. CG and LH $\beta$ genes are different in primates. In horse, however, a single gene encodes both eCG and eLH $\beta$ -subunits. The subunit mRNA levels seem to be independently regulated and their imbalance may account for differences in the quantities of $\alpha$ - and $\beta$-subunits in the placenta and pituitary. The dual activities of eCG could be separated by removal of the N-linked oligosaccharide on the $\alpha$-subunit Asn 56 or CTP-associated O-linked oligosaccharides. The tethered-eCG was efficiently secreted and showed similar LH-like activity to the dimeric eCG. Interestingly, the FSH-like activity of the tethered-eCG was increased markedly in comparison with the native and wild type eCG. These results also suggest that this molecular can implay particular models of FSH-like activity not LH-like activity in the eCG/indicate that the constructs of tethered molecule will be useful in the study of mutants that affect subunit association and/or secretion. A single-chain analog can also be constructed to include additional hormone-specific bioactive generating potentially efficacious compounds that have only FSH-like activity. The LH/CG receptor (LH/CGR), a membrane glycoprotein that is present on testicular Leydig cells and ovarian theca, granulosa, luteal, and interstitial cells, plays a pivotal role in the regulation of gonadal development and function in males as well as in nonpregnant and pregnant females. The LH/CGR is a member of the family of G protein-coupled receptors and its structure is predicted to of a large extracellular domain connected to a bundle of seven membrane-spanning a-helices. The LH/CGR phosphorylation can be induced with a phorbol ester, but not with a calcium ionophore. The truncated form of LHR also was down-regulated normally in response to hCG stimulation. In contrast, the cell lines expressing LHR-t631 or LHR-628, the two phosphorylation-negative receptor mutant, showed a delay in the early phase of hCG-induced desensitization, a complete loss of PMA-induced desensitization, and an increase in the rate of hCG-induced receptor down-regulation. These results clearly show that residues 632~653 in the C-terminal tail of the LHR are involved in PMA-induced desensitization, hCG-induced desensitization, and hCG-induced down-regulation. Recently, constitutively activating mutations of the receptor have been identified that are associated with familial male-precocious puberty. Cells expressing LHR-D556Y bind hCG with normal affinity, exhibit a 25-fold increase in basal cAMP and respond to hCG with a normal increase in cAMP accumulation. This mutation enhances the internalization of the free and agoinst-occupied receptors ~2- and ~17- fold, respectively. We conclude that the state of activation of the LHR can modulate its basal and/or agonist-stimulated internalization. Since the internalization of hCG is involved in the termination of hCG actions, we suggest that the lack of responsiveness detected in cells expressing LHR-L435R is due to the fast rate of internalization of the bound hCG. This statement is supported by the finding that hCG responsiveness is restored when the cells are lysed and signal transduction is measured in a subcellular fraction (membranes) that cannot internalize the bound hormone.

• 한국식품과학회지
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• 제34권3호
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• pp.425-430
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• 2002

본 연구에서는 축산 부산물인 닭발을 산과 알칼리에 각각 침지하여 제조한 젤라틴의 이화학적 특성을 조사하였다. 침지조건에 따른 아미노산 함량은 큰 차이가 없었으나 상업적으로 판매되는 돈피나 소가죽 젤라틴과는 그 조성이 달랐다. 이는 원료의 특성과 제조공정의 차이에서 기인하는 것으로 사료된다. 젤라틴의 수율과 젤라틴 겔의 경도는 산성 조건에서 24시간 침지한 경우에, 알칼리 조건에서 1주 침지한 경우에 가장 높다고 평가되었다. 점도와 투명도는 침지기간이 증가함에 따라 산성 조건에서는 증가한 반면 알칼리 조건에서는 감소하였다. 색도는 산처리 닭발 젤라틴 겔이 알칼리처리 젤라팅 겔에 비해 L값은 높고, a값과 b값이 낮아 미관상 더 바람직하다. 이화학적 특성이 우수한 산처리 24시간 침지 시료와 알칼리 처리 1주 침지 시료의 분자량 분포를 Gel permeation chromatography로 살펴본 결과, 두 시료 모두 12개의 획분으로 구성되었다. 가장 많이 발견되는 분자량은 669 kD으로 알칼리 처리 시료가 산처리 시료에 비해 두 배나 많았으며, 300 kD 이하는 산처리 시료가 알칼리 처리군에 비해 현저히 많아 산처리 시료가 알칼리 처리군에 비해 상대적으로 저분자화 되어있었다.

