• Journal of Plant Biology
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• 제24권4호
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• pp.171-179
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• 1981

옥수수 뿌리로부터 분리한 13,000g pellet과 13,000~80,000g pellet 내에 있는 membrane-bound ATPases의 특성을 구명하였다. 13,000g pellet과 13,000~80,000g pellet의 membrane-bound ATPases의 최적 pH는 5와 9였다. Discontinuous sucrose gradient centrifugation에 의한 13,000g pellet의 분획중 Fraction C는 pH 5에서, Fraction D, E 및 F는 pH 5에서보다 pH 9에서 더높은 활성을 나타냈다. 13,000~80,000g pellet의 분획에서 보면, Fraction A, C는 pH 9보다 pH 5에서, Fraction B, D, E 및 F는 pH 5보다 pH 9에서 더 높은 활성을 가겠다. pH 5와 pH 9에서 membrane-bound ATPases의 기질포화 농도는 3~5 mM이며 ATP에 대한 Km 값은 모두 0.25 mM이였다. Vmax 값은 8.0~55.6 $\mu$M Pi/mg membrane protein/hr의 범위에 있었다. Membrane-bound ATPase의 활성은 $K^+$ 이온에 의해 증가되었다.

• 한국동물학회지
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• 제28권1호
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• pp.31-43
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• 1985

$(Ca^{2+}+Mg^{2+})$-ATPase를 쥐의 근소포체로부터 sucrose density gradient centrifugation의 방법을 사용하여 분리 정제하였다. 정제된 효소를 폴리아크릴 아마이드 젤에서 전기영동한 결과, 토끼와 닭의 경우에서와 같이 분자량 115,000인 단일 단백질 띠로 나타났다. 정제된 이 효소의 활설도는 50 $\\muM$의 $Mg^{2+}, Ca^{2+}, Co^{2+}, Fe^{2+}, Min^{2+}$에 의해서는 증가되었고, 같은 농도의 $Zn^{2+}, Cu^{2+}, Hg^{2+}$에 의해서는 감소되었다. Quinine와 quinacrine 같은 antimalarial drug는 이 효소의 활성도에 큰 영향을 주지 않았으나, p-hydroxymercuric benzoate와 phenylmethylsulfonylfluoride는 이 효소의 활성을 억제하였다. 이 효소는 pH 6과 7 사이에서 가장 높은 활성을 나타내었고, ATP를 기질로 사용하였을 때 Km 값은 98 $\\muM$이었다. $(Ca^{2+}+Mg^{2+})$-ATPase는 microsomal fraction에서 선택적으로 분해되었다. $^{3}H-casein$ 이나 ^{125}I-insulin같은 방사성 동위원소로 표지된 기질을 사용하여 단백질 분해에 대한 활성도를 조사해 본 결과, microsomal preparation에 metalloendoprotease가 존재하였다. 그러나 아직까지는 그 효소가 $(Ca^{2+}+Mg^{2+})$-ATPase를 분해하는지는 확실하지 않다.

• BMB Reports
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• 제29권3호
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• pp.253-260
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• 1996

An enzyme that hydrolyzes flavin-adenine dinucleotide (FAD) was found to be present in rat liver lysosomal membrane prepared from Triton WR-1339 filled lysosomes (tritosomes) purified by flotation on sucrose. This FAD phosphohydrolase (FADase) exhibited optimal activity at pH 8.5 and had an apparent Km of approximately 3.3 mM. The activity was decreased 50~70% by dialysis against EDTA and this was restored by $Zn^{2+}$, $Mg^{+2}$, $Hg^{+2}$, and $Ca^{+2}$ ions inhibited the enzyme, but $F^-$ and molybdate had no effect. The enzyme was also inhibited by p-chloromercuribenzoate (pCMB), reduced glutathione and other thiols, cyanide, and ascorbate. The presence of ATP, ADP, AMP. ${\alpha}-{\beta}-methylene$ ATP, AMP-p-nitrophenyl phosphate (PNP), GMP, and coenzyme A (CoA) decreased the activity on FAD, but pyrimidine nucleotides, adenosine, adenine, or $NAD^+$ were without effect. Phosphate stimulated the activity slightly. FAD phosphohydrolase activity was separated from ATPase and inorganic pyrophosphatase activities by solubilization with detergents and polyacrylamide gel electrophoresis and by linear sucrose density gradient centrifugation suggesting that the enzyme is different from ATPase, inorganic pyrophosphatase, and soluble lysosomal FAD pyrophosphatase. Paper chromatography showed that FAD was hydrolyzed to flavin mononucleotide (FMN) and AMP which were further hydrolyzed to riboflavin and AMP by phosphatases known to be present in lysosomal membranes. Incubation of the intact Iysosomes with pronase showed that the active site of FAD phosphohydrolase must be oriented to the cytosol. The FAD hydrolyzing activity was detected in Golgi, microsome, and plasma membrane, but not in mitochondria or soluble lysosomal preparations.

