98 BIOCHIMICA ET BIOPHYSICA ACTA VOL. 12 (1953) 



REVERSIBILITY OF GLUCOSE-6-PHOSPHATE OXIDATION 



by 



B. L. HORECKER and P. Z. SMYRNIOTIS 



National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, 

 United Stales Public Health Service, Bethesda, Maryland {U.S.A.) 



The work of Warburg and his collaborators^ has established that glucose-6- 

 phosphate is oxidized by triphosphopyridine nucleotide (TPN) in the presence of an 

 enzyme which they called Zwischenferment. The reaction product was identified as 6- 

 phosphogluconate^. The oxidation of glucose-6-phosphate proceeds essentially to com- 

 pletion and can be used for the spectrophotometric determination of this substance^. 

 Interest in the reversal of this reaction has been stimulated by the observation that COo 

 fixation can occur by the reversal of the oxidative decarboxylation of 6-phospho- 

 gluconate^. Recently Cori and Lipmann^ have shown the primary product oiZivischen- 

 fcrment action on glucose-6-phosphate to be the S-lactone of 6-phosphogluconic acid, 

 rather than the free acid. As Lipmann^ has pointed out, from the thermodynamic stand- 

 point, the lactone should be more readily reduced to the glucose level than the free 

 carboxyl form. An analogy may be drawn with the oxidation of glucose. Bentley and 

 Neuberger'^ found the primary product of glucose oxidation by notatin (glucose 

 oxidase) to be the S-lactone. Strecker and Korkes^ obtained the same product with 

 liver glucose dehydrogenase and demonstrated the reversibility of the reaction. In 

 the present paper the reduction of 6-phosphogluconolactone to glucose-6-phosphate b}' 

 reduced triphosphopyridine nucleotide (TPNH) in the presence of Zwischenferment is 

 reported. 



METHODS 



Crystalline barium glucose-6-phosphate. The barium salt of glucose-6-phosphate has previously 

 been available only as an amorphous, water-soluble product. A crj'stalline heptahydrate has now 

 been obtained* which has a very limited solubility in water and can be prepared in essentially pure 

 form. The crystalline salt lias a solubility of 0.5 mg per ml and crystallization is readily induced even 

 in dilute solution by the addition of a small seed crystal. Barium glucose-6-pho3phate synthesized 

 according to Seegmiller .\nd Horecker^ or Lardy .and Fischer^ was purified by crystallization 

 from water. Small amounts of fructose-6-pliosphate present in one of these preparations^ remained 

 in the mother liquor. Recrystallization was carried out by solution of the crystals with the aid of 

 dilute acetic acid followed by neutralization with dilute ammonium hydroxide. Tlie following analytt 

 ical data were obtained**. 



(CeHiiOgP Ba-yH^O) Calculated Ba 2O.2, P 5.94, HgO 24.1 



Found Ba 25.9, P 5.79, HgO 24.4 



Enzymic assay with Zwischenferment and TPN gave a purity of 98 per cent. 



The monopotassium salt of glucose-6-pho3phate was prepared from the barium salt as previously 

 described^. 



A similar crystalline product has been obtained independently bj' the Schwarz Laboratories 

 (personal communication). 



** The analyses for Ba, P, and weight loss in high vacuum at 100° were carried out by the National 

 Institutes of Health Microanalytical Laboratory under the direction of Dr Wm C. Alford. 



References p. 102. 



