이 작품은 NSF 상을 # 0,920,274 및 사우스 캐롤라이나 실험 역 프로젝트 (SC - 1700340) KSS에 의해 지원되었다. 본 논문은 클렘슨 대학 실험 역 5929 기술 공헌 번호입니다.

이 프로토콜의 전체 구조는 그림 1에 설명되어있다. 1. 표준 곡선 및 Assays을위한 솔루션 준비 <ol><li> 히드록 실 아민 - HCL의 2 몰 / L 솔루션의 100 ML를 준비합니다. 히드록 실 아민 하이드로 클로라이드의 13.9 g (MW 69.49 g / 몰)을 무게와 약 50 ML 증류수 - 탈이온수 (ddH 2 O)에 용해. 수산화 칼륨의 알약이나 집중 솔루션을 사용하여 7.0 산도를 조정합니다. 100 ML에 최종 볼륨을 가져와. 솔루션은 최대 30 일 또는 4 ° C 90 일까지에 대한 상온에서 저장할 수 있습니다. </li><li> 산화철 염화 / 염산 용액 100 ML을 준비합니다. 13.5 g 산화철 염화물 (MW 270.32 g / 몰)을 무게와 약 50 ML ddH 2 O.에 용해 41.3 ML 집중 염산 (12.1 몰 / L)을 추가 100 ML의 최종 볼륨 가져와. 산화철 염화의 최종 농도는 0.5 몰 / L와 염산의 최종 농도는 5 몰 될 것입니다/ L. 솔루션은 상온에서 저장됩니다. </li><li> trichoroacetic 산의 1.84 몰 / L 솔루션의 100 ML를 준비합니다. trichloroacetic 산 (MW 163.39 g / 몰)의 30.0 g를 무게, ddH 2 O에 용해 100 ML에 최종 볼륨을 가지고. 솔루션은 상온에서 저장됩니다. </li><li> 0.1 몰 / L 아세틸 인산 용액 5 ML을 준비합니다. 0.092 g 아세틸 인산이 전분 (MW 184.06 g / 몰)을 무게, ddH 2 O에 용해 5 ML에 최종 볼륨을 가지고. 나누어지는 microcentrifuge 튜브 (튜브 당 0.25 ML)에 솔루션을 사용하기 전까지 -20 ° C에서 동결. 해동 솔루션은 얼음에 배치해야하고 약 1 시간 동안 사용할 수 있습니다. 해동되었습니다 아세틸 인산 솔루션은 refrozen하고 다시 사용하지 않을 것입니다. </li><li> ADP의 0.1 몰 / L 솔루션의 5 ML을 준비합니다. ADP의 0.24 g을 (MW 472.5 g / 몰) 달아서 나누다, ddH 2 O에 용해, 5 ML에 최종 볼륨을 가지고. -20에서 나누어지는 microcentrifuge 튜브에 용액 (튜브 당 0.5 ML)과 냉동 ° C까지사용합니다. 사용 전까지 얼음에 해동 솔루션 보관하십시오. </li><li> 트리스의 1 몰 / L 용액의 50 ML을 준비합니다. 6.06 g 트리스 (MW 121.14 g / 몰)을 무게, 40 ML ddH 2 O.에 용해 염산을 사용하여 7.0 솔루션의 산도를 조정 50 ML에 최종 볼륨을 가지고. 상온에서 저장 솔루션입니다. </li><li> 1 몰 / L 마그네슘 염화물 솔루션 10 ML을 준비합니다. 2.03 g의 염화 마그네슘 (MW 203.31 g / 몰)을 무게, ddH 2 O에 용해, 10 ML에 최종 볼륨을 가지고. 상온에서 솔루션을 저장합니다. </li><li> 산화철 염화 / 염산 용액과 산성 trichloroacetic 솔루션의 동등 권을 혼합 25 ML 개발 솔루션을 준비합니다. 개발 솔루션은 매일 신선한 준비하고 실온에서 보관해야합니다. </li></ol> 2. 