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Heptapeptide rkrsrae

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Heptapeptide (RKRSRAE) is a short sequence of seven amino acids. It serves as a core structural component in various biochemical applications.

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Lab products found in correlation

Protease inhibitor cocktail, by Merck Group (4 mentions) Wallac 1409 liquid scintillation counter, by Hidex (3 mentions) P81 filters, by Cytiva (2 mentions) 32p atp, by GE Healthcare (1 mentions) γ 32p atp, by Cytiva (1 mentions)

5 protocols using heptapeptide rkrsrae

1

Measuring Myocardial Protein Kinase G Activity

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Myocardial protein kinase G (PKG) activity was assessed in homogenized tissue samples. Tissues samples were homogenized in 25 mmol/l Tris (pH 7.4), 1 mmol/l EDTA, 2 mmol/l EGTA, 5 mmol/l dithiothreitol (DTT), 0.05% Triton X‐100, and protease inhibitor cocktail (Sigma Aldrich) and centrifuged for 5 minutes. Supernatants containing equal amounts of total protein were analyzed for PKG activity. The reaction mixture contained (at final concentration): 40 mmol/l Tris‐HCl (pH·7.4), 20 mmol/l magnesium acetate, 0.2 mmol/l [32P] ATP (500–1,000 c.p.m.·pmol−1) (GE Healthcare LifeScience, Little Chalfont, U.K.), 113 mg/ml heptapeptide (RKRSRAE), 3 mmol/l cGMP (Promega, Madison, WI) and a highly specific inhibitor of cAMP‐dependent protein kinase (5–24, Merck Millipore). The reaction mixtures were incubated at 30°C for 10 minutes, followed by termination of the reaction by spotting 70·μl of the reaction mix onto Whatman P‐81 filters, which were then soaked with 75·mmol/l H3PO4 for 5 minutes and washed three times with 75·mmol/l H3PO4 to remove any unbound [32P] ATP. Filters were rinsed with 100% ethanol and air dried before quantification. For quantification of PKG activity, counts were taken in a Wallac 1409 Liquid Scintillation Counter using universal scintillation cocktail. Specific activity of PKG was expressed as pmol of [32P] incorporated into the substrate (pmol/minute/mg protein).
Van Linthout S., Hamdani N., Miteva K., Koschel A., Müller I., Pinzur L., Aberman Z., Pappritz K., Linke W.A, & Tschöpe C. (2017). Placenta‐Derived Adherent Stromal Cells Improve Diabetes Mellitus‐Associated Left Ventricular Diastolic Performance. Stem Cells Translational Medicine, 6(12), 2135-2145.
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2

Quantification of PKG Activity

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To assess PKG activity tissue samples (n=6–7 LV samples/steady state) were homogenized in a buffer (in mM: 25 Tris (pH 7.4), 1 EDTA, 2 EGTA, and 5 DTT; 0.05% Triton X-100; protease inhibitor cocktail from Sigma-Aldrich, Munich, Germany), followed by centrifugation for 5 min. Supernatants containing equal amounts of total protein were analyzed for PKG activity. The reaction mixture contained (at final concentration): 40 mM Tris-HCl (pH 7.4), 20 mM magnesium acetate, 0.2 mM [γ−32P]ATP (500–1,000 cpm/pM; Amersham Pharmacia Biotech, Freiburg, Germany), 113 mg/mL heptapeptide (RKRSRAE), 3 mM cGMP (Promega, Madison, WI, USA) and a highly specific inhibitor of cAMP-dependent protein kinase (5–24, catalogue #116805; Calbiochem, San Diego, CA, USA). The reaction mixtures were incubated for 10 min at 30°C, followed by termination of the reaction by spotting 70 μl of the reaction mix onto Whatman P-81 filters, which were then soaked with 75 mM H3PO4 for 5 min and washed three times with 75 mM H3PO4 to remove any unbound [γ−32P]ATP. Filters were rinsed with 100% ethanol and air dried before quantification. For quantification of PKG activity, counts were taken in a Wallac 1409 Liquid 4 Scintillation Counter using universal scintillation cocktail (ICN). Specific activity of PKG was expressed as pM of 32P incorporated into the substrate (pM/min/mg protein).
Reil J.C., Reil G.H., Kovács Á., Sequeira V., Waddingham M.T., Lodi M., Herwig M., Ghaderi S., Kreusser M.M., Papp Z., Voigt N., Dobrev D., Meyhöfer S., Langer H.F., Maier L.S., Linz D., Mügge A., Hohl M., Steendijk P, & Hamdani N. (2020). CaMKII activity contributes to Homeometric Autoregulation of the heart: a novel mechanism for the Anrep effect. The Journal of physiology, 598(15), 3129-3153.
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3

