The authors would like to acknowledge funding from a National Institutes of Health grant RO1AI049294 (to M. S.).

1. 정제 된 단백질과 ATP 가수 분해 반응을 수행 <ol><li> 정제 된 단백질과 부화에 필요한 모든 시약의 주식을 준비합니다. <ol><li> 100 mM의 HEPES pH가 8.5, 65 mM의 염화나트륨, 5 % 글리세롤 (또는 다른 분석 완충액 적절한)을 함유하는 배 HEPES / 염화나트륨 / 글리세롤 (HNG) 버퍼를 준비한다. </li><li> 물 (ATP 아제 금속에 의존하는 경우, 또는 다른 금속) 100 밀리미터의 MgCl 2를 준비합니다. </li><li> 고순도 ATP를 사용 (추가하여 pH를 조정하지 않음) 200 mM 트리스 자료에서 신선한 100 mM의 ATP를 준비합니다. 나누어지는이 ATP가 시간이 지남에 따라 분해되므로, 몇 주 이상 더 이상 -20 ° C에서 ATP 재고를 저장하고, 동결 및 ATP 재고를 해동을 삼가합니다. </li><li> 1에서 프리믹스의 MgCl 2, ATP : 1의 비율로 바로 ATP의 가수 분해 반응을 설정하기 전에. </li></ol></li><li> 조제 및 반응에 걸쳐 일정한 간격으로 샘플을 수집하는 1.5 ㎖의 튜브 라벨. 시간 0 시간 15, 30에서 샘플을 수집하기 위해 튜브를 준비,45 및 60 분. 참고 : 다른 방법으로, 유일한 시간 0 엔드 포인트에서 샘플을 수집합니다. <ol><li> ATP에 가수 분해 반응은 1:50에서 수집 한 샘플을 희석하기 위해 각 튜브에 245 μL의 1X HNG 버퍼를 추가한다. </li></ol></li><li> 신속하게 반응을 정지 샘플을 동결을 위해 드라이 아이스와 에탄올의 목욕을 준비합니다. 고무 얼음 양동이 또는 다른 안전 (비 플라스틱) 용기에 드라이 아이스의 여러 조각을 추가하고 조심스럽게 드라이 아이스를 커버하기에 충분한 70 ~ 100 에탄올을 부어. </li><li> 적절한 (일반적으로 5 ~ 10 μM)로, 1 배 HNG 버퍼에 정제 된 단백질을 희석하고 얼음에 보관하십시오. </li><li> ATP에 가수 분해 반응을 설정합니다. <ol><li> 각 샘플에 대해 별도의 0.5 ml의 튜브에서, (순서대로) 다음과 같은 시약을 추가, 6 μl의 5 배 HNG 또는 다른 버퍼, 3 μL 100 밀리미터의 MgCl 2 -ATP 혼합물 (최종 30 μL의 볼륨까지) H 2 O, 및 0.25-5 μm의 단백질. </li><li> 어떤 단백질을 첨가하지 않은 버퍼 전용 대조군 조건을 포함한다.</li></ol></li><li> 시간 0에서 반응 5 μL를 제거 HNG 245 μL의 버퍼를 포함하는 제조 1.5 ㎖의 튜브에 1:50로 희석하고, 즉시 드라이 아이스 / 에탄올 욕에서 샘플을 동결. </li><li> 37 ° C에서 반응은 ATP 가수 분해는 1 시간 동안 발생할 수 있도록 인큐베이션. 각각의 구간 (15, 30, 45, 60 분), 반응을 5 ㎕의 분취 액을 제거하고, 단계 160에서와 같이 HNG 버퍼를 함유하는 표지 된 샘플 튜브에 추가. </li><li> ATP에 가수 분해 반응의 끝에서, 저장을 위해 -80 ° C에 냉동 희석 샘플을 이동. 