아세틸 인산 표준 곡선의 준비 <ol><li> 아세틸 인산 표준 곡선은 아세틸 인산 알려진 금액을 사용하여 생성됩니다. eigh 6의 표준 곡선t 포인트 적합합니다. 기준은 0.03 μmoles에서 0.9 μmoles로 0.1-3 mmol / L.의 최종 농도로 300 μL 상호를 범위해야 300 μL의 최종 볼륨 100 MM 트리스 솔루션에 적절한 농도로 아세틸 인산 재고 솔루션을 희석. 아세틸 인산이없는 컨트롤 샘플도 준비되어야합니다. </li><li> ° C 열 블록에서 1 분 37 각 표준 및 컨트롤을 품어. </li><li> 각 표준에 히드록 실 아민 하이드로 클로라이드 솔루션과 세 번 흔들어하여 제어 및 혼합 50 μL를 추가합니다. 60 ° C 열 블록에 표준을 놓고 5 분 동안 품어. </li><li> 100 표준 개발 솔루션의 μL 및 제어, 세 번 흔들어하여 믹스를 추가하고 색상 실온에서 1 분 이상을 위해 개발 수 있습니다. </li><li> 1 분 18,000 XG에 microcentrifuge의 모든 기준과 컨트롤을 원심. </li><li> spectrophotometrically 샘플을 측정분광 광도법을위한 빈과 같은 컨트롤을 사용하여 1mL 플라스틱 cuvettes를 사용하여 t 540 NM. </li><li> 적절한 데이터 분석 및 그래프 소프트웨어 (예 : Kaleidagraph, 시너지 소프트웨어)를 사용하여 표준 곡선을 생성합니다. 0.99 이상의 R 2 값은 매력입니다. 데이터는 아세틸 인산 현재의 μmoles 비해 540 nm의 흡광도에서로 꾸몄다 있습니다. </li></ol> 3. 아세테이트 키나제 활동에 대한 분석 <ol><li> 설명된 솔루션을 사용하여, 0.1 몰 / L 트리스 버퍼, 10 mmol / L 마그네슘 염화물, 5 mmol / L ADP, 2 mmol / L 아세틸 인산의 최종 농도를 포함하는 반응 혼합물을 준비합니다. 10 ML 반응 혼합물은 8.2 ML ddH 2 O, 1.0 ML 트리스 솔루션, 0.1 ML 마그네슘 염화물 용액, 0.5 ML ADP 솔루션, 그리고 0.2 ML 아세틸 인산 솔루션을 필요합니다. microcentrifuge 튜브 300 μl aliquots의 반응 믹스를 배포합니다. 기판 농도의 운동 매개 변수와 최적화 결정에 대한 하나의 기판 수 있습니다다양하고 다른 반응에 일정한 농도에서 열립니다. </li><li> 37 일분 대한 반응을 미리 품어 ° C 열 블록에. </li><li> 반응 효소의 원하는 금액을 추가하고 즉시 37 ° C 열 블록에 튜브를 반환합니다. 또한는 효소가 추가되지되는 컨트롤에 대한 반응을 포함합니다. 여러 개의 반응을 수행할 경우, 특정 시간 간격으로 튜브에 효소를 추가합니다. 시간 추적해야합니다. (참고 : 사용할 수 효소의 양은 아세틸 인산이나 ADP 기판의 모든이 소진되지 않을 것임을 보장하기 위해 최적화해야합니다 - 토론 섹션을 참조) </li><li> 5 분 후에 각각의 반응과 제어, 혼합에 히드록 실 아민 하이드로 클로라이드 솔루션의 50μl를 추가하고, 60 ° C 열 블록에 튜브를 삽입합니다. 여러 가지 반응이 수행되는 경우에는 효소가 추가되면서, 이것은 동일한 시간 간격으로 수행되어야합니다. </li><li> 60에서 5 분 동안 반응과 컨트롤을 품어 ° C. </li><li> 100 및 추가무, 각각의 반응과 실험실 벤치에서 튜브 랙에 제어, 믹스, 그리고 장소에 개발 솔루션의 L. 색상은 적어도 1 분 개발할 수 있습니다. 