1. Coenzyme A Glutathione Disulfide
by Hartmut Schlüter, Michael Meissner, Marcus van der Giet, Martin Tepel, Jürgen Bachmann, Isolde Groß, Eckhard Nordhoff, Michael Karas, Claus Spieker, Herbert Witzel, and Walter Zidek
Circulation Research
Volume 76(4):
April 1, 1995
Copyright © American Heart Association, Inc. All rights reserved.

2. Purification of coenzyme A glutathione disulfide from bovine adrenal glands.
Purification of coenzyme A glutathione disulfide from bovine adrenal glands. Vasopressor activity in the isolated perfused kidney is indicated by horizontal bars or by arrows. A, Size-exclusion chromatography (flow, 1 mL/min; column dimension, 26×950 mm; Sephacryl 100 HR gel, Pharmacia) of the delipidated methanol extract of adrenal glands dissolved in 5 mL eluent and 1 mol/L acetic acid. The open bar indicates vasopressor activity inhibitable by 10−6 mol/L phentolamine. B, Original tracings of the four fractions showing vasopressor activity in the presence of phentolamine. P indicates perfusion pressure. C, Chromatography of the fractions showing phentolamine-resistant vasopressor activity in panel A dissolved in 5 mL of 20 mmol/L triethylammonium acetate in water (eluent A) with a C4 reversed-phase column (flow, 1.5 mL/min; column dimension, 22×250 mm; Protein-Plus, Zorbax) in the displacement mode (displacer, 200 mmol/L n-butanol in eluent A). D, Chromatography of the active fractions in panel C on a reversed-phase high-performance liquid chromatographic (HPLC) column (flow, 0.5 mL/min; column dimension, 250×4 mm; Lichrosorb RP-C18, Merck) with the following gradient: 0 to 10 minutes, 100% eluent A (20 mmol/L triethylammonium acetate in water); 10 to 50 minutes, 0% to 20% eluent B (acetonitrile). E, Chromatography of the active fractions in panel D with a reversed-phase HPLC column (flow, 0.5 mL/min; column dimension, 250×4 mm; Superspher RP-C18 endcapped, Merck) with the following gradient: 0 to 10 minutes, 100% to 96% eluent C (0.1% trifluoroacetic acid in water); 10 to 60 minutes, 4% to 12% eluent B (acetonitrile). F, Chromatography of the active fraction in panel E on a reversed-phase HPLC column (flow, 0.5 mL/min; Lichrospher 60 RP-select B, Merck) with the following gradient: 0 to 4 minutes, 100% to 94% eluent A (20 mmol/L triethylammonium acetate in water); 4 to 64 minutes, 4% to 10% eluent B (acetonitrile). Only the fraction containing the UV peak showed vasopressor activity.
Hartmut Schlüter et al. Circ Res. 1995;76:
Copyright © American Heart Association, Inc. All rights reserved.

3. A, UV spectrum of the active fraction shown in Fig 1F.
A, UV spectrum of the active fraction shown in Fig 1F. Ordinate indicates absorbance; abscissa, wavelength (nanometers). B, Matrix-assisted laser desorption/ionization mass spectrometry of the active fraction shown in Fig 1F. Ordinate indicates relative intensity; abscissa, relative molecular mass/charge (m/z, in daltons). C, Matrix-assisted laser desorption/ionization mass spectrometry of the active fraction shown in Fig 1F in the presence of 10 mmol/L KCl. Ordinate indicates relative intensity; abscissa, relative molecular mass/charge, m/z (in daltons).
Hartmut Schlüter et al. Circ Res. 1995;76:
Copyright © American Heart Association, Inc. All rights reserved.

4. A, Chromatography of the active fraction in Fig 1F (upper tracing, control) after incubation with alkaline phosphatase (middle tracing) and after sequential incubation first with alkaline phosphatase and then with 5′ phosphodiesterase (lower tracing); conditions were as given for step 5 (see Fig 1F).
A, Chromatography of the active fraction in Fig 1F (upper tracing, control) after incubation with alkaline phosphatase (middle tracing) and after sequential incubation first with alkaline phosphatase and then with 5′ phosphodiesterase (lower tracing); conditions were as given for step 5 (see Fig 1F). B, Chromatography of the active fraction shown in Fig 1F after incubation with glutathione reductase; conditions were as described for step 3 (see Fig 1D). C, Chromatography of the fractions eluting from 4 to 15 minutes obtained from the chromatography shown in panel B after derivatization with ortho-phthaldialdehyde with a reversed-phase column (0.5 mL/min, Nucleosil C18, Macherey-Nagel). Eluent D consisted of 0.2 mol/L potassium acetate (pH 6) in water, and eluent E consisted of methanol. A gradient with 0% to 21% eluent D in 0 to 10 minutes, 21% to 21% eluent D in 10 to 33 minutes, and 21% to 36% eluent D in 33 to 40 minutes was developed. Fluorescence was monitored with 330-nm excitation and 445-nm emission.
Hartmut Schlüter et al. Circ Res. 1995;76:
Copyright © American Heart Association, Inc. All rights reserved.

5. A, Chromatography of chromaffin granules isolated from adrenal glands; conditions were as described for step 3 (see Fig 1D).
A, Chromatography of chromaffin granules isolated from adrenal glands; conditions were as described for step 3 (see Fig 1D). B and C, Chromatography of the supernatant from adrenal medulla slices, which is the last step of the purification procedure with a Superspher C18 reversed-phase column (Merck). In panel B, slices were stimulated with 10−4 mol/L carbachol; the peak identified as coenzyme A glutathione disulfide (CoASSG) is indicated with an arrow. Panel C shows chromatography of a supernatant from adrenal medulla slices in the absence of carbachol.
Hartmut Schlüter et al. Circ Res. 1995;76:
Copyright © American Heart Association, Inc. All rights reserved.

6. Graphs showing the vascular effects of authentic coenzyme A glutathione disulfide (CoASSG).
Graphs showing the vascular effects of authentic coenzyme A glutathione disulfide (CoASSG). A, Perfusion pressure (P) of an isolated rat kidney perfused with constant flow during perfusion with 10−12 mol/L CoASSG (horizontal line). Angiotensin II (AII, 1 pmol) or physiological salt solution (NaCl) was administered in a bolus (100 μL, arrows). B, Concentration-response curve of the experiments shown in panel A (mean±SEM, each n=6). • indicates vasopressor effect of CoASSG; ○, increase of the AII effect as percentage of the baseline response to 1 pmol AII. C, Changes in perfusion pressure (ordinate, ΔP) of an isolated perfused mesenteric vascular bed after sequential bolus injection of 500 fmol, 5 nmol, and 50 nmol CoASSG (arrows) and of 1 nmol AII. D, Mean arterial pressure (MAP) in a rat after intra-aortic injection (arrows) of 5×10−10 mol CoASSG and 5×10−9 mol AII, each dissolved in 100 μL physiological salt solution, and injection of 100 μL physiological salt solution as control (NaCl). E, Changes in [Ca2+]i induced by 10−9 mol/L CoASSG and 10−7 mol/L AII in the presence and absence of 10−9 mol/L CoASSG (horizontal line) measured with fura 2 in cultured rat aortic smooth muscle cells.
Hartmut Schlüter et al. Circ Res. 1995;76:
Copyright © American Heart Association, Inc. All rights reserved.