Kinetic properties of mammalian histidine decarboxylase

EUROPEAN JOURNAL OF PHARMACOLOGY 1 (1967) 42-46. NORTH-HOLLAND.PUBL. COMP., AMSTERDAM

KINETIC

PROPERTIES

OF

MAMMALIAN HISTIDINE DECARBOXYLASE

*

Rolf HAKANSON Department of Pharmacology, University of Lund, Lund, Sweden Accepted 8 November 1966

R. H/~KANSON, Kinetic properties of mammalian histidine decarboxylase, European J. Pharmacol. 1 (1967) 42-46. Semi-purified preparations of histidine decarboxylase from hamster placenta and from fetal rat tissues have been studied. The properties of the histamine-forming enzyme from hamster placenta are very similar to those of fetal rat histidine deearboxylase. Both enzymes can be extracted and purified by the same technique and their kinetic properties appear identical. With saturating concentrations of histidine the apparent maximal reaction velocity is around pH 5.5, but it could be established that the pH optimum of the reaction is inversely related to the substrate concentration. The apparent K m decreases with increasing pH. Enzymes with similar properties have been demonstrated in some other tissues of the rat and mouse; the apparent substrate specificity seems to justify the use of the name histidine decarboxylase to denote this enzyme, or group of enzymes. Fetal rat

Hamster placenta

Histidine decarboxylase

1. INTRODUCTION Since 1936 when W e r l e d e m o n s t r a t e d the h i s t a m i n e - f o r m i n g capacity of s o m e m a m m a l i a n t i s s u e s , h i s t i d i n e d e c a r b o x y l a s e has a t t r a c t e d the attention of many w o r k e r s . The d i s c r e p a n c i e s am o n g e a r l i e r r e p o r t s on the p r o p e r t i e s of m a m m a l i a n histidine d e c a r b o x y l a s e (Schayer, 1957; Hagen, W e i n e r , Ono and Lee, 1960; M a c kay, R i l e y and Shepherd, 1961; Ganrot, R o s e n g r e n and R o s e n g r e n , 1961; W e i s s b a c h , L o v e n b e r g and Udenfriend, 1961; B u r k h a l t e r , 1962; H£kanson, 1963) m a y be p a r t l y explained by the e x i s t e n c e of s e v e r a l h i s t a m i n e - f o r m i n g i s o e n z y m e s with d i f f e r e n t c h a r a c t e r i s t i c s . Abundant e v i d e n c e in f a v o u r of this view has been p r e sented (Ganrot et al., 1961; W e i s s b a c h et al., 1961; T e l f o r d and West, ]961; B u r k h a l t e r , ]962; H£kanson, 1963, 1964, 1966a, ]967; HZtkanson and Owman, 1963; W e r l e and L o r e n z , 1966). High h i s t i d i n e d e c a r b o x y l a s e a c t i v i t y has been d e m o n s t r a t e d in s o m e t i s s u e s c h a r a c t e r i z e d by ra pid growth (Kahlson, R o s e n g r e n and White, 1960; Kahlson, N i l s s o n , R o s e n g r e n and Z e d e r feldt, 1960; Mackay, M a r s h a l l and Riley, 1960; H~.kanson, 1961, 1966a, 1967); a m o n g t h e s e the fetal r a t l i v e r p r o v e d to be a c o n s p ic u o u s l y r i c h * Supported by grants from the Swedish Medical Research Council {No. 12x-1007-01) and from Alfred Oste rlunds Foundation.