허용 </li><li> 1 분 18,000 XG에 microcentrifuge의 모든 튜브를 원심 분리기. </li><li> 측정 샘플 spectrophotometrically 1mL 플라스틱 cuvettes를 사용하여 540 nm의에서, 아세틸 인산 표준 곡선과 비교하여 아세틸 인산 현재의 양을 결정합니다. </li><li> 고갈 아세틸 인산 기판의 크기는 조절 부족한 효소에있는 아세틸 인산이 전분의 양을에서 효소 반응 후 남아있는 아세틸 인산이 전분의 양을 빼서에 의해 결정하실 수 있습니다. 적절한 데이터 및 그래프 소프트웨어 (예 : Kaleidagraph, 시너지 소프트웨어)을 사용하여 데이터 분석을 수행합니다. 0.97보다 큰의 R 2 값은 매력입니다. </li></ol> 4. 대표 결과 : 이 분석의 목적은 직접에 아세트산 키나제의 활동을 측정하는 것입니다아세테이트 형성 이온. 아세테이트 생산에 대한 쉬운, 직접 측정이없는 등이, 아세틸 인산 기판의 소비를 측정하여 이루어집니다. 효소 반응 동안 주어진 시간 이후에 남아있는 아세틸 인산이 아세틸 hydroxamate하고 이후 540 nm의에서 측정할 수있는 붉은 색 (그림 2)이 산화철 hydroxamate 단지로 변환됩니다. 표준 μmoles 아세틸 인산 선물 대 흡광도를 계획하고 표준 곡선은 각 반응에 남아 아세틸 인산이 전분의 양을 결정하는 데 사용됩니다. 표준 곡선은 영점을 통해 선형되어야합니다. 그림 3과 같이 대표적인 표준 곡선은 데이터를 잘 선형 방정식에 맞는을 나타내는 μmole 아세틸 인산 제시하고 0.99의 R 2 값 당 1.2332 흡광도 단위의 슬로프가 있습니다. 표준 곡선 데이터에 선형 적합 방정식은 반응에 남아 아세틸 인산의 수량을 계산하는 데 사용됩니다. μmoles소비 아세틸 인산이 전분의 효소 및 효소 반응 후 남아있는 아세틸 인산의 μmoles을 부족한 제어 반응에있는 아세틸 인산의 μmoles 차이로 결정됩니다. 정화, 재조합 M. thermophila 아세트산 키나제는 설명 프로토콜을 사용 assayed했다. 그림 4에 표시된 실험, ADP의 농도 5 mmol / L에서 지속적인 개최되었으며 반응으로 아세틸 인산의 농도는 아세틸 인산에 대한 K M을 결정하기 위해 다양한되었습니다. 예상했던대로,이 실험은 효소가 각 기판에 대한 표준 Michaelis - Menten 같은 속도론을 다음과 아세틸 인산 농도 비교 활동을하려 할 때 쌍곡선 포화 곡선을 생산 것을 보여줍니다. 아세틸 인산이 전분에 대한 명백한 K m 값은 0.27 ± 0.47의 게시된 K m 값 유리와 비교 0.01 mmol / L, ± 결합으로 사용하는 0.01 mmol / L되었습니다13 말한다. 직접 분석은 또한 표준 결합 분석을 사용 불가능 ATP 이외의 기판을 활용 아세테이트 kinases의 활동을 측정하는 데 사용할 수 있습니다. 재조합 E.을 정화 아세테이트 - 성형 방향으로 PP 전보다는 ATP를 생산 histolytica 아세테이트 키나제 16, ADP 대신에 인산 나트륨을 사용하여이 분석의 대상이되었다. 반응에서 인산 나트륨의 농도는 0.2 몰 / L에서 지속적인 개최되었다 그리고 아세틸 인산 농도가 다양했습니다. M.에 대해서는 thermophila 아세트산 키나제, E. histolytica 아세트산 키나제는 아세틸 인산에 대한 Michaelis - Menten 같은 속도론을 따라하고 쌍곡선 포화 곡선은 (그림 5) 관찰되었다. 