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Interference by reactions of kynurenine metabolism in the estimation of tryptophan pyrrolase in rat-liver homogenate
SHORT CO.MMt, N IC:~TION.%
513
t ~,V. R. Cflv.ssRo AND J. H. Eva.~s, B~e~htm. B*ophys. Acta. 58 (zq6z) 538. = G. M. HIt.L~, B,ochem. J., 34 (I940} tO57t W . R. CH~SBRO, Coat. J..%f/~ra~toL, 7 (tO6,} ')32i A. At~I~M.~, R. MCNAMARA AND F. l~. JOHNSON. J. Bi,,l~ Chant.. z3.5 (t~6o} 3659 + V. A. KNlVI6X'T. Bio~hem. J., 56 (I954} 60/o. i H. a . ROBERTS AND M. (~. K t ) L O R . . 4 h a l (;hem.. 31 ( I 9 , $ 9 ] .~6-~. 1' J . } t E I L M A N N , J . }.{AROLI,||'R AND E . W A T Y K I ~ , Z , P~V$lOt r Chem.. 300 (Iq.58} 2 t 9 . H . R O S E N B ~ R G , A . H . E N N O R A.~D J . ~ ' . ~ I o R ~ I ~ O ~ . ~tlCCJ~cot+ J.. 6 3 ~l~S¢~t z53; ~_,. M . ~ I ~ t ' t T AND M . ( ~ , R H A K i ) T , J. Dacteri,d, 7 d , ( ] 9 5 8 } 2 7 6 . "
Receivcxl November
xgth, i96,: D,,.c;*:m .uiophys..-ida. ¢'7 (x963) 511-~t3
s¢x lO33 Interference by reactions of kynurenine metabolism in t h e estimation of tryptophan pyrrolase in rat-liver homogenate
In an investigati,m of aspects of the mechanism of trypto.phan ps~rol,xse induction u s i n g a c o n t i n u o u s - p e r f , t s i o n t e c h n i q u e t. t h e Ye.sult~ o f w h i c h a r e t o b e p u b l i s h e d e l s e w h e r e , a ~ r y p t o p h a n p y t - r o t a ~ e ~ , s a y w a s ~ s t a b l i s h e d u.~in~ m i n o r m o d i f i c a t i o n s o f t h e m e t h o d o u t l i n e d b y K . ~ o x *. T h e s i g n i f i c a n t m o d i f i c a t i o n w a s t h e u s e o f a Dounce, hand-operated h o m o g e x , i z e r * i n s t e a d ,~f a W a v i n g b l e n d e r f o r d i s p e . r s i o n o f t h e t i s s u e . U n d e r t h e ~ e c o n d i t i o n s , in c o n t r a . ~ t t c t h o s e o f p r e v i o u s w o r k c r ~ 4.s. it was found that the kynurenine synthesized during incubation of the homogenate with tryptophan w a s b e i n g f u r t h e r m e t a b o l i z e d . T h e r e f o r e , the. k v n u r e n i n e c o n t e n t o f t h e i n c u b a t i o n m i x t u r e a t t h e e n d o f t h e i n c t t b a t i o n p e r i o d w~.~ n o t a t r u e m e a s u r e of the tryptophan pyrrolase activity. The incubation mixture (total volume 3.omt a n d f i n a l p H 7.o) c o n t a i n e d 9/~moles L-tryptophan. x5o/~moles sodium phosphate buffer ali:t I25 mg rat liver as an homogenate ( z 2 . 5 ~ ( , , w / v , in o . I 4 M KC]). I n c u b a t i o n w a z f o r 2 h a t 37°.
