VOL. 12 (1953) ENZYMIC SYNTHESIS AND BREAKDOWN OF OROTIC ACID I 



231 



bond, was observed spectrophotometrically (the ultraviolet absorption of 0.4 micromole 

 of the fusion product disappeared in 20 minutes when incubated in 3.0 ml of 3.3 • 10^ M 

 cysteine at neutral pH). It appears 

 doubtful that a similar reaction occurs 

 with the enzymic compound since it is 

 produced in the presence of cysteine 

 and is oxidized to orotate in the pre- 

 sence of cysteine. 5. Enzymic oxidation 

 to orotate (by dihydro-orotic dehydro- 

 genase) was observed only with the 

 natural product ; the synthetic product 

 was inert. 6. Growth factor activity for 

 L. bulgaricus og was not detectable 

 with the fusion product while the bio- 

 logical product completely replaced the 

 orotate requirement of the bacterium 

 (see Fig. 5-) 



Enzymic dihydro-orotic acid as a 

 growth factor for Lactobacillus bulgaricus 

 og. Since this strain has been shown' 

 to require orotic acid or ureidosuccinic 

 acid for growth, it was of interest to 

 determine the effects of enzymic dihy- 

 dro-orotic acid and the urea-maleic acid 

 fusion product upon this organism. 



Solutions of the compounds under 

 test were sterilized by autoclaving* and 

 varying amounts of each were asepti- 

 cally added to the double strength basal 

 medium of Wright et al?. The inoculum 



was prepared as described by Wright et alJ and bacterial growth was measured at 64 

 hours turbidimetrically and by titrating aliquots of the culture with o.oio N NaOH 

 using phenol red as indicator. Growth was complete at 64 hours but none was apparent 

 in the control tubes which received no growth factor additions. 



From the results of the titrimetric determinations (Fig. 5), it can be seen that 

 enzymic dihydro-orotate completely replaced the orotate requirement of the lactobacilli 

 while growth was not supported by the synthetic compound. The synthetic product 

 did not inhibit the activity of added orotate. Thus, with 0.12 micromole of the synthetic 

 compound and 0.05 micromole of orotate per ml. of medium, 42.6 micromoles of acid 

 per ml were formed, as compared with 43.7 micromoles of acid with orotate alone. 



0.2 03 0.4 



Growth factor added (n mo/es/mO 



Fig. 5. The production of acid by Lactobacillus 

 bulgaricus og in the presence of orotic acid and 

 related compounds. 



To 6.0 ml of double strength medium' were 

 added aseptically the indicated amounts of the 

 test compounds and sterile water to 7.0 ml. After 

 inoculation of the organism'', incubation was car- 

 ried out at 37° C for 64 hours. Aliquots from each 

 tube were titrated with 0.0 1 N NaOH using a 

 microburette. A blank value of 18.3 micromoles 

 per ml, the titratable material present in the con- 

 trol tubes receiving no growth factor additions, 

 was subtracted. 



* Synthetic dihydro-orotic acid has the same ultraviolet absorption spectrum before and after 

 autoclaving. This was taken to indicate that it had not been altered during the sterilization process. 

 After autoclaving, enzymic dihydro-orotic acid behaved identically with the untreated material, 

 both enzymically and spectrophotometrically. 



References p. 2J4. 