아세틸 인산이 전분에 대한 명백한 K m 값은 0.50 ± 0.006 mmol / L.되었습니다 리브스와 오클라호마 주 15 원래 효소에 대한 아세틸 인산에 대한 0.06 mmol / L의 K m 값을 결정; 방법<html> 도, 그들의 효소가 부분적으로만 정제 및 분석도 일부만 정화 4 명이 결합 효소를 참여했습니다. 그러므로, 그것은 바로 이러한 가치를 비교하기가 어렵습니다. 프로토콜 제도 <ol><li> 아세틸 인산 표준 곡선을 생성 </li><li> 원하는 농도에 효소를 희석 </li><li> microcentrifuge 튜브에 반응 혼합물과 나누어지는 300 μl 준비 </li><li> 1 분 37 Preinc​​ubate 반응 ° C </li><li> 특정 시간 간격으로 효소를 추가하여 반응을 시작합니다 </li><li> 반응을 중지하고 60 부화 같은 간격으로 히드록 실 아민을 추가 ° C를 5 분 </li><li> 산화철 염화물 / 컬러 개발을위한 trichloroacetic 산 솔루션 추가 </li><li> 1 분 18,000 XG에 튜브를 원심 분리기 </li><li> 540 nm의 흡광도를 측정에서 </li><li> 표준 곡선과 비교하여 소비 아세틸 인산이 전분의 양을 결정 </li></ol>tp_upload/3474/3474fig1.jpg &quot;/&gt; 그림 1. 프로토콜 계획 <img alt="그림 2" src="/files/ftp_upload/3474/3474fig2.jpg" /> 그림 2. 아세틸 인산 기준. 아세틸 인산의 농도 증가가 설명된대로 히드록 실 아민 및 색상이 개발로 반응했다. 아세틸 인산이 부족한 컨트롤은 밝은 노란색이며, 아세틸 인산의 증가 금액을 포함하는 반응 컬러 연속 어두운됩니다. 이러한 색상 변경은 540 nm의에서 측정됩니다. <img alt="그림 3" src="/files/ftp_upload/3474/3474fig3.jpg" /> 그림 3. 샘플 아세틸 인산 표준 곡선. 아세틸 인산이 전분의 다양한 양의 히드록 실 아민과 반응되었으며 색상 개발 솔루션과 흡광도가 540 NM에서 측정되었다. μmoles 아세틸 인산이 전분 대 540 nm의 흡광도에서이 역모되었고 데이터에 선형에 맞게이 적용되었습니다. &lt;/ P&gt; <img alt="그림 4" src="/files/ftp_upload/3474/3474fig4.jpg" /> 그림 4. ATP 생산 M.에 의해 아세틸 인산의 이용 thermophila 아세트산 키나제. 효소 활동은 아세틸 인산의 다양한 농도 5 mmol / L ADP의 면전에서 아세테이트 형성의 방향으로 결정되었다. 아세틸 인산 소비의 양은 반응으로 아세틸 인산의 시작 금액 및 효소 반응 후 남아있는 아세틸 인산의 양에 차이로 결정되었습니다. Assays이 표시 오차 막대와 세중의에서 수행되었습니다. <img alt="그림 5" src="/files/ftp_upload/3474/3474fig5.jpg" /> 그림 5. PP에 의해 아세틸 인산의 이용 전 생산하는 E. histolytica 아세트산 키나제. 아세테이트 형성의 방향으로 효소 활동은 0의 존재에서 산출된 것입니다.아세틸 인산의 농도를 변화 2 몰 / L 인산 나트륨. 아세틸 인산 소비의 양은 반응으로 아세틸 인산의 시작 금액 및 효소 반응 후 남아있는 아세틸 인산의 양에 차이로 결정되었습니다. Assays이 표시 오차 막대와 세중의에서 수행되었습니다.

