Name: in vitro assay to measure phosphatidylethanolamine methyltransferase activity
Uploaded: 2016-01-05T13:00:01.000+00:00
Duration: 9 min 33 s
Description: Watch this Scientific Journal Video about In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity at JoVE.com

This work was supported by NIH grants ARRA RO3 AI078145 and 1SC3GM113743 to RZ.

הכנת תמצית 1. תא <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לגדול תאי שושנת יריחו בבקבוק סטרילי פלסטיק האטומים עם כובע הדוק אוויר ב 26 מעלות צלזיוס במדיום עשוי 1x M199 בתוספת 20 מ&quot;מ HEPES pH7.4, 100 U / פניצילין מיליליטר, 100 מיקרוגרם / מיליליטר סטרפטומיצין, 5 מיקרוגרם / מיליליטר heme , 0.35 גר &#39;/ L Naco 2 H, 0.1 אדנין מ&quot;מ, ו -2 מיקרוגרם / מיליליטר biopterin בלי ללחוץ. קציר התאים על ידי צנטריפוגה ב 1500 גרם במשך 5 דקות ב 4 מעלות צלזיוס, כאשר הם מגיעים צפיפות תאים של 1-2 x 10 7 / מיליליטר. </li><li style=";text-align:right;direction:rtl"> בטל supernatant עם טפטפת ולשטוף את התאים על ידי resuspending התא גלולה עם pipet סרולוגיות במחצית של תרבות הנפח של פוספט הקר שנאגרו (137 מ&quot;מ NaCl, 2.7 מ&quot;מ KCl, 10 מ&quot;מ Na 2 4 HPO, 1.8 מ&quot;מ KH 2 4 PO, pH7.4). השלך supernatants תא לפי הנחיות בטיחות BL2. </li><li style=";text-align:right;direction:rtl"> תאי צנטריפוגה שוב ב 1500 גרם במשך 5 דקות ב 4 מעלות צלזיוס. בטל supernatant עם טפטפת. המשך to השלב הבא או הצמד להקפיא את התא גלולה בחנקן נוזלי לאחסון לטווח ארוך ב -80 מעלות צלזיוס (עד שלושה חודשים). </li><li style=";text-align:right;direction:rtl"> הכן מאגר תמוגה 2x (0.5 M סוכרוז, 0.1 מ &#39;TrisHCl, pH7.5, 2 מ&quot;מ EDTA, ומעכב פרוטאז 2x קוקטייל) ולשמור אותו על 4 מעלות צלזיוס על קרח. </li><li style=";text-align:right;direction:rtl"> Resuspend התא גלולה (טרי או קפוא) בנפח שווה של חיץ תמוגה 2x. להוסיף נפח 1x של חרוזי זכוכית. וורטקס במרץ ב 4 מעלות צלזיוס במשך 10 דקות. </li><li style=";text-align:right;direction:rtl"> הוסף 2 כרכים של חיץ תמוגה 1x ומערבבים. תמציות תא צנטריפוגה ב 1500 XG ב 4 מעלות צלזיוס במשך 10 דקות עד גלולה תאים וגרעינים רצופים. </li><li style=";text-align:right;direction:rtl"> מעבירים את supernatant עם pipet לתוך צינור צנטריפוגות מגניב טרי ולשמור תמציות תא על קרח עד להשלמת הניסוי. </li></ol> 2. לקבוע את ריכוז החלבון של תמצית הנייד באמצעות ערכת הערכת חלבון כמו Bicinchoninic החומצה Assay <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> פתרון להכין את חומצת bicinchoninic (BCA) (1 מיליליטר / צינור) על ידי ערבוב BCA והנחושת (II) שלulfate ביחס של 49: 1 (V / V). </li><li style=";text-align:right;direction:rtl"> הכן את הסטנדרטים החלבון של 0, 10, 20, 30, 40, 50, ו -60 מיקרוגרם / מיליליטר ידי דילול 10 מ&quot;ג / פתרון מניות אלבומין בסרום שור (BSA) מיליליטר לתוך 1 מיליליטר aliquots של פתרון BCA. </li><li style=";text-align:right;direction:rtl"> הוסף 2 μl של תמציות תאים ב 1 מיליליטר של פתרון BCA בכפילויות. דגירה סטנדרטים ודגימות חלבון במשך 10 דקות באמבט מים C מראש חימם 60 °. </li><li style=";text-align:right;direction:rtl"> להעברת דגימות קרח במשך 3 דקות. מדוד את הספיגה של הסטנדרטים ודגימות החלבון עם ספקטרופוטומטר באורך גל של 562 ננומטר. </li><li style=";text-align:right;direction:rtl"> חשב את ריכוז החלבון של תמציות התא באמצעות התקן כהתייחסות כמתואר בפרוטוקול של היצרן BSA. לדלל תמציות תא לריכוז חלבון של 10 מ&quot;ג / מיליליטר עם חיץ תמוגה 1x. </li></ol> 3. אנזימתי Assay ב200 μl לכל צינור הערה: קח את השלבים הבאים במנדף כימי. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בדוק כל דגימה בשני עותקים ב15 מיליליטרצינור חרוטי. הכן 20 μl 1 M TrisHCl pH 7.5 לכל צינור ולשמור אותו על קרח. להכין 2 מיליליטר של כלורופורם / מתנול (1: 1 (V / V); עצירת פתרון) ב RT עבור כל צינור. </li><li style=";text-align:right;direction:rtl"> Pipet 20 μl של 1 M TrisHCl pH 7.5 בכל צינור חרוטי 15 מיליליטר על קרח. </li><li style=";text-align:right;direction:rtl"> פעל לפי הנחיות בטיחות קרינה מכאן והלאה. הוסף את המקבילה של 0.06 מיקרומטר (0.2 μCi) S - [Methyl- H 3] adenosyl-L-מתיונין ו -50 מיקרומטר SAM הקר לכל צינור לסך של 50.06 מיקרומטר של SAM. להוסיף x μl של מים קרים שבו x = 200 (20 (לחיץ) + 20 (לתמציות תא) + נפח של SAM הקר ורדיואקטיבי) לכל צינור. </li><li style=";text-align:right;direction:rtl"> העבר את כל צינור חרוטי לאמבטית מים 30 מעלות צלזיוס מראש חימם. הוסף לכל צינור 20 μl של תמציות תא (שווה ערך של 200 מיקרוגרם של חלבון) כדי להתחיל את התגובה. דגירה לזמן הרצוי (0-45 דקות). </li><li style=";text-align:right;direction:rtl"> לעצור את התגובה על ידי הוספת 2 מיליליטר של כלורופורם / מתנול (1: 1; / V V; פתרון עצירה) על צינור אחד. העבר את צינור חרוטי לRT (20-256; ג). </li></ol> 4. שומנים הפקה הערה: קח את השלבים הבאים במנדף כימי. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להוסיף 700 μl של מים לכל צינור המכיל מדגם התגובה האנזימטית. וורטקס במרץ למשך 30 שניות. צנטריפוגה ב 1500 XG במשך 5 דקות על RT להפריד אורגנית מהשלב המים. </li><li style=";text-align:right;direction:rtl"> מעבירים את השלב האורגני הנמוך לתוך צינור חרוטי 15 מיליליטר חדש עם טפטפת. הוסף 1 מיליליטר של מים לכל &quot;שלב תחתון&quot; המכיל צינור ומערבולת במרץ למשך 30 שניות. צנטריפוגה שוב ב 1500 XG במשך 5 דקות להפריד אורגנית מהשלב המים. </li><li style=";text-align:right;direction:rtl"> מעבירים את השלב האורגני הנמוך לתוך צינור נצנץ עם טפטפת. דגימות יבשים תחת זרם של N 2. השלך שלבי מים המכילים SAM-רדיואקטיבי שולב אינו וצינורות חרוטי רדיואקטיבי לפי הנחיות קרינה. </li><li style=";text-align:right;direction:rtl"> הוסף 2 מיליליטר / שפופרת של נוזל נצנץ. מדוד את הרדיואקטיביות שולבה עם cou נצנץnter על פי הפרוטוקול של היצרנים והשימוש של המכשיר. </li><li style=";text-align:right;direction:rtl"> לחשב את הפעילות האנזימטית בnmol / חלבון מ&quot;ג באמצעות המשוואה הכללית הבאה: [ריכוז מוחלט (רדיואקטיבי וקר) של SAM (מ&quot;מ)] ערך לדקה x 10 x 3 x 5 פעילות ספציפית של SAM רדיואקטיבי (Ci / mmol) x [הריכוז של SAM רדיואקטיבי (מ&quot;מ)] הערה: הפרוטוקול לעיל ניתן לשנות לריכוז תלוי SAM או assay PEMT תלוי חלבון. עבור assay האנזימטית התלוי ריכוז SAM, הזמן נשמר (15 דקות, שהוא בטווח ליניארי) קבועות וכמויות שונות של SAM הקר מתווספים assay ואילו עבור assay החלבון התלוי PEMT, ריכוז SAM (בחרנו 0.05 מ&quot;מ) וזמן נשמרים (15 דקות) קבועות. גם את ה- pH של המאגר ניתן לשנות בהתאם לצורך, אם יש לי כמה אנזימים PEMT ערך ה- pH אופטימלי שונה. </li></ol>

