This work received support through grant HA 6136/1-1 from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) to MH. JAT is supported by Program and Project Grants from the National Health and Medical Research Council of Australia.

참고 : 마우스에서 파생 된 비장 (나이 6-10 주) 모든 동물 실험은 피터 맥 칼럼 암 센터 (E486)의 동물 윤리 지침에 따라 수행 하였다. 샘플 1. 준비. <ol><li> 활성화 마우스 NK 세포의 준비. <ol><li> 시판되는 키트를 사용하여 음성 선별에 의해 (GzmB 유전자 NULL) 마우스 - / - C57BL / 6 또는 B6.GrzmB의 비장의 단일 세포 현탁액으로부터 기본 나이브 NK 세포를 분리. </li><li> 제조 업체의 프로토콜을 따르십시오. 요약하면, 이러한 자기 T 및 B 세포, 수지상 세포, 과립구, 대 식세포, 및 특정 표면 마커에 대해 바이오틴 - 컨쥬 게이트 항체를 사용하여 적혈구 세포와 같은 &quot;비 - NK&quot;세포를 라벨. &quot;비 NK&quot;세포를 고갈 방지 비오틴 구슬 자기 분리를 사용합니다. </li><li> 배양은 24 웰 플레이트에서 700,000 세포 / ml에서 IL-2 (1000 U / ㎖)를 함유하는 미디어 5-7 일간 NK 세포를 격리37 &#160; ° C. </li><li> 대안 적 사용은 19 T 세포, OT1 T 세포 수용체 트랜스 제닉 마우스 (20)로부터 기본 항원 제한된 CTL이 CTL 분비 GzmB (19)의 행동을 결정하기 위하여 활성화 된 NK 세포로부터 혼합 림프구 배양 물 (21), 또는 상층 액에 생성 된 활성화. </li></ol></li><li> 인간 NK 세포주 및 일차 NK 세포의 분리의 유지. <ol><li> 10 % 우태 혈청으로 보충 된 로스웰 파크 메모리얼 연구소 (RPMI) 배지에서 인간 NK 백혈병 (YT 세포)를 유지한다. 또한, 정상인의 말초 혈액에서 차 NK 세포를 정화하고 이전 22 설명 된 바와 같이 IL-2 100 U / ml의에서이 함유 배지 2 ~ 3 일 동안 자극한다. </li></ol></li><li> 세포 용 해물의 생성. <ol><li> PBS (PH 7.4)에 세포를 세 번 세척 한 후 Nonidet P-40 용해 버퍼에 일시 중단 (0.1 % NP-40, 250 mM의 염화나트륨, 25 mM의 헤 페스, 2.5 mM의 EDTA). 이상적으로는, 5 × 106의 최소 용해 </sNK 세포, 또는 100 μL 버퍼에 1 × 10 7 T 세포&gt;까지. 프로테아제 억제제를 추가하지 마십시오, 많은는 GzmA 취소 할 수없는 세린 프로테아제 억제제, 특히 tryptases에 있습니다. 얼음에서 20 분 동안 인큐베이션 한 후 10 분간 15,000 XG에 미세 원심에서 핵 펠렛. 핵 펠렛을 취소하고 새로운 튜브에 뜨는을 전송합니다. </li></ol></li><li> 해물의 단백질 농도를 결정합니다. <ol><li> 이러한 브래드 bicinchoninic 또는 산 (BCA)과 같은 선택의 시판 프로토콜을 사용하여 단백질 농도를 결정한다. 농도가 1 ~ 2 ㎎ / ㎖ 최소 있는지 확인하십시오. 필요한 때까지 -20 ° C에서 보관 용 해물 (그랜 자임 활동은 -20 ° C에서 몇 달 동안 안정). </li></ol></li></ol> 2. 그랜 자임 B 활동 분석 <ol><li> 시약의 조제 <ol><li> DMSO에 기판 (BOC-AAD-S-BZL 또는 AC-IEPD-pNA를)의 10 mM의 주식을 준비합니다 (~ 5 ㎎ / ㎖) -20 &amp; #에서 분취에 저장176; C. 갓 작업 희석을 준비하고 BOC-AAD-S-BZL 부분적으로 DMSO에 저장에 가수 분해로, 하루 후 폐기합니다. </li><li> -20 ° C에서 250 밀리미터의 DMSO 비색 시약 (5,5&#39;- 디티 오 - 비스 (2- 니트로 벤조산), DTNB)의 재고 (0.09 g / mL) 및 저장소를 준비한다. 매일 신선한 작업 희석을 준비합니다. 이 시약은 SBzl 표시가 아닌 그룹 pNA를 가진 기판이 필요합니다. </li><li> 신선한 버퍼 (0.1M HEPES, 0.05 M의 MgCl 2, pH를 7.3) 2 ~ 3 주까지합니다. </li></ol></li><li> 실온으로 DTNB 및 합성 기판을 준비한다. 희석 기판 (1시 16분, 예를 들어 1.6 버퍼 용액에 100 μL)과 DTNB (1 : 250, 예를 들어 10 2.5 ml의 버퍼의 μL) 잘 섞는다. </li><li> 160 μL의 최종 부피 버퍼에 각 시료 100 μg의 단백질 용 해물의 최소 희석. 멀티 채널 피펫을 사용하여 &#39;&#39;U &#39;&#39;바닥 비닐 96 웰 플레이트 (~ 33.3 ㎍ / 웰) 세중 잘 당 50 μl를 추가합니다.&quot;버퍼 전용&quot;제어를 포함 (또한 세중). </li><li> AC-IEPD-pNA를 사용하여 작업 할 때 샘플 / 버퍼 제어로 희석 DTNB 100 μl를 추가합니다 (이 단계를 생략합니다. 참고 : GzmB에 의해 AC-IEPD-pNA를의 쪼개짐 직접 형광 pNA를 (그림 4B)를 발표한다. </li><li> 빨리 다음 버퍼 부정적인 컨트롤로 시작하는 예상 양의 샘플을 마무리, 삼중의 각 세트로 희석 기판의 50 μl를 추가하는 멀티 채널 피펫을 사용합니다. </li><li> 플레이트 리더에서 접시를 놓습니다. OD 405 nm에서 마이크로 플레이트 리더에 BOC-알라 - 알라 -의 ASP-S-BZL / AC-IEPD-pNA를 가수 분해하여 ASP를-ASE 활동을 측정한다. 25 판독, 매 15 초 (약 6 분 총 독서 시간)을 가지고, 운동 분석 프로토콜을 사용합니다. </li><li> 플레이트 리더 (VMAX)의 소프트웨어에 대한 값을 생성 할 수없는 경우 직접 기판의 절단 속도를 계산하기 위해 각 시점에서 생성 흡광도 판독 값을 사용한다. </li></ol> 다 3. 분석타 참고 : 시간이 지남에 OD의 변화 (MOD / 분)로 정의 된 최대 속도로 제시됩니다 생성 된 데이터,. <ol><li> 데이터를 분석하는, 흡광도의 변화율로부터 산출되는 기판 절단의 최대 속도를 결정한다. 자동 일반적으로이 옵션이 플레이트 리더 소프트웨어를 사용하여이 작업을 수행합니다. </li><li> 또는 수동으로 플레이트 리더에 운동 분석 중에 생성되는 흡광도의 변화의 운동 플롯을 본 후 가파른 직선을주는 데이터 포인트를 선택합니다. 대부분의 상기 분석 최대 레이트로는 분석의 제 2 분 이내에 달성된다. </li><li> 시간 간격으로이 직선과 분열의 가장 높은 OD 독서에서 초기 OD 읽기를 뺍니다. 통계 분석 및 그래픽 디스플레이 용 Microsoft Excel / 그래프 패드에 원시 데이터를 전송합니다. </li></ol>