(14 ~ 2
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.320
330
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Wavelength (mpL) Fig. i. A b s o r p t i o n s p e c t r a o f t h e r e a c t i o n p r o d u c t s o f the t r y p t o p h a n pyrrola.~e assay using an h o m o g e n a t e fre~h]y p r e p a r e d w i t h a Dounce homt~genizer a n d using p e a t of t h e ~utne h o m o g e n a t e a f t e r i t M d been frozen in a d r y ice--acetone m i x t u r e a n d i m m e d i a t e l y thawed. Conditions o f tummy were as outltr-~d in the text. Value~ represent the m e a n s of three d e t e r m i n a t i o n s . C>~ O, freshly prepaxed h o m o g c n a t e ; H , frozen a n d t h a w e d h o m o g e n a t e ; . . . . . . authentic L-kynurenitt e. B~och.m. Biophys. Actao 67 (x963) 5 x 3 - ~ t 6
SHORT
5t4
COMMUNICATIONS
A f t e r d e p r o t e i n i z a t i o n a n d n e u t r a l i z a t i o n , t h e a b s o r b a n c v o f t h e supe_rnatant w a s d e t e r n d n e d at i n t e r v a l s t h r o u g h o u t t h e r a n g e o f w a v e l e n g t h s 3 o 9 - 3 7 5 rap. N o difference in levels o f a c " - ' i t y w a s f o u n d b e t w e e n m i x t u r e s i n c u b a t e d in a n a t m o s p h e r e o f air a n d t h o s e i n c u b a t e d in o x y g e n . All v a I u e s g i v e n were c o r r e c t e d for c o n t r o l s i n c u b a t e d w i t h o u t s u b s t r a t e a n d e a c h d e t e r m i n a t i o n w&~ c a r r i e d o u t in triplicate. As s h o w n in Fig. x, C u r v e I. t h e p r o d u c t s o f t h e reacti<>n i n c l u d e d o t h e r c o m p o u n d s besides k y n u r e n i n e w h i c h s h o w e d a b s o r p t i o n w i t h i n t h i s r a n g e . Th~-~e c o m l ~ u n d s were f o u n d t o he t h e p r o d u c t s o f k y n u r e n i n e m e t a b o l i s m . I t w a s f o u n d t h a t s u c h f u r t h e r m e t a b o l i s m o f k y n u r e n i n e or " . ~ c o n d a r y r e a c t i o n s " c o u l d be i n h i b i t e d t o a l a r g e e x t e n t b y f r e e z i n g a n d t h a w i n g t h e h o m o g e n a t e b e f o r e i n c u b a t i o n w i t h s u b s t r a t e (Fig. I, C u r v e 2). T h u s . o n t h e b a s i s o f t h e A ~ 0 ta, TABLE I E F F E C T O F FREI~.ZING A N D T H A W I N G O N T H E A P P A R ] L N T L E V F L O F A C T I V I T Y O F I " R Y P T O P H A N P Y R R O L A S E IN RAT-LXV:ER H O M O G E ~ A ' I ' P . ~
]Ra,.-liver homogcnates were prepaxed using & Dounce, band-operated homogenizer. Tryptoph~n p~Trola~e a~say a.~ outlined in the text was carried out on the homogenates when freshly prepared and after freeziag and thawing, The L-kynurenine sFTtthe~ized ~ _ ~ t-~tlmAted from Atomm~ v,~lucs ush~g a rnolecttlar extinction coefficient of 4.55' ;o~. Nitrogen wa,q eatirnated by nesslerization* and ~ae determinations were t~trried ,nut on trichloroacetie acid precipitates of the homoSenates. VMues represent the mcan~ with ~tandaxd deviation; of 5 determir~tion~. kymur~ ~ Hom~gt~wte
per ~ lim¢~ ( t e a ,t¢.)
~'r t p ~ mS N
.................................
Freshly Frozen
prepared and
thawed
~ . ~ 2 z~ o . t 4
o÷o 5 ~
0.o06
2.48
0.o~
0.008
~_ 0 . 2
~
o f t h e r e a c t i o n p r o d u c t s , it s e e m e d t h a t t h e t r y p t o p h a n p y r r o l a s e a c t i v i t y o f a f r o z e n a n d t h a w e d h o m o g e n a t e w a s a b o u t t w i c e t h a t s h o w n b e f o r e f r e e z i n g ( T a b l e I). H o w e v e r , compa_rLson o f t h e a b s o r p t i o n SlXctra (Fig. x a n d T a b l e l I ) L n d i c a t c d t h a t t h i s w a s n o t n e c e s s a r i l y so. I t is k n o w n t h a t r a t liver c o n t a i n s r e l a t i v e l y h i g h levels o f k y n u r e n i n e t r , m s a m i n a s e ( E C ~.6.1.7) a n d k y n u r e n i n a _ ~ ~ a n d t h e s e c o u l d t h e r e f o r e lead t o t h e p r o d u c t i o n o f k~qxurenic a c i d a n d a n t h x a n i l i c a c i d f r o m k y a ] u r e n i n e . T A B L E II &I$SO)CI~A.~CY RA~']OS OF T H E P R O D U C T ~ O F T H E T R Y I ' 1 " O P H A N P Y R k O L A S E A S S A Y
Homogerutte.~ of normal rat ~ivcr were prepared a.q indicated below and assa .,ed for tryptophan pyrrolase az outlined in the text. Th0 , ~ . . , , . :~ ¢tue Mince* en+irely to L-kynurenine. ~ r ~
~
As a~IAlr. ~
Asm mpJAstt ~#
A ~ m ~ A , ~ t ,~
Dou nee homogenate
Freshly prepared Frozen and t ~ w e d Waring-blendor homosenate 0, 5 rain 2 ~
Authentic z.-kyaurenine
0.85 0.28
z,zz 0.78
1,24 (9} l.zL (lb}
0.43 0.29
0.90 0.78
X.22 (2) 1.20 (I)
o.z6
0.72
s .2o
Biochim. B/opJ~ys. A m , 67 |t963) 5x3-~t¢,
SHOI~.-f C¢')M.MI;N:Z A FI()Ns
~v 5
Assuming the p r e ~ n c e o f kynltr~-r, i u e . k y n u r e n k : a c i d a n d a n t h r a n i l i c ac;.d, t h ~ vahte~ Atea ma, A a a 3 . ~. a n d A ~ , , ~ , we.re r~_.~.,_J...,e¢t using simultaneous cqu~_tionsL The calculated total molar value.~ of kynnrenine, kwturenic acid and anthranilic acid from i n c u b a t i o n m i x t u r e s using freshly prepared homogenate¢, were very. close t o t h o s e o f t h e k y n u r e n i n e p r o d u c e d u ~ m g f r o z e n a n d t h a w e d b, o m o g e n a t e s . H o m o g e n a t e s f r e s h l y p r e p a r e d fl'oln l i v e r s tff r a t s in w h i c h a a~igh l e v e l o f t r y p t o p h a n t)_vrrola*e b a d b e e n i n d u c e d b y p r i o r i n j e c t i o n o f t r y p t o p h a n ° aL~t, s h o w - : . ; further metalxdlsm of the kynurenine synthesized during the assay. FreeT;..g and
t h a w i n g the homoge,aates l~fore assay again greatly reduced the~- "secondary react ions' '. P r e p a r a t i o n of rat-Liver homogenates using a ~3.'aring blendor *-~ for x rain l a r g e l y i n a c t i v a t e d these. " s e c o n d a r y " r e a c t i o n s a s .~l,own in T a b l e I ! , w h i l e u~;e o f an all-glass, machine-driven. Potter-Elvchjem homogenizer did not. However, in the absence of added tryptophan, t i s s u e cli.~per~inn u ~ i n g e i t h e r a V v ' a r i n g b l e n d , r
or a P o t t e r - E l v e h j e m homugenizcr caused rzpid inactivatitm - f tile t,y.i~Lul~hml pyrrokL~e a~s shown in Table III. aT.XBLE I i I F~FI-'Ec'r OF
DII'FERE-'~'T OF
METHtII)9.
TRYPTOP1~IAN
tlv
tiSSUE
G:" T|IE
L~ISPERt/Iu.a
PY . ~ R g | I . A S K
IN
RAT
hCTIV|T"~"
LIVER
Ho,a~t)gelt~t~ o f normal r a t Livers were prc|~n~d a~ in¢licated below a n d assayed for t r y p t o p h a n pyrrola-se as o u t l i n e d in the te x t . During tli,persion using t h e Dounce a n d t h e P o t t e c - E l v c h j e m homogenizers t h e t e m p e r a t u r e of the suspension was maint,xined a t o - z ~. Although chilh;d a t f r e q u e n t interx, ats. the c o n t e n t s o f t h e Waxing blendc, r rose to 3.5 ° after o.5 rain, 7.5 ° after t 1sin. 14 ~ a f t er z rain anti zo" =fter 3 rain. A sample of the o.5-min ht)mogenattt: was prcincubat,:d for z min at zo ~ before addition o f aubstarate. T h e homog, erk~tes prepaxet" with the Dounce and tht: P o t t e r - E l v e h j e m homogenizers were frozt:n and t h a w e d before assay. A c t i v i t y is expressed as 2amoltm k y n u r e n i n e synthesized/g liver {wet wt.)!h. Dq~.per*z~,n lit¢,l~ ( r a i n /
"F:¢~ a / h 0 ~ t n * e e r ~.j
.,
M~,chine drivert Potter- Elvehjem
2~2
1.4
VCaring b le n d e r
x-4
2-7
1.6 °
2. 4
.t. 5
.z
?
I÷tJ
z-5
~.z
z. 4
I÷9
1.3
z+z
z.3
2,5°§ Dounce0
z.4"
hand-opur~t~rd
z. 4
z~3
homogenizJzr § P r e i n c u b a t e d 2 rain at 2 o ~. " Pr¢paxed f r o m th e s a m e pool o f tg~ue~
Storage
of homogenates
at
--20 ° fur period~ from
z h u p t o :3 d a y s
did not
affect t h e t r y . ' q p h a n pyrrolase a c t i v i t y provided each sample was frozen and t h a w e d o n l y t, ...'ce. Freezing and t h a w i n g h o m o g e n a t e s a second and third time c a u s e d a decrease in t r y p t o p h a n pyrrota_se a c t i v i t y of 3 - 4 % each time. In view of these results, h o m o g e n a t e s for u m in t r y p t o p h a n pyrrolase assays were r o u t i n e l y frozen and thawed before incub=_tion w i t h s~bstrate. H o m n g e n a t e s were prepared u s h , g a Dounce, hastd-opernted homogenizer kept at o - z ° in an ice~3iochim, Bi~hys.