모든 샘플이 완전히 고정되어 있는지 확인하려면 계속하기 전에 적어도 10 분을 기다립니다. </li></ol> 검출 시약과 무료 파이를 포함하는 2 품어 샘플 <ol><li> 각 시점에서의 ATP 가수 분해 반응의 분취 량을 함유하는 희석 된 샘플 녹여 실온에서 (단계 1.6 및 1.7에서 수득). </li><li> 샘플 및 포스페이트 표준을 함유하는 96 웰 플레이트를 설치했다. <ol><li> 0.5 ml의 TU에서800 μM 표준 104.5 μL에 HNG 완충액 5.5 μl를 첨가하여 800 μM 40 μM 내지 (PI 검출 시약으로 제공됨) 포스페이트 표준 희석 될. 잘 혼합하고, 96 웰 플레이트의 웰에 A1을이 40 μM 파이 표준의 100 μl를 추가합니다. </li><li> 1 우물 B1-H1에 50 μL의 HNG 버퍼를 추가 파이 표준의 1 직렬 희석. </li><li> 잘 A1에서 40 μM 파이의 50 μl를 제거하고 잘 B1에 50 μl를 분석 버퍼에 추가, 혼합, 잘 B1에서 50 μl를 제거하고 잘 G1을 통해 잘 C1에 계속 희석을 추가합니다. 각 웰은 동일한 볼륨이 보장하는 혼합하여 잘 G1 50 μl를 폐기하십시오. 잘 H1 40 0​​ μM에 파이 표준을 만들 수있는 유일한 버퍼를 포함해야합니다. 참고 : 추가 요소 (예 : 억제제 등) 샘플에 추가 된 경우, 그 조건 하에서 인산 릴리스 또는 흡광도의 변화를 제어하는​​ 요소를 포함하는 표준 곡선을 작성합니다. </li><li> 각 SAMP의 50 μl를 추가접시에 중복으로 제작. 서로 다른 시점 수평, 열에서 같은 시점 ​​수직 (샘플 2 시간 0 = B2, B3 샘플 1 시간 0 = A2, A3가)에서 샘플을 추가합니다. 이는 최대 8 샘플 및 플레이트 당 5 시간 지점 수 있습니다. </li></ol></li><li> 시료와 표준을 포함​​하는 웰 (시약 필요 (㎖) = 시료와 표준의 0.1 × 번호) 각 100 μl를 추가하기에 충분한 말라카이트 그린 / 몰리브덴 파이 검출 시약을 제거하기 위해 멸균 피펫을 사용하여 쉽게 피펫 팅에 대한 접시에 추가 멀티 채널 피펫을 사용. 파이 오염이 발생하기 쉽다 같이 접시에 직접 검출 시약을 부어 마십시오. </li><li> 멀티 채널 피펫을 사용하여, 처음 포인트 전회 점에서 바람직 위해 도입 거품 않고 조심스럽게 피펫으로 시간과 일관된 번호 아래 각 웰에 파이 검출 시약 100 μL를 추가 혼합한다. </li><li> 실온에서 25 분 동안 플레이트를 인큐베이션, 또는 MA에 따라nufacturer의 방향. </li></ol> 마이크로 플레이트 리더를 사용하여 3. 정량 결과 <ol><li> 흡광도 마이크로 플레이트 리더를 이용하여 650 nm에서 시료의 흡광도를 참조하십시오. </li><li> 파이 표준 곡선을 확인합니다. 그래프 소프트웨어를 사용하여 각 샘플에 인산의 양에 대해 해결하기 위해 사용 된 식을 찾기 위해 농도 대 파이 표준 샘플에 대한 흡광도 값을 그래프. </li><li> 포스페이트 표준을 교정하여 각 샘플에 대한 ATP 아제 활성을 계산한다. 인산 출시 = (OD 650 - Y 절편) / 경사. <ol><li> 각 샘플의 중복에서 전체 파이를 평균. 이 번호에서 버퍼 전용 컨트롤의 흡광도 판독 값을 뺍니다. 희석 인자 (예에서는 50)를 곱하면. </li><li> μmol 단백질 당 발표 nmol의 파이를 결정합니다. 역학적 분석에서 각 시점에 대한 이들 값을 그래프로하여 적어도 R = 0.99 (도 1)의 선형 ​​발작을 수득한다; N 경우OT는 분석은 더 많거나 적은 단백질 또는 길거나 짧은 배양 시간이 조정될 수있다. </li></ol></li><li> nmol의 PI / 단백질 μmol / min으로 같은 결과를 원하는 경우, 또는 nmol의 PI / μg의 단백질 / 분 (3,도 2 등)를 나타낸다. </li></ol>

