Hormone Action, Part C: Cyclic Nucleotides by Nathan P. Kaplan, Nathan P. Colowick, Joel G. Hardman, Bert

By Nathan P. Kaplan, Nathan P. Colowick, Joel G. Hardman, Bert W. O'Malley

The severely acclaimed laboratory general, equipment in Enzymology, is without doubt one of the such a lot hugely revered courses within the box of biochemistry. considering the fact that 1955, every one quantity has been eagerly awaited, often consulted, and praised by way of researchers and reviewers alike. The sequence comprises a lot fabric nonetheless suitable this present day - really an important e-book for researchers in all fields of lifestyles sciences.

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A procedure for this dilution has been described. ~ The second factor to be considered is the method of stopping a cyclase reaction. We had originally used EDTA or pyrophosphate to chelate magnesium or manganese ions and then heated the reaction mixture to precipitate protein. As mentioned earlier, in order to correct for losses in the subsequent chromatography, tritiated cyclic nucleotide is incorporated into the stop solution. However, we have found that when [sH]c~MP was heated at 100° for 2 minutes in a simulated reaction mixture, about 4% of the total radioactivity was converted into material that did not behave chromatographically like cGMP (it could be guanine and/or guanosine).

Front - Guo I . . . . . . . . . . -0 ........ =igin • - FI~. 2. 6 M ammonium sulfate (schematically). Open spots refer to positions of the compounds after the first and second run; filled spots, to final positions after the third run. [4] THIN-LAYER CHROMATOGRAPHY 35 LiC1 or a m m o n i u m sulfate are applied as solvent. Optimal separations of c G M P are obtained by successive developments with 50 m M acetic acid, distilled water, and 45 m M LiC1 (Fig. 6 M a m m o n i u m sulfate (Fig.

2 M NaC1 was added to the HC1 solution. 001 • 0 minutes FIo. 2. High pressure chromatograph of 43 pmoles of cyclic AMP. From G. Brooker, Anal. Chem. 43, 1095 (1971). OOC minutes 6 FIG. 3. High-pressure chromatograph of 50 pmoles of cyclic GMP. From G. Brooker, J. Biol. Chem. 246, 7810 (1971), point, and cGMP is eluted at about 5 minutes as shown in Fig. 3. In addition this buffer system has been used to measure cAMP analogs. 4 The unknown samples were injected in volumes of 25 ~l or less, the system was closed, and the flow started.

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