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F., Watts, R. W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5&#39;-Methylthioadenosine+(MTA)+phosphorylase+from+promastigote+of+Leishmania+donovani.">5&#39;-Methylthioadenosine (MTA) phosphorylase from promastigote of Leishmania donovani.</a> Purine and Pyrimidine Metabolism in Man V, Adv Exp Med Biol. 131, 559-563 (1986).</li><li>Biastoff, S., Teuber, M., Zhou, Z. S., Dr&#228;ger, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetric+activity+measurement+of+a+recombinant+putrescine+N.-methyltransferase+from+Datura+stramonium.">Colorimetric activity measurement of a recombinant putrescine N.-methyltransferase from Datura stramonium.</a> Planta Med. 72 (12), 1136-1141 (2006).</li><li>Hendricks, C. L., Ross, J. R., Pichersky, E., Noel, J. P., Zhou, Z. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enzyme-coupled+colorimetric+assay+for+S.-adenosylmethionine-dependent+methyltransferases.">An enzyme-coupled colorimetric assay for S.-adenosylmethionine-dependent methyltransferases.</a> Anal Biochem. 326 (1), 100-105 (2004).</li><li>Cannon, L. M., Butler, F. N., Wan, W., Zhou, Z. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+stereospecific+colorimetric+assay+for+(S.,S.)-adenosylmethionine+quantification+based+on+thiopurine+methyltransferase-catalyzed+thiol+methylation.">A stereospecific colorimetric assay for (S.,S.)-adenosylmethionine quantification based on thiopurine methyltransferase-catalyzed thiol methylation.</a> Anal Biochem. 308 (2), 358-363 (2002).</li><li>Hoffman, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatographic+analysis+of+the+chiral+and+covalent+instability+of+S.-adenosyl-L-methionine.">Chromatographic analysis of the chiral and covalent instability of S.-adenosyl-L-methionine.</a> Biochemistry. 25 (15), 4444-4449 (1986).</li><li>Wu, S. E., Huskey, W. P., Borchardt, R. T., Schowen, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chiral+instability+at+sulfur+of+S.-adenosylmethionine.">Chiral instability at sulfur of S.-adenosylmethionine.</a> Biochemistry. 22 (12), 2828-2832 (1983).</li><li>Dorgan, K. 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=An+enzyme-coupled+continuous+spectrophotometric+assay+for+S.-adenosylmethionine-dependent+methyltransferases.">An enzyme-coupled continuous spectrophotometric assay for S.-adenosylmethionine-dependent methyltransferases.</a> Anal Biochem. 350 (2), 249-255 (2006).</li></ol>