• BMB Reports
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• 제35권5호
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• pp.518-523
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• 2002

Nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase, inhibits the secretion of proteins and causes the redistribution of resident Golgi proteins into the endoplasmic reticulum (ER). In this study, the effect of NDGA on lipoprotein lipase (LPL) secretion was investigated in 3T3-L1 adipocytes, and compared with those of brefeldin A (BFA), a well-known fungal metabolite that exhibits similar ER-Golgi redistribution. Both BFA and NDGA blocked secretions of LPL. In the presence of BFA, the active and dimeric LPL was accumulated in adipocytes. After endoglycosidase H (endo H) digestion, the proportion of LPL subunits with partially endo H-sensitive oligosaccharide was significantly increased with BFA. However, in the presence of NDGA, the cellular LPL became inactive, and only the endo H-sensitive fraction of the LPL subunit was observed. An increase of the aggregated forms was observed in the fractions of the sucrose-density gradient ultracentrifugation. These properties of LPL in the NDGA-treated cells were similar to those of LPL that is retained in ER, and the effects of NDGA could not be reversed by BFA. These results indicate that the inhibitory mechanism of NDGA on the LPL secretion is functionally different from the ER-Golgi redistribution that is induced by BFA.

• Journal of the Korean Wood Science and Technology
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• 제17권3호
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• pp.61-66
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• 1989

포플라 세포(細胞)의 Sucrose gradient 원심분리(遠心分離)에 나타난 세포(細胞)분획은 6개의 band 및 침전으로 분리(分離) 되었다. Peroxidase의 활성변화(活性變化)는 휴면기(休眼期)에 높아졌으며, 자발적(自發的) 휴면(休眠)이 타파되면서 낮아지기 시작하였다. 휴면기(休眠期)에 급격히 높아진 Peroxidase 활성(活性)은 Peroxidative 활성(活性)을 지닌 세포내(細胞內) Microbody에 존재(存在)하는 것으로 사료된다. Catalase의 활성(活性)은 11월(月), 12 월(月) 1월(月)의 휴면기(休眼期)에 활성(活性)이 낮았으며. IAA의 농도는 휴면초기(休眼初期)에 감소하기 시작하여, 12월(月)에 최저(最低)가 되었다. 자연휴면(自然休眠)이 타파된 1월(月)부터는 IAA 농도가 증가하기 시작하여 4월(月)의 발아기(發芽期)까지 계속되었다. 이와같은 Peroxidase, Catalase를 비롯한 IAA의 변화(變化)는 봄철의 발아(發芽) 및 재생장(再生長)과 가을철 휴면조절(休眠調節)에 영향을 미치는 것으로 사료된다.

• BMB Reports
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• 제28권2호
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• pp.129-137
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• 1995

The ryanodine-receptor $Ca^{2+}$ release channel protein in the sarcoplasmic reticulum membrane of rabbit skeletal muscle plays an important role in muscle exitation-contraction (E-C) coupling. Various types of detergents were tested, including Chaps, cholate, octylglucoside, Zwittergents, Mega-9, Lubrol PX, and Triton X-100 for solubilization of this protein. Among these, Chaps and Triton X-100 were found to optionally solubilize the channel complex. Optimum conditions for this solubilization were pH 7.4 with a salt concentration of 1 M. The addition of phospholipid in the solubilization step helped in stabilizing the protein. The purification of the receptor was performed using sucrose density gradient centrifugation. Various methods [dilution, freeze-thaw, adsorption (Biobeads), and dialysis] were investigated to incorporate the Chaps-solubilized and purified $Ca^{2+}$ release channel protein into liposomes made from different types of phospholipids. Of these, a combined method consisting of a dialysis, freeze-thaw and sonication steps yielded the best results. Reconstituted vesicles produced by this method with 95% phosphatidylcholine (from soybean extract) had good function.