Kinetic properties

s o u r c e of this e n z y m e (Kahlson et al., 1960), Ganr o t et al. (1961) w e r e able to show that the p r o p e r t i e s of the e n z y m e f r o m fetal r at l i v e r w e r e d i f f er en t f r o m those of the h i s t a m i n e - f o r m i n g e n z y m e p r e s e n t in the r a b b i t kidney co r t ex . While the fetal e n z y m e was found to be specific, the r e n a l e n z y m e was cap ab l e of d e c a r b o x y l a t i n g s e v e r a l a r o m a t i c am i n o aci d s b e s i d e s histidine (cf. R o s e n g r e n , 1960; L o v e n b e r g , W e i s s b a c h and Udenfriend, 1962). Some p r o p e r t i e s of a p a r t i a l ly p u r i f i e d p r e p a r a t i o n of f et al h i st i d i n e d e c a r b o x y l a s e have b e e n d e s c r i b e d (H{tkanson, 1963). The pH o p t i m u m of the e n z y m e was found to d epend upon the s u b s t r a t e c o n c e n t r a t i o n ; at high histidine c o n c e n t r a t i o n the pH o p t i m u m was around 6, at v e r y low c o n c e n t r a t i o n s the optim u m was c l o s e to 7.2. Th e a p p a r e n t K m inc r e a s e d with d e c r e a s i n g pH. It was s u g g e s t e d f r o m t h e s e o b s e r v a t i o n s that the e n z y m e at t ack s the anionic f o r m of h i s t i d i n e (H~kanson, 1963, 1966a). An e n z y m e with p r o p e r t i e s s i m i l a r to those of fetal r a t h i st i d i n e d e c a r b o x y l a s e has been d e m o n s t r a t e d a l s o in the f et al m o u s e (H£kanson, 1967) and in s o m e t i s s u e s of the adult r at (H~kanson, 1964; H~kanson and Owman, 1966) and m o u s e (H~kanson, 1967). H a m s t e r p l a c e n t a contains high am o u n t s of a h i s t a m i n e - f o r m i n g enz y m e ; at the 16th day of p r e g n a n c y h a m s t e r p l a cental t i s s u e has b e e n found ten t i m e s m o r e potent in f o r m i n g h i s t a m i n e than f et al r a t l i v e r

MAMMALIAN HISTIDINE DECARBOXYLASE

b a t e d a t r o o m t e m p e r a t u r e f o r 15 m i n b e f o r e t h e a d d i t i o n of s u b s t r a t e . T h e m o l a r c o n c e n t r a t i o n of c o e n z y m e w a s a l w a y s l e s s t h a n t h a t of h i s t i d i n e . V e r y s m a l l a m o u n t s of p y r i d o x a l ' - 5 - p h o s p h a t e w e r e n e e d e d to s a t u r a t e t h e e n z y m e (H~tkanson, 1963; H ~ k a n s o n , to b e p u b l i s h e d ) ; i n a l l e x p e r i ments excess amounts were added. Some diffic u l t i e s w e r e o b s e r v e d in m e a s u r i n g t h e e n z y m e a c t i v i t y a t e x t r e m e pH v a l u e s ; a t h i g h pH v a l u e s ( a b o v e 8) t h e e n z y m e a c t i v i t y i s v e r y low a n d d i f f i c u l t to a s s a y e v e n w i t h s e n s i t i v e r a d i o m e t r i c m e t h o d s ; a t low pH v a l u e s (below 5.5) t h e e n z y m e d e m a n d s a v e r y h i g h c o n c e n t r a t i o n of h i s t i d i n e . A f t e r a d d i t i o n of l a r g e a m o u n t s of s u b s t r a t e t h e b u f f e r c a p a c i t y of t h e i n c u b a t i o n m e d i u m i s i n a d e q u a t e a n d t h e pH of t h e s a m p l e s h a s to b e a d j u s t e d to t h e d e s i r e d v a l u e w i t h s m a l l a m o u n t s of 0.1 N HC1. S o m e e x p e r i m e n t s w i t h h i g h h i s t i d i n e c o n c e n t r a t i o n s w e r e p e r f o r m e d in 0.4 M p h o s p h a t e b u f f e r ; u n d e r t h e s e c o n d i t i o n s a d j u s t m e n t of t h e pH w a s o f t e n u n n e c e s s a r y . T h e i o n i c c o m p o s i t i o n of t h e i n c u b a t i o n m e d i u m a f f e c t s t h e r e a c t i o n to s o m e e x t e n t ( W e r l e a n d L o r e n z , 1966; H £ k a n s o n , u n p u b l i s h e d d a t a ) b u t in t h e s e e x p e r i m e n t s t h e r e w a s no e v i d e n c e t h a t t h e elevated ionic strength caused any significant d i s t o r t i o n of t h e r e s u l t s . W i t h v e r y low a m o u n t s of s u b s t r a t e (10 -6 - 10 -7 M) t h e c o n c e n t r a t i o n of e n z y m e w a s c o r r e s p o n d i n g l y d e c r e a s e d to a v o i d first-order reactions. Enzyme activity was ex-

(Rosengren, 1964). T h i s p a p e r d e s c r i b e s t h e k i n e t i c p r o p e r t i e s of s e m i - p u r i f i e d p r e p a r a t i o n s of h i s t i d i n e d e c a r b o x y l a s e f r o m f e t a l r a t a n d from hamster placenta.