אין ניגוד העניינים הצהיר

assay זה פשוט, מהיר PEMT מאפשר כימות של צורות מפוגלים של PE שנובע מהעברה של קבוצות מתיל רדיואקטיביים מSAM על PE באמצעות תמצית תא כולו כמקור חלבון. הוא מהיר, רגיש, לשחזור, ומתאים גם לאנזימים מטוהרים 17. ניתן להוסיף Monomethyl- או דימתיל-PE לassay אם methyltransferase של עניין הוא ספציפי למצע...

methyltransferase Phosphatidylethanolamine אנזימים (PEMT) לזרז את הקובץ המצורף קוולנטיים של קבוצות מתיל אחד או יותר באמצעות -adenosylmethionine S (SAM) כתורם קבוצה מתיל על PE, monomethyl-PE או דימתיל-PE לתת monomethyl-PE, דימתיל-PE ו / או phosphatidylcholine (PC). אנזימים אלה הם כמעט בכל מקום בתאים של בעלי חיים ופטריות. ניתן גם למצוא אותם בכמה צמחים 1 וכ -10% מחיידקים, במיוחד אלה שאינטראקציה עם אאוקריוטים 2. PEMTs רלוונטי לביולוגיה של התא לא רק על ידי תרומה לייצור של מחשב, אשר הוא ברמת שומנים בדם העיקרית בתאים של בעלי חיים, אלא גם על ידי מילוי תפקודים תאיים חשובים נוספים. ביונקים, PEMTs באים לידי ביטוי בעיקר בכבד שבו הם נדרשים להפרשה נורמלית של ליפופרוטאין בצפיפות נמוכה מאוד, והם גם תורמים להשמנה יתר הנגרם דיאטה 3, טרשת עורקים 4, ואינסולין להתנגדאהה 5. בנוסף, PEMT יונקים גם באו לידי ביטוי בתאי שומן, אם כי לרמות נמוכות יותר, ולהשתתף בתצהיר שומן 6, 7. תפקיד PEMT בהתפתחות סרטן 8, 9 אפופטוזיס, ו- 10 צמיחת תאים גם הוכח. בחיידקים, אנזימי PEMT הוכחו להיות חשוב לצמיחה נורמלית של תאי 2, ארסיות 2, וסימביוזה עם צמח המארח 11. המטרה והרציונל של הפרוטוקול הנוכחי הוא למדוד פעילות PEMT מתמציות תא שלמות ללא הצורך לטהר את האנזים. שני פרוטוקולים שונים פותחו כדי למדוד את פעילות PEMT. אחד הראשון והנפוץ ביותר מודד את העברת קבוצה מתיל tritiated מSAM רדיואקטיבי על PE, אשר הוא הנושא של מאמר זה. פרוטוקול זה פותח במקור כדי למדוד את פעילות PEMT משמרים 12 ותאי יונקים (כבד) 13 כדי לקבל understanding של ביוסינתזה מחשב בתאים אלה, כמו גם כדי לקבוע את הספציפיות של אנזימים אלה. מאוחר יותר, בטכניקה זו יושמה לסוגי תאים אחרים, כגון חיידקים 2 (באמצעות ערך ה- pH בסיסי עבור assay למרות 15) וטפילי protozoan 14. טכניקה זו יכולה לשמש עם תמציות תא שלמות, כמו גם אנזים מטוהר, וניתן ליישם פוטנציאלי לכל מערכת תמצית תא. Assay רדיואקטיבי שאינו גם תוכנן שמסתמך על הכימות האנזימטית של -adenosylhomocysteine ​​S, מוצר transmethylation של SAM 16. Assay האחרון עשוי להיות נוח יותר כפי שהוא אינו כרוך בקרינה רדיואקטיבית, אבל זה מתאים רק לאנזימים מטוהרים.