• Journal of Microbiology and Biotechnology
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• 제15권4호
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• pp.767-771
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• 2005

Expression in yeast may prove more amenable to generating large amounts of viral antigens for a vaccine candidate. We, therefore, cloned the gene encoding the Hepatitis C virus (HCV) structural proteins (C-El-E2, c740) fused in-frame with, and immediately 3' to, the chicken-lysozyme signal peptide (C-SIG) gene and under the control of the yeast glyceraldehyde-3-phosphate dehydrogenase gene promoter. In yeast, the HCV structural proteins were expressed in two different forms: a processed and a nonprocessed aggregated form. Biophysical characterization by sucrose linear gradient centrifugation revealed that both forms were present in the same fractions with a buoyant density of 1.127-1.176 g/$cm^3$. These findings suggest that the efficient synthesis of HCV structural proteins in yeast may be an important tool to study virus assembly and may lead to the development of an HCV vaccine.

• Journal of Microbiology and Biotechnology
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• 제15권6호
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• pp.1229-1239
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• 2005

Human immunodeficiency virus-like particles (HIVVLPs) with native conformations similar to that of the wild-type virion could be valid candidates for vaccine development. To this end, we used a Semliki Forest Virus (SFV) expression system to produce HIV- VLPs containing high quantities of native envelope proteins. Here, we described a single SFV replicon containing the HIV gagpol and env genes under the control of separate subgenomic promoters. Mature VLPs incorporating the Gag and Env proteins were detected in the supernatant of replicon-expressing cells by Western blot analysis. The HIV-VLPs showed the expected molecular density (1.14-1.18 g/ml) on a $20-60\%$ sucrose gradient; the particles were 100-120 nm in diameter and Env proteins were observed on their surfaces by immunogold electron microscopy. RT-PCR analysis of VLP-associated RNAs in mature HIV-VLPs revealed two SF V-derived RNA species (full-length and subgenomic). Immunization studies in Balb/c mice showed that these HIV-VLPs were capable of inducing both HIV-specific antibodies and cell-mediated immune responses. Taken together, our results indicate that the SFV replicon system is useful for the production of HIV-VLPs, which may be valuable candidates for an HIV vaccine.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 1986년도 추계학술대회
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• pp.512.1-512
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• 1986

The cdd gene of Bacillus subtilis, encoding the deoxycytidinecytidine deaminase of pyrimidine nucleotide biosynthesis has been cloned into the EcoRl site of pBR322. The recombinant plasmid, pSol, promoted the synthesis of 100-140 fold elevated levels of the enzyme. A comparison of the polypeptides encoded by cdd complementing and non-complementing plasmids in the mini cell showed the gene product to have a molecular mass of approximately 14 kDa. The nucleotide sequence of the gene and 460 base pairs upstream from the coding region was determined. An open-reading frame, encoding a protein with a calculated molecular mass of 14337 Da, was deduced to be the coding region for cdd. However, the enzyme has an apparent molecular mass of 54 kDa as determined by gel filteration, whereas sucrose density gradient centrifugation shows 58 kDa. It means that the enzyme could be forming a tetramer in a physiological state. About 28 amino acids of the N-tetramer in a physiological state. About 28 amino acids of the N-terminal presumably form a signal for membrane translocation and six cystein residues are contained in the structure. S1 nuclease mapping indicated that transcription of cdd is initiated 17 base pairs upstream from the translational start. The structural characterization of the odd gene was performed.

• Asian-Australasian Journal of Animal Sciences
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• 제20권10호
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• pp.1594-1599
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• 2007

Plasma membrane proteins from pig adipocytes, brain, heart, kidney, liver and spleen were isolated using a 32% sucrose gradient. An adult male sheep was immunized three times at 3-wk intervals with the purified pig adipocyte plasma membrane (APM) proteins. Blood samples were taken from the immunized sheep 12 d after the third immunization. Antiserum showed strong reactivity with APM proteins determined by ELISA, and the reactivity could be detected at dilutions in excess of 1:128,000. Antiserum showed very low binding affinity with proteins isolated from brain, heart, kidney, liver or spleen. Ninety weanling pigs were allocated randomly to three treatment groups and were injected i.p. with 40 ml of antiserum (n = 30) or 20 ml of lyophilized antiserum (21.5 mg/ml; n = 30). A control group (n = 30) received 40 ml of saline, and all pigs were slaughtered at 24 wk of age. The polyclonal antiserum did not change BW or ADG. Carcass percentage of pigs was numerically increased by the antiserum treatment compared with control. Both antiserum treatments did not significantly (p>0.05) affect body composition, including body fat content, relative to the control group.