<ol>
	<li>Trapani, J. A., Browne, K. A., Dawson, M., Smyth, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunopurification+of+functional+Asp-ase+(natural+killer+cell+granzyme+B)+using+a+monoclonal+antibody).">Immunopurification of functional Asp-ase (natural killer cell granzyme B) using a monoclonal antibody).</a> Biochem Biophys Res Commun. 195 (2), 910-920 (1993).</li><li>Ewen, C. L., Kane, K. P., Bleackley, R. 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+quarter+century+of+granzymes.">A quarter century of granzymes.</a> Cell Death Differ. 19 (1), 28-35 (2012).</li><li>Hoves, S., Trapani, J. A., Voskoboinik, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+battlefield+of+perforin/granzyme+cell+death+pathways.">The battlefield of perforin/granzyme cell death pathways.</a> J Leukoc Biol. , (2009).</li><li>Peters, P. 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=Cytotoxic+T+lymphocyte+granules+are+secretory+lysosomes,+containing+both+perforin+and+granzymes.">Cytotoxic T lymphocyte granules are secretory lysosomes, containing both perforin and granzymes.</a> J Exp Med. 173 (5), 1099-1109 (1991).</li><li>Berthou, C., 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=Acquisition+of+granzyme+B+and+Fas+ligand+proteins+by+human+keratinocytes+contributes+to+epidermal+cell+defense.">Acquisition of granzyme B and Fas ligand proteins by human keratinocytes contributes to epidermal cell defense.</a> J Immunol. 159 (11), 5293-5300 (1997).</li><li>Tschopp, C. M., 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=Granzyme+B,+a+novel+mediator+of+allergic+inflammation:+its+induction+and+release+in+blood+basophils+and+human+asthma.">Granzyme B, a novel mediator of allergic inflammation: its induction and release in blood basophils and human asthma.</a> Blood. 108 (7), 2290-2299 (2006).</li><li>Strik, M. C., 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=Human+mast+cells+produce+and+release+the+cytotoxic+lymphocyte+associated+protease+granzyme+B+upon+activation.">Human mast cells produce and release the cytotoxic lymphocyte associated protease granzyme B upon activation.</a> Mol Immunol. 44 (14), 3462-3472 (2007).</li><li>Jahrsdorfer, B., 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=Granzyme+B+produced+by+human+plasmacytoid+dendritic+cells+suppresses+T+cell+expansion.">Granzyme B produced by human plasmacytoid dendritic cells suppresses T cell expansion.</a> Blood. , (2009).</li><li>Hagn, M., 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=Human+B+cells+secrete+granzyme+B+when+recognizing+viral+antigens+in+the+context+of+the+acute+phase+cytokine+IL-21.">Human B cells secrete granzyme B when recognizing viral antigens in the context of the acute phase cytokine IL-21.</a> J Immunol. 183 (3), 1838-1845 (2009).</li><li>Hagn, M., 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=Human+B+cells+differentiate+into+granzyme+B-secreting+cytotoxic+B+lymphocytes+upon+incomplete+T-cell+help.">Human B cells differentiate into granzyme B-secreting cytotoxic B lymphocytes upon incomplete T-cell help.</a> Immunol Cell Biol. 90 (4), 457-467 (2012).</li><li>Froelich, C. J., Pardo, J., Simon, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granule-associated+serine+proteases:+granzymes+might+not+just+be+killer+proteases.">Granule-associated serine proteases: granzymes might not just be killer proteases.</a> Trends Immunol. 30 (3), 117-123 (2009).</li><li>Hagn, M., Jahrsdorfer, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+do+human+B+cells+secrete+granzyme+B?+Insights+into+a+novel+B-cell+differentiation+pathway.">Why do human B cells secrete granzyme B? Insights into a novel B-cell differentiation pathway.</a> Oncoimmunology. 1 (8), 1368-1375 (2012).</li><li>Susanto, O., Trapani, J. A., Brasacchio, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controversies+in+granzyme+biology.">Controversies in granzyme biology.</a> Tissue Antigens. 80 (6), 477-487 (2012).</li><li>Walch, M., 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=Cytotoxic+cells+kill+intracellular+bacteria+through+granulysin-mediated+delivery+of+granzymes.">Cytotoxic cells kill intracellular bacteria through granulysin-mediated delivery of granzymes.</a> Cell. 157 (6), 1309-1323 (2014).</li><li>Odake, S., 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=Human+and+murine+cytotoxic+T+lymphocyte+serine+proteases:+subsite+mapping+with+peptide+thioester+substrates+and+inhibition+of+enzyme+activity+and+cytolysis+by+isocoumarins.">Human and murine cytotoxic T lymphocyte serine proteases: subsite mapping with peptide thioester substrates and inhibition of enzyme activity and cytolysis by isocoumarins.</a> Biochemistry. 30 (8), 2217-2227 (1991).</li><li>Casciola-Rosen, L., 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=Mouse+and+human+granzyme+B+have+distinct+tetrapeptide+specificities+and+abilities+to+recruit+the+bid+pathway.">Mouse and human granzyme B have distinct tetrapeptide specificities and abilities to recruit the bid pathway.</a> J Biol Chem. 282 (7), 4545-4552 (2007).