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Characterization of histidine decarboxylase from rat peritoneal fluid mast cells
392
mOCalmC..~ ET tlIOPt'IYSlC.-k ACTA
CHARACTERIZATION FROM
OF HISTIDINE
l¢2kT P E R I T O N E A L
A 1 ) O l . l r O M. R O T I L S C H I L I ) *
VOL, 34 (x959)
DECARBOXYLASE
FLUID
AND R I C H A R D
MAST CELLS XV. S C H A Y E R
The ,lIerck I~stift~le #~' Th~,vi,peutic l¢esearch, Rahovay, . \ ' . j . (U..S'..4 .) ( R e e c i v e d O c t o b e r 7 t h , 1<158t
SUMMARY
Following fractionation of the cells of rat peritoneal fluid it was d e m o n s t r a t e d t h a t histidine decarboxylase a c t i v i t y is almost e n t i r e l y a_nsociated w i t h t h e m a s t cells. This permits use of u n f r a e t i o n a t e d peritoneal fluid cells as a source of m a s t cell histidine decarboxylase. Pyridoxal p h o s p h a t e is the coenzyme. Results of o t h e r e x p e r i m e n t s on the c h a r a c t e r i z a t i o n of this enzynlc arc reported. I NTROL~UCT.ION
The m a s t cell is a major repository of histamine in mammals~, 2. Release of histamine, along w i t h o t h e r substances from the m a s t celt, is believed to be of i m p o r t a n c e in certain i n f l a m m a t o r y , aUergie, a n d o t h e r pathological states. C o n s e q u e n t l y , characterization of Mstidine decarboxylase from ma.qt cells m a y h a v e a practical as well as a theoretical value. I n a previous publication, one of us z reported t h a t suspensions of r a t p e r i t o n e a l fluid cells were able to decm'boxylate L-[2-1~C]histidine a n d to bind the resulting [v~C]histamilm in a stable mamler. A soluble histidine decarboxy!ase could be o b t a i n e d from these ceils b u t v i r t u a l l y no work could be done on it owing to its low a c t i v i t y in t~m absence of the i n t a c t cell. The a v a i l a b i l i t y of liquid scintillation c o u n t i n g now makes 2urther experiments possible. One purpose of this paper is to present evidence t h a t the mast cells are responsible for virtually all of the histidine d e c a r b o x y l a s e a c t i v i t y of peritoneal fluid'*. H a v i n g d e m o n s t r a t e d this, it was possible to use t h e m i x t u r e of ceils for experiments characterizing this e n z y m e . METHODS
P;'cparagion aJrd assay o/histidine deearboxylase [tom periloJ~ul coils Male, \Vistar r a t s (2oo-3oo g) were injected i n t r a p e r i t o n e a l l y w i t h 4 ml of " T h e p r i n c i p a l p~jrtion of t h i s r e s e a r c h w a s d o n e a t tire R h e u m a t i c F e v e r l l e s e a r c h I n s t i t u t e , Chicagr~, Jllinai.~. F o r t h i s p e r i o d o n e of us (A. M. R o x i t s c m L o ) g r a t e f u l l y a c k n o w l e d g e s t h e g e n e r o u s a s s i s t a n c e ot t i m J o h n S i m o n G u g g e n h e i m F o u n d a t i o n a n d tile C o n s e l h o N a c i o n a l d e Pesquiza.% R i o d e J a n e i r o . t3*asil. " E v i d e n c e o n t h i s p o i n t w a ~ p r e s e n t e d i n t h e caviler p a p e r z. I t w a s s h o w n t h a t willie p e r i t o n e a l l t u i d cells were a c t i v e , r a t b u f i y c o a t h a d v e r y tow a c t i v i t y . T h i s r e a s o n i n g w a s l a t e r c r i t i c i z e d o n t h e b a s i ~ t h a t t h e d i s t r i b u t i o n oI t y p e s of l e u c o c y t e s i a b l o o d is q u i t e difl~erent t r o m t h a t i n p e r i t o n e a l ttutd. A c c o r d i n g l y , we h a v e u n e d a m o r e d i r e c t a p p r o a c h t o t h e p r o b l e m of r e l a t i n g t h e h i s t i d i n e d e c a r b o x y l a s e * t e t i v i t y of p e r i t o n e a l f l u i d t o cell t y p e ,
Relere~ces p. 397[39.7.
e e l . 34 (1959)
rtiSTH)~,~V_ DIt.C.\RI]OXYL.\SY.
393
heparinized saline, d e c a p i t a t e d , exsanguinated, and the a b d o m i n a l wall massaged to dislodge most of the free peritoneal cells. After opening the abdominal c a v i t y the cell suspension was r e n e w e d with a dropper, centrifuged and resuspended in isotonic p h o s p h a t e buffer of p H 7.4 containing o.2% gluc(~.~.. The buffered ~nspension was frozen a n d t h a w e d 5 or 6 time.,; a n d centrifuged to remove cell fragments. The m e t h o d of a_~say of histidine decarboxvla.ge has been described t, s. It involves i n c u b a t i o n of the e n z y m e preparation with L-'~C~tfisti¢line and subsequent determin a t i o n of [~4C]histamine b y isotope dilution. Carrier histamine is isolated,* c o n v e r t e d to d i b e n z e n e s u l f o n y l h i s t a m i n c (13tSH)'and c o u n t e d in a P a c k a r d Tri-Carb Liquid Scintillation Spectrometer. Enzymic, activities are t-xpressed as counts per m i n u t e per xoo mg of B S H , attd are corrected I,)r b a c k g r o u n d and for blank values o b t a i n e d from incubates run w i t h h e a t - i n a c t i v a t e d er, zymc~. 5ttb~tr~.tt," conccntn~tiou was o,o65 /~moles of L-~4C]histidine per ml in all expcrimer~ts.
Cou~t#t~g cell populatio~t o[ rat peritoneal [tHid Mast-cell c o n t e n t s of the suspensions were e s t i m a t e d after dilution and staining w i t h a solution of toluidine bhte in saline. In the perito~eal fluid o[ W i s t a r rats, m a s t ceils c o n s t i t u t e _, to 4% of the total ceils. Total leucocyte c o n t e n t s were obtained following dilution w i t h o. • % acetic acid containing crystal violet. Dilutions were m~de in a white blood cell pipette; aliquots were a d d e d to both chambers of a %pester h e m o c y t o m e t e r a n d the cells counted. The values t)rt~sent~d represent the averages of the c o u n t s o b t a i n e d from duplicate samples of each suspension. The counts agreed w i t h i n 3 to 7?o w i t h each other.
Identi,".'cation o] thc ¢dhtlar ]ractiott. contai~b~g l~istidi~t~" d,.carboxylase Methods for the complete ~eparation of m a s t cells from the remaining peritoneai fluid cells of the rat have been de.~cribed~,v. T h e y employ differential centrifi~gation in h y p e r t o n i c sucrose a n d o b t a i n layers of cells of differellt specific g r a v i t y . We f o u n d t h a t m a s t ceils separated in this m a n n e r h a d w r y little extractable hisfidine deearboxylase a c t i v i t y . This was due to a d a m a g i n g action of the fractionation m e d i u m on t h e cells as is shown b y the following observations: (I} when all of the cells of t h e peritoneal fluid were suspended for 30 rain in the hypertonie sucrose m e d i u m a n d then completely s e d i m e n t e d b y cvntrifugation in the cold, a large a m o u n t of the e~zymic aeti~ i t y leaked out of ti~e cells a n d could be recovered in the s u p e r n a t a n t . I n c o n t r a s t , less t h a n ~o% of the t o t a l a c t i v i t y was found in the s e d i m e n t e d ceils. (2) Sucrose in 'Xself did not affect the a c t i v i t y o[ the eazvme once it had be~-n released from the cells. No changes in a c t i v i t y were .~bserved when sucrose, ill a m o u n t s up to 75 mg/ml, was a d d e d to ce].l-free e x t r a c t s obtained i~x the usual m a n n e r . In an a t t e m p t to find a suitable m e t h o d for separating active m a s t cells, concent r a t e d serum albnnfin solutions were tried but did not show promise. Therefi)re, a partial separation of tna~t cells from leucocytes in balanced salt solution w,~s used. Pooled peritoneal fluid cells were suspended in i-~ ~.~-K'~ balaaced ~:~it solution s, 3 ml being used for the cells from one rat. T~vo ml of this suspension were placed in tapered, blood-cen~rifug,." tubes of ~-5 m! capacity, a n d centrifuged for 3-5 min at a p p r o x i m a t e l y 500 rev./min in a horizontal I n t e r n a t i o n a l CKnical Centrifuge. The lowest o.~ ml of the suspensiot~ weft: collected with a drawn-out mcdicil~e dropper. This fraction, called (A), c o n t a i n e d from 5o to 65% of the total mast-cell c o n t e n t .l¢elare~ces p. 397/39,~.