A~a, 6 7 ( x 9 6 3 ) 5 t 3 - S x 6
51~
SHORT COMMUNICATIONS
w a t e r b a t h , aa~d a f t e r f i l t r a t i o n t h r o u g h f o u r thick_nesscs o f c h e e s e - c l o t h w e r e rapidly frozen in a dxy ice--acetone bath. Frozen homogenates were either stored a t - - z o ~ f o r s u b s e q u e n t u s e o r i m m e d i a t e l y th:~wed r a p i d l y b y i n t e r m i t t e n t i m m e r sion ~ i t h ~-igorous s h a k i n g in a w a t e r b a t h a t 37*- C a r e w a s t a k e n n o t t o a l l o w t h e t e m p e r a t u r e o f t h e h o m o g e n a t e t o rise a b o v c 4 - 5 " - T h a w e d h o m o g e n a t e s w e r e immediately transferred to an ice-water bath. When homogenates were used without s t o r a g e , i n c u b a t i o n w i t h su[ s t r a t e w a s u s u a l l y b e g u n ,~ithin z o - 3 o rain a f t e r t h e death of the animal. W e w i s h t o t h a n k Misses K. F o ~ . ~ v , I. L t ) x ~ x s a n d M. MORAS f o r t e c h n i c a l a s s i s t a n c e . T h i s i n v e s t i g a t i o n w a s s u p p o r t e d in p a r t b y a g r a n t {C-536o ) f r o m t h e N a t i o n a l C a n c e r I n s t i t u t e , N a t i o n a l I n s t i t u t e s o f H e a l t h , U.S. P u b l i c H e a l t h S e r v i c e . a n d in p a r t b y t h e A n t i - C a n c e r C o u n c i l o f V i c t o r i a , A u s t r a l i a . M. V. JACO .}. F. NELSON S. R o s e
Department of Physiology, U n i v e r s i t y o f M dbot¢r~te, Pavt~,ille, l"ict. ( A u s t r a l i a )
i j . R. LtxrL}:. G. BRECeZk. T. R. Bt~At>L~V ^~x) S. ROSZ, Dlood, I9 (t96z} 236. "- W. E. KNox. in S. P. COLOW[CK At;D N. O. KAPL^~'. Methods in Enzyrnology, Vol. z, Academic Press, N e w York, x955, p. a'42A+ L. L~O~'NCE, R+ ~'. V¢ITTI~R, K . J . MO.~TY, S+ PATI~ AND M. A+ f.~OTXONI~, J . I.liopJ#)'$+ B i o c ; ~ m ,
Gytol., ( I 9 5 5 ) z39,
• W. E. KNOX ^.'~n V. H. ACERn^cX, J. Biol. Chem., 2x4 (t9551 307 • P. FeIGv.t.so~ AND O. GI~ZV,I~G*.t~D. J. Biol. Chem., Z36 I!061) 153. e F. T Di~. C^.~rl~o, R. R÷ BRowr¢ ^.~D J. M. PRicv, J÷ Biol. Chem.. zzS (I957) 777. I. I.. Ml: ~.r~R. M. TStJCnX])A A S D E. A. ADELBVSlIG, J. Biol. Ch$~l.. 203 (i95.~) 205. E. ~,V. K.~ox, Brit. J. Exptl. Pathol., 3~ (~95t) 46~• I{. K. MORTON, BiO~I#e~. l . , 60 (195~) 57~"
R e c e i v e d O c t o b e r zStb, x96z Biochim. Bio.phys. Acfa. 07 ~t963) 513-5z6
sc xIo39
Studies of small inr.estine during d e v e l o p m e n t !1. The intracellular location of intestinal ~-iala~tosidase R e c e n t s t u d i e s h a v e i n d i c a t e d t h a t i n t e s t i n a l ~q-galactosidasc ( E C 3 . z . x . z 3 ) c a n b e scdimentcd with the "nucleax" and microsomal fractions derived from an homogenate ofintest~:~al m u c o s a ~. I n l i v e r s e v e r a l a c i d h y d r o l a s e s axe c o n t a i n e d w i t h i n a l y s o s o m e , w h i c h w h e n d i s r u p t e d will r e l e a s e t h e a c t i v e enz)~rnes s. T h i s s t u d y wa~ d e s i g n e d t o determine more accurately the distribution of //-galactosidase by comparing its c e l l u l a r l o c a t i o n w i t h t h a t o f a c i d p h o s p h a t a s e ( E C 3.x.3.z) a n d ~ - g ] u c u r o n i d a s e (EC3.z.x.3;) (typical lysosomal enzymes) and also with alkaline phosphatase ( E C 3.z.3.x). a n e n z y m e a s s o c i a t e d w i t h t h e b r u s h b o r d e r s. R a t s o f ~ e W t s t a x strait,, z - 4 d a y s o f a g e w e r e u s e d . T h e m e t h o d o f p r e p a r a t i o n of the intestinal homogenate, the centrifugal fraetionation (except for the use of a s o m e w h a t h i g h e r force, 600 x g, f o r . ~ d i m e n t a t i o n o f t h e n u c l e a r f r a c t i o n ) a n d t h e /~/-galactosidase a s s a y , u s i n g l a c t o s e as s u b s t r a t e h a v e b e e n d e s c r i b e d 1. H o m o g e n a t e s Biockim. Bio~hys. AcCa, 67 (x~$) 5s6--St9
513
t ~,V. R. Cflv.ssRo AND J. H. Eva.~s, B~e~htm. B*ophys. Acta. 58 (zq6z) 538. = G. M. HIt.L~, B,ochem. J., 34 (I940} tO57t W . R. CH~SBRO, Coat. J..%f/~ra~toL, 7 (tO6,} ')32i A. At~I~M.~, R. MCNAMARA AND F. l~. JOHNSON. J. Bi,,l~ Chant.. z3.5 (t~6o} 3659 + V. A. KNlVI6X'T. Bio~hem. J., 56 (I954} 60/o. i H. a . ROBERTS AND M. (~. K t ) L O R . . 4 h a l (;hem.. 31 ( I 9 , $ 9 ] .~6-~. 1' J . } t E I L M A N N , J . }.{AROLI,||'R AND E . W A T Y K I ~ , Z , P~V$lOt r Chem.. 300 (Iq.58} 2 t 9 . H . R O S E N B ~ R G , A . H . E N N O R A.~D J . ~ ' . ~ I o R ~ I ~ O ~ . ~tlCCJ~cot+ J.. 6 3 ~l~S¢~t z53; ~_,. M . ~ I ~ t ' t T AND M . ( ~ , R H A K i ) T , J. Dacteri,d, 7 d , ( ] 9 5 8 } 2 7 6 . "
Receivcxl November
xgth, i96,: D,,.c;*:m .uiophys..-ida. ¢'7 (x963) 511-~t3
s¢x lO33 Interference by reactions of kynurenine metabolism in t h e estimation of tryptophan pyrrolase in rat-liver homogenate
In an investigati,m of aspects of the mechanism of trypto.phan ps~rol,xse induction u s i n g a c o n t i n u o u s - p e r f , t s i o n t e c h n i q u e t. t h e Ye.sult~ o f w h i c h a r e t o b e p u b l i s h e d e l s e w h e r e , a ~ r y p t o p h a n p y t - r o t a ~ e ~ , s a y w a s ~ s t a b l i s h e d u.~in~ m i n o r m o d i f i c a t i o n s o f t h e m e t h o d o u t l i n e d b y K . ~ o x *. T h e s i g n i f i c a n t m o d i f i c a t i o n w a s t h e u s e o f a Dounce, hand-operated h o m o g e x , i z e r * i n s t e a d ,~f a W a v i n g b l e n d e r f o r d i s p e . r s i o n o f t h e t i s s u e . U n d e r t h e ~ e c o n d i t i o n s , in c o n t r a . ~ t t c t h o s e o f p r e v i o u s w o r k c r ~ 4.s. it was found that the kynurenine synthesized during incubation of the homogenate with tryptophan w a s b e i n g f u r t h e r m e t a b o l i z e d . T h e r e f o r e , the. k v n u r e n i n e c o n t e n t o f t h e i n c u b a t i o n m i x t u r e a t t h e e n d o f t h e i n c t t b a t i o n p e r i o d w~.~ n o t a t r u e m e a s u r e of the tryptophan pyrrolase activity. The incubation mixture (total volume 3.omt a n d f i n a l p H 7.o) c o n t a i n e d 9/~moles L-tryptophan. x5o/~moles sodium phosphate buffer ali:t I25 mg rat liver as an homogenate ( z 2 . 5 ~ ( , , w / v , in o . I 4 M KC]). I n c u b a t i o n w a z f o r 2 h a t 37°.
(14 ~ 2
F"
§a3
C'
_..-.-a~.- " ~ -
t
3~0
I
.320
330
_i
.~
340
......
____~'_
350
. .
"°'°'o,
t
360
!
