• Bulletin of the Korean Chemical Society
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• 제12권6호
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• pp.714-716
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• 1991

• BMB Reports
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• 제40권5호
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• pp.617-624
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• 2007

Protein arginine methylation is a posttranslational modification involved in various cellular functions including cell signaling, protein subcellular localization and transcriptional regulation. We analyze the protein arginine methyltransferases (PRMTs) that catalyze the formation of methylarginines in porcine brain. We fractionated the brain extracts and determined the PRMT activities as well as the distribution of different PRMT proteins in subcellular fractions of porcine brain. The majority of the type I methyltransferase activities that catalyze the formation of asymmetric dimethylarginines was in the cytosolic S3 fraction. High specific activity of the methyltransferase was detected in the S4 fraction (high-salt stripping of the ultracentrifugation precipitant P3 fraction), indicating that part of the PRMT was peripherally associated with membrane and ribosomal fractions. The amount and distribution of PRMT1 are consistent with the catalytic activity. The elution patterns from gel filtration and anion exchange chromatography also indicate that the type I activity in S3 and S4 are mostly from PRMT1. Our results suggest that part of the type I arginine methyltransferases in brains, mainly PRMT1, are sequestered in an inactive form as they associated with membranes or large subcellular complexes. Our biochemical analyses confirmed the complex distribution of different PRMTs and implicate their regulation and catalytic activities in brain.

• 약학회지
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• 제43권6호
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• pp.709-715
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• 1999

Polyphenol oxidase (PPO) activity in 200×g (cell wall), 4,000×g (plastid), 100,000×g (mitochondrial) and soluble fractions of the perilla leaves was monitored in the upper, middle and lower sections of the plant. In the course of plant growth, PPO activities in plastid and mitochondrial fractions were decreased, while those in cell wall fraction were maintained. During growing process, specific activities and PPO activities of each fraction were decreased, while total phenol content were decreased in middle (middle) and then increased in later stage (lower). Cell wall, plastid, mitochondrial (pellet) and soluble fraction had slightly different pH optima and substrate specificities. Isoenzyme patterns were identical in two bands for PPO activity in different subcellular fractions. Their molecular weights were 37KD and 48KD respectively.

• 대한의생명과학회지
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• 제18권2호
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• pp.160-164
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• 2012

The baculovirus/insect cell expression system is of great value for the large-scale production of normal and mutant mammalian passive glucose-transport proteins heterologously for structural and functional studies. In most mammalian cells that express HepG2, this transporter isoform is predominantly located at the cell surface. However, it had been reported that heterologous expression of other membrane proteins using the baculovirus system induced highly vacuolated cytoplasmic membranes. Therefore, how a cell responds to the synthesis of large amounts of a glycoprotein could be an interesting area for investigation. In order to examine the subcellular location of the human HepG2 transport proteins when expressed in insect cells, immunofluorescence studies were carried out. Insect cells were infected with the recombinant baculovirus AcNPVHIS-GT or with wild-type virus at a MOI of 5, or were not exposed to viral infection. A high level of fluorescence displayed in cells infected with the recombinant virus indicated that transporters are expressed abundantly and present on the surface of infected Sf21 cells. The evidence for the specificity of the immunostaining was strengthened by the negative results shown in the negative controls. Distribution of the transporter protein expressed in insect cells was further revealed by making a series of optical sections through an AcNPVHIS-GT-infected cell using a confocal microscope, which permits optical sectioning of cell sample. These sections displayed intense cytoplasmic immunofluorecence surrounding the region occupied by the enlarged nucleus, indicating that the expressed protein was present not only at the cell surface but also throughout the cytoplasmic membranous structures.