394
;~. m. I¢OTHSCHILD, R. "~V.SCHAYER
VOL. t g (X959) "
of t h e o r i g i n a l s u s p e n s i o n , a n d s h o w e d a 2.5-fold r e l a t i v e i n c r e a s e in its m a s t cell] leucocyte ratio when c o m p a r e d with the u n f r a c t i o n a t e d suspension. To obtain a f r a c t i o n c o n t a i n i n g a l o w e r e d m a s t c e l l / l e u c o c y t e r a t i o , t h e r e m a i n i n g p a r t of t h e cell s u s p e n s i o n was c e n t r i f u g e d a t t h e s a m e t~peed f o r a n a d d i t i o n a l 5 rain a n d t h e Imvest o . : m l a g a i n r e m o v e d . This f r a c t i o n w a s d i s c a r d e d . T h e r e m a i n i n g u p p e r l a y e r s f l o m s e v c r a l t u b e s w e r e p o o l e d m i d c e n t r i f u g e d a t 18oo r e v . / m i n f o r 5 rain. ] ' h i s r e s u l t e d in t h e s e d i m e n t a t i o n of all t h e r e m a i n i n g ceils, a n d a f r a c t i o n , c a l l e d (B), h a v i n g a mast cell/leucocyte ratio 4 to 5 times lower t h a n t h a t of the u n f r a c t i o n a t e d fluid, w a s o b t a i n e d . H i s t i d i n e d e c a r b o x y l a s e w a s e x t r a c t e d f r o m t h e ceils of e a c h o f t h e s e f r a c t i o n s a n d a s s a y e d in t h e m a n n e r d e s c r i b e d . EXPERIMENTS AND RESULTS
Correlation botweeJr histfil.in.e decarboxylase activity and mast-cell content o/rat peritoneal ~qzdd cell ]factions. I n T a b l e I t h e e n z y m i c a c t i v i t y e x t r a c t a b l e f r o m a m i x e d cell f r a c t i o n , (A), h a v i n g a m a s t c e l l / l e u c o c y t e r a t i o of a b o u t t : i o , is c o m p a r e d w i t h t h a t o b t a i n e d f r o m a f r a c t i o n , (B), h a v i n g a m a s t c e i l ] l e u c o c y t e r a t i o of less t h a n I :IOO. T h e n u m b e r of f r a c t i o n t3 cells w e r e so c h o s e n t h a t e q u a l a m o u n t s o f l e u c o c y t e s w e r e u s e d for e x t r a c t i o n in b o t h cases. T h e r e s u l t s of t h e t h r e e e x p e r i m e n t s s h o w n i n d i c a t e t h a t a m - o h higher histidine d e c a r b o x y I a s e a c t i v i t y was associated with the m a s t cell e n r i c h e d f r a c t i o n . T h e low ~ c t i v i t i e s f o u n d in t h e (B) f r a c t i o n s c a n be a t t r i b u t e d a l m o s t e n t i r e l y t o t h e few m a s t cells p r e s e n t . TABLE I I-tlSTII)INIg OBTAINE[) FROM
l-xpt.
1
II Ii1
Crr! /ro*tion
DI,:CARBOXYLASE ACTI'CIT~t" I,..~RTIAI.I.%" t"RACTION.e~TED
Ctllular c*mlct*t be/ore extraction .llast dells LeU¢oc~,tes
OF" C I ~ L L - } ' R E E E X T I ~ A C ' F . ~, RAT PERITO'?gEAL FLIa'll) CIgLLS
Rati,~: ,~,tI~$l On,-.. i h iI(6L)'It'S
Enzymic aaivitv (¢Oflflf.S;J~li~tllO0 n~g ~JS~'~)
A
6, 7 . to:'
7 7 ' ~o~
J:n1
B
0. 7. ~o:'
76. ~o~'
2 : uo8
(,
A
7.7" ~°~
S4- Io ~
r : ux
84
t-~
O , 7 " I o "5
S 4 ' I0 ~
I ; 1 2¢)
17
5.2. lO-'
51- no"~
n : no
i_~5
I :210
92
A l'~
0,~5"
IO5
33"
I0"~
t i
7
Ctmracterizatio~, o / ~ a s t cell histidi~,e decarboxylas~* ; effect o / p H . A c t i v i t y o f t h e e n z y m e of t h e ceil-free e x t l a c t was o p t i m a l o v e r a f a i r l y b r o a d z o n e , r a n g i n g f r o m p H 6. 5 t o 7.6. O u t s i d e of t h e s e limits t h e a c t i v i t y d r o p p e d r a p i d l y a n d w a s l o w a t p H 6.0 a n d 8.o. Mast cell histidine d e c a r b o x y l a s e d i d n o t s h o w a s h a r p m a x i m u m a c t i v i t y as d o histidine d e c a r b o x y l a s e p r e p a r a t i o n s f r o m o t h e r sources~, 1°. All s t u d i e s r e p o r t e d in this p a p e r were d o n e a t p H 7.4. " Some of the techniques commonly used in the characterization of enzymes had to be omitted roving to the e.~tremely low substrate concentration used. This low substrate concentration is essentiaJ_ to the detection and q~a,~Littttivc estimation of mammalian histidine decarboxylase from man) sonrees ~. For example metal ions such as cobalt, and sulfhydryl blocking agents such as p-chloromerct,r'_,ber.zoate might give 9ptlrioll~ enzyme-inhibiting effects when they are in fact forming complexes with the substrate.
Re[ere~,ces p. 397 /39;~'.
VOL. 114 (1959)
HISTIDINE m'C.Xnt~OX-YL.xsr
395
Effect o/e~zvme concentration.. F;g. 1 s h o w s t h e effect of i n c r e a s i n g a m o u n t s of e n z y m e o n t h e d e c a r b o x y l a t i o n of a eol~stant a m o u n t of s u b s t r a t e . I n c r e a s e s in e n z y m e c o n c e n t r a t i o n o v e r a f o u r - f o l d r a n g e , p r o d u c e d p r o p o r t i o n a l increases in activity. "'160 ur) 140
OE12Q 0 .~G10C £ 80 0
60 4c
•~ 9O < 0
|
0.2
I
I
0.4
0.6 ml
!
08
enzyme
F i g , i. E l f e e t t)[ c o n c e n t r a t i o n of t),jstit|ine £1ecarl.)¢)xyluse f r o m r a t perittn~enl ttuid masL c e l l s ¢)n histamine formation.