<ol>
	<li>Hanson, P. I., Whiteheart, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AAA++proteins:+have+engine,+will+work.">AAA+ proteins: have engine, will work.</a> Nat Rev Mol Cell Biol. 6 (7), 519-529 (2005).</li><li>Baker, T. A., Sauer, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ClpXP,+an+ATP-powered+unfolding+and+protein-degradation+machine.">ClpXP, an ATP-powered unfolding and protein-degradation machine.</a> Biochimica et Biophysica Acta. 1823 (1), 15-28 (2012).</li><li>Maxson, M. E., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vacuolar-type+H+-ATPase+at+a+glance+-+more+than+a+proton+pump.">The vacuolar-type H+-ATPase at a glance - more than a proton pump.</a> J Cell Sci. 127 (23), 4987-4993 (2014).</li><li>Planet, P. J., Kachlany, S. C., DeSalle, R., Figurski, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogeny+of+genes+for+secretion+NTPases:+Identification+of+the+widespread+tadA+subfamily+and+development+of+a+diagnostic+key+for+gene+classification.">Phylogeny of genes for secretion NTPases: Identification of the widespread tadA subfamily and development of a diagnostic key for gene classification.</a> Proc Natl Acad Sci U S A. 98 (5), 2503-2508 (2001).</li><li>Robien, M. A., Krumm, B. E., Sandkvist, M., Hol, W. G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+Structure+of+the+Extracellular+Protein+Secretion+NTPase+EpsE+of+Vibrio+cholerae.">Crystal Structure of the Extracellular Protein Secretion NTPase EpsE of Vibrio cholerae.</a> J Mol Biol. 333 (3), 657-674 (2003).</li><li>Camberg, J. L., Sandkvist, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+analysis+of+the+Vibrio+cholerae+type+II+secretion+ATPase+EpsE.">Molecular analysis of the Vibrio cholerae type II secretion ATPase EpsE.</a> J Bacteriol. 187 (1), 249-256 (2005).</li><li>Sandkvist, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+II+secretion+and+pathogenesis.">Type II secretion and pathogenesis.</a> Infect Immun. 69 (6), 3523-3535 (2001).</li><li>Brune, M., Hunter, J. L., Corrie, J. E. T., Webb, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct,+Real-time+measurement+of+rapid+inorganic+phosphate+release+using+a+novel+fluorescent+probe+and+its+application+to+actomyosin+subfragment+1+ATPase.">Direct, Real-time measurement of rapid inorganic phosphate release using a novel fluorescent probe and its application to actomyosin subfragment 1 ATPase.</a> Biochemistry. 33 (27), 8262-8271 (1994).</li><li>Carter, S. G., Karl, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inorganic+phosphate+assay+with+malachite+green:+An+improvement+and+evaluation.">Inorganic phosphate assay with malachite green: An improvement and evaluation.</a> J Biochem Biophys Methods. 7 (1), 7-13 (1982).</li><li>Henkel, R. D., VandeBerg, J. L., Walsh, R. 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+microassay+for+ATPase.">A microassay for ATPase.</a> Anal Biochem. 169 (2), 312-318 (1988).</li><li>Harder, K. W., Owen, P., Wong, L. K. H., Aebersold, R., Clark-Lewis, I., Jirik, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+kinetic+analysis+of+the+intracellular+domain+of+human+protein+tyrosine+phosphatase+&#946;+(HPTP+&#946;)+using+synthetic+phosphopeptides.">Characterization and kinetic analysis of the intracellular domain of human protein tyrosine phosphatase &#946; (HPTP &#946;) using synthetic phosphopeptides.</a> Biochem J. 298 (2), 395-401 (1994).</li><li>Camberg, J. L., Johnson, T. L., Patrick, M., Abendroth, J., Hol, W. G., Sandkvist, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+stimulation+of+EpsE+ATP+hydrolysis+by+EpsL+and+acidic+phospholipids.">Synergistic stimulation of EpsE ATP hydrolysis by EpsL and acidic phospholipids.</a> EMBO J. 26 (1), 19-27 (2006).</li><li>McLaughlin, S. H., Smith, H. W., Jackson, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulation+of+the+weak+ATPase+activity+of+human+Hsp90+by+a+client+protein.">Stimulation of the weak ATPase activity of human Hsp90 by a client protein.</a> J Mol Biol. 315 (4), 787-798 (2002).</li><li>Shiue, S., Kao, K., Leu, W., Chen, L., Chan, N., Hu, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XpsE+oligomerization+triggered+by+ATP+binding,+not+hydrolysis,+leads+to+its+association+with+XpsL.">XpsE oligomerization triggered by ATP binding, not hydrolysis, leads to its association with XpsL.</a> EMBO J. 25 (7), 1426-1435 (2006).</li><li>Ghosh, A., Hartung, S., van der Does, C., Tainer, J. A., Albers, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Archaeal+flagellar+ATPase+motor+shows+ATP-dependent+hexameric+assembly+and+activity+stimulation+by+specific+lipid+binding.">Archaeal flagellar ATPase motor shows ATP-dependent hexameric assembly and activity stimulation by specific lipid binding.</a> Biochem J. 437 (1), 43-52 (2011).</li><li>Savvides, S. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VirB11+ATPases+are+dynamic+hexameric+assemblies:+new+insights+into+bacterial+type+IV+secretion.">VirB11 ATPases are dynamic hexameric assemblies: new insights into bacterial type IV secretion.</a> EMBO J. 22 (9), 1969-1980 (2003).</li><li>Sanghera, J., Li, R., Yan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+luminescent+ADP-Glo+assay+to+a+standard+radiometric+assay+for+measurement+of+protein+kinase+activity.">Comparison of the luminescent ADP-Glo assay to a standard radiometric assay for measurement of protein kinase activity.</a> Assay Drug Dev Techn. 7 (6), 615-622 (2009).</li><li>Sherman, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decoupling+catalytic+activity+from+biological+function+of+the+ATPase+that+powers+lipopolysaccharide+transport.">Decoupling catalytic activity from biological function of the ATPase that powers lipopolysaccharide transport.</a> Proc Natl Acad Sci U S A. 111 (13), 4982-4987 (2014).</li><li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+cofactor+regulation+with+disease-associated+p97/VCP+mutations.">Altered cofactor regulation with disease-associated p97/VCP mutations.</a> Proc Natl Acad Sci U S A. 112 (14), E1705-E1714 (2015).</li><li>Rowlands, M. G., Newbatt, Y. M., Prodromou, C., Pearl, L. H., Workman, P., Aherne, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening+assay+for+inhibitors+of+heat-shock+protein+90+ATPase+activity.">High-throughput screening assay for inhibitors of heat-shock protein 90 ATPase activity.</a> Anal Biochem. 327 (2), 176-183 (2004).</li></ol>