Measurement of Protein Kinase G Activity

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LV tissues samples (n = 8–10 samples) were homogenized in 25 mM Tris-HCl (pH 7.4), 1 mM EDTA, 2 mM EGTA, 5 mM DTT, 0.05% Triton X-100, and protease inhibitor cocktail (all from Sigma-Aldrich) and centrifuged for 5 min. Supernatants containing equal amounts of total protein were analyzed for PKG activity as described previously (Franssen et al., 2016 (link)). Briefly, reaction mixtures were incubated at 30°C for 10 min. Reaction mixtures contained 40 mM Tris-HCl (pH 7.4), 20 mM Mg(CH3COO)2, 0.2 mM [32P] adenosine triphosphate (ATP) (500–1,000 cpm pM–1; Amersham PLC, Little Chalfont, United Kingdom), 113 mg/ml heptapeptide (RKRSRAE), and 3 μM cGMP (both from Promega Corp., Madison, WI, United States), and a highly specific inhibitor of cyclic adenosine monophosphate-dependent protein kinase (5–24; Calbiochem, San Diego, CA, United States). The reaction was terminated by spotting 70 μl onto Whatman P-81 filters (MACHEREY-NAGEL). Samples were subsequently incubated and washed with 75 mM H3PO4 for 5 min to remove unbound ATP. Filters were then washed with 100% ethanol and air-dried before quantification. PKG activity was quantified using a Wallac 1409 Liquid Scintillation Counter (Hidex Oy, Turku, Finland). Specific activity of PKG was expressed as pM of 32P incorporated into the substrate (pM/min/mg protein).
Kolijn D., Kovács Á., Herwig M., Lódi M., Sieme M., Alhaj A., Sandner P., Papp Z., Reusch P.H., Haldenwang P., Falcão-Pires I., Linke W.A., Jaquet K., Van Linthout S., Mügge A., Tschöpe C, & Hamdani N. (2020). Enhanced Cardiomyocyte Function in Hypertensive Rats With Diastolic Dysfunction and Human Heart Failure Patients After Acute Treatment With Soluble Guanylyl Cyclase (sGC) Activator. Frontiers in Physiology, 11, 345.
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4

Quantifying PKG Activity in LV Tissue

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PKG activity was analyzed using a radioactive assay. LV tissue samples were homogenized in a buffer and centrifugated for 5 min as described before [18 (link),32 (link),33 (link)]. Reaction solutions containing 40 mM Tris-HCl (pH 7.4), 20 mM Mg (CH3COO)2, 0.2 mM [32P] adenosine triphosphate (ATP) (500–1000 cpm pM–1; Amersham PLC, Little Chalfont, United Kingdom), 113 mg/mL heptapeptide (RKRSRAE), and 3 μM cGMP (both from Promega Corp., Madison, WI, United States), and a highly specific inhibitor of cyclic adenosine monophosphate-dependent protein kinase (5–24; Calbiochem, San Diego, CA, United States) were incubated at 30 °C for 10 min. The reaction was subsequently terminated by adding 70 μL onto Whatman P-81 filters (MACHEREY-NAGEL). The samples were incubated and washed with 75 mM H3PO4 for 5 min to remove unbound ATP. The filters were washed with 100% ethanol and air-dried. For the measurement of PKG activity, a Wallac 1409 liquid scintillation counter (Hidex Oy, Turku, Finland) was used. Here, the specific activity of PKG was analyzed by quantification of the incorporated pM of 32P into the substrate (pM/min/mg protein).
Budde H., Hassoun R., Tangos M., Zhazykbayeva S., Herwig M., Varatnitskaya M., Sieme M., Delalat S., Sultana I., Kolijn D., Gömöri K., Jarkas M., Lódi M., Jaquet K., Kovács Á., Mannherz H.G., Sequeira V., Mügge A., Leichert L.I., Sossalla S, & Hamdani N. (2021). The Interplay between S-Glutathionylation and Phosphorylation of Cardiac Troponin I and Myosin Binding Protein C in End-Stage Human Failing Hearts. Antioxidants, 10(7), 1134.
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5

Quantifying Protein Kinase G Activity

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RV and LV tissues samples (n = 3 in triplicates/group) were homogenized in 25 mM Tris-HCl (pH 7.4), 1 mM EDTA, 2 mM EGTA, 5 mM DTT, 0.05% Triton X-100, and protease inhibitor cocktail (all from Sigma-Aldrich, St. Louis, MO, USA) and centrifuged for 5 min. Supernatants containing equal amounts of total protein were analyzed for PKG activity as described previously [24 (link)]. Briefly, reaction mixtures were incubated at 30 °C for 10 min. Reaction mixtures contained 40 mM Tris-HCl (pH 7.4), 20 mM Mg(CH3COO)2, 0.2 mM [32P] adenosine triphosphate (ATP) (500–1000 cpm pM–1; Amersham, Little Chalfont, UK), 113 mg/mL heptapeptide (RKRSRAE), and 3 μM cGMP (both from Promega, Madison, WI, USA), and a highly specific inhibitor of cyclic adenosine monophosphate-dependent protein kinase (5–24; Calbiochem, San Diego, CA, USA). The reaction was terminated by spotting 70 μL onto Whatman P-81 filters (MACHEREY-NAGEL). Samples were subsequently incubated and washed with 75 mM H3PO4 for 5 min to remove unbound ATP. Filters were then washed with 100% ethanol and air dried before quantification. PKG activity was quantified using a Wallac 1409 Liquid Scintillation Counter (Hidex Oy, Turku, Finland). Specific activity of PKG was expressed as pM of 32P incorporated into the substrate (pM/min/mg protein). Results of triplicate determinations were averaged.
Kovács Á., Herwig M., Budde H., Delalat S., Kolijn D., Bódi B., Hassoun R., Tangos M., Zhazykbayeva S., Balogh Á., Czuriga D., Van Linthout S., Tschöpe C., Dhalla N.S., Mügge A., Tóth A., Papp Z., Barta J, & Hamdani N. (2021). Interventricular Differences of Signaling Pathways-Mediated Regulation of Cardiomyocyte Function in Response to High Oxidative Stress in the Post-Ischemic Failing Rat Heart. Antioxidants, 10(6), 964.
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