</li><li>Kaiserman, D., 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=The+major+human+and+mouse+granzymes+are+structurally+and+functionally+divergent.">The major human and mouse granzymes are structurally and functionally divergent.</a> J Cell Biol. 175 (4), 619-630 (2006).</li><li>Powers, J. C., Kam, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+thioester+substrates+for+serine+peptidases+and+metalloendopeptidases.">Peptide thioester substrates for serine peptidases and metalloendopeptidases.</a> Methods Enzymol. 248, 3-18 (1995).</li><li>Hagn, M., 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=Activated+mouse+B+cells+lack+expression+of+granzyme.">Activated mouse B cells lack expression of granzyme.</a> B. J Immunol. 188 (2), 3886-3892 (2012).</li><li>Konjar, S., 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=Human+and+mouse+perforin+are+processed+in+part+through+cleavage+by+the+lysosomal+cysteine+proteinase+cathepsin+L.">Human and mouse perforin are processed in part through cleavage by the lysosomal cysteine proteinase cathepsin L.</a> Immunology. 131 (2), 257-267 (2010).</li><li>Sutton, V. R., 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=Residual+active+granzyme+B+in+cathepsin+C-null+lymphocytes+is+sufficient+for+perforin-dependent+target+cell+apoptosis.">Residual active granzyme B in cathepsin C-null lymphocytes is sufficient for perforin-dependent target cell apoptosis.</a> J Cell Biol. 176 (4), 425-433 (2007).</li><li>Trapani, J. A., Smyth, M. J., Apostolidis, V. A., Dawson, M., Browne, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granule+serine+proteases+are+normal+nuclear+constituents+of+natural+killer+cells.">Granule serine proteases are normal nuclear constituents of natural killer cells.</a> J Biol Chem. 269 (28), 18359-18365 (1994).</li><li>Cullen, S. P., Adrain, C., Luthi, A. U., Duriez, P. J., Martin, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+and+murine+granzyme+B+exhibit+divergent+substrate+preferences.">Human and murine granzyme B exhibit divergent substrate preferences.</a> J Cell Biol. 176 (4), 435-444 (2007).</li><li>Thornberry, N. A., 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=A+combinatorial+approach+defines+specificities+of+members+of+the+caspase+family+and+granzyme+B.+Functional+relationships+established+for+key+mediators+of+apoptosis.">A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis.</a> J Biol Chem. 272 (29), 17907-17911 (1997).</li><li>Bird, C. H., 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=Selective+regulation+of+apoptosis:+the+cytotoxic+lymphocyte+serpin+proteinase+inhibitor+9+protects+against+granzyme+B-mediated+apoptosis+without+perturbing+the+Fas+cell+death+pathway.">Selective regulation of apoptosis: the cytotoxic lymphocyte serpin proteinase inhibitor 9 protects against granzyme B-mediated apoptosis without perturbing the Fas cell death pathway.</a> Mol Cell Biol. 18 (11), 6387-6398 (1998).</li><li>Bots, M., Medema, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serpins+in+T+cell+immunity.">Serpins in T cell immunity.</a> J Leukoc Biol. 84 (5), 1238-1247 (2008).</li><li>Sun, 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=A+new+family+of+10+murine+ovalbumin+serpins+includes+two+homologs+of+proteinase+inhibitor+8+and+two+homologs+of+the+granzyme+B+inhibitor+(proteinase+inhibitor+9).">A new family of 10 murine ovalbumin serpins includes two homologs of proteinase inhibitor 8 and two homologs of the granzyme B inhibitor (proteinase inhibitor 9).</a> J Biol Chem. 272 (24), 15434-15441 (1997).</li><li>Bird, C. H., Hitchen, C., Prescott, M., Harper, I., Bird, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunodetection+of+granzyme+B+tissue+distribution+and+cellular+localisation.">Immunodetection of granzyme B tissue distribution and cellular localisation.</a> Methods Mol Biol. 844, 237-250 (2012).</li><li>Jenkins, M. R., 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=Visualizing+CTL+activity+for+different+CD8++effector+T+cells+supports+the+idea+that+lower+TCR/epitope+avidity+may+be+advantageous+for+target+cell+killing.">Visualizing CTL activity for different CD8+ effector T cells supports the idea that lower TCR/epitope avidity may be advantageous for target cell killing.</a> Cell Death Differ. 16 (4), 537-542 (2009).</li><li>Edwards, K. M., Kam, C. M., Powers, J. C., Trapani, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+cytotoxic+T+cell+granule+serine+protease+granzyme+H+has+chymotrypsin-like+(chymase)+activity+and+is+taken+up+into+cytoplasmic+vesicles+reminiscent+of+granzyme+B-containing+endosomes.">The human cytotoxic T cell granule serine protease granzyme H has chymotrypsin-like (chymase) activity and is taken up into cytoplasmic vesicles reminiscent of granzyme B-containing endosomes.</a> J Biol Chem. 274 (43), 30468-30473 (1999).</li><li>Sutton, V. R., 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=Initiation+of+apoptosis+by+granzyme+B+requires+direct+cleavage+of+bid,+but+not+direct+granzyme+B-mediated+caspase+activation.">Initiation of apoptosis by granzyme B requires direct cleavage of bid, but not direct granzyme B-mediated caspase activation.</a> J Exp Med. 192 (10), 1403-1414 (2000).</li></ol>