The authors have nothing to disclose.

이 생화학 적 특성에 대한 정제 된 단백질의 생체 외 ATP 아제 활동 측정하는 일반적인 프로토콜입니다. 이 방법은 쉽게 최적화되어 있습니다; 예를 들어, 단백질, 완충 염 및 조성물, 온도, 및 상기 분석과 다양한 길이 (간격의 총 개수를 증가 포함) 간격의 양을 조절하는 활성을 정량을 향상시킬 수있다. 시판 말라카이트 그린 기반의 시약은 매우 민감, 무료 인산 소량 검출 할 수있다 ...

ATPases are integral enzymes in many processes across all kingdoms of life. ATPases act as molecular motors that use the energy of ATP hydrolysis to power such diverse reactions as protein trafficking, unfolding, and assembly; replication and transcription; cellular metabolism; muscle movement; cell motility; and ion pumping1-3. Some ATPases are transmembrane proteins involved in transporting solutes across membranes, others are cytoplasmic and may be associated with a biological membrane such as the plasma membrane or those of organelles.
AAA+ ATPases (ATPases associated with various cellular activities) make up a large group of ATPases that share some sequence and structural conservation. These proteins contain conserved nucleotide binding motifs such as Walker-A and -B boxes and form oligomers (generally hexamers) in their active state1. Large conformational changes in these proteins upon nucleotide binding have been characterized among diverse members of the AAA+ family. EpsE is a AAA+ ATPase and member of the bacterial Type II/IV secretion subfamily of NTPases4-6. EpsE powers Type II Secretion (T2S) in Vibrio cholerae, the causative agent of cholera. The T2S system is responsible for the secretion of a wide variety of proteins, such as the virulence factor cholera toxin that causes profuse watery diarrhea when V. cholerae colonizes the human small intestine7. 
Techniques for quantitating in vitro ATPase activity are varied, but commonly measure phosphate release using colorimetric, fluorescent, or radioactive substrates8-11. We describe a basic method for determining in vitro ATPase activity of purified proteins using a colorimetric assay based on a commercially available malachite green-containing substrate that measures liberated inorganic phosphate (Pi). At low pH, malachite green molybdate forms a complex with Pi and the level of complex formation can be measured at 650 nm. This simple and sensitive assay may be used to functionally characterize new ATPases and to evaluate the roles of potential activators or inhibitors, to determine the importance of domains and/or specific residues, or to assess the effect of particular treatments on enzymatic activity.