<table><tbody><tr><td>S-[Methyl-&lt;sup&gt;3&lt;/sup&gt;H]adenosyl-L-methionine (specific activity of 5-15 Ci/mMole)</td><td>Perkin Elmer</td><td>NET155050UC</td><td>Aliquot the reagent and freeze at -20 &amp;deg;C; follow radiation safety guidelines while using this reagent</td></tr><tr><td>Protease inhibitor cocktail</td><td>Roche Life Sciences</td><td>11836170001</td><td>dilute it fresh&amp;nbsp;</td></tr><tr><td>Glass beads, acid washed, 425-600 mm</td><td>Sigma Aldrich</td><td>G8772</td><td></td></tr><tr><td>Bicinchoninic acid solution</td><td>Sigma Aldrich</td><td>B9643</td><td></td></tr><tr><td>Copper (II) sulfate</td><td>Sigma Aldrich</td><td>C2284</td><td></td></tr><tr><td>Scintillation counter MicroBeta2 with 1-detector</td><td>Perkin Elmer&amp;nbsp;</td><td>2450-0010</td><td></td></tr><tr><td>Spectrophotometer Biomate 3</td><td>Thermo Scientific</td><td>840208300</td><td></td></tr><tr><td>BSA stock solution (10 mg/ml)</td><td>New England Biolabs</td><td>B9001S</td><td></td></tr><tr><td>Scintillation liquid&amp;nbsp;</td><td>Research Product International Corp</td><td>111198</td><td></td></tr><tr><td>S-(5&#039;-Adenosyl)-L-methionine chloride (hydrochloride)&amp;nbsp;</td><td>Cayman Chemicals</td><td>13956</td><td>dilute the reagent in 20 mM HCl and freeze aliquots at -80 &amp;deg;C&amp;nbsp;</td></tr></tbody></table>

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.

איור 1 מציג assay PEMT התלוי זמן, שבוצע עם תמצית כל תא שושנת יריחו כמקור אנזים באמצעות PE אנדוגני כמצע. כמות הרדיואקטיביות בשלב האורגני הייתה לכמת ידי ספירת נצנץ. מספרים וכתוצאה מכך נוצלו כדי לחשב את הסכום של קבוצות מתיל tritiated מועברות על גבי PE...

Watch this Scientific Journal Video about In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity at JoVE.com

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.

במבחנה Assay למדוד Phosphatidylethanolamine Methyltransferase פעילות

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from ...

in-vitro-assay-to-measure-phosphatidylethanolamine-methyltransferase

Research

JoVE Journal

Biology

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

9.8K Views.  St John's University. 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. 

Video: In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

Defining Substrate Specificities for Lipase and Phospholipase Candidates

Microorganisms produce a wide spectrum of (phospho)lipases that are secreted in order to make external substrates available for the organism. Alternatively, other (phospho)lipases may be physically associated with the producing organism causing a turnover of intrinsic lipids and frequently giving rise to a remodeling of the cellular membranes. Although potential (phospho)lipases can be predicted with a number of algorithms when the gene/protein sequence is available, experimental proof of the enzyme activities, substrate specificities, and potential physiological functions has frequently not been obtained. This manuscript describes the optimization of assay conditions for prospective (phospho)lipases with unknown substrate specificities and how to employ these optimized conditions in the search for the natural substrate of a respective (phospho)lipase. Using artificial chromogenic substrates, such as p-nitrophenyl derivatives, may help to detect a minor enzymatic activity for a predicted (phospho)lipase under standard conditions. Having encountered such a minor enzymatic activity, the distinct parameters of an enzyme assay can be varied in order to obtain a more efficient hydrolysis of the artificial substrate. After having determined the conditions under which an enzyme works well, a variety of potential natural substrates should be assayed for their degradation, a process that can be followed employing distinct chromatographic methods. The definition of substrate specificities for new enzymes, often provides hypotheses for a potential physiological role of these enzymes, which then can be tested experimentally. Following these guidelines, we were able to identify a phospholipase C (SMc00171) that degrades phosphatidylcholine to phosphocholine and diacylglycerol, in a crucial step for the remodeling of membranes in the bacterium Sinorhizobium meliloti upon phosphorus-limiting conditions of growth. For two predicted patatin-like phospholipases (SMc00930 and SMc01003) of the same organism, we could redefine their substrate specificities and clarify that SMc01003 is a diacylglycerol lipase.

Many predicted (phospho)lipases are poorly characterized with regard to their substrate specificities and physiological functions. Here we provide a protocol to optimize enzyme activities, search for natural substrates, and propose physiological functions for these enzymes.

הגדרת מצע ספציפי עבור מועמדי lipase ו פוספוליפאז

Microorganisms produce a wide spectrum of (phospho)lipases that are secreted in order to make external substrates available for the organism. ...

Biochemistry

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 (Ehmeth) Enzyme

Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of the three most common causes of death from parasitic disease. The agent of amebiasis is the amoeba parasite Entamoeba histolytica that exists under two stages: the infective cyst found in food or water and the invasive trophozoite living in the intestine. The clinical manifestations of amebiasis range from being asymptomatic to colitis, dysentery or liver abscesses. E. histolytica is one of the rare unicellular parasite with 5-methylcytosine (5mC) in its genome. 1, 2 It contains a single DNA methyltransferase, Ehmeth, that belongs to the Dnmt2 family. 2 A role for Dnmt2 in the control of repetitive elements has been established in E. histolytica, 3 Dictyostelium discoideum 4,5 and Drosophila. 6 Our recent work has shown that Ehmeth methylates tRNAAsp, and this finding indicates that this enzyme has a dual DNA/tRNAAsp methyltransferase activity. 7 This observation is in agreement with the dual activity that has been reported for D. discoideum and D. melanogaster. 8 The functional significance of the DNA/tRNA specificity of Dnmt2 enzymes is still unknown. To address this question, a method to determine the tRNA methyltransferase activity of Dnmt2 proteins was established. In this video, we describe a straightforward approach to prepare an adequate tRNA substrate for Dnmt2 and a method to measure its tRNA methyltransferase activity.

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 Ehmeth Enzyme

This protocol describes the preparation of a synthetic tRNA substrate for the Entamoeba histolytica DNA/tRNA methyltransferase 2 (Dnmt2) homolog Ehmeth and the measure of its methyltransferase activity. This experimental approach can be used for investigating the activity of other Dnmt2 proteins.

ב Assay tRNA מתילציה במבחנה עם histolytica Entamoeba דנ&quot;א Dnmt2 tRNA methyltransferase (Ehmeth) אנזימים

Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of ...

Immunology and Infection

Antibody-Free Assay for RNA Methyltransferase Activity Analysis

There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and especially methylations, has re-emerged due to the important roles played by the enzymes that write and erase them in biological processes relevant to disease and cancer. Here, a sensitive in vitro assay for accurate analysis of RNA methylation writer activity on synthetic or in vitro transcribed RNAs is provided. This assay uses a tritiated form of S-adenosyl-methionine, resulting in direct labeling of methylated RNA with tritium. The low energy of tritium radiation makes the method safe, and pre-existing methods of tritium signal amplification, make it possible to quantify and to visualize the methylated RNA without the use of antibodies, which are commonly prone to artifacts. While this method is written for RNA methylation, few tweaks make it applicable to the study of other RNA modifications that can be radioactively labeled, such as RNA acetylation with 14C acetyl coenzyme A. Overall, this assay allows to quickly assess RNA methylation conditions, inhibition with small molecule inhibitors, or the effect of RNA or enzyme mutants, and provides a powerful tool to validate and expand results obtained in cells.

Here, an antibody-free in vitro assay for direct analysis of methyltransferase activity on synthetic or in vitro transcribed RNA is described.

טיפול ללא נוגדנים עבור RNA מתילטרנספראז ניתוח פעילות

There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and ...

Genetics

Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins

Activity-dependent alterations in the levels of synaptic AMPA receptors (AMPARs) within the postsynaptic density (PSD) is thought to represent a cellular mechanism for learning and memory. Palmitoylation regulates localization and function of many synaptic proteins including AMPA-Rs, auxiliary factors and synaptic scaffolds in an activity-dependent manner. We identified the synapse differentiation induced gene (SynDIG) family of four genes (SynDIG1-4) encoding brain-specific transmembrane proteins that associate with AMPARs and regulate synapse strength. SynDIG1 is palmitoylated at two cysteine residues located at positions 191 and 192 in the juxta-transmembrane region important for activity-dependent excitatory synapse development. Here, we describe an innovative biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay, to investigate the palmitoylation state of any protein of interest and demonstrate its utility with the SynDIG family of proteins in mouse brain lysates.