• 미생물학회지
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• 제42권4호
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• pp.271-276
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• 2006

이전의 연구 결과 어류 rhabdovirus에 감염된 세포에서 virus-inducible stress protein (VISP)의 발현이 증가함을 확인하였다. 본 연구에서는 VISP의 세포내 위치를 확인하였으며, 또한 세포내 위치 결정에 중요한 역할을 담당하는 VISP의 부위를 확인하였다. 먼저 endogenous VISP의 세포내 위치를 확인하기 위하여 CHSE-214 세포를 VISP에 대한 단클론항체를 사용하여 염색한 후 confocal microscope로 관찰하였다. 그 결과 VISP가 세포의 핵 주변에 점구조를 형성함이 확인되었다. 이를 확인하기 위하여 VISP에 enhanced green fluorescent protein (EGFP)이 붙은 fusion gene을 발현하는 plasmid를 제조하였다. EGFP-VISP를 발현하는 plasmid 벡터를 세포에 transfection 시킨 후 confocal microscope로 관찰한 결과 핵 주변에 점구조를 형성함이 확인되었다. VISP의 아미노산서열 중 핵 주변의 점구조 형성에 관여하는 부분을 확인하기 위하여 VISP의 다양한 deletion mutant들을 제조하였다. 이 mutant를 사용한 transfection 실험 결과 VISP의 C-terminal 부위(aa 612-710)가 핵주변의 점구조 형성에 중요한 역할을 담당함이 확인되었으며, 이 부분의 functional motif 분석결과 691-TLTSLLL-697 부위에 nuclear receptor binding motif가 존재함이 확인되었다. 이와 같은 결과들을 종합하면, VISP는 핵 주변에 존재하며 VISP의 C-terminal부위가 혀 주위 분포에 중요한 역할을 담당함을 알 수 있었다. 이후의 연구로부터 VISP의 핵 주위 분포가 IHNV의 성장에 미치는 영향이 확인되면 IHNV 병원성의 새로운 기작을 밝혀내는 중요한 자료가 될 것이다.

• 생명과학회지
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• 제9권6호
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• pp.722-727
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• 1999

본 연구는 연어 뇌 연접의 단백질구성에 대한 기초연구로서, 기억형성에 중요한 역활을 하는 NMDA 수용체의 분포와 PTK에 의한 인산화에 대하여 조사하였다. 본 실험에 사용한 연어 뇌의 PSD 분획에서는 Coomassie로 염색할 경우 20여개 분명한 단백질띠를 확인할 수 있었으며, 소량으로 존재하여 전체적으로 도말되어 보이는 펩타이드의 수는 알 수 없었다. 이들 중 180kD 크기의 단백질은 phosphotyrosine 특이성 항체에 상대적으로 잘 인식이 되었다. 또한 이 180 kDa의 단백질들은 쥐에 있어서의 NR2A 및 NR2B의 위치인 분자량 약 180kD의 위치와 동일한 것으로 보아 연어에 있어서도 후각기능에 관계하는 NR2A 및 NR2B가 존재함을 추정할 수 있다.

• 대한수의학회지
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• 제39권1호
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• pp.45-54
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• 1999

In the present study we have analyzed the characteristics and distribution of the mu-opioid receptor(MOR) by raising anti-peptide antisera to the C-terminal peptide of MOR. The antisera against MOR was produced in New Zealand White rabbit against 15 residue corresponding to amino acids, 384-398 of the cloned rat MOR. The antigenic peptide was synthesized using an Applied Biosystems 432 solid-phase peptide synthesizer. The specificity and identification of the antisera were tested by analysis of transfected cells, epitope mapping and immunohistochemical method. COS-7 cells electroporated with MOR cDNA were used to evaluate the characteristics and subcellular distribution of MOR. MOR immunoreactivity was prodominent in the plasmalemma and subcellular compartments such as endoplasmic reticulum, Golgi apparatus and vesicle like structure. Furthermore, both tissue sections and transfected cell lines could be immunostained with these antisera and the immunoreactivity was abolished when anti-MOR sera were preincubated with the peptide against which they were raised. Based on epitope mapping analysis, all antisera appeared to have a similar epitope, which was determined to be within the last amino acid, 391-398. Moreover, immunohistochemistry showed that MOR immunoreactivity was observed in many brain areas including cerebral cortex, striatum, hippocampus, locus coeruleus and the superficial laminae of the dorsal horn. These stained spinal cord and brain areas showed the mirrored pattern observed in auto radiographic studies of mu-opioid binding as well as a pattern similar to that seen by is situ hybridization for MOR. Thus, several lines of evidence support the conclusion that the antisera produced in the present study most likely recognize mu-opioid receptor. These results suggest that MOR antisera may be utilized as useful tool to analyze the physiological and pharmacological studies for mu-opioid receptor in the future.

