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

1. تنفيذ ATP الحلمأة رد الفعل مع البروتين النقي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد مخزونات جميع الكواشف اللازمة لحضانة مع البروتين النقي. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد عازلة 5X HEPES / كلوريد الصوديوم / الجلسرين (هنج) تحتوي على 100 ملي HEPES الرقم الهيدروجيني 8.5، 65 مم كلوريد الصوديوم، و 5٪ الجلسرين (أو عازلة فحص آخر حسب الاقتضاء). </li><li style=";text-align:right;direction:rtl"> إعداد 100 ملي MgCl 2 (أو المعادن الأخرى، إذا أتباز يعتمد المعادن) في الماء. </li><li style=";text-align:right;direction:rtl"> إعداد الطازجة 100 ملي اعبي التنس المحترفين في 200 ملي قاعدة تريس (لا ضبط درجة الحموضة مزيد) باستخدام عالية ATP النقاء. قسامة وتخزين الأوراق المالية اعبي التنس المحترفين في -20 درجة مئوية لمدة لا تزيد عن بضعة أسابيع، كما ATP سوف تنهار مع مرور الوقت، والامتناع عن تجميد وذوبان الأسهم اعبي التنس المحترفين. </li><li style=";text-align:right;direction:rtl"> بريمكس MgCl 2 و ATP في نسبة 1: 1 قبل وضع رد فعل التحلل اعبي التنس المحترفين. </li></ol></li><li style=";text-align:right;direction:rtl"> تحضير تسمية 1.5 مل أنابيب لجمع العينات على فترات منتظمة طوال رد فعل. إعداد أنابيب جمع العينات في وقت 0 وفي وقت 15، 30،45، و 60 دقيقة. ملاحظة: وكبديل، وجمع عينات فقط في وقت 0 ونقطة النهاية. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إضافة 245 ميكرولتر العازلة 1X هنغ إلى كل أنبوب من أجل تمييع العينات التي تم جمعها من ردود الفعل المائي ATP 01:50. </li></ol></li><li style=";text-align:right;direction:rtl"> يعد حمام من الثلج الجاف والايثانول لتجميد بسرعة عينات لوقف التفاعل. في دلو المطاط الجليد أو آمنة الأخرى (غير البلاستيك) حاوية، إضافة عدة قطع من الثلج الجاف وتصب بعناية ما يكفي من 70-100٪ من الإيثانول لتغطية الثلج الجاف. </li><li style=";text-align:right;direction:rtl"> تمييع البروتين النقي في المخزن هنغ 1X، حسب الاقتضاء (عادة 5-10 ميكرون)، والحفاظ على الجليد. </li><li style=";text-align:right;direction:rtl"> إعداد ردود فعل التحلل اعبي التنس المحترفين. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> في منفصلة 0.5 مل أنابيب لكل عينة، إضافة الكواشف التالية (بالترتيب): H 2 O (تصل إلى الحجم النهائي من 30 ميكرولتر)، 6 ميكرولتر 5X هنغ أو عازلة آخرين، 3 ميكرولتر 100 ملي MgCl 2 -ATP الخليط، و،25-5 البروتين ميكرومتر. </li><li style=";text-align:right;direction:rtl"> تشمل منطقة عازلة فقط حالة السيطرة السلبية التي لم تتم إضافة البروتين.</li></ol></li><li style=";text-align:right;direction:rtl"> إزالة 5 ميكرولتر من رد فعل في وقت 0، وتمييع 01:50 في أنبوب 1.5 مل استعداد تحتوي على 245 عازلة هنغ ميكرولتر، وتجميد فورا العينة في حمام الثلج / الايثانول الجاف. </li><li style=";text-align:right;direction:rtl"> احتضان ردود الفعل عند 37 درجة مئوية للسماح للاعبي التنس المحترفين المائي للتحدث لمدة 1 ساعة. في كل فترة (15، 30، 45، و 60 دقيقة)، وإزالة 5 مكل من التفاعل وإضافة إلى أنابيب العينات وصفت تحتوي عازلة هنغ كما في الخطوة 1.6. </li><li style=";text-align:right;direction:rtl"> في نهاية التفاعل المائي ATP، نقل عينات المخفف إلى -80 درجة مئوية الثلاجة لمدة التخزين. لضمان تجميد جميع العينات تماما، انتظر على الأقل 10 دقيقة قبل المتابعة. </li></ol> 2. عينات احتضان يتضمن بي الحرة مع كشف الكاشف <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> عينات ذوبان الجليد المخففة التي تحتوي على ATP قسامات رد فعل التحلل في كل نقطة زمنية (تم الحصول عليها من الخطوات 1.6 و 1.7) في درجة حرارة الغرفة. </li><li style=";text-align:right;direction:rtl"> إعداد لوحة 96-جيدا تحتوي على عينات والمعايير الفوسفات. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> في مل تو 0.5يكون، يخفف من مستوى الفوسفات (المتوفرة مع بي كشف كاشف) من 800 ميكرومتر إلى 40 ميكرومتر بإضافة 5.5 ميكرولتر من المخزن المؤقت هنغ 800 ميكرومتر القياسية إلى 104.5 ميكرولتر. تخلط جيدا، وإضافة 100 ميكرولتر من هذا 40 ميكرومتر بي المعيار إلى ما A1 من لوحة 96-جيدا. </li><li style=";text-align:right;direction:rtl"> إضافة العازلة هنغ 50 ميكرولتر من الآبار B1-H1 لل1: 1 التخفيفات التسلسلي للمعيار بي. </li><li style=";text-align:right;direction:rtl"> إزالة 50 ميكرولتر من 40 ميكرومتر بي من A1 جيدا وإضافة إلى عازلة فحص 50 ميكرولتر في B1 جيدا، خلط، وإزالة 50 ميكرولتر من B1 جيدا وإضافة إلى ما C1، التخفيفات المستمر من خلال G1 جيدا. تجاهل 50 ميكرولتر من G1 جيدا بعد خلطه لضمان كل بئر لديه نفس الحجم. كذلك يجب أن تحتوي H1 عازلة الوحيد لخلق معيار بي 40-0 ميكرومتر. ملاحظة: إذا تمت إضافة عامل إضافي (مثل مثبطات) للعينات، وخلق منحنى القياسية التي تحتوي على هذا العامل للسيطرة على التغيرات في الافراج عن الفوسفات أو الامتصاصية في ظل هذه الظروف. </li><li style=";text-align:right;direction:rtl"> إضافة 50 ميكرولتر من كل عصيدةلو في نسختين لوحة. إضافة عينات من نقطة زمنية واحدة في الأعمدة عموديا (عينة 1 مرة 0 = A2، A3، عينة 2 مرة 0 = B2، B3) ونقاط زمنية مختلفة أفقيا. هذا يسمح لمدة تصل إلى 8 عينات و 5 نقاط الوقت لكل لوحة. </li></ol></li><li style=";text-align:right;direction:rtl"> استخدام الماصة العقيمة لإزالة ما يكفي من المرمر الأخضر / موليبدات بي كشف كاشف لإضافة 100 ميكرولتر إلى كل من الآبار التي تحتوي على عينات والمعايير (كاشف اللازمة (مل) = 0.1 × عدد العينات والمعايير) وإضافة إلى طبق لسهولة pipetting ل باستخدام الماصة متعدد القنوات. لا صب كاشف الكشف مباشرة في طبق، حيث من المحتمل أن يحدث تلوث بي. </li><li style=";text-align:right;direction:rtl"> باستخدام الماصة متعدد القنوات، إضافة 100 ميكرولتر من كشف كاشف بي إلى كل بئر وتخلط بواسطة pipetting بعناية صعودا وهبوطا عدد ثابت من الأوقات دون فقاعات إدخال، ويفضل أن يكون في أمر من النقاط الزمنية الماضية إلى نقطة لأول مرة. </li><li style=";text-align:right;direction:rtl"> احتضان لوحة لمدة 25 دقيقة في درجة حرارة الغرفة، أو وفقا لأماهالاتجاهات nufacturer و. </li></ol> 3. النتائج Quantitate باستخدام قارئ صفيحة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قراءة الامتصاصية من العينات في 650 نانومتر باستخدام قارئ صفيحة ميكروسكوبية الامتصاصية. </li><li style=";text-align:right;direction:rtl"> جعل منحنى القياسية بي. باستخدام برنامج الرسوم البيانية، رسم بياني لقيم الامتصاصية للعينات بي القياسية مقابل تركيز من أجل إيجاد المعادلة المستخدمة في حل لكمية الفوسفات في كل عينة. </li><li style=";text-align:right;direction:rtl"> حساب النشاط أتباز لكل عينة من معايرة مع معيار الفوسفات. الفوسفات صدر = (OD 650 - Y التقاطع) / المنحدر. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> متوسط ​​مجموع بي من التكرارات من كل عينة. طرح القراءة الامتصاصية سيطرة عازلة الوحيد من هذا الرقم. اضرب هذا من قبل عامل التخفيف (50 في مثالنا). </li><li style=";text-align:right;direction:rtl"> تحديد بي نانومول صدر في البروتين مكرومول. يجب أن الرسوم البيانية هذه القيم لكل نقطة زمنية في مقايسة الحركية تسفر عن نوبات الخطية على الأقل R = 0.99 (الشكل 1). إذا كان nبعد التمديد، ويمكن تعديل مقايسة مع أكثر أو أقل من البروتين أو فترة حضانة أطول أو أقصر. </li></ol></li><li style=";text-align:right;direction:rtl"> تمثل النتائج على النحو نانومول بي / مكرومول بروتين / دقيقة (الشكلان 2 و 3)، أو البروتين نانومول بي / ميكروغرام / دقيقة إذا رغبت في ذلك. </li></ol>

