1578 PHOTOCHEMISTRY OF CHLOROPHYLL CHAP. 35 



(prepared from pigeon liver) and Mn++ ions (which this enzyme specifically 

 requires), malic acid is formed. This formation can be demonstrated after 

 exposure to hght (but not in darkness) by means of a specific enzymatic test 

 (hberation of CO2 by mafic decarboxylase). In a second test, tracer C*02 

 was employed, and practically all of the C^^ fixed in Hght was found in 

 malate, with 75% of it localized in the /3-carboxyl group. 



Similar results were obtained ^\dth a-ketoglutaric acid and bicarbonate 

 in the presence of chloroplasts, TPN and appropriate enzymes. Other 

 pyridine nucleotide-specific reductions also could be carried out with the 

 help of illuminated chloroplast preparations, including the reduction of 

 pyruvate to lactate by DPN and lactic dehydrogenase: 



chloroplasts 



(35.34Aa) DPN + H.O — > DPNH. + V2O2 



light 



(35.34Ab) CH3COCOOH + DPNH.2 > CH3CHOHCOOH + DPN 



pyruvate lactate 



also reduction of oxalacetate to malate in the presence of DPN and mafic 

 dehydrogenase, reductive amination of a-ketoglutarate to glutamic acid 

 by ammonia and DPN and reduction of fumaric acid to succinic acid in 

 the presence of DPN and extracts from Escherichia coli (a reaction which 

 can be brought about in the dark by molecular hydrogen) . 



All the above reductions (except that of fumarate) involve the hydro- 



\ \ / 



genation of the carhonyl group, C=0 (a C=C group is hydrogenated m 



/ / ^ 



fumarate). As stated above, these reductions can be achieved by pyridine 



nucleotides without the assistance of high energy phosphate. The 

 reduction of a carboxyl group requires a stronger reducing agent than 

 TPNH2 or DPNHo (because, as mentioned on pages 215-219, C— O bonds 

 are stabilized by accumulation at a single C atom). As was said before, 

 in order to reduce a — COOH group by hydrogen available in reduced 

 pyridine nucleotide, a high energy phosphate ester must be supplied, and 

 its degradation to a "low energy" phosphate coupled with the reduction. 

 (As an example, oxidation of glyceraldehyde to glyceric acid by DPN 

 becomes reversible by oxidizing and phosphorylating a "low energy" 

 triose monophosphate to "high energy" diphosphoglycerate.) By com- 

 bining the reversal of this reaction with the (reversible) glycolytic reactions, 

 it is possible to start with PGA, ATP and DPNH2 {i. e., DPN + chloro- 

 plasts in fight), and end up with hexose diphosphate. Ochoa suggested 

 (as did earfier Puben, Kok, van der Veen and others, cf. pages 1116-1117) 

 that the high energy phosphate needed for this synthesis of hexose from 

 PGA can be supplied, in fight, by reversal of a part of the photochemical 

 reaction, e. g., by allowing some of the DPNHa formed in fight to be oxi- 