2. M A T E R I A L AND M E T H O D S

2.1. Preparation of enzyme The enzyme extracts used were prepared from fetal rat tissues (collected 16-20 days after m a t i n g , u s u a l l y 5 0 - 7 0 g) o r h a m s t e r p l a c e n t a e (collected 15-18 days after mating, usually 5-10 g) by e x t r a c t i o n w i t h 2 v o l u m e s of 0.1 M s o d i u m a c e t a t e - a c e t i c a c i d b u f f e r , pH 4.5, h e a t t r e a t m e n t a t 52o f o r 5 m i n a n d r e p e a t e d f r a c t i o n a t i o n with ammonium sulphate; histidine decarboxylase from both tissues was precipitated at bet w e e n 25 a n d 40o/0 s a t u r a t i o n i n t h e f i r s t f r a c t i o n a t i o n , a n d b e t w e e n 28 a n d 45% i n t h e s e c o n d (cf. H £ k a n s o n , 1963). T h e v o l u m e of t h e f i n a l e x tract was approx. 5 ml. 2.2. Enzyme assays If n o t o t h e r w i s e s t a t e d a l i q u o t s ( 1 0 - 1 0 0 #1) w e r e i n c u b a t e d u n d e r w i t h h i s t i d i n e i n t h e p r e s e n c e of p y r i d o x a l v - 5 - p h o s p h a t e , i n 0.1 M f e r i n a t o t a l v o l u m e of a b o u t 1 d i e n t s of t h e i n c u b a t i o n m i x t u r e

I

[

I

i

i

i

i

i

i

i

of t h e e n z y m e n i t r o g e n a t 37o the coenzyme, phosphate bufml. All ingrewere preincu-

i

i

i

[

i

43

t

i

i

i

L

I

I

I

I

i

HISTIDINE DECARBOXYLASE FROM FETAL RATS

W

ZD n 0 Fr 13_

4

w z 2~

3

G) -r

2

=1-

i

I

I

i

6.0

I

I

I

I

I

6.5

I

I

i

i

i

7.0

pH

i

i

i

i

i

7.5

i

I

i

i

i

8.0

i

i

i

Fig. 1. The change in pH optimum with different substrate concentrations. Enzyme activity is e x p r e s s e d as # g h i s tamine formed. The conditions of the experiment are given in the text. *- -*, histidine 5 × 10 -2 M, pyridoxal 5-phosphate 10 # g / ml; o o, histidine 5 × 10-3 M, pyridoxal 5-phosphate 10 # g / ml; -% histidine 5 x 10 -4 M, pyridoxal 5-phosphate 10 # g / ml.

44

R. H~,KANSON

p r e s s e d a s /lg h i s t a m i n e ( f r e e b a s e } p r o d u c e d in o n e h o u r . T h e a m o u n t of h i s t a m i n e f o r m e d was determined fluorometrically by the method of S h o r e , B u r k h a l t e r a n d C o h n (1958) a f t e r o r g a n i c e x t r a c t i o n of t h e a m i n e ( B u r k h a l t e r , 1962). In e x p e r i m e n t s where higher sensitivity was n e e d e d 1 4 C - h i s t i d i n e w a s u s e d . T h e a m o u n t of 1 4 C - h i s t a m i n e f o r m e d w a s d e t e r m i n e d by a r a d i o m e t r i c t e c h n i q u e ( H £ k a n s o n , 1966b).

w e a v e r a n d B u r k , 1934) c h a n g e d w i t h pH a s s h o w n i n f i g s . 4 a n d 5; c a l c u l a t e d m a x i m a l r e a c t i o n v e l o c i t y w a s o b s e r v e d a t a b o u t pH 5.5.

7.5

Q.

-6

The histamine-forming e n z y m e of h a m s t e r placenta was purified as described for fetal rat histidine decarboxylase ( H ~ k a n s o n , 1963); t h e " s a l t i n g - o u t " p r o f i l e s of t h e s e e n z y m e s w e r e identical. The enzyme extract prepared from hamster placentae had 5-10 times more histidine decarboxylase activity (per unit protein) than the usual fetal rat extract. W i t h b o t h e n z y m e s t h e pH o p t i m u m of t h e r e a c t i o n w a s f o u n d to v a r y w i t h t h e s u b s t r a t e c o n c e n t r a t i o n (figs. 1 - 3 ) ; w i t h v e r y h i g h h i s t i d i n e concentrations optimal reaction velocity was obs e r v e d a t a b o u t pH 6.0; w i t h d i m i n i s h i n g s u b s t r a t e c o n c e n t r a t i o n s t h e pH o p t i m u m s h i f t e d t o wards more alkaline values. The apparent K m and apparent Vma x (both d e t e r m i n e d b y t h e g r a p h i c p r o c e d u r e of L i n e -

