Reaktion #1287277

Reaktionsgleichung

adenine

beta-estradiol diacetate

isobutylmethylxanthine

KCl

glucose

disodium hydrogen phosphate

CaCl2

NaCl

MgSO4

phenol red

→

cyclic AMP

Lösungsmittel

CO2

acetic acid

Reaktionsbedingungen

Aufarbeitung

1. 1
   SonstigeUterus homogenate is prepared
2. 2
   FiltrationThe tissue is now filtered
3. 3
   SonstigeAfter a preincubation period of 5 minutes
4. 4
   workup.ADDITION2 are added
5. 5
   FiltrationIt is then filtered again
6. 6
   Temperaturthe tissue is deep-frozen in liquid nitrogen
7. 7
   Sonstigecrushed
8. 8
   SonstigeCyclic AMP is subsequently separated out
9. 9
   Sonstigeresults

Vorschrift

Uterus homogenate is prepared as described in Example 5 from rats treated with beta-estradiol diacetate. The buffer used for the homogenization is composed of 0.14 mol/l NaCl, 5 mmol/l KCl, 10 mmol/l glucose, 10 mmol/l acetic acid, 2 mmol/l disodium hydrogen phosphate, 20 mmol/l tris-HCl, 0.001% phenol red, 1.2 mol/l MgSO4, 0.8 mmol/l CaCl2 and 0.01 mmol/l isobutylmethylxanthine. Before use, this buffer is brought to pH 7.4 by passing in CO2 gas. 10 ml of the uterus homogenate are now incubated with 0.3 mmol/l tritiated adenine at 37° C. for 40 minutes. The tissue is now filtered, again taken up in the above buffer, and subsequently divided up in such a way that each portion is suspended in 10 ml of the said buffer. After a preincubation period of 5 minutes, the active substances specified in Tab. 2 are added, and the mixture is incubated for a further 100 minutes. It is then filtered again, the tissue is deep-frozen in liquid nitrogen and crushed, and the resulting powder is homogenized in 1 ml of 6% strength trichloroacetic acid. Cyclic AMP is subsequently separated out and quantified by column chromatography as in Analytical Biochemistry 58, (1974) 541-548 (in particular Method C, page 543). Tab. 2 shows the results:

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