• Parasites, Hosts and Diseases
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• 제21권1호
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• pp.41-48
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• 1983

The activity and distribution of aspartate aminotransferase (EC 2.6. 1. 1) and alanine aminotransferase (EC 2.6.1.2) in adult Fascicle hepatica have been studied. Fasciola hepatica was fractionated by differential centrifugation into nuclear, mitochondrial and cytosolic fractions. The activity of GOT and GPT was measured by the method of Reitman and Frankel. Isozyme patterns of those enzyme were also examined by DEAE-cellulose column chromatography. The results obtained were as follows; 1. The activity of aspartate and alanine aminotransferase was about 0.55 unit and 0.92 unit per 1g of Fascicle hepatica, respectively. 2. The activity of those enzymes was relatively low compared with those in mammalian tissues. 3. The distribution of aspartate aminotransferase in the subcellular organelles showed that 71% of the activity was in cytosolic, 24% in mitochondrial and 5% was in nuclear fraction. 4. About 22% of the total alanine aminotransferase activity was found in the mitochondrial fratstion, about 66% in the cytosolic fraction. 5. Aspartate aminotransferase from cytosolic fraction was separated into two types of isozymes, whereas alanine aminotransferase from cytosolic fraction gave only one active peak on DEAE-cellulose column chromatography.

• Molecules and Cells
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• 제22권1호
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• pp.97-103
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• 2006

Phosphoinositides are critical regulators of ion channel and transporter activity. There are multiple isomers of biologically active phosphoinositides in the plasma membrane and the different lipid species are non-randomly distributed. However, the mechanism by which cells impose selectivity and directionality on lipid movements and so generate a non-random lipid distribution remains unclear. In the present study we investigated which structural elements of phosphoinositides are responsible for their subcellular location and movement. We incubated phosphatidylinositol (PI), phosphatidylinositol 4-monophosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate ($PI(4,5)P_2$) with short or long acyl chains in CHO and HEK cells. We show that phosphate number and acyl chain length determine cellular location and translocation movement. In CHO cells, $PI(4,5)P_2$ with a long acyl chain was released into the cytosol easily because of a low partition coefficient whereas long chain PI was released more slowly because of a high partition coefficient. In HEK cells, the cellular location and translocation movement of PI were similar to those of PI in CHO cells, whereas those of $PI(4,5)P_2$ were different; some mechanism restricted the translocation movement of $PI(4,5)P_2$, and this is in good agreement with the extremely low lateral diffusion of $PI(4,5)P_2$. In contrast to the dependence on the number of phosphates of the phospholipid head group of long acyl chain analogs, short acyl chain phospholipids easily undergo translocation movement regardless of cell type and number of phosphates in the lipid headgroup.

• 지구물리와물리탐사
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• 제4권2호
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• pp.45-54
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• 2001

지표 레이다(Ground Penetrating Radar; GPR) 탐사를 모사하기 위해서, 시간 영역 유한 차분법을 이용하여 송수신 안테나를 고려한 3차원 모델링 알고리듬을 개발하였다. 국내에서 주로 사용되는 GPR탐사기기인 쌍극자 안테나를 모사하고, 안테나 끝점에서의 내부반사(ringing) 현상을 줄이기 위해 subcellular법을 사용하였다. 반무한 매질에서 종단 저항에 따른 출력 전압의 변화를 비교하여, 종단 저항의 수가 많아지면 내부반사가 줄어드는 반면 출력 전압이 작아진다는 사실을 알 수 있었고, 이 결과를 토대로 내부반사를 줄이는 쌍극자 안테나를 구현하였다. 각도에 따른 전기장 분포를 알기 위해 안테나에 직교하는 면과 안테나를 포함하는 면에서 방사 패턴을 구하였다. 안테나를 포함하는 면의 전기장 에너지가 안테나에 직교하는 면보다 안테나 직하부로 집중되는 현상을 볼 수 있었다.