. Cell chemistry; a collection of papers dedicated to Otto Warburg on the occasion of his 70th birthday. Warburg, Otto Heinrich, 1883-; Biochemistry. ? 2 4 6 8 10 12 MINUTES Fig. g. Optical /?-keto reductase test. Pyrophosphate buffer pH , 50 ixM; DPNH, fxH; S-aceto- acetyl-N-acetyl thioethanolamine, jxM. Volume, ml; d = cm; temp. 25°. O 2CH3—C—S—CoA: O HS—CoA+CH3—CO—CH 2—O—S—Co A (thiolase) O CH3—CO—CHj—C—S—C0A + DPNH + H+ ^ CH3—CHOH—CH,—C—S—C0A + DPN+ (reductase) O Sum: 2 CH3—C—S—Co A + DPNH + Hh O II CH3—CHOH—CH2—C—S—CoA + HS—CoA + DPN+ The formation of HS-CoA can be fol
. Cell chemistry; a collection of papers dedicated to Otto Warburg on the occasion of his 70th birthday. Warburg, Otto Heinrich, 1883-; Biochemistry. ? 2 4 6 8 10 12 MINUTES Fig. g. Optical /?-keto reductase test. Pyrophosphate buffer pH , 50 ixM; DPNH, fxH; S-aceto- acetyl-N-acetyl thioethanolamine, jxM. Volume, ml; d = cm; temp. 25°. O 2CH3—C—S—CoA: O HS—CoA+CH3—CO—CH 2—O—S—Co A (thiolase) O CH3—CO—CHj—C—S—C0A + DPNH + H+ ^ CH3—CHOH—CH,—C—S—C0A + DPN+ (reductase) O Sum: 2 CH3—C—S—Co A + DPNH + Hh O II CH3—CHOH—CH2—C—S—CoA + HS—CoA + DPN+ The formation of HS-CoA can be followed through the appearance of sulfhydryl groups. /?-hydroxybutyryl-S-CoA was extracted from the acidified reaction mixture with /j-cresol and converted into the corresponding hydroxamic acid by reaction with hydroxylamine. The j3-hydroxybutyrohydrox- amic acid was identified by paper chro- matography {Rp in aqueous butanol, ). On incubation of the natural j8-hydroxy- butyryl-S-CoA with DPN+ and purified ^- ketoreductase at pH , the reduction of DPN+, followed at 340 m/x, is accompanied by the formation of acetoacetyl-S-CoA as shown by the increase in optical density at 303 mju,. As previously mentioned both j3-hydroxy- butyryl- and acetoacetyl-S-CoA have recently become available synthetically. The course of the j3-ketoreductase reaction with these two compounds*^^ is shown in Fig. 10. Lehninger AND Greville*^ have recent- ly reported the interesting observation that liver contains two different /D-ketoreduc- tases. One of them catalyzes the reversible oxidation of free /-hydroxybutyrate by DPN+, the other catalyzes the reversible oxidation. Fig. 10. Optical experiments with /S-keto reductase. Pyrophosphate bufter pH , 100(iM; DPN+, ; S-;3-hydroxybutyryl CoA, [xM. Volume, ml; A, 366 raij,; d = cm; temp. 21°. /3-keto reductase ( mg of protein) added at the first arrow. S-acetoacetyl CoA
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