The authors have nothing to disclose.

역사적으로 granzymes은 표적 세포의 급속한 세포 사멸 죽음을 유도 할 수있는 세포 독성 림프구 (CTL 및 NK 세포)의 주요 이펙터 분자로 확인되었다. 이는 아스 파르 테이트 (D) 잔기에 타깃 기판 분자를 절단함으로써 그 하류 표적 여러뿐만 아니라, 모두 절단 프로 카스파하여 카스파 제 캐스케이드를 활성화 할 수 있었다 GzmB의 작용에 주로이었다. 그러나, 지금 GzmB 발현 림프구 세포 독성 세포에 국한...

Granzymes 자연 킬러 (NK) 세포 및 세포 독성 T 림프구 (CTL) (1)의 분비 리소좀에서 발견되는 세린 프로테아제 패밀리이다. 다섯 가지 granzymes 인간 (A, B, H, K 및 M)에 존재하고, 마우스에서 십 (A - G, K, M 및 N) 2,3-. 그랜 자임과 그랜 자임 B (GzmA, GzmB)는 가장 풍부하고 광범위하게 인간과 설치류 설정에서 조사되었다. GzmB의 고전 함수는 표적 세포 사이토 졸로 액세스 4 그랜 자임을 허용 기공 형성 단백질 퍼포과 함께 실행되는 표적 세포에서 아폽토시스의 유도이다. GzmB 발현 명백하게 세포 상해 성 림프구에서 발견되지만, 최근의 연구는 호염기구 (6), 비만 세포 (7) 형질 수지상 세포 (8) 및 B 세포 (9)를 포함하지만, 케 라티노 사이트 (5)에 한정되지 않고, 다른 GzmB 발현 세포 유형의 다양한 어드레싱되었습니다 10.이러한 맥락에서, 비 세포 사멸 GzmB 기능은 염증 과정, 조직 리모델링과 다른 면역 특성 11-14 참여에 이르기까지 드러났다. 폭 넓은 생물학적 역할이 이전에 의심보다 GzmB 제안 된 것을 감안할 때, 연구자는 검출 신뢰성과 특정 도구가 필요합니다. 이점 진핵 세린 프로테아제 (15) 사이에서 고유 아스파르트 산 잔기, 속성의 카복실 측 쪼개지 GzmB의 특정 요구 사항이다. 마우스는 인간과 쥐 GzmB 그러나 마우스 GzmB의 확장 기질 특이성 터미널 (P1)에서의 ASP와 특정 일반 기판 GzmB 효율적으로 절단 할 수 있음을 의미 모두 인간과 쥐 16과는 미묘하게 다른, 구조적으로 매우 유사 세 종, 상류 P1의 더 복잡한 시퀀스와 다른 기판 반면 널리 변수 결과를 제공 할 수 있습니다. 과거 최신 리튬 둘terature,이 사실은주의 깊게 통제에도 불구하고, 상당한 혼란과 일부 실험 결과의 생물학적 의미의 오해가 발생했습니다, 운동 연구는 상황 (17)를 해결하기 위해 노력했다. 이 논문에서 우리는 두 가지 상업적으로 이용 가능한 기판, 즉 BOC-알라 - 알라 -의 ASP SBzl 및 N- 아세틸 일드 서열 1 프로의 ASP-P-니트로 아닐리드를 사용하여 이러한 점을 설명하기 위해 노력했다. 이 시약은 분열 (형광 무료 paranitroanilide 대 무료 sulphydryl) 다음과 같은 서로 다른 반응 그룹을 생성 할 수 있지만,이 단백질 분해 분열에 영향 무엇이든이 없습니다. 기술 된 프로토콜은 매우 오래 프로토콜 (18)의 현대 적응이지만 또한 GzmA 및 GzmH 다른 granzymes의 활성을 검출하기위한 방법 론적 틀을 제공하면서 조사관, 적절히 다른 GzmB 기판을 사용하는 것이 도움이 될 것이다.