Effect o/ carbonyl group reagents. As s h o w n in T a b l e II, m a s t cell histidine d e e a r b o x y l a s e is m a r k e d l y a f f e c t e d b y s e m i c a r b a z i d e a n d b y h y d r o x y l a m i n e , b o t h o f w h i c h a r e p o t e n t b l o c k i n g a g e n t s for c a r b o n y l g r o u p s . T h i s i n h i b i t i o n , w h i c h is o b s e r v e d w i t h m a n y a m i n o acid d e c a r b o x y l a s e s , s u g g e s t e d t h a t p y r i d o x a l p h o s p h a t e m i g h t be t h e c o e n z y m e . The p y r i d o x a l p h o s p h a t e r e q u i r e m e n t of m a s t ceil h i s t i d i n e decarboxylase was therefore investigated. TABLb5 II LFFEGT
OF RA]:
tt~'DROXYLAMINE PERITONEAL
FLIYll)
AND
OF
S['2.MIGARt:I:XZll)E ON
.MA,GT C E L L
IliSTII)INE
THE
ACT[V1TY
OF
D~CARBOX~,'LASi':
Enzyme and inhibitor were pre-incubated for t 5 rain at 3? " pri,~r to the'addition of the st, b.~trate. E n z y m i c activ~ly (~ol|lll.~¢~litl~ lr~o mg / q N H /
[..lhibitor
~u 12tki:n'Ha~
Nolle
2 1 tl
--
Hydroxylamine, re -4 31 Hydrt~xyhtnllne, t o -~ .1I
3 124
too 4l
None
202
Semicarbazide. Semicarbazide, Semicarbazide,
xo -a 31 t o~t 3/ lo "'a .Xl
o 33 104
=~-
Ioo .q8 26
Effecl o/pyridoxal phosphate o~ the aativily o/ dialyzed #r@arations o/ mast cell histidiue decarboxylase. Cell-free e x t r a c t s of r a t p e r i t o n e a l fluid were d i a l y z e d for I 8 h a g a i n s t o . I A I p h o s p h a t e b u f f e r in t h e cold. A c t i v i t y w a s t h e n m e a s u r e d a n d c o m p a r e d x ~ t h t h a t o b t a i n e d f o l l o w i n g t h e a d d i t i o n of o.12o ~ m o l e s / m l of p y r i d o x a l - 5 - p h o s phate to the dialyzed extract. R e s u l t s (eounts/min:'IOO lng B S H ) obtM~.led in d u p l i c a t e w i t h t h e d i a l y z e d e x -
Re]erences f . .197] 3 9 A'.
396
. x . M . R O T H S C H I L D , R, W . NCIt:kYER
VOL, ~ 4
(t959)
tracts were 4 ° and 12o averaging 80; after adding pyridoxal p h o s p h a t e activities
were IIiO and II8O, averaging xx5o. Another experiment confirmed these results. Thus,
a marked
potentiating
effect of this coenzyme
was evident'.
R e v e r s a l o~ h y d r o x y l a ~ i ~ e - i n d u v e d i ~ d d b i t i o ~ o f ~ t a s t ~ell h i s t i d i n e d e c a r b o x y l a s e b y p y r i d o x a l phosphate,. T h e e f f e c t o f p y r i d o x a l p h o s p h a t e on inhibition of mast cell
histldine decarboxylase by hydroxylamine was tested. Results (counts/min/Ioo mg BSH) of the four groups of the experiment arc: (a) enzyme only; 126, ]26 and 134: average 129 counts/rain. (b) enzyme plus hydroxylamine IO -'t M ; all samples had zero activity. (c) enzyme plus hydroxylamine Io -4 M plus pyridoxal phosphate, approximately 1o ~ M; all samples had negligible activity, 4 counts/rain average. (d) enzyme plus hydroxglamine IO-~ M plus pyridoxal phosphate, 4.~o -a M ; 156, IS6 and 2t 7 counts/rain, average x86 °* Another experiment confirmed these results. "1",,\ B L E tNHIIII'I'IflN
el:
R..ST .MA.NT ( 'I ~I .L
tI|NTIllINI~
I11
~)I':C,~,]~.f|OX'~'LA.qE ~[1"~" A R O M A T I C
AMINO
AI.'I|)-N
% lnhib;tiol~*
.4 m itzo acid k
l~:.-5-hydroyy Tryptophan
itL-3"ryl~tophan zJL-TVrosino
0. 4 . 0.8" t.5, 3.2,
t o - ~ .11 IO -a 3I 10 -a 31 tO - ~ . 1 /
25 3~
5o 7 ~,
o . ~ - l o -n 31
to
3,2" lo -'~ .lI
2-
3.2"
40
t
o -a .lI
DL'i>h°||vl~tl:tlli|le 3 . ; " ] ° - a -1'1
]t
" T h e a v e r a g ~ , d r e s u l t s oI two, o r t h r t ' e s e p a r a t e e x p e r i m e l l t s , r n n in duplieatL,, a r e r e p o r t e d . ~lht-sc e x p e r i m e n t s ga~'e e s s e n t i a l l y i d e n t i c a l r e s u l t s , l n h i t d t o r a n d s u b s t r a t e w e r e m i x e d p r i o r t o Ilm a¢tdi~i,~n of e n z y n l e .
h~hibition of mast c¢ll histidD,c ,tecarboxylase activity by aromatic a~in,a achts. V¢i:rlA.: .~xD Koctt tt showed that tile activity of guinea pig kidney histidine decarboxylase could be inhibited to v a r y i n g e x t e n t s b y different a m i n o acids. R i n g h y d r o x y l a t i o n increased the i n h i b i t o r y c a p a c i t y of the aromatic a m i n o acids, t y r o s i n e being a c o m p a r a t i v e l y weak inhibitor while d i h y d r o x y p h e n y l a l a n i n e was a strong one. W e have found a similar effect on mast cell histidine decarboxylase. The d a t a of Table 1I I s h o w t h a t the h y d r o x y l - s u b s t i t u t e d a m i n o acids are m u c h stronger inhibitors
than unsubstituted ones. " A p o t e n t i a t i n ~ e f f e c t of p y r i d o x a l p h o s p h a t e w a s a l s o n o t e d a f t e r i t s a d d i t i o n t o a m a s t cell e x t r a c t ~ h i c h h a d b e e n f r o z e n tor 18 la. T h e i r o z e n e n z y m e g a v e h i s t i d m e d e c a r b o x y l a s e a c t i v i t i e s of 4 5 0 :and 47 ° c o u n t s / n u n ; a d d i t i o n o l t h e c o e n z y m e r a i s e d t h e s e t o 97 ° a n d l o 8 o . "i-hus, t o s s of p y r i d ( m a l p h o s p h a t e p r o b a b l y a c c o u n t s for n~..uch of t h e d r o p i n a c t i v i t y o b s e r v e d in f r o z e n m a s t cell h i s t i d i n e d e c a r b o x y l a s e p r e p a r a t i o n s 3, *" T h e e n h a n c e m e n t o f a c t i v i t y t o a l e v e l h i g h e r t h a n t h a t of t h e c o n t r o l s h a s b e e n o b s e r v e d r e p e a t e d l y a n d i n d i c a t e s t h a t e v e n t h e f r e s h e n ~ y n l e is n o t s a t u r a t e d w i t h c o e n z y m e , T h i s is p r o b a b l y d u e t o t h e p r e s e n c e in t h e c r u d e e x t r a c t o f p h o s p h a t a s e s w h i c i l d e s t r o y p y r i d o x a l phnsphate.