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The authors have nothing to disclose.

هذا هو بروتوكول عام لقياس النشاط أتباز المختبر من البروتينات النقاء لتوصيف والكيمياء الحيوية. هذه الطريقة بسهولة الأمثل. على سبيل المثال، وضبط كمية من البروتين، العازلة والتراكيب الملح، ودرجة الحرارة، وتتراوح مدة الفحص وفترات (بما في ذلك زيادة عدد فترات) ?...

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

الأدينوساين الإنزيمات hydrolyzing ثلاثي الفوسفات، أو ATPases، تلعب دورا حاسما في مجموعة متنوعة من الوظائف الخلوية. ويمكن لهذه البروتينات ديناميكية توليد الطاقة عن الأعمال الميكانيكية، مثل الاتجار البروتين وتدهور والنقل المذاب، والحركات الخلوية. بروتوكول الموصوفة هنا هو فحص أساسي لقياس النشاط في المختبر من ATPases تنقيته لتوصيف وظيفي. البروتينات يتحلل اعبي التنس المحترفين في رد الفعل الذي يؤدي إلى الإفراج الفوسفات غير العضوية، وكمية من الفوسفات تحرير ثم quantitated باستخدام مقايسة اللونية. هذا البروتوكول العالية للتكيف يمكن تعديلها لقياس النشاط أتباز في المقايسات الحركية أو نقطة النهاية. ويرد بروتوكول التمثيلي هنا على أساس النشاط ومتطلبات EpsE، تشارك AAA + أتباز في النوع الثاني إفراز في بكتيريا ضمة الكوليرا. كمية من البروتين النقي اللازمة لقياس النشاط ومدة الفحص وتوقيت وعدد من سافترات mpling، العازلة وتكوين الملح، ودرجة الحرارة، والعوامل المشتركة، والمنشطات (إن وجدت)، وما إلى ذلك قد تختلف عن تلك المذكورة هنا، وبالتالي بعض التحسين قد تكون ضرورية. يوفر هذا البروتوكول إطارا أساسيا لATPases تميز ويمكن أن يؤديها بسرعة وتعديلها بسهولة عند الضرورة.