<table border="0" cellpadding="0" cellspacing="0" width="1133">
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			<td height="21" style="height:21px;width:284px;">Product</td>
			<td style="width:241px;">Company</td>
			<td style="width:181px;">Catalogue number</td>
			<td style="width:427px;">Comment/description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Boc-Ala-Ala-Asp-S-Bzl</td>
			<td>SM Biochemicals LLC, CA</td>
			<td>SMSB05</td>
			<td>Granzyme B substrate (mouse and human)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ac-IEPD-pNA&#160;</td>
			<td>SM Biochemicals LLC, CA</td>
			<td>SMPNA009</td>
			<td>Granzyme B substrate (only human)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-a-CBZ-L-lysine-S-Bzl</td>
			<td>Sigma-Aldrich</td>
			<td>C3647</td>
			<td>Granzyme A substrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Suc-Phe-Leu-Phe-S-Bzl</td>
			<td>SM Biochemicals LLC, CA</td>
			<td>SB025</td>
			<td>Granzyme H substrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5,5&#8217;-dithio-bis(2-nitrobenzoic acid)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>D8130</td>
			<td>&#160;DTNB, Ellman&#8217;s Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NK cell isolation kit II mouse</td>
			<td>Miltenyi Biotec GmbH</td>
			<td style="width:181px;">130-096-892</td>
			<td style="width:427px;">negative selection kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NK cell isolation kit human</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-092-657</td>
			<td style="width:427px;">negative selection kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Plate reader</td>
			<td>Biorad iMark</td>
			<td></td>
			<td>Biorad Microplate Manager Software Version MPM6.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Serocluster U-bottom vinyl 96-well plate</td>
			<td>Corning, MA, USA</td>
			<td>2797</td>
			<td></td>
		</tr>
	</tbody>
</table>

a colorimetric assay that specifically measures granzyme b proteolytic activity: hydrolysis of boc-ala-ala-asp-s-bzl

The serine protease Granzyme B (GzmB) mediates target cell apoptosis when released by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. GzmB is the most studied granzyme in humans and mice and therefore, researchers need specific and reliable tools to study its function and role in pathophysiology. This especially necessitates assays that do not recognize proteases such as caspases or other granzymes that are structurally or functionally related. Here, we apply GzmB&#8217;s preference for cleavage after aspartic acid residues in a colorimetric assay using the peptide thioester Boc-Ala-Ala-Asp-S-Bzl. GzmB is the only mammalian serine protease capable of cleaving this substrate. The substrate is cleaved with similar efficiency by human, mouse and rat GzmB, a property not shared by other commercially available peptide substrates, even some that are advertised as being suitable for this purpose. This protocol is demonstrated using unfractionated lysates from activated NK cells or CTL and is also suitable for recombinant proteases generated in a variety of prokaryotic and eukaryotic systems, provided the correct controls are used. This assay is a highly specific method to ascertain the potential pro-apoptotic activity of cytotoxic molecules in mammalian lymphocytes, and of their recombinant counterparts expressed by a variety of methodologies.

BOC-AAD-S-BZL 기판 GzmB에 대한 특정 세린 프로테아제 GzmB 세포 독성 림프구 (CTL 및 NK 세포)의 주요 구성 성분이며, 바이러스 감염 또는 형질 전환 세포와 같은 표적 세포에 빠른 세포 사멸 죽음을 유도 주로 담당하고 있습니다. 이 때문에 선택된 단백질의 특정 특정 아스파 테이트 잔류 후 분열에 대한 기판 선호도에 크게이며, 속성은 아스 파르 테이트 잔류 후 절단?...

Watch this Scientific Journal Video about A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl at JoVE.com

A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl

We describe a simple, quantitative colorimetric assay that specifically measures the proteolytic activity of human, mouse or rat Granzyme B (GzmB). This protocol can be easily adapted for determining protease activity of other granule serine proteases by the hydrolysis of other synthetic peptide substrates with an appropriate recognition sequence.

BOC-알라 - 알라 -의 ASP-S-BZL의 가수 분해 : 색채 특히 그랜 자임 B 단백질 분해 활동를 측정 분석

The serine protease Granzyme B (GzmB) mediates target cell apoptosis when released by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. GzmB is ...

a-colorimetric-assay-that-specifically-measures-granzyme-b

Research

JoVE Journal

Chemistry

12.9K 조회수.  Peter MacCallum Cancer Centre. We describe a simple, quantitative colorimetric assay that specifically measures the proteolytic activity of human, mouse or rat Granzyme B (GzmB). This protocol can be easily adapted for determining protease activity of other granule serine proteases by the hydrolysis of other synthetic peptide substrates with an appropriate recognition sequence.