<ol>
	<li>Bork, P., Sander, C., Valencia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ATPase+domain+common+to+prokaryotic+cell+cycle+proteins,+sugar+kinases,+actin,+and+hsp70+heat+shock+proteins.">An ATPase domain common to prokaryotic cell cycle proteins, sugar kinases, actin, and hsp70 heat shock proteins.</a> Proc. Natl. Acad. Sci. U. S. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning,+sequence+analysis,+and+hyperexpression+of+the+genes+encoding+phosphotransacetylase+and+acetate+kinase+from+Methanosarcina+thermophila.">Cloning, sequence analysis, and hyperexpression of the genes encoding phosphotransacetylase and acetate kinase from Methanosarcina thermophila.</a> J. Bacteriol. 175, 6822-6829 (1993).</li><li>Ingram-Smith, C., Barber, R. D., Ferry, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+histidines+in+the+acetate+kinase+from+Methanosarcina+thermophila.">The role of histidines in the acetate kinase from Methanosarcina thermophila.</a> J. Biol. Chem. 275, 33765-33770 (2000).</li><li>Miles, R. D., Iyer, P. P., Ferry, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-directed+mutational+analysis+of+active+site+residues+in+the+acetate+kinase+from+Methanosarcina+thermophila.">Site-directed mutational analysis of active site residues in the acetate kinase from Methanosarcina thermophila.</a> J. Biol. Chem. 276, 45059-45064 (2001).</li><li>Miles, R. D., Gorrell, A., Ferry, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+a+transition+state+analog,+MgADP-aluminum+fluoride-acetate,+in+acetate+kinase+from+Methanosarcina+thermophila.">Evidence for a transition state analog, MgADP-aluminum fluoride-acetate, in acetate kinase from Methanosarcina thermophila.</a> J. Biol. Chem. 277, 22547-22552 (2002).</li><li>Ingram-Smith, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+acetate+binding+pocket+in+the+Methanosarcina+thermophila+acetate+kinase.">Characterization of the acetate binding pocket in the Methanosarcina thermophila acetate kinase.</a> J. Bacteriol. 187, 2386-2394 (2005).</li><li>Gorrell, A., Lawrence, S. H., Ferry, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+kinetic+analyses+of+arginine+residues+in+the+active+site+of+the+acetate+kinase+from+Methanosarcina+thermophila.">Structural and kinetic analyses of arginine residues in the active site of the acetate kinase from Methanosarcina thermophila.</a> J. Biol. Chem. 280, 10731-10742 (2005).</li><li>Gorrell, A., Ferry, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+the+Methanosarcina+thermophila+acetate+kinase+mechanism+by+fluorescence+quenching.">Investigation of the Methanosarcina thermophila acetate kinase mechanism by fluorescence quenching.</a> Biochemistry. 46, 14170-14176 (2007).</li><li>Reeves, R. E., Guthrie, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetate+kinase+(pyrophosphate).+A+fourth+pyrophosphate-dependent+kinase+from+Entamoeba+histolytica.">Acetate kinase (pyrophosphate). A fourth pyrophosphate-dependent kinase from Entamoeba histolytica.</a> Biochem. Biophys. Res. Commun. 66, 1389-1395 (1975).</li><li>Fowler, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+and+structural+characterization+of+the+novel+PPi-dependent+acetate+kinase+from+the+parasite+Entamoeba+histolytica.">Kinetic and structural characterization of the novel PPi-dependent acetate kinase from the parasite Entamoeba histolytica.</a> , (2011).</li><li>Lipmann, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+synthesis+of+acetyl+phosphate.">Enzymatic synthesis of acetyl phosphate.</a> J. Biol. Chem. 155, 55-70 (1944).</li><li>Lipmann, F., Tuttle, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+specific+micromethod+for+determination+of+acyl+phosphates.">A specific micromethod for determination of acyl phosphates.</a> J. Biol. Chem. 159, 21-28 (1945).</li><li>Lipmann, F., Jones, M. E., Black, S., Flynn, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+the+ATP-CoA-acetate+reaction.">The mechanism of the ATP-CoA-acetate reaction.</a> J. Cell. Physiol. Suppl. 41, 109-112 (1953).</li><li>Rose, I. A., Grunberg-Manago, M., Korey, S. F., Ochoa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+phosphorylation+of+acetate.">Enzymatic phosphorylation of acetate.</a> J. Biol. Chem. 211, 737-756 (1954).</li><li>Allen, S. H., Kellermeyer, R. W., Stjernholm, R. L., Wood, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+properties+of+enzymes+involved+in+the+proponic+acid+fermentation.">Purification and properties of enzymes involved in the proponic acid fermentation.</a> Journal of Bacteriology. 87, 171-187 (1964).</li><li>Clarke, P. M., Payton, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enzymatic+assay+for+acetate+in+spent+bacterial+culture+supernatants.">An enzymatic assay for acetate in spent bacterial culture supernatants.</a> Anal. Biochem. 130, 402-405 (1983).</li><li>Mukhopadhyay, S., Hasson, M. S., Sanders, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+continuous+assay+of+acetate+kinase+activity:+measurement+of+inorganic+phosphate+release+generated+by+hydroxylaminolysis+of+acetyl+phosphate.">A continuous assay of acetate kinase activity: measurement of inorganic phosphate release generated by hydroxylaminolysis of acetyl phosphate.</a> Bioorg. Chem. 36, 65-69 (2008).</li><li>Nakajima, H., Suzuki, K., Imahori, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+properties+of+acetate+kinase+from+Bacillus+stearothermophilus.">Purification and properties of acetate kinase from Bacillus stearothermophilus.</a> J. Biochem. 84, 193-203 (1978).</li></ol>

관심 없음 충돌 선언하지 않습니다.

이 분석에서 아세틸 인산의 감지는 히드록 실 아민 하이드로 클로라이드의 충분한 농도와 산화철 염화물 용액의 농도와 산도에 의존적일 수 밖에 없습니다. 분석 볼륨의 변경은 이러한 구성 요소 모두의 재검토가 필요합니다. 효소 반응이 여기에 설명된 37 실시했다 ° 5 분 ° C 60 수행한 히드록 실 아민의 종료와 함께 C. 이것은 높은 온도는 아세틸 hydroxamate에 남아있는 아세틸 인산의 급속한 전환 ...

<table border="1"><tr><td width="160"> 시약의 이름 </td><td width="108"> 회사 </td><td width="134"> 카탈로그 번호 </td><td width="103"> 댓글 </td></tr><tr><td> 아세틸 인산 </td><td> 시그마 알드리치 </td><td> 01,409 </td><td> 리튬 소금 (97 %) </td></tr><tr><td> 나트륨 인산염 일염기의 (탈수) </td><td> ThermoFisher </td><td> S381 </td><td></td></tr><tr><td> 나트륨 인산염 이염 (무수) </td><td> ThermoFisher </td><td> S374 </td><td></td></tr><tr><td> 마그네슘 염화 (hexahydrate) </td><td> ThermoFisher </td><td> M33 </td><td></td></tr><tr><td> 트리스베이스 </td><td> ThermoFisher </td><td> B152 </td><td></td></tr><tr><td> 산화철 염화물 (hexahydrate) </td><td> ThermoFisher </td><td> I88 </td><td></td></tr><tr><td> Trichloroacetic 산 </td><td> ThermoFisher </td><td> A324 </td><td></td></tr><tr><td> 히드록 실 아민 하이드로 클로라이드 </td><td> ThermoFisher </td><td> H330 </td><td></td></tr><tr><td> 5&#39; - diphosphate을 아데노신 나트륨 소금 </td><td> 시그마 알드리치 </td><td> A2754 </td><td></td></tr><tr><td> Biomate III 분광 </td><td> ThermoFisher </td><td> 142,982,082 </td><td> 표준 UV / VIS 분광 광도계 </td></tr></table>

direct detection of the acetate-forming activity  of the enzyme acetate kinase

아세테이트 키나제, 아세테이트, 설탕 키나제 - Hsp70 - 굴지 (ASKHA) 효소 superfamily 1-5의 회원은 기판으로 ATP를 이용 아세틸 인산에 아세테이트의 인산화 가역에 대한 책임이 있습니다. 아세테이트 kinases는 Archaea 중 하나 속에서 발견, 박테리아에 유비 쿼터스하고 있으며, 또한 Eukarya 6 미생물에 존재한다. 가장 잘 특징 아세테이트 키나제는 그 메탄 생산 archaeon Methanosarcina thermophila 7-14에서. 단 인산염 - 성형 아세틸 방향으로 ATP하지 i를 PP 활용할 수도 있지만 아세테이트 키나제는 Entamoeba histolytica, 아메바 이질의 원인이되는 대리인으로부터 격리되어 있으며, 지금까지이 속 15,16에서만 발견되었습니다. 아세틸 인산 형성의 방향에서 아세테이트 키나제 활동이 일반적으로 첫번째 Lipmann에 의해 설명 hydroxamate 분석을 사용하여 측정17-20, 효소의 pyruvate의 키나 아제 및 젖산 탈수소 효소 21,22에 의해 ADP로 ATP의 전환이 NAD으로 NADH의 산화에 결합되는 결합 분석 +, 또는 아세틸 인산 제품의 반응 후 무기 인산염의 릴리스를 측정 분석 히드록 실 아민 23. 반대로 활동은 아세테이트 - 형성 방향은 ADP에서 NADP의 감소 + 효소의 hexokinase 및 포도당 6 인산 탈수소 효소에 의해 24 NADPH로하는 ATP 형성을 결합하여 측정됩니다. 여기 커플링 효소를 필요로하지 않는 아세테이트 형성의 방향으로 아세테이트 키나제 활동의 검출하는 방법을 설명하는 대신 아세틸 인산 소비의 직접적인 결정에 따라 달라집니다. 효소 반응 후 남아있는 아세틸 인산이 hydroxamate 분석에 관해서 spectrophotometrically 측정할 수있는 푹 삭은 hydroxamate 복합 변환됩니다. 따라서 일와는 달리ADP에서 ATP의 생산에 의존이이 방향에 대한 andard 결합 분석,이 직접적인 분석은 ATP 또는 PP i를 생산 아세테이트 kinases 사용할 수 있습니다.

Watch this Scientific Journal Video about Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase at JoVE.com

Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase

아세테이트 키나제 활동의 결정을위한 방법을 설명합니다. 이 분석은 다른 phosphoryl acceptors와 아세테이트 - 성형 방향으로 아세테이트 키나제 효소의 활동과 속도론을 결정하기위한 직접적인 반응을 활용합니다. 또한,이 방법은 다른 아세틸 인산 또는 아세틸 - COA 활용한 효소를 시금 활용하실 수 있습니다.

효소 아세테이트 키나제의 아세테이트 - 형성 활동의 직접적인 탐지

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of ...

direct-detection-acetate-forming-activity-enzyme-acetate

Research

JoVE Journal

Biology

23.0K 조회수.  Clemson University. 아세테이트 키나제 활동의 결정을위한 방법을 설명합니다. 이 분석은 다른 phosphoryl acceptors와 아세테이트 - 성형 방향으로 아세테이트 키나제 효소의 활동과 속도론을 결정하기위한 직접적인 반응을 활용합니다. 또한,이 방법은 다른 아세틸 인산 또는 아세틸 - COA 활용한 효소를 시금 활용하실 수 있습니다.

비디오: Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

단백질 Ubiquitination의 감지

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

연구

생물학

Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect protease activity uses casein labeled with fluorescein isothiocyanate (FITC) as the substrate.
Protease activity results in the cleavage of the FITC-labeled casein substrate into smaller fragments, which do not precipitate under acidic conditions. After incubation of the protease sample and substrate, the reaction is acidified with the addition of trichloroacetic acid (TCA). The mixture is then centrifuged with the undigested substrate forming a pellet and the smaller, acid soluble fragments remaining in solution. The supernatant is neutralized and the fluorescence of the FITC-labeled fragments is measured.
The described kit procedure detects the trypsin protease control at a concentration of approximately 0.5 &mu;g/ml (5 ng of trypsin added to the assay). This sensitivity can be increased with a longer incubation time, up to 24 hours. The assay is performed in microcentrifuge tubes and procedures are provided for fluorescence detection using either cuvettes or multiwell plates.

The Protease Fluorescent Detection Kit is designed for the measurement of protease activity using fluorometry. It is also suitable for detection of trace amounts of protease contamination. The method is based on the proteolytic hydroysis of a proprietary formulation of a FITC-labeled casein substrate.

단백 분해 효소의 활동을 확인하는 프로 테아제 형광 검출 키트의 사용

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect ...

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous mycotoxins in food commodities. In this protocol we show how to fabricate and interrogate an electrochemical competitive Enzyme linked immunomagnetic assay based on the use of magnetic beads as solid support for the immunochemical chain3 and screen printed electrodes as sensing platform.
Our method aims to determine the total amount of HT-2 and T-2 toxins, mycotoxins belonging to the trichothecenes family and of great concern for human health4. The use of an antibody clone with a cross reactivity of 100% towards HT-2 and T-2 allows to simultaneously detect both toxins with similar sensitivity5.
The first step of our assay is the coating step where we immobilize HT2-KLH conjugate toxin on the surface of magnetic beads. After a blocking step, necessary to avoid non-specific absorptions, the addition of a monoclonal antibody allows the competition between immobilized HT-2 and free HT-2 or T-2 present in the sample or dissolved in a standard solution.
At the end of the competition step, the amount of monoclonal antibody linked to the immobilized HT-2 will be inversely proportional to the amount of toxin in the sample solution.
A secondary antibody labeled with alkaline phosphatase (AP) is used to reveal the binding between the specific antibody and the immobilized HT-2. The final measurement step is performed by dropping an aliquot of magnetic bead suspension, corresponding to a specific sample/standard solution, on the surface of a screen-printed working electrode; magnetic beads are immobilized and concentrated by means of a magnet placed precisely under the screen-printed electrode. After two minutes of incubation between magnetic beads and a substrate for AP, the enzymatic product is detected by Differential Pulse Voltammetry (DPV) using a portable instrument (PalmSens) also able to initiate automatically eight measurements within an interval of few seconds.

ELIME Enzyme Linked Immuno Magnetic Electrochemical Method for Mycotoxin Detection

A protocol to detect trichothecenes (mycotoxins of concern for human health) using a newly developed screening method based on a competitive immunochemical method and a final electrochemical detection is demonstrated.

진균 독소의 검출을위한 ELIME (효소가 단백질 자기 전기를 연결) 방법

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

트렌스 제닉 생산 Xenopus laevis 제한 효소 중재 통합 및 핵 이식에 의해

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability1, 2. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation3, and ultimately to oxidative stress leading to cell injury or death4. 
Superoxide radical anion (O2&bull;-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O2&bull;- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O2&bull;- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O2&bull;- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase5. 
Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO6. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O2&bull;- in a process called uncoupling, which is believed to be caused by oxidation of heme7 or the co-factor, tetrahydrobiopterin (BH4)8.
There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production9.
The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO10, the phosphoryl-substituted DEPMPO11, and the ester-substituted, EMPO12 and BMPO13, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O2&bull;- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct14.

Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.

질소 산화물과 세포로부터 전자 상자 성체의 프로그램 공명 분광학에 의한 초과 산화물 음이온 급진적가 탐지 스핀 트랩을 사용하여

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in ...