<table><tbody><tr><td>HEPES buffer</td><td>Fisher</td><td>BP310-500</td><td></td></tr><tr><td>Sodium chloride</td><td>Fisher</td><td>BP358-212</td><td></td></tr><tr><td>Magnesium chloride</td><td>Fisher</td><td>BP214-500</td><td></td></tr><tr><td>Adenosine triphosphate (ATP)</td><td>Fisher</td><td>BP41325</td><td></td></tr><tr><td>96-well plates (clear, flat-bottom)</td><td>VWR</td><td>82050-760</td><td></td></tr><tr><td>BIOMOL Green</td><td>Enzo Life Sciences</td><td>BML-AK111</td><td>Preferred phosphate detection reagent. Caution: irritant.</td></tr><tr><td>Microplate reader</td><td></td><td></td><td>BioTek Synergy or comparable</td></tr><tr><td>Prism 5</td><td>GraphPad Software</td><td></td><td></td></tr></tbody></table>

measuring in vitro atpase activity for enzymatic characterization

아데노신 삼인산 가수 분해 효소, 또는 -ATPase를는 세포 기능의 다양한 배열에 중요한 역할을한다. 이러한 동적 단백질은 단백질 인신 매매 및 저하, 용질 수송, 및 세포의 움직임과 같은 기계적인 작업을위한 에너지를 생성 할 수 있습니다. 여기에 설명 된 프로토콜은 기능적 특성화 -ATPase를 정제의 시험 관내 활성을 측정하기위한 기본적인 분석이다. 단백질은 무기 포스페이트 방출을 초래 반응의 ATP 가수 분해 및 유리 된 인산의 양은 다음 비색 정량 분석​​법을 사용한다. 이것은 고도로 적응 프로토콜 운동 또는 엔드 포인트 분석에서 ATP 아제 활성을 측정하기 위해 조정될 수있다. 대표적인 프로토콜이 활동과 EPSE의 요구 사항에 따라 여기에 제공되면, AAA + ATP 아제는 박테리아 비브리오 콜레라를 입력 II 분비에 관여. 활성을 측정하기 위해 필요로 정제 된 단백질의 양을 분석의 길이와 SA의 타이밍 및 횟수mpling 간격, 버퍼 소금 조성, 온도, 공동 요인, 각성제 (있는 경우) 등이 여기에 설명하는 것과 다를 수 있습니다, 따라서 약간의 최적화가 필요할 수 있습니다. 이 프로토콜의 특성 -ATPase를위한 기본 틀을 제공하고 신속하게 수행하고 쉽게 필요한 조절 될 수있다.

T2S ATP 아제 EPSE의 생체 외 활동은 세포질 EpsL (EPSE-cytoEpsL)의 도메인과 산성 인지질 카디오 리핀 (12)의 추가와 함께 EPSE의 copurification에 의해 자극 될 수있다. 또한이 분석을 사용하여 단백질의 변이체 형태를 야생형 (WT)의 활성을 비교함으로써 ATP 가수 분해에 EPSE 특정 잔기의 역할을 결정하는 것도 가능하다. 여기서, EPSE 아연 결합 도메인에 두 개의 리신 잔기...

Watch this Scientific Journal Video about Measuring In Vitro ATPase Activity for Enzymatic Characterization at JoVE.com

Measuring In Vitro ATPase Activity for Enzymatic Characterization

We describe a basic protocol for quantitating in vitro ATPase activity. This protocol can be optimized based on the level of activity and requirements for a given purified ATPase.

자질<em&gt; 체외</em효소 특성에 대한&gt; ATP 아제 활동

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate ...

measuring-in-vitro-atpase-activity-for-enzymatic-characterization

Research

JoVE Journal

Biology

Measuring In Vitro ATPase Activity for Enzymatic Characterization

18.0K 조회수.  University of Michigan. We describe a basic protocol for quantitating in vitro ATPase activity. This protocol can be optimized based on the level of activity and requirements for a given purified ATPase.