비디오: A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl

Rapid Colorimetric Assays to Qualitatively Distinguish RNA and DNA in Biomolecular Samples

Biochemical experimentation generally requires accurate knowledge, at an early stage, of the nucleic acid, protein, and other biomolecular components in potentially heterogeneous specimens. Nucleic acids can be detected via several established approaches, including analytical methods that are spectrophotometric (e.g., A260), fluorometric (e.g., binding of fluorescent dyes), or colorimetric (nucleoside-specific chromogenic chemical reactions).1 Though it cannot readily distinguish RNA from DNA, the A260/A280 ratio is commonly employed, as it offers a simple and rapid2 assessment of the relative content of nucleic acid, which absorbs predominantly near 260 nm and protein, which absorbs primarily near 280 nm. Ratios &lt; 0.8 are taken as indicative of 'pure' protein specimens, while pure nucleic acid (NA) is characterized by ratios &gt; 1.53.
However, there are scenarios in which the protein/NA content cannot be as clearly or reliably inferred from simple uv-vis spectrophotometric measurements. For instance, (i) samples may contain one or more proteins which are relatively devoid of the aromatic amino acids responsible for absorption at &asymp;280 nm (Trp, Tyr, Phe), as is the case with some small RNA-binding proteins, and (ii) samples can exhibit intermediate A260/A280 ratios (~0.8 &lt; ~1.5), where the protein/NA content is far less clear and may even reflect some high-affinity association between the protein and NA components. For such scenarios, we describe herein a suite of colorimetric assays to rapidly distinguish RNA, DNA, and reducing sugars in a potentially mixed sample of biomolecules. The methods rely on the differential sensitivity of pentoses and other carbohydrates to Benedict's, Bial's (orcinol), and Dische's (diphenylamine) reagents; the streamlined protocols can be completed in a matter of minutes, without any additional steps of having to isolate the components. The assays can be performed in parallel to differentiate between RNA and DNA, as well as indicate the presence of free reducing sugars such as glucose, fructose, and ribose (Figure 1).

A suite of colorimetric assays is described for rapidly distinguishing protein, RNA, DNA, and reducing sugars in potentially heterogeneous biomolecular samples.

신속한 Colorimetric Assays는 질적 Biomolecular 샘플에서 RNA와 DNA를 구별 할

Biochemical experimentation generally requires accurate knowledge, at an early stage, of the nucleic acid, protein, and other biomolecular components in ...

연구

화학

Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization of OGG1 is undertaken to measure the rates of 8-oxoG excision and product release. When the OGG1 concentration is lower than substrate DNA, time courses of product formation are biphasic; a rapid exponential phase (i.e. burst) of product formation is followed by a linear steady-state phase. The initial burst of product formation corresponds to the concentration of enzyme properly engaged on the substrate, and the burst amplitude depends on the concentration of enzyme. The first-order rate constant of the burst corresponds to the intrinsic rate of 8-oxoG excision and the slower steady-state rate measures the rate of product release (product DNA dissociation rate constant, koff). Here, we describe steady-state, pre-steady-state, and single-turnover approaches to isolate and measure specific steps during OGG1 catalytic cycling. A fluorescent labeled lesion-containing oligonucleotide and purified OGG1 are used to facilitate precise kinetic measurements. Since low enzyme concentrations are used to make steady-state measurements, manual mixing of reagents and quenching of the reaction can be performed to ascertain the steady-state rate (koff). Additionally, extrapolation of the steady-state rate to a point on the ordinate at zero time indicates that a burst of product formation occurred during the first turnover (i.e. y-intercept is positive). The first-order rate constant of the exponential burst phase can be measured using a rapid mixing and quenching technique that examines the amount of product formed at short time intervals (&lt;1 sec) before the steady-state phase and corresponds to the rate of 8-oxoG excision (i.e. chemistry). The chemical step can also be measured using a single-turnover approach where catalytic cycling is prevented by saturating substrate DNA with enzyme (E&gt;S). These approaches can measure elementary rate constants that influence the efficiency of removal of a DNA lesion.

Time courses for the glycosylase activity of 8-oxoguanine DNA glycosylase are biphasic exhibiting a burst of product formation and a linear steady-state phase. Utilizing quench-flow techniques, the burst and the steady-state rates can be measured, which correspond to excision of 8-oxoguanine and release of the glycosylase from the product DNA, respectively.

DNA의 Glycosylase 활동에 대한 정상 상태, 전 정상 상태 및 단일 매출 운동 측정

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization ...

Fluorescence-based Monitoring of PAD4 Activity via a Pro-fluorescence Substrate Analog

Post-translational modifications may lead to altered protein functional states by increasing the covalent variations on the side chains of many protein substrates. The histone tails represent one of the most heavily modified stretches within all human proteins. Peptidyl-arginine deiminase 4 (PAD4) has been shown to convert arginine residues into the non-genetically encoded citrulline residue. Few assays described to date have been operationally facile with satisfactory sensitivity. Thus, the lack of adequate assays has likely contributed to the absence of potent non-covalent PAD4 inhibitors. Herein a novel fluorescence-based assay that allows for the monitoring of PAD4 activity is described. A pro-fluorescent substrate analog was designed to link PAD4 enzymatic activity to fluorescence liberation upon the addition of the protease trypsin. It was shown that the assay is compatible with high-throughput screening conditions and has a strong signal-to-noise ratio. Furthermore, the assay can also be performed with crude cell lysates containing over-expressed PAD4.

PAD4 is an enzyme responsible for the conversion of peptidyl-arginine to peptidyl-citrulline. Dysregulation of PAD4 has been implicated in a number of human diseases. A facile and high-throughput compatible fluorescence based PAD4 assay is described.

프로 형광 기판 아날로그를 통해 PAD4 활동의 형광 기반의 모니터링

Post-translational modifications may lead to altered protein functional states by increasing the covalent variations on the side chains of many protein ...

