/
6 0
5 0
LU4 0
3 0
'A 2 0 cc/ /
0-2
00-2
0-4
0-6
0-8
Re c i p r o c a l of s u b s t r a t e c o n c e n tr a ti o n ( m m o l l ' )
The problem o f ammonia contam ination o f amino a c id s was a g ain encountered (p 58.) and was d e te c te d m ainly because o f the d if f e r e n t shape o f the re a c tio n tra c e in re s p e c t of the la g re a c tio n .
Subsequently a l l amino acid s o lu tio n s were checked fo r the presen ce of ammonia,using the same re a c tio n m ixture b ut la c k in g the aminp acid o x id a se . Those amino a c id s which co n tain ed only sm all amounts o f ammonia when so te s te d }were allow ed to r e a c t u n t i l a s ta b le
absorbance was achieved and i f th e re was s t i l l an adequate amount of NADHjas in d ic a te d by the absorbance v a lu e , the assay re a c tio n was i n i t i a t e d by th e a d d itio n o f the t e s t enzyme. A sp a rtic a c id ,d u e to th e r e la tiv e ly la rg e amount o f ammonia p re se n t, was tre a te d in b u lk w ith glutam ate dehydrogenase, o x o g lu ta ra te and MDH b efo re checking
" lA
ag ain ^th e manner in d ic a te d .
The s p e c ific itie s o f D-amino a cid oxidase from both l i v e r and kidney were examined in th is way and proved to be very s im ila r
and th e r e s u lts a re l i s t e d in Table 13. (p 60) to g e th e r w ith th e d a ta from the commercial enzyme p re p a ra tio n .
3*3. Amino a c id oxidase co n ten t o f human tis s u e s .
One o f th e i n i t i a l o b je c tiv e s o f th is re se a rc h was to in v e s tig a te the d is tr ib u tio n o f th e amino a cid ox idases in human tis s u e s , w ith a view to c la r if y in g any p o te n tia l r e la tio n s h ip betw een enzyme d is tr ib u tio n and tis s u e d y sfu n c tio n . However
o rg a n is a tio n a l d i f f i c u l t i e s in the l a t e r sta g e s of th e work made i t d i f f i c u l t to o b ta in th e wide range of s u ita b le specimens i n i t i a l l y en v isag ed . As a re s u lt,th e ex p erim en tatio n had to be r e s t r i c t e d to an exam ination o f th e d is tr ib u tio n o f the am ino*acid o x id ases in a lim ite d number of tis s u e s .
The tis s u e s examined were b ra in , h e a rt, kidney, liv e r , lung, and sp leen and were random specimens obtained from post mortems a t a la rg e g e n e ra l h o s p ita l,w ith no p r io r knowledge of th e p re c is e d ise a se p ro cesses in v o lv e d . The tis s u e s were tak en w ith in 12 hours of death and im m ediately fro zen a t -20°C. All th e tis s u e s s e le c te d were s u p e r f ic ia lly norm al in appearance and were from donors in the
55 - 65 y e a rs of age range, death b ein g as a r e s u lt of e ith e r , v a sc u la r d ise a se or secondary m a lig n a n c ie s. In al l te n s e ts o f
tis s u e s were examined.
Homogenates were p repared in the manner d escrib ed (Method 6 . p12 and assayed im m ediately fo r th e presence of D- and L-amino acid
o x id ases usin g bo th the k in e tic and flu o rim e tric assay m ethods. Subsequently th e p ro te in co n ten t o f the homogenates was determ ined u sin g th e ty ro s in e method (p 0126)and th e enzyme co n ten t o f each tis s u e expressed as the number of In te rn a tio n a l U n its of a c ti v ity p e r gram o f so lu b le p ro te in (Table 29. p 105).
The r e s u lts showed th a t of th e six tis s u e s examined , in each case, only th re e c o n s is te n tly showed any amino acid oxidase a c t i v i t y . Kidney g e n e ra lly had th e h ig h e st co n ten t o f D-amino a cid oxidase w ith liv e r c o n ta in in g s im ila r, although u s u a lly low er a c t i v i t i e s . The samples o f b ra in examined showed an a c ti v ity o f D-amino a c id oxidase which was approxim ately one te n th th a t o f k id n ey . Only kidney and
liv e r co n tained any L-amino a cid oxidase, the a c tiv ity o f which was r e la tiv e ly low.
Five sam ples of fr e s h ly sep arated serum from normal a d u lts f a ile d to dem onstrate any D- or L-amino acid oxidase a c ti v ity .
4 . D iscussiono
A m ajor problem encountered d u rin g th e assay o f tis s u e homogenates was th e ra p id u t i l i z a t i o n o f NADH in the presen ce o f th e homogenate a t pH 7.5- The subsequent d e c is io n to measure th e a c ti v ity o f L-amino acid oxidase a t pH o f 7«5 only circum vented the problem w ithout id e n tify in g th e cause. The s e le c tio n o f pH 8 .3 as the assay pH fo r th e enzyme reduced th e observed r a te o f re a c tio n by approxim ately 25$ (F igure 6. p 47) which alth o u g h u n d e sira b le , did n o t have a serio u s e f f e c t on fundam ental s e n s itiv ity o f th e method. C e rta in ly compared w ith the use o f pH 7 .5 th e s e le c tio n o f pH 8 .3 s ig n if ic a n tly improved the s e n s iti v ity (Table 2 4 . p 96) and a lso co n sid erab ly s im p lifie d th e assay procedure in th a t on ly one b u ffe r was re q u ire d fo r b o th a ss a y s .
The presence of g ly c y lg ly c in e was obviously e s s e n tia l fo r th is enzymic g e n e ra tio n o f ammonia although i t s ex act ro le was n o t c le a r . The most lik e ly fu n c tio n was th a t o f s u b s tra te ,a lth o u g h i t a ls o could