12e]eren~es p. 397¢' ~9a',
VOL. ~ 4
(r959)
HIS'rIDINE
D E C . \ R I ' O X Y I ASE
397
Effect o/benzene. The histidine decarboxylase a c t i v i t y of e x t r a c t s of rat peritoneal fluid m a s t ceils is inhibited to the e x t e n t of 87% when one drop of benzene is a d d e d to the incubation m e d i u m . The m a s t cell e n z y m e differs, therefore, from rabbit k i d n e y histidine deearboxy!ase, which, as shown b2,: W.vrox '~, is e n h a n c e d in a c t i v i t y b y the a d d i t i o n of benzene. The mast cell enzyme hehaves in this respect like rat s t o m a c h histidine decarbox3?lase, which is also s t r o n g l y iahibited by benzene ~. DI~(; t7 SSI{ }N
We have d e m o n s t r a t e d t h a t in rat peritoneal fluid the mast ceils arc a m u c h rictlt~r source of histidine decarbo~ylase t h a n the wl-,ite cells. Thus it seems valid to interpret findings on histidine decarboxylast., of perittmeaI fluid a.~ being due almost exclusively to the e n z y m e in the m a s t ceils. P y r i d o x a ! p h o s p h a t e is the cocz~zvme of rat mast cell hisfidine decarboxylase. It has been de.scribed as the coenzyme of m a n y a m i a o acid decarboxy!ases, a n d its effect in p o t e n t i a t i n g the formation of histamine t)y e x t r a c t s from guinea pig a n d pig k i d n e y i n c u b a t e d with L-histidine has been reported b y WERLE u and HOLrrZVL However, these k k l n e y e n z y m e s differ considerably in properties from m a s t celt histidine decarboxylase a n d findings from studies on t h e m are not necessarily valid for e n z y m e from o t h e r sources% P y r i d o x a l phosphate seems to be m u c h more firmly b o u n d to the k i d n e y e n z y m e t h a n to the mast cell enzwne. W,.'RLE failed to obserw. a drop in a c t i v i t y of dialyzed rabbit k i d n e y histidine decarboxvlase~L The effect of this eoenzyme, in reversing the blocking of mast cell t,istidine decarboxylase b y h y d r o x y l a m i n e suggests t h a t the carbonyt group of "pyridoxal p h o s p h a t e is involved in the enzymic d e c a r b o x y l a t i o n 1-'. Mast cell histidine decarboxylase interacts with other amino acids besides histidine: this can result in r e t a r d a t i o n of the rate of d e c a r b o x y l a t i o n of Ldfistidine {Table I I l ) . METZLER~" has reported the a b i l i t y of p y r i d o x a l phosphate to combine a t n e u t r a l p H w i t h a m i n o acids and aanines to h)rm imino c o m p o u n d s (Schiff bases) which arc p r o b a b l y i n t e r m e d i a t e s in the enzymic d e c a r b o x y l a t i o n of the amino acids. Such complexes could inhibit the flmction ;}f the enzyme. The m a r k e d effect of ring h y d r o x y t a t i o n on the i n h i b i t o r y c a p a c i t y of the a m i n o acids tested could be explained as being due to an additional, direct binding of the amino acid to the e n z y m e via t h e phenolic h y d r o x y l group. Finally, our finding t h a t addition of pyridoxal phosphate increases a c t i v i t y to above t h a t of the fresh e n z y m e suggests t h a t the coenzyme is loosely bound in histidine decarboxylase a n d t h a t in future studie.~ exce.*s coenzyme should be a d d e d to insure m a x i m u m a c t i v i t y .
I l t ' H c I " R E N t" 1";.%
1 j . R , R1L~ZY .X.~D G. 1~. W E S T , J . Phvsi~)t., I_'o (i,)33) 5zS. H . T . GR.~,HAM, O . H . I.(.,WRY, N. VCAHL :',.~o ~I. K. [:'Rtl~'B."-rr, [. ENI~H..'Lied., 1o2 (19.55) 307 , I~.. W . SCnAYER, .-Ira. J . . P h y s i o [ , , IS*> (t~)5O) r u e . •t R . ~,V. SC|IAYER, K , J, I]tAVl~ AND 1¢. l...%~,tlLE',-, .4m. g Physi~,t., I.~2 ( I 9 5 5 ) 54R . I,V. SeHA'~'F.R A,-,'o A. C. iv'.-, .Ira. J . t"~y~i'q., :~q (:-~37) 3(x~ . II j . PADAWER AND A. GOlCl;ON, Pr, w. Noc. l'~xptt, i t i O l . . l i e d . , 82 (1055) 20. 7 O . GLICK, S. L. BONTING .XXD I). Dt.:N Ht)ER, Prec..b'¢~c. Expt[. B i o l . . l i e d . . 02 {~95~) 357.
398
a.
M, R O T H S C H I L D ,
R . W . S(3HAYi?-I~
VOL. 34
(x959)
J, ltA.~K, J. Cellular Comp. PhysioL, 3t (1948) -'35. 0 R. \V. SCH.a,V~n.Am, J. Physical., tg9 (I957) 533.
~0 1{, \~'. S C H A Y E R AND Y . | ( O B A Y A S H I . P¥OC. Soc. k'xptl. Biol. ,lled., 92" (19,q6~ 6 5 3 . 11 |~, '~.~:'EI'.tLF.. AND X,,~.:'. [{O'Ctl, Biochel~. Z . , 3 I O (l(.j',l(~) 3 0 5 . x,2 N. G. XV^ToX, Biochem. ,],, 64 (1950) 3 I8. I.O p . }~IOLTZ. A , E N G E L H A R D T A . ~ l ) G . T}IltCLI~,CKE, ~\'aturu2issenst:hapen, 3 9 ( I 0 . 5 2 ) 2 6 0 . 14 E , ~?ERLF, AND K , K R A U T Z U S , 13iochem. Z . , "-96 (1938) 3 1 5 . la 1), AIE'FZLER, ~[, IKAVCA AND E . E . SN1~LL, J . ,4~I. Chel~t. S o t . , 7 ° (I~)~4) 0 4 8 . '16 1). 31ETZLER, J . Jlp$t. CJl,.ln. Sot-., 7~) ( 1 9 5 7 } .1.85 •
PRODUCTIQN
OF PENTOSE OF NOCARDIA
SAPROPHYTIC
INTERMEDIATES OPACA
SOIL NOCARDIAS
DURING
GROWTH
AND OTHER AND MYCOBACTERIA
R. B. DUFF AND D. M1 W K ] " ] ] . } ~ l
r
l)epartme~2t o] Biochemistry a*~d Section o/ A4ic~,obiology, The a~fa6c*~lay Institute /or So~l Research, A bevdee~z (Great Britain)
(Received October ] st, x958)
SUMMARY :. During g r o w t h in simple media N o c a r d i a oi)aca (strain Tt6 ) a n d some o t h e r s a p r o p h y t i c soil nocardias a n d m y c o b a c t e r i a liberate quau/zities ¢i s e d o h e p t u l o s e a n d ribulose which can be d e t e c t e d b y a p p l y i n g as little as lO-2o /~l of m e d i u m directly to the paper c h r o m a t o g r a m . An u n k n o w n s u b s t a n c e which gave a s p e c t r u m similar to t h a t of pentuiose in the c y s t e i n e - c a r b a z o l e reaction was also f o r m e d b u t this was neither ribulose xylulose, nor erythro-3-pentulose. 2. Liberation of the pentose cycle c o m p o n e n t s occurs more q u i c k l y a n d generaUy in greater a m o u n t s when the m e d i u m is kept a t a b o u t p H 7,o (e.g, b y a d d i t i o n of calcium carbonate, b y using an increased p h o s p h a t e buffer c o n c e n t r a t i o n , b y using sodium n i t r a t e r a t h e r t h a n a m m o n i u m s u l p h a t e as a nitrogen source or by using sodium gluconate as a source of carbon). There seems no reason to suppose t h a t calcium ion has a n y specific effect on t h e s y s t e m . 3 - S e d o h e p t u l o s e and ribulo,~e from the above cultures were identified a n d estimated by paper c h r o m a t o g r a p h y a n d by application of t h e cy÷:tehle-sulphuric acid and orcinol reactions respectively to larger q u a n t i t i e s isolated b y c o l u m n c h r o m a t o g r a p h y . D i h y d r o x y a c e t o n e (characterised as t h e 0~acetate) was o b t a i n e d o n l y when the cultures were kept at a b o u t p H 7 . o (i.e. b y a d d i t i o n of calcium carbonate}. This a n d the failure to produce d i h y d r o x y a c e t o n e from sodium g l u c o n a t e suggests t h a t the triose is m a i n l y a product of the E m d e n - M e y c r h o f - p a t h w a y . Aldolase (with fructose diphosphate as a substrate) is present in cell-free e x t r a c t s of *he bacteria.