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

안달 Ratiometric하여 세포주기를 통해 키나제와 인산 가수 분해 효소 활동을 모니터링

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

LRRK2의 키나제 활동 시금 체외에서</em

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. Current strategies for microbial analysis include both traditional laboratory culture-dependent techniques and those based on direct extraction and determination of certain biomarkers1, 2. Few among the diversity of microbial species inhabiting soil can be cultured, so culture-dependent methods introduce significant biases, a limitation absent in biomarker analysis.
The glucosamine, mannosamine, galactosamine and muramic acid have been well served as measures of both the living and dead microbial mass, of these the glucosamine (most abundant) and muramic acid (uniquely from bacterial cell) are most important constituents in the soil systems3, 4. However, the lack of knowledge on the analysis restricts the wide popularization among scientific peers. Among all existing analytical methods, derivatization to aldononitrile acetates followed by GC-based analysis has emerged as a good option with respect to optimally balancing precision, sensitivity, simplicity, good chromatographic separation, and stability upon sample storage5.
Here, we present a detailed protocol for a reliable and relatively simple analysis of glucosamine and muramic acid from soil after their conversion to aldononitrile acetates. The protocol mainly comprises four steps: acid digestion, sample purification, derivatization and GC determination. The step-by-step procedure is modified according to former publications6, 7. In addition, we present a strategy to structurally validate the molecular ion of the derivative and its ion fragments formed upon electron ionization. We applied GC-EI-MS-SIM, LC-ESI-TOF-MS and isotopically labeled reagents to determine the molecular weight of aldononitrile acetate derivatized glucosamine and muramic acid; we used the mass shift of isotope-labeled derivatives in the ion spectrum to investigate ion fragments of each derivatives8. In addition to the theoretical elucidation, the validation of molecular ion of the derivative and its ion fragments will be useful to researchers using &delta;13C or ion fragments of these biomarkers in biogeochemical studies9, 10.

We describe a method protocol for the GC-based analysis of the aldonitrile acetate derivatives of glucosamine and muramic acid extracted from soil. For elucidation of the chemical mechanism, we also present a strategy to confirm the structure of the derivative and the ion fragments formed upon electron ionization.

토양에 미생물 잔류 결정을위한 Aldononitrile 아세트산 Derivati​​zed 글루코사민과 Muramic 산성의 GC 기반 탐지

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. ...

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

에서 애벌레 활동의 측정<em&gt; 초파리</em&gt; 활동 모니터

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...

Studying the Activity of Neuropeptides and Other Regulators of the Excretory System in the Adult Mosquito

Studies of insect physiology, particularly in those species that are vectors of pathogens causing disease in humans and other vertebrates, provide the foundation to develop novel strategies for pest control. Here, a series of methods are described that are routinely utilized to determine the functional roles of neuropeptides and other neuronal factors (i.e., biogenic amines) on the excretory system of the mosquito, Aedes aegypti. The Malpighian tubules (MTs), responsible for primary urine formation, can continue functioning for hours when removed from the mosquito, allowing for fluid secretion measurements following hormone treatments. As such, the Ramsay assay is a useful technique to measure secretion rates from isolated MTs. Ion-selective microelectrodes (ISME) can sequentially be used to measure ion concentrations (i.e., Na+ and K+) in the secreted fluid. This assay allows for the measurement of several MTs at a given time, determining the effects of various hormones and drugs. The Scanning Ion-selective Electrode Technique uses ISME to measure voltage representative of ionic activity in the unstirred layer adjacent to the surface of ion transporting organs to determine transepithelial transport of ions in near real time. This method can be used to understand the role of hormones and other regulators on ion absorption or secretion across epithelia. Hindgut contraction assays are also a useful tool to characterize myoactive neuropeptides, that may enhance or reduce the ability of this organ to remove excess fluid and waste. Collectively, these methods provide insight into how the excretory system is regulated in adult mosquitoes. This is important because functional coordination of the excretory organs is crucial in overcoming challenges such as desiccation stress after eclosion and before finding a suitable vertebrate host to obtain a bloodmeal.

This protocol outlines methodologies behind the Ramsay assay, ion-selective microelectrodes, Scanning Ion-selective Electrode Technique (SIET) and in vitro contraction assays, applied to study the adult mosquito excretory system, comprised of the Malpighian tubules and hindgut, to collectively measure ion and fluid secretion rates, contractile activity, and transepithelial ion transport.

성인 모기에 있는 배설 시스템의 Neuropeptides 및 그밖 규제기관의 활동을 공부하기

Studies of insect physiology, particularly in those species that are vectors of pathogens causing disease in humans and other vertebrates, provide the ...