i

i

7.0

-r

3. R E S U L T S

i

i

i

I

i

i

1

,

~

i

HISTIDINE D E C A R B O X Y L A S E

i

i

i

i

FROM

i

i

E

o 6.5

6o

0

I

i

I

I

I

I

I

2

5

4

5

6

7

pS Fig. 2. The influence of s u b s t r a t e concentration (exp r e s s e d as pS) on the pH optimum of the reactions. The highest s u b s t r a t e concentration used may be more c o r r e c t l y d e s c r i b e d as a saturated histidine solution. o o, histidine decarboxylase from fetal r a t s ; e e, histidine decarboxylase from h a m s t e r placentae.

i

i

i

HAMSTER

i

i

i

i

i

i

PLACENTA

5

a w ~D a o LL W Z

2

I

O

I

6'0

6-5

7"0

pH

7-5

80

I

1

Fig. 3. The change in pH optimum with different s u b s t r a t e concentrations. & - - - ~ , histidine 5 × 10 -2 M, pyridoxal 5-phosphate 1 / l g / m l ; o o, histidine 5 × 10-3 M, pyridoxal 5 phosphate 1 /lg/ml; ; ; , histidine 5 x 10-4 M, pyridoxal 5-phosphate 1 / l g / m l ; A A, histidine 5 × 10-5 M, pyridoxal 5-phosphate 1 /2g/ml.

M A M M A L I A N HISTIDINE D E C A R B O X Y L A S E

6

r

45

i

E 4

- ,¢- -

3

(D E~

o o

c~-2
0

I

i

I

I

I

I

I

I

4

5

6

T

8

9

t

i

pH i

,

Fig. 4. T h e i n f l u e n c e of pH on a p p a r e n t K m (the c u r v e w a s fitted visually}. The following b u f f e r s w e r e e m p l o y e d : pH 3-4.5, H3PO 4 + K H 2 P O 4; pH 4 . 5 9, K H 2 P O 4 + Na2HPO4; above pH 9, Na2HPO 4 + N a 3 P O 4. o o, h i s t i d i n e c a r b o x y l a s e f r o m fetal r a t s ; = =, h i s t i d i n e d e c a r boxylase from hamster placentae.

I

'-,i

Q.

]

]

]

I

I

i

4

5

6

7

8

9

pH

Fig. 5. A p p a r e n t Vma x { e x p r e s s e d a s % of the m a x i m u m v a l u e w i t h in the e x p e r i m e n t a l pH r e g i o n ) calculated from double-reciprocal p l o t s of i n i t i a l v e l o c i t i e s v e r s u s substrate concentration. Buffers a s in fig. 4. o o, histidine decarboxylase from fetal rats; © =, h i s t i d i n e decarboxylase from hamster placentae.

46

R. HAKANSON

4. DISCUSSION T h e h i s t a m i n e - f o r m i n g e n z y m e of h a m s t e r p l a c e n t a a p p e a r s to b e i d e n t i c a l w i t h , o r v e r y s i m i l a r to f e t a l r a t 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 v i o u s s t u d i e s on c e r t a i n t i s s u e s of t h e r a t a n d m o u s e h a v e d e m o n s t r a t e d t h e p r e s e n c e of h i s t a m i n e - f o r m i n g e n z y m e s c l o s e l y r e l a t e d to f e t a l r a t h i s t i d i n e d e c a r b o x y l a s e ( H a k a n s o n , 1964; H a k a n s o n and O w m a n , 1966; H a k a n s o n , 1966a, 1967). T h e a p p a r e n t s u b s t r a t e s p e c i f i c i t y s e e m s to j u s t i f y the u s e of t h e n a m e h i s t i d i n e d e c a r o o x y l a s e to d e n o t e t h i s e n z y m e o r g r o u p of e n zymes. M a m m a l i a n histidine d e c a r b o x y l a s e has the following c h a r a c t e r i s t i c s : 1) M a x i m a l r e a c t i o n velocity (calculated from Lineweaver-Burk plots) i s o b s e r v e d at a b o u t pH 5.5; 2) t h e pH o p t i m u m of the r e a c t i o n i s i n v e r s e l y r e l a t e d to the s u b s t r a t e c o n c e n t r a t i o n : w i t h v e r y low h i s t i d i n e c o n c e n t r a t i o n the m a x i m a l r e a c t i o n v e l o c i t y i s c l o s e to pH 7: 3) t h e a p p a r e n t K m d e c r e a s e s w i t h i n c r e a s i n g pH. It h a s b e e n s u g g e s t e d in p r e v i o u s c o m m u n i c a t i o n s t h a t t h e k i n e t i c p r o p e r t i e s of m a m m a l i a n h i s t i d i n e d e c a r b o x y l a s e may be p a r t l y explained by the a s s u m p t i o n t h a t t h e e n z y m e a t t a c k s a s p e c i f i c i o n i c v a r i e t y of h i s t i d i n e (H~kanson, 1963, 1966a). A m o r e e l a b o r a t e m o d e l f o r t h e m o l e c u l a r m e c h a n i s m of h i s t a m i n e f o r m a t i o n w i l l b e p r e s e n t e d e l s e w h e r e ( H ~ k a n s o n , to b e published).