Re/ece~tces p. 4o6.
mOCalmC..~ ET tlIOPt'IYSlC.-k ACTA
CHARACTERIZATION FROM
OF HISTIDINE
l¢2kT P E R I T O N E A L
A 1 ) O l . l r O M. R O T I L S C H I L I ) *
VOL, 34 (x959)
DECARBOXYLASE
FLUID
AND R I C H A R D
MAST CELLS XV. S C H A Y E R
The ,lIerck I~stift~le #~' Th~,vi,peutic l¢esearch, Rahovay, . \ ' . j . (U..S'..4 .) ( R e e c i v e d O c t o b e r 7 t h , 1<158t
SUMMARY
Following fractionation of the cells of rat peritoneal fluid it was d e m o n s t r a t e d t h a t histidine decarboxylase a c t i v i t y is almost e n t i r e l y a_nsociated w i t h t h e m a s t cells. This permits use of u n f r a e t i o n a t e d peritoneal fluid cells as a source of m a s t cell histidine decarboxylase. Pyridoxal p h o s p h a t e is the coenzyme. Results of o t h e r e x p e r i m e n t s on the c h a r a c t e r i z a t i o n of this enzynlc arc reported. I NTROL~UCT.ION
The m a s t cell is a major repository of histamine in mammals~, 2. Release of histamine, along w i t h o t h e r substances from the m a s t celt, is believed to be of i m p o r t a n c e in certain i n f l a m m a t o r y , aUergie, a n d o t h e r pathological states. C o n s e q u e n t l y , characterization of Mstidine decarboxylase from ma.qt cells m a y h a v e a practical as well as a theoretical value. I n a previous publication, one of us z reported t h a t suspensions of r a t p e r i t o n e a l fluid cells were able to decm'boxylate L-[2-1~C]histidine a n d to bind the resulting [v~C]histamilm in a stable mamler. A soluble histidine decarboxy!ase could be o b t a i n e d from these ceils b u t v i r t u a l l y no work could be done on it owing to its low a c t i v i t y in t~m absence of the i n t a c t cell. The a v a i l a b i l i t y of liquid scintillation c o u n t i n g now makes 2urther experiments possible. One purpose of this paper is to present evidence t h a t the mast cells are responsible for virtually all of the histidine d e c a r b o x y l a s e a c t i v i t y of peritoneal fluid'*. H a v i n g d e m o n s t r a t e d this, it was possible to use t h e m i x t u r e of ceils for experiments characterizing this e n z y m e . METHODS
P;'cparagion aJrd assay o/histidine deearboxylase [tom periloJ~ul coils Male, \Vistar r a t s (2oo-3oo g) were injected i n t r a p e r i t o n e a l l y w i t h 4 ml of " T h e p r i n c i p a l p~jrtion of t h i s r e s e a r c h w a s d o n e a t tire R h e u m a t i c F e v e r l l e s e a r c h I n s t i t u t e , Chicagr~, Jllinai.~. F o r t h i s p e r i o d o n e of us (A. M. R o x i t s c m L o ) g r a t e f u l l y a c k n o w l e d g e s t h e g e n e r o u s a s s i s t a n c e ot t i m J o h n S i m o n G u g g e n h e i m F o u n d a t i o n a n d tile C o n s e l h o N a c i o n a l d e Pesquiza.% R i o d e J a n e i r o . t3*asil. " E v i d e n c e o n t h i s p o i n t w a ~ p r e s e n t e d i n t h e caviler p a p e r z. I t w a s s h o w n t h a t willie p e r i t o n e a l l t u i d cells were a c t i v e , r a t b u f i y c o a t h a d v e r y tow a c t i v i t y . T h i s r e a s o n i n g w a s l a t e r c r i t i c i z e d o n t h e b a s i ~ t h a t t h e d i s t r i b u t i o n oI t y p e s of l e u c o c y t e s i a b l o o d is q u i t e difl~erent t r o m t h a t i n p e r i t o n e a l ttutd. A c c o r d i n g l y , we h a v e u n e d a m o r e d i r e c t a p p r o a c h t o t h e p r o b l e m of r e l a t i n g t h e h i s t i d i n e d e c a r b o x y l a s e * t e t i v i t y of p e r i t o n e a l f l u i d t o cell t y p e ,
Relere~ces p. 397[39.7.
e e l . 34 (1959)
rtiSTH)~,~V_ DIt.C.\RI]OXYL.\SY.
393
heparinized saline, d e c a p i t a t e d , exsanguinated, and the a b d o m i n a l wall massaged to dislodge most of the free peritoneal cells. After opening the abdominal c a v i t y the cell suspension was r e n e w e d with a dropper, centrifuged and resuspended in isotonic p h o s p h a t e buffer of p H 7.4 containing o.2% gluc(~.~.. The buffered ~nspension was frozen a n d t h a w e d 5 or 6 time.,; a n d centrifuged to remove cell fragments. The m e t h o d of a_~say of histidine decarboxvla.ge has been described t, s. It involves i n c u b a t i o n of the e n z y m e preparation with L-'~C~tfisti¢line and subsequent determin a t i o n of [~4C]histamine b y isotope dilution. Carrier histamine is isolated,* c o n v e r t e d to d i b e n z e n e s u l f o n y l h i s t a m i n c (13tSH)'and c o u n t e d in a P a c k a r d Tri-Carb Liquid Scintillation Spectrometer. Enzymic, activities are t-xpressed as counts per m i n u t e per xoo mg of B S H , attd are corrected I,)r b a c k g r o u n d and for blank values o b t a i n e d from incubates run w i t h h e a t - i n a c t i v a t e d er, zymc~. 5ttb~tr~.tt," conccntn~tiou was o,o65 /~moles of L-~4C]histidine per ml in all expcrimer~ts.
Cou~t#t~g cell populatio~t o[ rat peritoneal [tHid Mast-cell c o n t e n t s of the suspensions were e s t i m a t e d after dilution and staining w i t h a solution of toluidine bhte in saline. In the perito~eal fluid o[ W i s t a r rats, m a s t ceils c o n s t i t u t e _, to 4% of the total ceils. Total leucocyte c o n t e n t s were obtained following dilution w i t h o. • % acetic acid containing crystal violet. Dilutions were m~de in a white blood cell pipette; aliquots were a d d e d to both chambers of a %pester h e m o c y t o m e t e r a n d the cells counted. The values t)rt~sent~d represent the averages of the c o u n t s o b t a i n e d from duplicate samples of each suspension. The counts agreed w i t h i n 3 to 7?o w i t h each other.
Identi,".'cation o] thc ¢dhtlar ]ractiott. contai~b~g l~istidi~t~" d,.carboxylase Methods for the complete ~eparation of m a s t cells from the remaining peritoneai fluid cells of the rat have been de.~cribed~,v. T h e y employ differential centrifi~gation in h y p e r t o n i c sucrose a n d o b t a i n layers of cells of differellt specific g r a v i t y . We f o u n d t h a t m a s t ceils separated in this m a n n e r h a d w r y little extractable hisfidine deearboxylase a c t i v i t y . This was due to a d a m a g i n g action of the fractionation m e d i u m on t h e cells as is shown b y the following observations: (I} when all of the cells of t h e peritoneal fluid were suspended for 30 rain in the hypertonie sucrose m e d i u m a n d then completely s e d i m e n t e d b y cvntrifugation in the cold, a large a m o u n t of the e~zymic aeti~ i t y leaked out of ti~e cells a n d could be recovered in the s u p e r n a t a n t . I n c o n t r a s t , less t h a n ~o% of the t o t a l a c t i v i t y was found in the s e d i m e n t e d ceils. (2) Sucrose in 'Xself did not affect the a c t i v i t y o[ the eazvme once it had be~-n released from the cells. No changes in a c t i v i t y were .~bserved when sucrose, ill a m o u n t s up to 75 mg/ml, was a d d e d to ce].l-free e x t r a c t s obtained i~x the usual m a n n e r . In an a t t e m p t to find a suitable m e t h o d for separating active m a s t cells, concent r a t e d serum albnnfin solutions were tried but did not show promise. Therefi)re, a partial separation of tna~t cells from leucocytes in balanced salt solution w,~s used. Pooled peritoneal fluid cells were suspended in i-~ ~.~-K'~ balaaced ~:~it solution s, 3 ml being used for the cells from one rat. T~vo ml of this suspension were placed in tapered, blood-cen~rifug,." tubes of ~-5 m! capacity, a n d centrifuged for 3-5 min at a p p r o x i m a t e l y 500 rev./min in a horizontal I n t e r n a t i o n a l CKnical Centrifuge. The lowest o.~ ml of the suspensiot~ weft: collected with a drawn-out mcdicil~e dropper. This fraction, called (A), c o n t a i n e d from 5o to 65% of the total mast-cell c o n t e n t .l¢elare~ces p. 397/39,~.
394
;~. m. I¢OTHSCHILD, R. "~V.SCHAYER
VOL. t g (X959) "
of t h e o r i g i n a l s u s p e n s i o n , a n d s h o w e d a 2.5-fold r e l a t i v e i n c r e a s e in its m a s t cell] leucocyte ratio when c o m p a r e d with the u n f r a c t i o n a t e d suspension. To obtain a f r a c t i o n c o n t a i n i n g a l o w e r e d m a s t c e l l / l e u c o c y t e r a t i o , t h e r e m a i n i n g p a r t of t h e cell s u s p e n s i o n was c e n t r i f u g e d a t t h e s a m e t~peed f o r a n a d d i t i o n a l 5 rain a n d t h e Imvest o . : m l a g a i n r e m o v e d . This f r a c t i o n w a s d i s c a r d e d . T h e r e m a i n i n g u p p e r l a y e r s f l o m s e v c r a l t u b e s w e r e p o o l e d m i d c e n t r i f u g e d a t 18oo r e v . / m i n f o r 5 rain. ] ' h i s r e s u l t e d in t h e s e d i m e n t a t i o n of all t h e r e m a i n i n g ceils, a n d a f r a c t i o n , c a l l e d (B), h a v i n g a mast cell/leucocyte ratio 4 to 5 times lower t h a n t h a t of the u n f r a c t i o n a t e d fluid, w a s o b t a i n e d . H i s t i d i n e d e c a r b o x y l a s e w a s e x t r a c t e d f r o m t h e ceils of e a c h o f t h e s e f r a c t i o n s a n d a s s a y e d in t h e m a n n e r d e s c r i b e d . EXPERIMENTS AND RESULTS
Correlation botweeJr histfil.in.e decarboxylase activity and mast-cell content o/rat peritoneal ~qzdd cell ]factions. I n T a b l e I t h e e n z y m i c a c t i v i t y e x t r a c t a b l e f r o m a m i x e d cell f r a c t i o n , (A), h a v i n g a m a s t c e l l / l e u c o c y t e r a t i o of a b o u t t : i o , is c o m p a r e d w i t h t h a t o b t a i n e d f r o m a f r a c t i o n , (B), h a v i n g a m a s t c e i l ] l e u c o c y t e r a t i o of less t h a n I :IOO. T h e n u m b e r of f r a c t i o n t3 cells w e r e so c h o s e n t h a t e q u a l a m o u n t s o f l e u c o c y t e s w e r e u s e d for e x t r a c t i o n in b o t h cases. T h e r e s u l t s of t h e t h r e e e x p e r i m e n t s s h o w n i n d i c a t e t h a t a m - o h higher histidine d e c a r b o x y I a s e a c t i v i t y was associated with the m a s t cell e n r i c h e d f r a c t i o n . T h e low ~ c t i v i t i e s f o u n d in t h e (B) f r a c t i o n s c a n be a t t r i b u t e d a l m o s t e n t i r e l y t o t h e few m a s t cells p r e s e n t . TABLE I I-tlSTII)INIg OBTAINE[) FROM
l-xpt.
1
II Ii1
Crr! /ro*tion
DI,:CARBOXYLASE ACTI'CIT~t" I,..~RTIAI.I.%" t"RACTION.e~TED
Ctllular c*mlct*t be/ore extraction .llast dells LeU¢oc~,tes
OF" C I ~ L L - } ' R E E E X T I ~ A C ' F . ~, RAT PERITO'?gEAL FLIa'll) CIgLLS
Rati,~: ,~,tI~$l On,-.. i h iI(6L)'It'S
Enzymic aaivitv (¢Oflflf.S;J~li~tllO0 n~g ~JS~'~)
A
6, 7 . to:'
7 7 ' ~o~
J:n1
B
0. 7. ~o:'
76. ~o~'
2 : uo8
(,
A
7.7" ~°~
S4- Io ~
r : ux
84
t-~
O , 7 " I o "5
S 4 ' I0 ~
I ; 1 2¢)
17
5.2. lO-'
51- no"~
n : no
i_~5
I :210
92
A l'~
0,~5"
IO5
33"
I0"~
t i
7
Ctmracterizatio~, o / ~ a s t cell histidi~,e decarboxylas~* ; effect o / p H . A c t i v i t y o f t h e e n z y m e of t h e ceil-free e x t l a c t was o p t i m a l o v e r a f a i r l y b r o a d z o n e , r a n g i n g f r o m p H 6. 5 t o 7.6. O u t s i d e of t h e s e limits t h e a c t i v i t y d r o p p e d r a p i d l y a n d w a s l o w a t p H 6.0 a n d 8.o. Mast cell histidine d e c a r b o x y l a s e d i d n o t s h o w a s h a r p m a x i m u m a c t i v i t y as d o histidine d e c a r b o x y l a s e p r e p a r a t i o n s f r o m o t h e r sources~, 1°. All s t u d i e s r e p o r t e d in this p a p e r were d o n e a t p H 7.4. " Some of the techniques commonly used in the characterization of enzymes had to be omitted roving to the e.~tremely low substrate concentration used. This low substrate concentration is essentiaJ_ to the detection and q~a,~Littttivc estimation of mammalian histidine decarboxylase from man) sonrees ~. For example metal ions such as cobalt, and sulfhydryl blocking agents such as p-chloromerct,r'_,ber.zoate might give 9ptlrioll~ enzyme-inhibiting effects when they are in fact forming complexes with the substrate.
Re[ere~,ces p. 397 /39;~'.
VOL. 114 (1959)
HISTIDINE m'C.Xnt~OX-YL.xsr
395
Effect o/e~zvme concentration.. F;g. 1 s h o w s t h e effect of i n c r e a s i n g a m o u n t s of e n z y m e o n t h e d e c a r b o x y l a t i o n of a eol~stant a m o u n t of s u b s t r a t e . I n c r e a s e s in e n z y m e c o n c e n t r a t i o n o v e r a f o u r - f o l d r a n g e , p r o d u c e d p r o p o r t i o n a l increases in activity. "'160 ur) 140
OE12Q 0 .~G10C £ 80 0
60 4c
•~ 9O < 0
|
0.2
I
I
0.4
0.6 ml
!
08
enzyme
F i g , i. E l f e e t t)[ c o n c e n t r a t i o n of t),jstit|ine £1ecarl.)¢)xyluse f r o m r a t perittn~enl ttuid masL c e l l s ¢)n histamine formation.
Effect o/ carbonyl group reagents. As s h o w n in T a b l e II, m a s t cell histidine d e e a r b o x y l a s e is m a r k e d l y a f f e c t e d b y s e m i c a r b a z i d e a n d b y h y d r o x y l a m i n e , b o t h o f w h i c h a r e p o t e n t b l o c k i n g a g e n t s for c a r b o n y l g r o u p s . T h i s i n h i b i t i o n , w h i c h is o b s e r v e d w i t h m a n y a m i n o acid d e c a r b o x y l a s e s , s u g g e s t e d t h a t p y r i d o x a l p h o s p h a t e m i g h t be t h e c o e n z y m e . The p y r i d o x a l p h o s p h a t e r e q u i r e m e n t of m a s t ceil h i s t i d i n e decarboxylase was therefore investigated. TABLb5 II LFFEGT
OF RA]:
tt~'DROXYLAMINE PERITONEAL
FLIYll)
AND
OF
S['2.MIGARt:I:XZll)E ON
.MA,GT C E L L
IliSTII)INE
THE
ACT[V1TY
OF
D~CARBOX~,'LASi':
Enzyme and inhibitor were pre-incubated for t 5 rain at 3? " pri,~r to the'addition of the st, b.~trate. E n z y m i c activ~ly (~ol|lll.~¢~litl~ lr~o mg / q N H /
[..lhibitor
~u 12tki:n'Ha~
Nolle
2 1 tl
--
Hydroxylamine, re -4 31 Hydrt~xyhtnllne, t o -~ .1I
3 124
too 4l
None
202
Semicarbazide. Semicarbazide, Semicarbazide,
xo -a 31 t o~t 3/ lo "'a .Xl
o 33 104
=~-
Ioo .q8 26
Effecl o/pyridoxal phosphate o~ the aativily o/ dialyzed #r@arations o/ mast cell histidiue decarboxylase. Cell-free e x t r a c t s of r a t p e r i t o n e a l fluid were d i a l y z e d for I 8 h a g a i n s t o . I A I p h o s p h a t e b u f f e r in t h e cold. A c t i v i t y w a s t h e n m e a s u r e d a n d c o m p a r e d x ~ t h t h a t o b t a i n e d f o l l o w i n g t h e a d d i t i o n of o.12o ~ m o l e s / m l of p y r i d o x a l - 5 - p h o s phate to the dialyzed extract. R e s u l t s (eounts/min:'IOO lng B S H ) obtM~.led in d u p l i c a t e w i t h t h e d i a l y z e d e x -
Re]erences f . .197] 3 9 A'.
396
. x . M . R O T H S C H I L D , R, W . NCIt:kYER
VOL, ~ 4
(t959)
tracts were 4 ° and 12o averaging 80; after adding pyridoxal p h o s p h a t e activities
were IIiO and II8O, averaging xx5o. Another experiment confirmed these results. Thus,
a marked
potentiating
effect of this coenzyme
was evident'.
R e v e r s a l o~ h y d r o x y l a ~ i ~ e - i n d u v e d i ~ d d b i t i o ~ o f ~ t a s t ~ell h i s t i d i n e d e c a r b o x y l a s e b y p y r i d o x a l phosphate,. T h e e f f e c t o f p y r i d o x a l p h o s p h a t e on inhibition of mast cell
histldine decarboxylase by hydroxylamine was tested. Results (counts/min/Ioo mg BSH) of the four groups of the experiment arc: (a) enzyme only; 126, ]26 and 134: average 129 counts/rain. (b) enzyme plus hydroxylamine IO -'t M ; all samples had zero activity. (c) enzyme plus hydroxylamine Io -4 M plus pyridoxal phosphate, approximately 1o ~ M; all samples had negligible activity, 4 counts/rain average. (d) enzyme plus hydroxglamine IO-~ M plus pyridoxal phosphate, 4.~o -a M ; 156, IS6 and 2t 7 counts/rain, average x86 °* Another experiment confirmed these results. "1",,\ B L E tNHIIII'I'IflN
el:
R..ST .MA.NT ( 'I ~I .L
tI|NTIllINI~
I11
~)I':C,~,]~.f|OX'~'LA.qE ~[1"~" A R O M A T I C
AMINO
AI.'I|)-N
% lnhib;tiol~*
.4 m itzo acid k
l~:.-5-hydroyy Tryptophan
itL-3"ryl~tophan zJL-TVrosino
0. 4 . 0.8" t.5, 3.2,
t o - ~ .11 IO -a 3I 10 -a 31 tO - ~ . 1 /
25 3~
5o 7 ~,
o . ~ - l o -n 31
to
3,2" lo -'~ .lI
2-
3.2"
40
t
o -a .lI
DL'i>h°||vl~tl:tlli|le 3 . ; " ] ° - a -1'1
]t
" T h e a v e r a g ~ , d r e s u l t s oI two, o r t h r t ' e s e p a r a t e e x p e r i m e l l t s , r n n in duplieatL,, a r e r e p o r t e d . ~lht-sc e x p e r i m e n t s ga~'e e s s e n t i a l l y i d e n t i c a l r e s u l t s , l n h i t d t o r a n d s u b s t r a t e w e r e m i x e d p r i o r t o Ilm a¢tdi~i,~n of e n z y n l e .
h~hibition of mast c¢ll histidD,c ,tecarboxylase activity by aromatic a~in,a achts. V¢i:rlA.: .~xD Koctt tt showed that tile activity of guinea pig kidney histidine decarboxylase could be inhibited to v a r y i n g e x t e n t s b y different a m i n o acids. R i n g h y d r o x y l a t i o n increased the i n h i b i t o r y c a p a c i t y of the aromatic a m i n o acids, t y r o s i n e being a c o m p a r a t i v e l y weak inhibitor while d i h y d r o x y p h e n y l a l a n i n e was a strong one. W e have found a similar effect on mast cell histidine decarboxylase. The d a t a of Table 1I I s h o w t h a t the h y d r o x y l - s u b s t i t u t e d a m i n o acids are m u c h stronger inhibitors
than unsubstituted ones. " A p o t e n t i a t i n ~ e f f e c t of p y r i d o x a l p h o s p h a t e w a s a l s o n o t e d a f t e r i t s a d d i t i o n t o a m a s t cell e x t r a c t ~ h i c h h a d b e e n f r o z e n tor 18 la. T h e i r o z e n e n z y m e g a v e h i s t i d m e d e c a r b o x y l a s e a c t i v i t i e s of 4 5 0 :and 47 ° c o u n t s / n u n ; a d d i t i o n o l t h e c o e n z y m e r a i s e d t h e s e t o 97 ° a n d l o 8 o . "i-hus, t o s s of p y r i d ( m a l p h o s p h a t e p r o b a b l y a c c o u n t s for n~..uch of t h e d r o p i n a c t i v i t y o b s e r v e d in f r o z e n m a s t cell h i s t i d i n e d e c a r b o x y l a s e p r e p a r a t i o n s 3, *" T h e e n h a n c e m e n t o f a c t i v i t y t o a l e v e l h i g h e r t h a n t h a t of t h e c o n t r o l s h a s b e e n o b s e r v e d r e p e a t e d l y a n d i n d i c a t e s t h a t e v e n t h e f r e s h e n ~ y n l e is n o t s a t u r a t e d w i t h c o e n z y m e , T h i s is p r o b a b l y d u e t o t h e p r e s e n c e in t h e c r u d e e x t r a c t o f p h o s p h a t a s e s w h i c i l d e s t r o y p y r i d o x a l phnsphate.
12e]eren~es p. 397¢' ~9a',
VOL. ~ 4
(r959)
HIS'rIDINE
D E C . \ R I ' O X Y I ASE
397
Effect o/benzene. The histidine decarboxylase a c t i v i t y of e x t r a c t s of rat peritoneal fluid m a s t ceils is inhibited to the e x t e n t of 87% when one drop of benzene is a d d e d to the incubation m e d i u m . The m a s t cell e n z y m e differs, therefore, from rabbit k i d n e y histidine deearboxy!ase, which, as shown b2,: W.vrox '~, is e n h a n c e d in a c t i v i t y b y the a d d i t i o n of benzene. The mast cell enzyme hehaves in this respect like rat s t o m a c h histidine decarbox3?lase, which is also s t r o n g l y iahibited by benzene ~. DI~(; t7 SSI{ }N
We have d e m o n s t r a t e d t h a t in rat peritoneal fluid the mast ceils arc a m u c h rictlt~r source of histidine decarbo~ylase t h a n the wl-,ite cells. Thus it seems valid to interpret findings on histidine decarboxylast., of perittmeaI fluid a.~ being due almost exclusively to the e n z y m e in the m a s t ceils. P y r i d o x a ! p h o s p h a t e is the cocz~zvme of rat mast cell hisfidine decarboxylase. It has been de.scribed as the coenzyme of m a n y a m i a o acid decarboxy!ases, a n d its effect in p o t e n t i a t i n g the formation of histamine t)y e x t r a c t s from guinea pig a n d pig k i d n e y i n c u b a t e d with L-histidine has been reported b y WERLE u and HOLrrZVL However, these k k l n e y e n z y m e s differ considerably in properties from m a s t celt histidine decarboxylase a n d findings from studies on t h e m are not necessarily valid for e n z y m e from o t h e r sources% P y r i d o x a l phosphate seems to be m u c h more firmly b o u n d to the k i d n e y e n z y m e t h a n to the mast cell enzwne. W,.'RLE failed to obserw. a drop in a c t i v i t y of dialyzed rabbit k i d n e y histidine decarboxvlase~L The effect of this eoenzyme, in reversing the blocking of mast cell t,istidine decarboxylase b y h y d r o x y l a m i n e suggests t h a t the carbonyt group of "pyridoxal p h o s p h a t e is involved in the enzymic d e c a r b o x y l a t i o n 1-'. Mast cell histidine decarboxylase interacts with other amino acids besides histidine: this can result in r e t a r d a t i o n of the rate of d e c a r b o x y l a t i o n of Ldfistidine {Table I I l ) . METZLER~" has reported the a b i l i t y of p y r i d o x a l phosphate to combine a t n e u t r a l p H w i t h a m i n o acids and aanines to h)rm imino c o m p o u n d s (Schiff bases) which arc p r o b a b l y i n t e r m e d i a t e s in the enzymic d e c a r b o x y l a t i o n of the amino acids. Such complexes could inhibit the flmction ;}f the enzyme. The m a r k e d effect of ring h y d r o x y t a t i o n on the i n h i b i t o r y c a p a c i t y of the a m i n o acids tested could be explained as being due to an additional, direct binding of the amino acid to the e n z y m e via t h e phenolic h y d r o x y l group. Finally, our finding t h a t addition of pyridoxal phosphate increases a c t i v i t y to above t h a t of the fresh e n z y m e suggests t h a t the coenzyme is loosely bound in histidine decarboxylase a n d t h a t in future studie.~ exce.*s coenzyme should be a d d e d to insure m a x i m u m a c t i v i t y .
I l t ' H c I " R E N t" 1";.%
1 j . R , R1L~ZY .X.~D G. 1~. W E S T , J . Phvsi~)t., I_'o (i,)33) 5zS. H . T . GR.~,HAM, O . H . I.(.,WRY, N. VCAHL :',.~o ~I. K. [:'Rtl~'B."-rr, [. ENI~H..'Lied., 1o2 (19.55) 307 , I~.. W . SCnAYER, .-Ira. J . . P h y s i o [ , , IS*> (t~)5O) r u e . •t R . ~,V. SC|IAYER, K , J, I]tAVl~ AND 1¢. l...%~,tlLE',-, .4m. g Physi~,t., I.~2 ( I 9 5 5 ) 54R . I,V. SeHA'~'F.R A,-,'o A. C. iv'.-, .Ira. J . t"~y~i'q., :~q (:-~37) 3(x~ . II j . PADAWER AND A. GOlCl;ON, Pr, w. Noc. l'~xptt, i t i O l . . l i e d . , 82 (1055) 20. 7 O . GLICK, S. L. BONTING .XXD I). Dt.:N Ht)ER, Prec..b'¢~c. Expt[. B i o l . . l i e d . . 02 {~95~) 357.
398
a.
M, R O T H S C H I L D ,
R . W . S(3HAYi?-I~
VOL. 34
(x959)
J, ltA.~K, J. Cellular Comp. PhysioL, 3t (1948) -'35. 0 R. \V. SCH.a,V~n.Am, J. Physical., tg9 (I957) 533.
~0 1{, \~'. S C H A Y E R AND Y . | ( O B A Y A S H I . P¥OC. Soc. k'xptl. Biol. ,lled., 92" (19,q6~ 6 5 3 . 11 |~, '~.~:'EI'.tLF.. AND X,,~.:'. [{O'Ctl, Biochel~. Z . , 3 I O (l(.j',l(~) 3 0 5 . x,2 N. G. XV^ToX, Biochem. ,],, 64 (1950) 3 I8. I.O p . }~IOLTZ. A , E N G E L H A R D T A . ~ l ) G . T}IltCLI~,CKE, ~\'aturu2issenst:hapen, 3 9 ( I 0 . 5 2 ) 2 6 0 . 14 E , ~?ERLF, AND K , K R A U T Z U S , 13iochem. Z . , "-96 (1938) 3 1 5 . la 1), AIE'FZLER, ~[, IKAVCA AND E . E . SN1~LL, J . ,4~I. Chel~t. S o t . , 7 ° (I~)~4) 0 4 8 . '16 1). 31ETZLER, J . Jlp$t. CJl,.ln. Sot-., 7~) ( 1 9 5 7 } .1.85 •
PRODUCTIQN
OF PENTOSE OF NOCARDIA
SAPROPHYTIC
INTERMEDIATES OPACA
SOIL NOCARDIAS
DURING
GROWTH
AND OTHER AND MYCOBACTERIA
R. B. DUFF AND D. M1 W K ] " ] ] . } ~ l
r
l)epartme~2t o] Biochemistry a*~d Section o/ A4ic~,obiology, The a~fa6c*~lay Institute /or So~l Research, A bevdee~z (Great Britain)
(Received October ] st, x958)
SUMMARY :. During g r o w t h in simple media N o c a r d i a oi)aca (strain Tt6 ) a n d some o t h e r s a p r o p h y t i c soil nocardias a n d m y c o b a c t e r i a liberate quau/zities ¢i s e d o h e p t u l o s e a n d ribulose which can be d e t e c t e d b y a p p l y i n g as little as lO-2o /~l of m e d i u m directly to the paper c h r o m a t o g r a m . An u n k n o w n s u b s t a n c e which gave a s p e c t r u m similar to t h a t of pentuiose in the c y s t e i n e - c a r b a z o l e reaction was also f o r m e d b u t this was neither ribulose xylulose, nor erythro-3-pentulose. 2. Liberation of the pentose cycle c o m p o n e n t s occurs more q u i c k l y a n d generaUy in greater a m o u n t s when the m e d i u m is kept a t a b o u t p H 7,o (e.g, b y a d d i t i o n of calcium carbonate, b y using an increased p h o s p h a t e buffer c o n c e n t r a t i o n , b y using sodium n i t r a t e r a t h e r t h a n a m m o n i u m s u l p h a t e as a nitrogen source or by using sodium gluconate as a source of carbon). There seems no reason to suppose t h a t calcium ion has a n y specific effect on t h e s y s t e m . 3 - S e d o h e p t u l o s e and ribulo,~e from the above cultures were identified a n d estimated by paper c h r o m a t o g r a p h y a n d by application of t h e cy÷:tehle-sulphuric acid and orcinol reactions respectively to larger q u a n t i t i e s isolated b y c o l u m n c h r o m a t o g r a p h y . D i h y d r o x y a c e t o n e (characterised as t h e 0~acetate) was o b t a i n e d o n l y when the cultures were kept at a b o u t p H 7 . o (i.e. b y a d d i t i o n of calcium carbonate}. This a n d the failure to produce d i h y d r o x y a c e t o n e from sodium g l u c o n a t e suggests t h a t the triose is m a i n l y a product of the E m d e n - M e y c r h o f - p a t h w a y . Aldolase (with fructose diphosphate as a substrate) is present in cell-free e x t r a c t s of *he bacteria.
Re/ece~tces p. 4o6.