105 

 between SP-I (M^ at 59,000 to 60,000) and SP-II (M^ at about 

 62,000) from gel filtration, SDS and silver stained PAGE. 

 Bands were also present at about 40,000 (SP-I) and at about 

 42,000 (SP-II). It was not clear what the lower molecular 

 mass bands represent. These bands could be contaminating 

 protein, isoforms of each protein, or proteolytically 

 damaged protein. Activity stained gels showed three bands 

 (each of which may contain two bands for a total of six 

 bands) of activity for SDH from crude extracts, while, 

 purified SP-I had two bands that migrated slower than the 

 two bands of SP-II. The difference in M,., may suggest these 

 proteins are isoenzymes. One possibility, is that SP-I, the 

 smaller M^ protein (the major fraction of activity that was 

 located on the Celite column) may be the mature chloroplast 

 isoenzyme with transit peptide removed and that SP-I, the 

 larger M^ protein (the minor fraction of activity from the 

 column) might be the cytosolic preprotein and isoenzyme that 

 functions in aromatic biosynthesis. A second differential 

 property, that of Km values was observed for SP-I and SP-II. 

 SP-II has greater affinity for SHK, DHS and DHQ, substrates 

 for both functional domain of the bifunctional protein. 

 Both proteins have about the same affinity for cofactor, 

 NADP* or NADPH. These Km values are in the range of Km 

 values found in the literature (listed in Table 1-1) . The 

 difference in affinity between SP-I and SP-II for substrates 

 is also suggestive of differentially located isoenzyme. 