비디오: Measuring In Vitro ATPase Activity for Enzymatic Characterization

Assaying Protein Kinase Activity with Radiolabeled ATP

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering allosteric details of their regulation. Kinases comprise signaling networks whose defects are often hallmarks of multiple forms of cancer and related diseases, making an assay platform amenable to manipulation of upstream regulatory factors and validation of reaction requirements critical in the search for improved therapeutics. Here, we describe a basic kinase assay that can be easily adapted to suit specific experimental questions including but not limited to testing the effects of biochemical and pharmacological agents, genetic manipulations such as mutation and deletion, as well as cell culture conditions and treatments to probe cell signaling mechanisms. This assay utilizes radiolabeled [&#947;-32P] ATP, which allows for quantitative comparisons and clear visualization of results, and can be modified for use with immunoprecipitated or recombinant kinase, specific or typified substrates, all over a wide range of reaction conditions.

Protein kinases are highly evolved signaling enzymes and scaffolds that are critical for inter- and intracellular signal transduction. We present a protocol for measuring kinase activity through the use of radiolabeled adenosine triphosphate ([&#947;-32P] ATP), a reliable method to aid in elucidation of cellular signaling regulation.

방사성 표지 된 ATP로 단백질 키나아제 활성 측정

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering ...

연구

생화학

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important roles for the cell and may unravel new cellular processes. Among proteins, kinases are major actors as they belong to cell signaling pathways and have the ability to switch on or off many processes crucial to the fate of the cell, such as cell growth, division, differentiation, motility, and death.
In this study, we focused on a new potential kinase protein, LIMK2-1. We demonstrated its existence by Western Blot using a specific antibody. We evaluated its interaction with an upstream regulating protein using coimmunoprecipitation experiments. Coimmunoprecipitation is a very powerful technique able to detect the interaction between two target proteins. It may also be used to detect new partners of a bait protein. The bait protein may be purified either via a tag engineered to its sequence or via an antibody specifically targeting it. These protein complexes may then be separated by SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel) and identified using mass spectrometry. Immunoprecipitated LIMK2-1 was also used to test its kinase activity in vitro by &#947;[32P] ATP labeling. This well-established assay may use many different substrates, and mutated versions of the bait may be used to assess the role of specific residues. The effects of pharmacological agents may also be evaluated since this technique is both highly sensitive and quantitative. Nonetheless, radioactivity handling requires particular caution. Kinase activity may also be assessed with specific antibodies targeting the phospho group of the modified amino acid. These kinds of antibodies are not commercially available for all the phospho modified residues.

We characterized a new kinase protein using robust biochemical approaches: Western Blot analysis with a dedicated specific antibody on different cell lines and tissues, interactions by coimmunoprecipitation experiments, kinase activity detected by Western Blot using a phospho-specific antibody and by &#947;[32P] ATP labeling.

새로운 키나제 단백질의 강력한 생화학 적 접근법을 사용하여 분자 수준에서특성화

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important ...

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 &#181;L per well. The platform should be applicable to virtually any ADP producing enzyme.

소분자 억제제 스크리닝을 위한 NADH 결합 ATPase 분석의 반 높은 처리량 적응

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

Nucleoside Triphosphate Hydrolases Assay in Toxoplasm gondii and Neospora caninum for High-Throughput Screening using a Robot Arm

Protozoan parasites infect humans and many warm-blooded animals. Toxoplasma gondii, a major protozoan parasite, is commonly found in HIV-positive patients, organ transplant recipients and pregnant women, resulting in the severe health condition, Toxoplasmosis. Another major protozoan, Neospora caninum, which bears many similarities to Toxoplasma gondii, causes serious diseases in animals, as does Encephalomyelitis and Myositis-Polyradiculitis in dogs and cows, resulting in stillborn calves. All these exhibited similar nucleoside triphosphate hydrolases (NTPase). Neospora caninum has a NcNTPase, while Toxoplasma gondii has a TgNTPase-I. The enzymes are thought to play crucial roles in propagation and survival. In order to establish compounds and/or extracts preventing protozoan infection, we targeted these enzymes for drug discovery. The next step was to establish a novel, highly sensitive, and highly accurate assay by combining a conventional biochemical enzyme assay with a fluorescent assay to determine ADP content. We also validated that the novel assay fulfills the criteria to carry out high-throughput screening (HTS) in the two protozoan enzymes. We performed HTS, identified 19 compounds and six extracts from two synthetic compound libraries and an extract library derived from marine bacteria, respectively. In this study, a detailed explanation has been introduced on how to carry out HTS, including information about the preparation of reagents, devices, robot arm, etc.