Palmitoylation entails the incorporation of a 16-carbon palmitate moiety to cysteine residues of target proteins in a reversible manner. Here, we describe a biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay, to investigate the palmitoylation state of any protein of interest in mouse brain lysates.

Acyl-PEGyl החליפין ג'ל שינוי שיטת להגדרה כמותית של פלמילציה של חלבונים קרום המוח

Activity-dependent alterations in the levels of synaptic AMPA receptors (AMPARs) within the postsynaptic density (PSD) is thought to represent a cellular ...

Click-Chemistry Based Fluorometric Assay for Apolipoprotein N-acyltransferase from Enzyme Characterization to High-Throughput Screening

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral membrane enzymes, resulting in triacylated proteins. The first step in the lipoprotein modification pathway involves the transfer of a diacylglyceryl group from phosphatidylglycerol onto the prolipoprotein, resulting in diacylglyceryl prolipoprotein. In the second step, the signal peptide of prolipoprotein is cleaved, forming an apolipoprotein, which in turn is modified by a third fatty acid derived from a phospholipid. This last step is catalyzed by apolipoprotein N-acyltransferase (Lnt). The lipoprotein modification pathway is essential in most &#947;-proteobacteria, making it a potential target for the development of novel antibacterial agents. Described here is a sensitive assay for Lnt that is compatible with high-throughput screening of small inhibitory molecules. The enzyme and substrates are membrane-embedded molecules; therefore, the development of an in vitro test is not straightforward. This includes the purification of the active enzyme in the presence of detergent, the availability of alkyne-phospholipids and diacylglyceryl peptide substrates, and the reaction conditions in mixed micelles. Furthermore, in order to use the activity test in a high-throughput screening (HTS) setup, direct readout of the reaction product is preferred over coupled enzymatic reactions. In this fluorometric enzyme assay, the alkyne-triacylated peptide product is rendered fluorescent through a click-chemistry reaction and detected in a multiwell plate format. This method is applicable to other acyltransferases that use fatty acid-containing substrates, including phospholipids and acyl-CoA.

Presented here is a sensitive fluorescence assay to monitor apolipoprotein N-acyltransferase activity using diacylglyceryl peptide and alkyne-phospholipids as substrates with click-chemistry.

בדיקה פלואורומטרית מבוססת קליק-כימיה עבור אפוליפופרוטאין N-אצילטרנספראז מאיפיון אנזים ועד סינון תפוקה גבוהה

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral ...

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) establish and maintain the DNA methylation pattern. Changes to the normal DNA methylation pattern are linked to cancer development and progression, making DNMTs potential cancer drug targets. Thus, identifying and characterizing novel small molecule inhibitors of these enzymes is of great importance. This paper presents a protocol that can be used to screen for DNA methyltransferase inhibitors. The continuous coupled kinetics assay allows for initial velocities of DNA methylation to be determined in the presence and absence of potential small molecule inhibitors. The assay uses the methyl-sensitive endonuclease Gla I to couple methylation of a hemimethylated DNA substrate to fluorescence generation.
This continuous assay allows for enzyme activity to be monitored in real time. Conducting the assay in small volumes in microtiter plates reduces the cost of reagents. Using this assay, a small example screen was conducted for inhibitors of DNMT1, the most abundant DNMT isozyme in humans. The highly substituted anthraquinone natural product, laccaic acid A, is a potent, DNA-competitive inhibitor of DNMT1. Here, we examine three potential small molecule inhibitors &#8212; anthraquinones or anthraquinone-like molecules with one to three substituents &#8212; at two concentrations to describe the assay protocol. Initial velocities are used to calculate the percent activity observed in the presence of each molecule. One of three compounds examined exhibits concentration-dependent inhibition of DNMT1 activity, indicating that it is a potential inhibitor of DNMT1.