Dynamic Electrochemical Measurement of Chloride Ions

This protocol describes the dynamic measurement of chloride ions using the transition time of a silver silver chloride (Ag/AgCl) electrode. Silver silver chloride electrode is used extensively for potentiometric measurement of chloride ions concentration in electrolyte. In this measurement, long-term and continuous monitoring is limited due to the inherent drift and the requirement of a stable reference electrode. We utilized the chronopotentiometric approach to minimize drift and avoid the use of a conventional reference electrode. A galvanostatic pulse is applied to an Ag/AgCl electrode which initiates a faradic reaction depleting the Cl&#713; ions near the electrode surface. The transition time, which is the time to completely deplete the ions near the electrode surface, is a function of the ion concentration, given by the Nernst equation. The square root of the transition time is in linear relation to the chloride ion concentration. Drift of the response over two weeks is negligible (59 &#181;M/day) when measuring 1 mM [Cl&#713;]using a current pulse of 10 Am-2. This is a dynamic measurement where the moment of transition time determines the response and thus is independent of the absolute potential. Any metal wire can be used as a pseudo-reference electrode, making this approach feasible for long-term measurement inside concrete structures.

Dynamic measurement of chloride ions is presented. Transition time of an Ag/AgCl electrode, during a chronopotentiometric technique, can give the concentration of chloride ions in electrolyte. This method does not require a stable conventional reference electrode.

This protocol describes the dynamic measurement of chloride ions using the transition time of a silver silver chloride (Ag/AgCl) electrode. Silver silver ...

A Method to Manipulate Surface Tension of a Liquid Metal via Surface Oxidation and Reduction

Controlling interfacial tension is an effective method for manipulating the shape, position, and flow of fluids at sub-millimeter length scales, where interfacial tension is a dominant force. A variety of methods exist for controlling the interfacial tension of aqueous and organic liquids on this scale; however, these techniques have limited utility for liquid metals due to their large interfacial tension.
Liquid metals can form soft, stretchable, and shape-reconfigurable components in electronic and electromagnetic devices. Although it is possible to manipulate these fluids via mechanical methods (e.g., pumping), electrical methods are easier to miniaturize, control, and implement. However, most electrical techniques have their own constraints: electrowetting-on-dielectric requires large (kV) potentials for modest actuation, electrocapillarity can affect relatively small changes in the interfacial tension, and continuous electrowetting is limited to plugs of the liquid metal in capillaries.
Here, we present a method for actuating gallium and gallium-based liquid metal alloys via an electrochemical surface reaction. Controlling the electrochemical potential on the surface of the liquid metal in electrolyte rapidly and reversibly changes the interfacial tension by over two orders of magnitude ( &#820;500 mN/m to near zero). Furthermore, this method requires only a very modest potential (&#60; 1 V) applied relative to a counter electrode. The resulting change in tension is due primarily to the electrochemical deposition of a surface oxide layer, which acts as a surfactant; removal of the oxide increases the interfacial tension, and vice versa. This technique can be applied in a wide variety of electrolytes and is independent of the substrate on which it rests.

We present a method to control the interfacial energy of a liquid metal in an electrolyte via electrochemical deposition (or removal) of a surface oxide layer. This simple method can control the capillary behavior of gallium-based liquid metals by tuning the interfacial energy rapidly, significantly, and reversibly using modest voltages.

방법은 표면 산화와 환원을 통해 액체 금속의 표면 장력을 조작

Controlling interfacial tension is an effective method for manipulating the shape, position, and flow of fluids at sub-millimeter length scales, where ...

Preparation and Reactivity of a Triphosphenium Bromide Salt: A Convenient and Stable Source of Phosphorus(I)

We present herein the optimized synthesis of a triphosphenium bromide salt. Apart from being a versatile metathesis reagent, this unusually stable low-valent-phosphorus-containing compound acts as a useful P+ transfer agent. Unlike traditional methods employed to access low-coordinate phosphorus species which usually require pyrophoric phosphorus-containing precursors (white phosphorus, Tris(trimethylsilyl)phosphine, etc.), or harsh reducing agents (alkali metals, potassium graphite, etc.), the current approach does not involve pyrophoric or explosive reagents and can be done on large scales (&#62;20 g) in excellent yields by undergraduates with basic air-free synthetic training. The bromide counter ion is readily exchanged with other anions such as tetraphenyl borate (described herein) using typical salt metathesis reagents to obtain materials with desired properties and reactivities. The versatility of this P+ transfer approach is exemplified by the reactions of these triphosphenium precursors with an N-heterocyclic carbene and an anionic bisphosphine, each of which readily displace the neutral bisphosphine to give an NHC-stabilized phosphorus(I) cation and a phosphorus(I) containing zwitterion, respectively.

Preparation and Reactivity of a Triphosphenium Bromide Salt: A Convenient and Stable Source of PhosphorusI

The synthesis of a triphosphenium bromide salt is described and its use as a P+ transfer agent is outlined by reactions with an N-heterocyclic carbene and an anionic bisphosphine, yielding an NHC-stabilized P(I) cation and a P(I) containing zwitterion, respectively.

Triphosphenium 브로마이드 소금의 제조 및 반응성 : 인의 편리하고 안정적인 소스 (I)

We present herein the optimized synthesis of a triphosphenium bromide salt. Apart from being a versatile metathesis reagent, this unusually stable ...

Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since the approval of romidepsin, a class I HDAC inhibitor for cancer therapy, and the clinical investigation of HDAC6-specific inhibitors for multiple myeloma. The present method is used to determine the inhibitory activity of test compounds on HDAC1 and HDAC6 in cells. The isoform activity is measured using the ultra-high-performance liquid chromatography &#8211; mass spectrometry (UHPLC-MS) analysis of specific substrates incubated with treated and untreated HeLa cells. The method has the advantage of reflecting the endogenous HDAC activity within the cell environment, in contrast to cell-free biochemical assays conducted on isolated isoforms. Moreover, because it is based on the quantification of synthetic substrates, the method does not require the antibody recognition of endogenous acetylated proteins. It is easily adaptable to several cell lines and an automated process. The method has already proved useful in finding HDAC6-selective compounds in neuroblasts. Representative results are shown here with the standard HDAC inhibitors trichostatin A (non-specific), MS275 (HDAC1-specific), and tubastatin A (HDAC6-specific) using HeLa cells.

The present method serves to identify isoform-specific inhibitors of histone deacetylases (HDAC) in HeLa cells by the UHPLC-MS analysis of multiple substrates. This is an antibody-free method developed to reflect HDAC1 and HDAC6 activity in the living cell environment, in contrast to single-isoform cell-free assays.

헬러의 HDAC1 및 HDAC6의 동시 측정 셀 사용 하 여 UHPLC MS

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since ...

Microfluidic Preparation of Liquid Crystalline Elastomer Actuators

This paper focuses on the microfluidic process (and its parameters) to prepare actuating particles from liquid crystalline elastomers. The preparation usually consists in the formation of droplets containing low molar mass liquid crystals at elevated temperatures. Subsequently, these particle precursors are oriented in the flow field of the capillary and solidified by a crosslinking polymerization, which produces the final actuating particles. The optimization of the process is necessary to obtain the actuating particles and the proper variation of the process parameters (temperature and flow rate) and allows variations of size and shape (from oblate to strongly prolate morphologies) as well as the magnitude of actuation. In addition, it is possible to vary the type of actuation from elongation to contraction depending on the director profile induced to the droplets during the flow in the capillary, which again depends on the microfluidic process and its parameters. Furthermore, particles of more complex shapes, like core-shell structures or Janus particles, can be prepared by adjusting the setup. By the variation of the chemical structure and the mode of crosslinking (solidification) of the liquid crystalline elastomer, it is also possible to prepare actuating particles triggered by heat or UV-vis irradiation.

This article describes the microfluidic process and parameters to prepare actuating particles from liquid crystalline elastomers. This process allows the preparation of actuating particles and the variation of their size and shape (from oblate to strongly prolate, core-shell, and Janus morphologies) as well as the magnitude of actuation.

액체 결정 탄성 액츄에이터의 미세 준비

This paper focuses on the microfluidic process (and its parameters) to prepare actuating particles from liquid crystalline elastomers. The preparation ...

Synthesis of Esters Via a Greener Steglich Esterification in Acetonitrile

The Steglich esterification is a widely-used reaction for the synthesis of esters from carboxylic acids and alcohols. While efficient and mild, the reaction is commonly performed using chlorinated or amide solvent systems, which are hazardous to human health and the environment. Our methodology utilizes acetonitrile as a greener and less hazardous solvent system. This protocol exhibits rates and yields that are comparable to traditional solvent systems and employs an extraction and wash sequence that eliminates the need for the purification of the ester product via column chromatography. This general method can be used to couple a variety of carboxylic acids with 1&#176; and 2&#176; aliphatic alcohols, benzylic and allylic alcohols, and phenols to obtain pure esters in high yields. The goal of the protocol detailed here is to provide a greener alternative to a common esterification reaction, which could serve useful for ester synthesis in both academic and industrial applications.

A modified Steglich esterification reaction was used to synthesize a small library of ester derivatives with primary and secondary alcohols. The methodology uses a non-halogenated and greener solvent, acetonitrile, and enables product isolation in high yields without the need for chromatographic purification.

이기에 친 환 경적 Steglich 에스테 르 화를 통해 에스테 르의 합성

The Steglich esterification is a widely-used reaction for the synthesis of esters from carboxylic acids and alcohols. While efficient and mild, the ...

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment screening. NMR-based activity assays are ideally suited to work at the higher concentrations of test compounds required to detect these weaker inhibitors. The dynamic range and chemical shift dispersion in an NMR experiment can easily resolve resonances from substrate, product, and test compounds. This contrasts with spectrophotometric assays, in which read-out interference problems often arise from compounds with overlapping UV-vis absorption profiles. In addition, since they lack reporter enzymes, the single-enzyme NMR assays are not prone to coupled-assay false positives. This attribute makes them useful as orthogonal assays, complementing traditional high throughput screening assays and benchtop triage assays. Detailed protocols are provided for initial compound assays at 500 &#956;M and 250 &#956;M, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells. The methods are demonstrated using two nucleoside ribohydrolase enzymes. The use of 1H NMR is shown for the purine-specific enzyme, while 19F NMR is shown for the pyrimidine-specific enzyme. The protocols are generally applicable to any enzyme where substrate and product resonances can be observed and distinguished by NMR spectroscopy. To be the most useful in the context of drug discovery, the final concentration of substrate should be no more than 2-3x its Km value. The choice of NMR experiment depends on the enzyme reaction and substrates available as well as available NMR instrumentation.

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

NMR-based activity assays have been developed to identify and characterize inhibitors of two nucleoside ribohydrolase enzymes. Protocols are provided for initial compound assays at 500 &#956;M and 250 &#956;M, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells.

화합물 억제, IC50 값, 관절 활성 및 뉴클레오시드 리보하이드라제의 전세포 활성을 결정하기 위한 NMR 기반 활성 반응

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment ...