150 



HARLYN HALVORSON 



Clucost-6-PO^ (purpit) 



2-ktto 9lMCOftolt{9'«m»h- 

 yellow) 



6-P04-2-lnfo 9lueonot* 

 (vioitt) 



Pyruvott 

 Gluconott 



(red) 

 (pwfpit) 



Clucost (purpi*) 



Fig. 7. Detection of end products of glucose oxidation by chromatog- 

 raphy. The reaction mixture of the experiment of Fig. 5 was deproteinized 

 after 60 minutes incubation. The chromatogram containing the reaction mix- 

 ture and knowns was run in an ethanol: methanol: H20(45: 45: 10) sys- 

 tem. After drying, the paper was developed by the method of DeLey (1953). 



the initial oxidation is not phosphate-dependent (Fig. 8). Dialyzed prepa- 

 rations suspended in a phosphate-free glycylglycene buffer contain an active 

 DPN-linked glucose dehydrogenase (Fig. 9). This enzyme is soluble, since 

 the total activity following centrifugation at 140,000 times gravity remains 

 in the supernatant fraction (Table I). In contrast with the glucose dehy- 

 drogenase of Pseudomonas fluorescens (Wood, 1955), which is particulate 

 and uses cytochromes b and c as hydrogen acceptors, the dehydrogenase 

 of spores, as that in liver, utilizes DPN as the hydrogen acceptor. 



The active G-6-P dehydrogenase in spore extracts (Fig. 10) is of the 

 usual microbial variety employing TPN as the hydrogen acceptor (DeLey, 

 1955). From the studies of Cori and Lipmann (1952) and Brodie and Lip- 

 mann (1955) it is probable that the reaction here proceeds in two steps: 

 (a) an oxidation of the pyranose ring of glucose-6-phosphate to 6-phospho- 

 ^-gluconolactone and (b) a hydrolysis by a delactonizing enzyme to 6-P-G. 



The reduction of DPN or TPN is not stimulated by the addition of phos- 

 phate. These findings are similar to those of Hachisuka et al (1956) who 

 observed that the oxidation of glucose to gluconate in transclucent spores 

 was not phosphate-dependent. It is interesting that although at least several 

 of the enzymes of the HMP pathway, G-6-P dehydrogenase and a TPN 