DNA methyltransferases are potential cancer drug targets. Here, a protocol is presented to assess small molecules for DNA methyltransferase inhibition. This assay utilizes an endonuclease to couple DNA methylation to fluorescence generation and allows for enzyme activity to be monitored in real time.&#160;

בדיקת מתילציה רציפה של דנ"א מצומד אנדונוקלאז מבוססת פלואורסצנציה לסינון מעכבי מתיל-טרנספראז של DNA

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to measure protein phosphorylation cannot preserve the heterogeneity in phosphorylation across individual proteins. The single-molecule pull-down (SiMPull) assay was developed to investigate the composition of macromolecular complexes via immunoprecipitation of proteins on a glass coverslip followed by single-molecule imaging. The current technique is an adaptation of SiMPull that provides robust quantification of the phosphorylation state of full-length membrane receptors at the single-molecule level. Imaging thousands of individual receptors in this way allows for quantifying protein phosphorylation patterns. The present protocol details the optimized SiMPull procedure, from sample preparation to imaging. Optimization of glass preparation and antibody fixation protocols further enhances data quality. The current protocol provides code for the single-molecule data analysis that calculates the fraction of receptors phosphorylated within a sample. While this work focuses on phosphorylation of the epidermal growth factor receptor (EGFR), the protocol can be generalized to other membrane receptors and cytosolic signaling molecules.

The present protocol describes sample preparation and data analysis to quantify protein phosphorylation using an improved single-molecule pull-down (SiMPull) assay.

בדיקה ממוטבת של משיכה במולקולה בודדת לכימות של זרחון חלבונים

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to ...

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.

ב חוץ גופית Assay פעילות עבור ppGpp (p) המעביר מ Clostridium difficile וטיהור

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

Measuring In Vitro ATPase Activity for Enzymatic Characterization

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate energy for mechanical work, such as protein trafficking and degradation, solute transport, and cellular movements. The protocol described here is a basic assay for measuring the in vitro activity of purified ATPases for functional characterization. Proteins hydrolyze ATP in a reaction that results in inorganic phosphate release, and the amount of phosphate liberated is then quantitated using a colorimetric assay. This highly adaptable protocol can be adjusted to measure ATPase activity in kinetic or endpoint assays. A representative protocol is provided here based on the activity and requirements of EpsE, the AAA+ ATPase involved in Type II Secretion in the bacterium Vibrio cholerae. The amount of purified protein needed to measure activity, length of the assay and the timing and number of sampling intervals, buffer and salt composition, temperature, co-factors, stimulants (if any), etc. may vary from those described here, and thus some optimization may be necessary. This protocol provides a basic framework for characterizing ATPases and can be performed quickly and easily adjusted as necessary.

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.

מדידה<em&gt; במבחנה</em&gt; פעילות ATPase עבור אנזימתי אפיון

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

Phospholipase Activity Assay: A Gel Diffusion Assay to Determine Phospholipase Activity in Crude Venom Extract from Anthopleura dowii

Source: Ram&#237;rez-Carreto, S., et al., <a href="https://www.jove.com/v/63630">Identification of Hemolytic and Phospholipase Activity in Crude Extracts from Sea Anemones by Straightforward Bioassays.</a> J. Vis. Exp. (2022)
In this video, we demonstrate an assay to establish the presence of phospholipase in the crude venom extract from Anthopleura dowii&#8212;a marine sea anemone. The assay utilizes an agarose gel diffusion technique, in which the enzyme phospholipase diffuses and breaks down phospholipids incorporated in the solid media in a radial manner.

בדיקת פעילות פוספוליפאז: בדיקת דיפוזיה בג'ל לקביעת פעילות פוספוליפאז בתמצית ארס גולמי מ - Anthopleura dowii

Source: Ram&#237;rez-Carreto, S., et al., Identification of Hemolytic and Phospholipase Activity in Crude Extracts from Sea Anemones by Straightforward ...

במבחנה Assay למדוד Phosphatidylethanolamine Methyltransferase פעילות

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