• 한국미생물·생명공학회지
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• 제23권4호
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• pp.446-453
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• 1995

$\alpha $-Arabinofuranosidase was produced by E. coli HB101 haboring the recombinant plasmid pKMG11 which contained the arfI gene of Bacillus stearothermophilus. The maximum production of the enzyme was observed when E. coli HB101 cells were grown at 37$\circ$C for 20 hours in the medium containing 0.5% arabinose, 1.0% tryptone, 0.5% yeast extract, and 1% NaCl. The $\ALPHA $-arabinofuranosidase produced was purified to homogeneity using a combination of 20-50% ammonium sulfate precipitation, DEAE-Sepharose CL-6B ion exchange column chromatography and Sepharose 6B-100 gel filtration. The purified enzyme was most active at 55$\circ$C and pH 6.5. The K$_{m}$ and V$_{max}$ values of the enzyme on $\rho $-nitrophenyl-$\alpha $-arabinofuranoside was determined to be 2.99 mM and 0.43 $\mu $mole/min (319.74 $\mu $mole/min/mg), respectively. The pI value was 4.5. The molecular weight of the native protein was estimated to be 289 kDa. The SDS-polyacrylamide gel clectrophoresis analysis suggested that the functional protein was a trimer of the 108 kDa identical subunits. The N-terminal amino acid sequence of the a-arabinofuranosidase was identified as X-Ser-Thr-Ala-Pro-Arg( \ulcorner )-Ala-Thr-Met-Val-Ile-Asp-X-Ala-Phe.

• KSBB Journal
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• 제31권2호
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• pp.105-112
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• 2016

Ferritin is known to regulate iron metabolism and maintain iron in a variety of the eukaryotic organisms. The region encoding the mature ferritin (0.47 kb, H-type) of Periserrula leucophryna was amplified using the designed primers including restriction enzyme site and termination codon and subcloned in frame to the pRBAS secretion vector containing the signal sequence, RBS, and promoter of amylase gene (E. coli-Bacillus shuttle vector), resulting in recombinant pRBAS-PLF vector. Recombinant ferritin (18 kDa) was correctly processed and secreted from Bacillus subtilis LKS strain harboring the pRBAS-PLF vector and quantitatively analyzed by SDS-PAGE and western blot, respectively. Secretion of the ferritin was optimized by culture conditions (host, medium, temperature, nitrogen source) in 3 L batch culture and 5 L jar fermenter. Finally. the ferritin was largely produced using 50 L fermenter as the following conditions; at $30^{\circ}C$, 150 rpm, 1 vvm in Bacillus subtilis LKS using PY medium. The secreted ferritin was maximally measured (approximately 177.6 ug/ml) when the cell density reached to 14.4 at $OD_{600}$ (20 h incubation). The iron binding activity was confirmed by Perls' staining in 7.5% non-denaturing gel, indicating that the multimeric ferritin (composed of 24 subunits) was formed in the culture broth after secretion. Biologically, the culture broth and powder type containing ferritin were tested for possibility as feed additive in chicken broiler. As a result, the ferritin stimulated the growth of chick broil and improved feed efficiency and production index.

• Journal of Microbiology and Biotechnology
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• 제9권6호
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• pp.854-860
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• 1999

Superoxide dismutase (SOD) was purified to homogeneity from fruiting bodies of edible mushroom, Lentinus edodes, by ammonium sulfate precipitation, diethylaminoethyl (DEAE)-Sepharose FF ion-exchange chromatography, Sephacryl S-200 gel filtration chromatography, and preparative PAGE. The molecular weight of the purified enzyme was estimated to be approximately 54 kDa by gel filtration chromatography, and the enzyme was shown to be consisted of two identical subunits of molecular weight 27 kDa by SDS-PAGE. The isoelectric point of the enzyme was 4.9 as determined by isoelectric focusing. The enzyme had optimal pH and temperature of pH 8.0 and $20^{\circ}C$, respectively. The activity of the enzyme was inhibited by hydrogen peroxide, but inhibited less by cyanide and azide. The native enzyme was found to contain 0.89g-atom of iron, 0.75g-atom of zinc, and 0.46g-atom of copper per mol of enzyme. Analysis of amino acids composition revealed that the SOD from L. edodes contained a relatively large amount of glutamic acid/glutamine, proline, cysteine, isoleucine, and leucine, but only a small amount of aspartic acid/asparagine, tyrosine, and tryptophan when compared to the other iron-containing SODs.