<meta charset="utf8"></head><body>로봇 팔을 사용한 고처리량 스크리닝을 위한 Toxoplasm gondii 및 Neospora caninum의 Nucleoside Triphosphate Hydrolases Assay

Toxoplasma gondii and Neospora caninum infections are found in humans and animals and lead to serious health issues. The two parasites share similar nucleoside triphosphate hydrolases and play important roles in propagation and survival. We established a high-standard assay of the enzymes requiring robot arm usage.

로봇 팔을 사용한 고처리량 스크리닝을 위한 Toxoplasm gondii 및 Neospora caninum 의 Nucleoside Triphosphate Hydrolases Assay

Protozoan parasites infect humans and many warm-blooded animals. Toxoplasma gondii, a major protozoan parasite, is commonly found in HIV-positive ...

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

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of acetate to acetyl phosphate utilizing ATP as a substrate. Acetate kinases are ubiquitous in the Bacteria, found in one genus of Archaea, and are also present in microbes of the Eukarya6. The most well characterized acetate kinase is that from the methane-producing archaeon Methanosarcina thermophila7-14. An acetate kinase which can only utilize PPi but not ATP in the acetyl phosphate-forming direction has been isolated from Entamoeba histolytica, the causative agent of amoebic dysentery, and has thus far only been found in this genus15,16.
In the direction of acetyl phosphate formation, acetate kinase activity is typically measured using the hydroxamate assay, first described by Lipmann17-20, a coupled assay in which conversion of ATP to ADP is coupled to oxidation of NADH to NAD+ by the enzymes pyruvate kinase and lactate dehydrogenase21,22, or an assay measuring release of inorganic phosphate after reaction of the acetyl phosphate product with hydroxylamine23. Activity in the opposite, acetate-forming direction is measured by coupling ATP formation from ADP to the reduction of NADP+ to NADPH by the enzymes hexokinase and glucose 6-phosphate dehydrogenase24.
Here we describe a method for the detection of acetate kinase activity in the direction of acetate formation that does not require coupling enzymes, but is instead based on direct determination of acetyl phosphate consumption. After the enzymatic reaction, remaining acetyl phosphate is converted to a ferric hydroxamate complex that can be measured spectrophotometrically, as for the hydroxamate assay. Thus, unlike the standard coupled assay for this direction that is dependent on the production of ATP from ADP, this direct assay can be used for acetate kinases that produce ATP or PPi.

A method for the determination of acetate kinase activity is described. This assay utilizes a direct reaction for determining enzyme activity and kinetics of acetate kinase in the acetate-forming direction with different phosphoryl acceptors. Furthermore, this method can be utilized for assaying other acetyl phosphate or acetyl-CoA utilizing enzymes.

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 ...

생물학

A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to substrates to create phosphorylated products. The use of &#947;-32-P labeled NTP precursors allows simultaneous monitoring of substrate utilization and product formation by radiography. Thin layer chromatography (TLC) on cellulose plates allows rapid separation and sensitive quantification of substrate and product. We present a method for utilizing the thin-layer chromatography to assay the pyrophosphokinase activity of a purified (p)ppGpp synthetase. This method has previously been used to characterize the activity of cyclic nucleotide and dinucleotide synthetases and is broadly suitable for characterizing the activity of any enzyme that hydrolyzes a nucleotide triphosphate bond or transfers a terminal phosphate from a phosphate donor to another molecule.

A Purification and In Vitro Activity Assay for a pppGpp Synthetase from Clostridium difficile

Here, we describe a method for purifying histidine-tagged pyrophosphokinase enzymes and utilizing thin layer chromatography of radiolabelled substrates and products to assay for the enzymatic activity in vitro. The enzyme activity assay is broadly applicable to any kinase, nucleotide cyclase, or phosphor-transfer reaction whose mechanism includes nucleotide triphosphate hydrolysis.

정화 및 체 외에서 활동 분석 결과 (p) ppGpp Clostridium 남과 어울리지 않 는에서 합성에 대 한

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to ...

면역학 및 감염병학

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-&epsilon; improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-&alpha; and PKC-&epsilon; are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-&epsilon; mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.
Here, we present methods for assessing the role of PKC-&epsilon; in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-&epsilon; is activated by overexpressing the constitutively active PKC-&epsilon; mutant. PKC-&epsilon; is inhibited by overexpressing the inactive mutant of PKC-&epsilon;. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.
These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.

