Component om itted from the assay m ixture Rate o f re a c tio n AE340nm kour~1 None 0.53 Homogenate sample 0.01 Glutamate dehydrogenase 0.06 2 -o x o g lu ta ra te 0.08 ADP 0.05 Glutamate dehydrogenase ) 2 -o x o g lu ta ra te ) 0.08 ADP )
The b lan k re a c tio n was m onitored as in d ic a te d in Method 2 when u sin g an assay pH o f 7»5 and tis s u e homogenate as th e source o f enzyme.
In the absence of ADP the s t a b i l i t y of the glutam ate dehydrogenase was m ain ta in e d by the in c o rp o ra tio n o f 1 .0 mol 1 NaCl s o lu tio n .
which had been dem onstrated to be e ffe c tiv e in p rev io u s s tu d ie s (p 30). The a d d itio n of e x tra NADH to the c o n tro l when a l l the i n i t i a l
coenzyme had been o x id iz e d ,re s u lte d in a re a c tio n r a te which was g re a te r th an th e i n i t i a l r a te and t h i s suggested th a t th e re was a p o ssib le b u ild up s itu a tio n which was not dependent upon th e presence o f NADH.
The only component n o t checked by t h i s s e r ie s o f experim ents was th e g ly c y lg ly c in e b u ffe r, and subsequent in v e s tig a tio n suggested
th a t t h i s compound was p rim a rily re s p o n sib le , in th e presence o f the homogenate, fo r th i s blan k re a c tio n ,, A se r ie s o f t e s t s were made in which th e g ly c y lg ly c in e b u ffe r was re p la ce d w ith 0.1 mol 1 phosphate b u ff e r pH 7 .5 and in which g ly c y lg ly c in e was in c o rp o ra te d in
c o n ce n tra tio n s v ary in g from 0 - 0.024 mol 1 . The f a l l in
absorbance was m onitored a f t e r the a d d itio n o f hom ogenate. Compared . -1
w ith a r a te o f 0.019 min in th e u su a l g ly c y lg ly c in e b u ffe r, th e r a te in th e phosphate b u ff e r was only 0.0050 min . The a d d itio n of
g ly c y lg ly c in e to the phosphate b u ffe r caused the re a c tio n r a te to
- 1 -1
in c re a se from a valu e of 0.0050 min to a value o f 0.018 min
“ 1
(a t a g ly c y lg ly c in e c o n c e n tra tio n o f 0.024 mol 1 ) . This was _ 1
comparable w ith th e maximum r a te o f 0.019 min achieved in th e g ly c y lg ly c in e b u f f e r . (Table 21. p 8 8 ).
The f a c t th a t th e r a te o f th e b lank re a c tio n a t pH 8 .3 was a ccep tab le, suggested th a t pH might be an im po rtan t fa c to r, and a study o f the pH p ro f ile o f the b la n k re a c tio n in g ly c y lg ly c in e b u ffe r over the pH range 7 .0 - 8 .5 in d ic a te d a maximum r a te a t about pH 7«5 (Figure 16. p 8 9 ). G-asometric s tu d ie s o f the re a c tio n u sin g s e v e ra l samples o f homogenates evidencing th e hig h b la n k re a c tio n f a ile d to dem onstrate any uptake of oxygen d u rin g th e re a c tio n , and thus excluded any p ro cess in v o lv in g the o x id a tio n o f re s id u a l L-amino a c id s . The use of d ia ly se d homogenates showed no s ig n if ic a n t dim inu tio n of the b lan k r e a c tio n whereas b o ilin g th e homogenate did e f f e c tiv e ly e lim in a te the r e a c tio n . These r e s u lts suggested, th a t w h ilst the re a c tio n was not due to the amino a c id o x id a s e s ,it was n e v e rth e le ss enzymic in n a tu re .