2f~
Wavelength (mpL) Fig. i. A b s o r p t i o n s p e c t r a o f t h e r e a c t i o n p r o d u c t s o f the t r y p t o p h a n pyrrola.~e assay using an h o m o g e n a t e fre~h]y p r e p a r e d w i t h a Dounce homt~genizer a n d using p e a t of t h e ~utne h o m o g e n a t e a f t e r i t M d been frozen in a d r y ice--acetone m i x t u r e a n d i m m e d i a t e l y thawed. Conditions o f tummy were as outltr-~d in the text. Value~ represent the m e a n s of three d e t e r m i n a t i o n s . C>~ O, freshly prepaxed h o m o g c n a t e ; H , frozen a n d t h a w e d h o m o g e n a t e ; . . . . . . authentic L-kynurenitt e. B~och.m. Biophys. Actao 67 (x963) 5 x 3 - ~ t 6
SHORT
5t4
COMMUNICATIONS
A f t e r d e p r o t e i n i z a t i o n a n d n e u t r a l i z a t i o n , t h e a b s o r b a n c v o f t h e supe_rnatant w a s d e t e r n d n e d at i n t e r v a l s t h r o u g h o u t t h e r a n g e o f w a v e l e n g t h s 3 o 9 - 3 7 5 rap. N o difference in levels o f a c " - ' i t y w a s f o u n d b e t w e e n m i x t u r e s i n c u b a t e d in a n a t m o s p h e r e o f air a n d t h o s e i n c u b a t e d in o x y g e n . All v a I u e s g i v e n were c o r r e c t e d for c o n t r o l s i n c u b a t e d w i t h o u t s u b s t r a t e a n d e a c h d e t e r m i n a t i o n w&~ c a r r i e d o u t in triplicate. As s h o w n in Fig. x, C u r v e I. t h e p r o d u c t s o f t h e reacti<>n i n c l u d e d o t h e r c o m p o u n d s besides k y n u r e n i n e w h i c h s h o w e d a b s o r p t i o n w i t h i n t h i s r a n g e . Th~-~e c o m l ~ u n d s were f o u n d t o he t h e p r o d u c t s o f k y n u r e n i n e m e t a b o l i s m . I t w a s f o u n d t h a t s u c h f u r t h e r m e t a b o l i s m o f k y n u r e n i n e or " . ~ c o n d a r y r e a c t i o n s " c o u l d be i n h i b i t e d t o a l a r g e e x t e n t b y f r e e z i n g a n d t h a w i n g t h e h o m o g e n a t e b e f o r e i n c u b a t i o n w i t h s u b s t r a t e (Fig. I, C u r v e 2). T h u s . o n t h e b a s i s o f t h e A ~ 0 ta, TABLE I E F F E C T O F FREI~.ZING A N D T H A W I N G O N T H E A P P A R ] L N T L E V F L O F A C T I V I T Y O F I " R Y P T O P H A N P Y R R O L A S E IN RAT-LXV:ER H O M O G E ~ A ' I ' P . ~
]Ra,.-liver homogcnates were prepaxed using & Dounce, band-operated homogenizer. Tryptoph~n p~Trola~e a~say a.~ outlined in the text was carried out on the homogenates when freshly prepared and after freeziag and thawing, The L-kynurenine sFTtthe~ized ~ _ ~ t-~tlmAted from Atomm~ v,~lucs ush~g a rnolecttlar extinction coefficient of 4.55' ;o~. Nitrogen wa,q eatirnated by nesslerization* and ~ae determinations were t~trried ,nut on trichloroacetie acid precipitates of the homoSenates. VMues represent the mcan~ with ~tandaxd deviation; of 5 determir~tion~. kymur~ ~ Hom~gt~wte
per ~ lim¢~ ( t e a ,t¢.)
~'r t p ~ mS N
.................................
Freshly Frozen
prepared and
thawed
~ . ~ 2 z~ o . t 4
o÷o 5 ~
0.o06
2.48
0.o~
0.008
~_ 0 . 2
~
o f t h e r e a c t i o n p r o d u c t s , it s e e m e d t h a t t h e t r y p t o p h a n p y r r o l a s e a c t i v i t y o f a f r o z e n a n d t h a w e d h o m o g e n a t e w a s a b o u t t w i c e t h a t s h o w n b e f o r e f r e e z i n g ( T a b l e I). H o w e v e r , compa_rLson o f t h e a b s o r p t i o n SlXctra (Fig. x a n d T a b l e l I ) L n d i c a t c d t h a t t h i s w a s n o t n e c e s s a r i l y so. I t is k n o w n t h a t r a t liver c o n t a i n s r e l a t i v e l y h i g h levels o f k y n u r e n i n e t r , m s a m i n a s e ( E C ~.6.1.7) a n d k y n u r e n i n a _ ~ ~ a n d t h e s e c o u l d t h e r e f o r e lead t o t h e p r o d u c t i o n o f k~qxurenic a c i d a n d a n t h x a n i l i c a c i d f r o m k y a ] u r e n i n e . T A B L E II &I$SO)CI~A.~CY RA~']OS OF T H E P R O D U C T ~ O F T H E T R Y I ' 1 " O P H A N P Y R k O L A S E A S S A Y