النشاط في المختبر من T2S أتباز EpsE يمكن يحفزه copurification من EpsE مع المجال حشوية من EpsL (EpsE-cytoEpsL) وإضافة كارديوليين فوسفورية الحمضية 12. ومن الممكن أيضا لتحديد دور معين بقايا EpsE في اعبي التنس المحترفين المائي من خلال مقارنة النشاط من النوع البري (WT) ?...

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.

قياس<em&gt; في المختبر</em&gt; أتباز آخر لالأنزيمية توصيف

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

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Biology

Measuring In Vitro ATPase Activity for Enzymatic Characterization

18.0K Views.  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.

Video: 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.

فحص البروتين كيناس النشاط مع راديولابيلد أتب

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 ...

Biochemistry

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.

توصيف على المستوى الجزيئي باستخدام نهج كيميائية حيوية قوية لبروتين كيناز جديد

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.

تكييف شبه عالي الإنتاجية لفحص ATPase المقترن بـ NADH لفحص مثبطات الجزيئات الصغيرة

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.

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

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.

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.

الكشف عن مباشرة النشاط خلات تشكيل لأنزيم كيناز خلات

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

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.

تنقية و في المختبر نشاط الإنزيم (ع) بجب سينثاتيز من المطثيّة العسيرة

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

Immunology and Infection

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.

تقييم وظائف الميتوكوندريا وبقاء الخلية في خلايا الكلى البروتين Overexpressing نظائر الانزيمات C كيناز

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.

فحوصات البيوكيميائية لتحليل أنشطة ATP التي تعتمد على لونين إعادة عرض الانزيمات

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.

في المختبر الفحص لقياس فسفاتيديل إيثانولامين ناقلة ميثيل آخر

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

Thin Layer Chromatography Based Enzyme Activity Assay: A Technique to Determine In Vitro Pyrophosphokinase Activity Using Thin Layer Chromatography

Source: Pokhrel, A., et al. A <a href="https://jove.unicartagenaproxy.elogim.com/v/58547">Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile</a>. J. Vis. Exp. (2018).
This video demonstrates a thin-layer chromatography-based enzyme assay to determine pyrophosphokinase activity using radiolabeled nucleotides. The method provides insights into nucleotide synthesis and phosphotransfer reactions.

فحص نشاط الإنزيم القائم على كروماتوغرافيا الطبقة الرقيقة: تقنية لتحديد نشاط بيروفوسفوكيناز في المختبر باستخدام كروماتوغرافيا الطبقة الرقيقة

Source: Pokhrel, A., et al. A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile. J. Vis. Exp. (2018).
This ...

قياس

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فحص البروتين كيناس النشاط مع راديولابيلد أتب

توصيف على المستوى الجزيئي باستخدام نهج كيميائية حيوية قوية لبروتين كيناز جديد

تكييف شبه عالي الإنتاجية لفحص ATPase المقترن بـ NADH لفحص مثبطات الجزيئات الصغيرة

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

الكشف عن مباشرة النشاط خلات تشكيل لأنزيم كيناز خلات

تنقية و في المختبر نشاط الإنزيم (ع) بجب سينثاتيز من المطثيّة العسيرة

تقييم وظائف الميتوكوندريا وبقاء الخلية في خلايا الكلى البروتين Overexpressing نظائر الانزيمات C كيناز

فحوصات البيوكيميائية لتحليل أنشطة ATP التي تعتمد على لونين إعادة عرض الانزيمات

في المختبر الفحص لقياس فسفاتيديل إيثانولامين ناقلة ميثيل آخر

فحص نشاط الإنزيم القائم على كروماتوغرافيا الطبقة الرقيقة: تقنية لتحديد نشاط بيروفوسفوكيناز في المختبر باستخدام كروماتوغرافيا الطبقة الرقيقة