The effects of activation of protein kinase C (PKC) isozymes on mitochondrial functions associated with respiration and oxidative phosphorylation and on cell viability are described. The approach adapts adenoviral technique to selectively overexpress PKC isozymes in primary cell culture and a variety of assays to determine mitochondrial functions and energy status of the cell.

신장 세포 Overexpressing 단백질 키나제 C Isozymes에 Mitochondrial 기능 및 세포 생존 능력의 평가

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Biochemically dissecting the contributions of individual subunits of such complexes to the multi-step ATP-dependent chromatin remodeling reaction requires the use of assays that monitor the production of reaction products and measure the formation of reaction intermediates. This JOVE protocol describes assays that allow one to measure the biochemical activities of chromatin remodeling complexes or subcomplexes containing various combinations of subunits. Chromatin remodeling is measured using an ATP-dependent nucleosome sliding assay, which monitors the movement of a nucleosome on a DNA molecule using an electrophoretic mobility shift assay (EMSA)-based method. Nucleosome binding activity is measured by monitoring the formation of remodeling complex-bound mononucleosomes using a similar EMSA-based method, and DNA- or nucleosome-dependent ATPase activity is assayed using thin layer chromatography (TLC) to measure the rate of conversion of ATP to ADP and phosphate in the presence of either DNA or nucleosomes. Using these assays, one can examine the functions of subunits of a chromatin remodeling complex by comparing the activities of the complete complex to those lacking one or more subunits. The human INO80 chromatin remodeling complex is used as an example; however, the methods described here can be adapted to the study of other chromatin remodeling complexes.

Here we describe biochemical assays that can be used to characterize ATP-dependent chromatin remodeling enzymes for their abilities to 1) catalyze ATP-dependent nucleosome sliding, 2) engage with nucleosome substrates, and 3) hydrolyze ATP in a nucleosome- or DNA-dependent manner.

ATP에 의존 염색질 리모델링 효소의 활동을 분석하는 생화학 분석 실험

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and ...

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from S-adenosyl-L-methionine onto phosphatidylethanolamine, monomethyl-phosphatidylethanolamine, or dimethyl-phosphatidylethanolamine to give either monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine or phosphatidylcholine. These enzymes are ubiquitous in animal cells, fungi, and are also found in approximately 10% of bacteria. They fulfill various important functions in cell physiology beyond their direct role in lipid metabolism such as in insulin resistance, diabetes, atherosclerosis, cell growth, or virulence. The present manuscript reports on a simple cell-free enzymatic assay that measures the transfer of tritiated methyl group(s) from S-[Methyl-3H]adenosyl-L-methionine onto phosphatidylethanolamine using whole cell extracts as an enzyme source. The resulting methylated forms of phosphatidylethanolamine are hydrophobic and thus, can be separated from water soluble S-[Methyl-3H]adenosyl-L-methionine by organic extraction. This assay can potentially be applied to any other cell types and used to test inhibitors/drugs specific to a phosphatidylethanolamine methyltransferase of interest without the need to purify the enzyme.

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

The present report describes an in vitro enzymatic assay to measure phosphatidylethanolamine methyltransferase activity using Leishmania cell extracts. This assay is based on the transfer of a radioactive methyl group from S-[Methyl-3H]adenosyl-L-methionine onto endogenous phosphatidylethanolamine.

자질

요약

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방사성 표지 된 ATP로 단백질 키나아제 활성 측정

새로운 키나제 단백질의 강력한 생화학 적 접근법을 사용하여 분자 수준에서특성화

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로봇 팔을 사용한 고처리량 스크리닝을 위한 Toxoplasm gondii 및 Neospora caninum 의 Nucleoside Triphosphate Hydrolases Assay

로봇 팔을 사용한 고처리량 스크리닝을 위한 Toxoplasm gondii 및 Neospora caninum 의 Nucleoside Triphosphate Hydrolases Assay

효소 아세테이트 키나제의 아세테이트 - 형성 활동의 직접적인 탐지

정화 및 체 외에서 활동 분석 결과 (p) ppGpp Clostridium 남과 어울리지 않 는에서 합성에 대 한

신장 세포 Overexpressing 단백질 키나제 C Isozymes에 Mitochondrial 기능 및 세포 생존 능력의 평가

ATP에 의존 염색질 리모델링 효소의 활동을 분석하는 생화학 분석 실험

시험 관내 분석에서 포스파티딜 메틸화 된 역가를 측정하는 방법