Homogerutte.~ of normal rat ~ivcr were prepared a.q indicated below and assa .,ed for tryptophan pyrrolase az outlined in the text. Th0 , ~ . . , , . :~ ¢tue Mince* en+irely to L-kynurenine. ~ r ~
~
As a~IAlr. ~
Asm mpJAstt ~#
A ~ m ~ A , ~ t ,~
Dou nee homogenate
Freshly prepared Frozen and t ~ w e d Waring-blendor homosenate 0, 5 rain 2 ~
Authentic z.-kyaurenine
0.85 0.28
z,zz 0.78
1,24 (9} l.zL (lb}
0.43 0.29
0.90 0.78
X.22 (2) 1.20 (I)
o.z6
0.72
s .2o
Biochim. B/opJ~ys. A m , 67 |t963) 5x3-~t¢,
SHOI~.-f C¢')M.MI;N:Z A FI()Ns
~v 5
Assuming the p r e ~ n c e o f kynltr~-r, i u e . k y n u r e n k : a c i d a n d a n t h r a n i l i c ac;.d, t h ~ vahte~ Atea ma, A a a 3 . ~. a n d A ~ , , ~ , we.re r~_.~.,_J...,e¢t using simultaneous cqu~_tionsL The calculated total molar value.~ of kynnrenine, kwturenic acid and anthranilic acid from i n c u b a t i o n m i x t u r e s using freshly prepared homogenate¢, were very. close t o t h o s e o f t h e k y n u r e n i n e p r o d u c e d u ~ m g f r o z e n a n d t h a w e d b, o m o g e n a t e s . H o m o g e n a t e s f r e s h l y p r e p a r e d fl'oln l i v e r s tff r a t s in w h i c h a a~igh l e v e l o f t r y p t o p h a n t)_vrrola*e b a d b e e n i n d u c e d b y p r i o r i n j e c t i o n o f t r y p t o p h a n ° aL~t, s h o w - : . ; further metalxdlsm of the kynurenine synthesized during the assay. FreeT;..g and
t h a w i n g the homoge,aates l~fore assay again greatly reduced the~- "secondary react ions' '. P r e p a r a t i o n of rat-Liver homogenates using a ~3.'aring blendor *-~ for x rain l a r g e l y i n a c t i v a t e d these. " s e c o n d a r y " r e a c t i o n s a s .~l,own in T a b l e I ! , w h i l e u~;e o f an all-glass, machine-driven. Potter-Elvchjem homogenizer did not. However, in the absence of added tryptophan, t i s s u e cli.~per~inn u ~ i n g e i t h e r a V v ' a r i n g b l e n d , r
or a P o t t e r - E l v e h j e m homugenizcr caused rzpid inactivatitm - f tile t,y.i~Lul~hml pyrrokL~e a~s shown in Table III. aT.XBLE I i I F~FI-'Ec'r OF
DII'FERE-'~'T OF
METHtII)9.
TRYPTOP1~IAN
tlv
tiSSUE
G:" T|IE
L~ISPERt/Iu.a
PY . ~ R g | I . A S K
IN
RAT
hCTIV|T"~"
LIVER
Ho,a~t)gelt~t~ o f normal r a t Livers were prc|~n~d a~ in¢licated below a n d assayed for t r y p t o p h a n pyrrola-se as o u t l i n e d in the te x t . During tli,persion using t h e Dounce a n d t h e P o t t e c - E l v c h j e m homogenizers t h e t e m p e r a t u r e of the suspension was maint,xined a t o - z ~. Although chilh;d a t f r e q u e n t interx, ats. the c o n t e n t s o f t h e Waxing blendc, r rose to 3.5 ° after o.5 rain, 7.5 ° after t 1sin. 14 ~ a f t er z rain anti zo" =fter 3 rain. A sample of the o.5-min ht)mogenattt: was prcincubat,:d for z min at zo ~ before addition o f aubstarate. T h e homog, erk~tes prepaxet" with the Dounce and tht: P o t t e r - E l v e h j e m homogenizers were frozt:n and t h a w e d before assay. A c t i v i t y is expressed as 2amoltm k y n u r e n i n e synthesized/g liver {wet wt.)!h. Dq~.per*z~,n lit¢,l~ ( r a i n /
"F:¢~ a / h 0 ~ t n * e e r ~.j
.,
M~,chine drivert Potter- Elvehjem
2~2
1.4
VCaring b le n d e r
x-4
2-7
1.6 °
2. 4
.t. 5
.z
?
I÷tJ
z-5
~.z
z. 4
I÷9
1.3
z+z
z.3
2,5°§ Dounce0
z.4"
hand-opur~t~rd
z. 4
z~3
homogenizJzr § P r e i n c u b a t e d 2 rain at 2 o ~. " Pr¢paxed f r o m th e s a m e pool o f tg~ue~
Storage
of homogenates
at
--20 ° fur period~ from
z h u p t o :3 d a y s
did not
affect t h e t r y . ' q p h a n pyrrolase a c t i v i t y provided each sample was frozen and t h a w e d o n l y t, ...'ce. Freezing and t h a w i n g h o m o g e n a t e s a second and third time c a u s e d a decrease in t r y p t o p h a n pyrrota_se a c t i v i t y of 3 - 4 % each time. In view of these results, h o m o g e n a t e s for u m in t r y p t o p h a n pyrrolase assays were r o u t i n e l y frozen and thawed before incub=_tion w i t h s~bstrate. H o m n g e n a t e s were prepared u s h , g a Dounce, hastd-opernted homogenizer kept at o - z ° in an ice~3iochim, Bi~hys.
