کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
7556770 | 1491296 | 2018 | 14 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
In situ measurements of mitochondrial matrix enzyme activities using plasma and mitochondrial membrane permeabilization agents
ترجمه فارسی عنوان
فعالیت های آنزیم ماتریکس میتوکندری در محیط های منطقه با استفاده از عوامل نفوذپذیری غشای پلاسما و میتوکندری
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کلمات کلیدی
موضوعات مرتبط
مهندسی و علوم پایه
شیمی
شیمی آنالیزی یا شیمی تجزیه
چکیده انگلیسی
Activities of enzymes localized to the mitochondrial matrix of mammalian cells are often critical regulatory steps in cellular metabolism. As such, measurement of matrix enzyme activities in response to genetic modifications or drug interventions is often desired. However, measurements in intact cells are often hampered by the presence of other isozymes in the cytoplasm as well as the inability to deliver enzyme substrates across cellular membranes. Classic approaches to liberate matrix enzymes utilize harsh treatments that disrupt intracellular architecture or require significant starting material to allow mitochondrial isolation prior to sample extraction. We describe a method using permeabilization reagents for both the plasma and mitochondrial membranes to allow in situ measurement of matrix enzyme activities. It is applied to adherent cell monolayers in 96-well plates treated with perfringolysin O to permeabilize the plasma membrane and alamethicin to permeabilize the mitochondrial inner membrane. We present three examples validated with inhibitor sensitivity: (i) Complex I-mediated oxygen consumption driven by NADH, (ii) ATP hydrolysis by the F1FO complex measuring pH changes in an Agilent Seahorse XF Analyzer, and (iii) Mitochondrial glutaminase (GLS1) activity in a coupled reaction monitoring NADH fluorescence in a plate reader.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 552, 1 July 2018, Pages 60-65
Journal: Analytical Biochemistry - Volume 552, 1 July 2018, Pages 60-65
نویسندگان
Ajit S. Divakaruni, Alexander Y. Andreyev, George W. Rogers, Anne N. Murphy,