654 



N. E. Tolbert 



Tolbert and G. Orth, unpublished). The activity of this phospha- 

 tase is equal to or exceeds that of other phosphatases in the 

 leaf. For example phosphoglycolate is hydrolyzed 5 times faster t 

 by wheat sap at pH 6.3 than most other phosphate esters (e.g. 3- 

 phosphoglycerate) . About 97o of the phosphoglycolate phosphatase 

 activity from spinach, tobacco or swiss chard leaves remains with 

 unwashed chloroplasts . Once washed chloroplasts have 2% the total 

 enzyme, and if washed chloroplasts are then put in water, the rest 

 of this phosphatase goes into solution, while the phosphatase re- 

 sponsible for hydrolysis of f ructose-1 , 6-diphosphate remains with 

 the particles. Preparation of chloroplasts in glycerol yields 

 similar results. The phosphatase is certainly not tightly bound 

 in the chloroplasts. 



Phosphoglycolate phosphatase is stable at pH 4, and it has been 

 purified 200-fold by acetone and ethanol fractionation and DEAE- 

 cellulose. The stability of the enzyme from wheat is dependent 

 upon the natural association with it of cis-aconitate. If the 

 factor were removed by Sephadex, the enzyme is stable at 0° for 

 only a short period of time. Stability at 40° is restored only 

 by cis-aconitate, citrate or isocitrate. Our most purified prep- 

 aration of the enzyme had both phosphatase and ATPase activity. 

 The ATPase was less active than the phosphatase. The enzyme also 

 forms methyl or ethyl phosphates when flooded with the alcohol, 

 but this is typical of phosphatases. In addition the protein 

 rapidly incorporated P^^-orthophosphate in an ATP dependent reac- 

 tion. This latter reaction was heat stable, and the protein was 

 not precipitated by heat. These properties are in part character- 

 istic of certain phosphatases or ATPase which may be involved in 

 transport phenomena. Our published procedure for isolating this 

 enzyme from tobacco leaves involved (NH4)2S04 treatment which 

 destroyed the ATPase and changed the pH optimum of the remaining 

 phosphoglycolate phosphatase from 6.3 to 5 or less (13). The 

 enzyme from most other leaves is inactivated by (NH4)2S04, 



Like the phosphatase, active forms of glycolate oxidase (31) 

 and a specific glyoxylate-serine transaminase (E. R. Waygood, 

 personal communication) are nearly absent in etiolated leaves and 

 are formed during greening. This characteristic is presumptive 

 evidence that they are associated with photosynthesis. Glycolate 

 oxidase also may be loosely associated with the chloroplast (22, 

 32, 33). There is also a NAD linked glyoxylate reductase v^ich is 

 probably cytoplasmic (34) and another glyoxylate reductase which 

 is NADP linked and located in the chloroplast (35). Further stud- 

 ies concerning the formation or activation of these enzymes during 

 greening of the plant should be pertinent to an understanding of 

 chloroplast structure and its association with the rest of the cell. 