A~a, 6 7 ( x 9 6 3 ) 5 t 3 - S x 6
51~
SHORT COMMUNICATIONS
w a t e r b a t h , aa~d a f t e r f i l t r a t i o n t h r o u g h f o u r thick_nesscs o f c h e e s e - c l o t h w e r e rapidly frozen in a dxy ice--acetone bath. Frozen homogenates were either stored a t - - z o ~ f o r s u b s e q u e n t u s e o r i m m e d i a t e l y th:~wed r a p i d l y b y i n t e r m i t t e n t i m m e r sion ~ i t h ~-igorous s h a k i n g in a w a t e r b a t h a t 37*- C a r e w a s t a k e n n o t t o a l l o w t h e t e m p e r a t u r e o f t h e h o m o g e n a t e t o rise a b o v c 4 - 5 " - T h a w e d h o m o g e n a t e s w e r e immediately transferred to an ice-water bath. When homogenates were used without s t o r a g e , i n c u b a t i o n w i t h su[ s t r a t e w a s u s u a l l y b e g u n ,~ithin z o - 3 o rain a f t e r t h e death of the animal. W e w i s h t o t h a n k Misses K. F o ~ . ~ v , I. L t ) x ~ x s a n d M. MORAS f o r t e c h n i c a l a s s i s t a n c e . T h i s i n v e s t i g a t i o n w a s s u p p o r t e d in p a r t b y a g r a n t {C-536o ) f r o m t h e N a t i o n a l C a n c e r I n s t i t u t e , N a t i o n a l I n s t i t u t e s o f H e a l t h , U.S. P u b l i c H e a l t h S e r v i c e . a n d in p a r t b y t h e A n t i - C a n c e r C o u n c i l o f V i c t o r i a , A u s t r a l i a . M. V. JACO .}. F. NELSON S. R o s e
Department of Physiology, U n i v e r s i t y o f M dbot¢r~te, Pavt~,ille, l"ict. ( A u s t r a l i a )
i j . R. LtxrL}:. G. BRECeZk. T. R. Bt~At>L~V ^~x) S. ROSZ, Dlood, I9 (t96z} 236. "- W. E. KNox. in S. P. COLOW[CK At;D N. O. KAPL^~'. Methods in Enzyrnology, Vol. z, Academic Press, N e w York, x955, p. a'42A+ L. L~O~'NCE, R+ ~'. V¢ITTI~R, K . J . MO.~TY, S+ PATI~ AND M. A+ f.~OTXONI~, J . I.liopJ#)'$+ B i o c ; ~ m ,
Gytol., ( I 9 5 5 ) z39,
• W. E. KNOX ^.'~n V. H. ACERn^cX, J. Biol. Chem., 2x4 (t9551 307 • P. FeIGv.t.so~ AND O. GI~ZV,I~G*.t~D. J. Biol. Chem., Z36 I!061) 153. e F. T Di~. C^.~rl~o, R. R÷ BRowr¢ ^.~D J. M. PRicv, J÷ Biol. Chem.. zzS (I957) 777. I. I.. Ml: ~.r~R. M. TStJCnX])A A S D E. A. ADELBVSlIG, J. Biol. Ch$~l.. 203 (i95.~) 205. E. ~,V. K.~ox, Brit. J. Exptl. Pathol., 3~ (~95t) 46~• I{. K. MORTON, BiO~I#e~. l . , 60 (195~) 57~"
R e c e i v e d O c t o b e r zStb, x96z Biochim. Bio.phys. Acfa. 07 ~t963) 513-5z6
sc xIo39
Studies of small inr.estine during d e v e l o p m e n t !1. The intracellular location of intestinal ~-iala~tosidase R e c e n t s t u d i e s h a v e i n d i c a t e d t h a t i n t e s t i n a l ~q-galactosidasc ( E C 3 . z . x . z 3 ) c a n b e scdimentcd with the "nucleax" and microsomal fractions derived from an homogenate ofintest~:~al m u c o s a ~. I n l i v e r s e v e r a l a c i d h y d r o l a s e s axe c o n t a i n e d w i t h i n a l y s o s o m e , w h i c h w h e n d i s r u p t e d will r e l e a s e t h e a c t i v e enz)~rnes s. T h i s s t u d y wa~ d e s i g n e d t o determine more accurately the distribution of //-galactosidase by comparing its c e l l u l a r l o c a t i o n w i t h t h a t o f a c i d p h o s p h a t a s e ( E C 3.x.3.z) a n d ~ - g ] u c u r o n i d a s e (EC3.z.x.3;) (typical lysosomal enzymes) and also with alkaline phosphatase ( E C 3.z.3.x). a n e n z y m e a s s o c i a t e d w i t h t h e b r u s h b o r d e r s. R a t s o f ~ e W t s t a x strait,, z - 4 d a y s o f a g e w e r e u s e d . T h e m e t h o d o f p r e p a r a t i o n of the intestinal homogenate, the centrifugal fraetionation (except for the use of a s o m e w h a t h i g h e r force, 600 x g, f o r . ~ d i m e n t a t i o n o f t h e n u c l e a r f r a c t i o n ) a n d t h e /~/-galactosidase a s s a y , u s i n g l a c t o s e as s u b s t r a t e h a v e b e e n d e s c r i b e d 1. H o m o g e n a t e s Biockim. Bio~hys. AcCa, 67 (x~$) 5s6--St9