AEFERENCES '~urkhalter, A., 1962, The formation of histamine by fetal rat liver, Biochem. Pharmacol. Ii, 315. ;anrot, P.O.. A. M. Rosengren and E.Rosengren, 1961, Ot~ the presence of different histidine decarboxylating enzymes in mammalian tissues, Experientia 17, 263. Hagen, P., N. Weiner, S. Ono and Fu-Li Lee, 1960, Amino acid decarboxylases of mouse mastocytoma tissue. J. ]?harmacol. Exptl. Therap. 130, 9.

H~kanson, R., 1961, Formation of histamine in t r a n s plants from a rat m a m m a r y carcinoma, Experientia 17, 402. H'~kanson, R., 1963, Histidine decarboxylase in the fetal rat, Biochem. Pharmaeol. 12, 1289. H~kanson, R., 1964, Histidine decarboxylase in the bone m a r r o w of the rat, Experientia 20, 205. H~tkanson, R., 1966a, Histidine decarboxylase in experimental tumours, J. ]?harm. ]?harmacol. 18, 769. H~kanson, R., 1966b, Radiometric micromethods for the study of some amino acid decarboxylases, Acta Pharmacol. Toxicol. (Kbh.) 24, 217. H~kanson, R., 1967, H i s t a m i n e - f o r m i n g isoenzymes in the fetal and adult mouse, European J. P h a r m a col. 1, 34. H~kanson, R. and Ch. Owman, 1966, Distribution and p r o p e r t i e s of amino acid decarboxylases in gastric mucosa, Biochem. ]?harmacol. 15, 489. Kahlson, G., E . R o s e n g r e n and T.White, 1960, The formation of histamine in rat foetus, J. ]?hysiol. 151, 131. Kahlson, E., K. Nilsson, E. Rosengren and B. Z e d e r feldt, 1960, Wound healing as dependent on rate of histamine formation, Lancet 279, 230. Lineweaver, H. and D. Burk, 1934, The determination of enzyme dissociation constants, J. Am. Chem. Soc. 56, 658. Lovenberg, W., H. Weissbach and S. Udenfriend, 1962, Aromatic L-amino acid decarboxylase, J. Biol. Chem. 237, 89. Mackay, D., ]?. B. Marshall and J.F. Riley, 1960, Histidine decarboxylase activity in a malignant rat hepatoma, J. Physiol. 153, 31 p. Mackay, D., J . F . Riley and D.M. Shepherd, 1961, Amino acid decarboxylase activities in rat hepatoma, J. ]?harm. ]?harmacol. 13, 257. Rosengren, E., 1960, Are dihydroxyphenylalanine decarboxylase and 5-hydroxytryptophan decarboxylase individual e n z y m e s ? Acta Physiol. Scand. 49, 364. Schayer, R.W., 1957, Histidine decarboxylase of rat stomach and other mammalian t i s s u e s , Am. J. Physiol. 189, 533. Shore, P . A . , A. Burkhalter and V. H. Cohn Jr.. 1959, A method for the fluorometric assay of histamine in t i s s u e s , J. ]?harmacol. Exptl. Therap. 127, 182. Telford, J . M . and G.B. West, 1961, The formation of histamine in the rat, J. ]?harm. Pharmacol. 13, 75. Weissbach, H., W. Lovenberg and S. Udenfriend, 1961, C h a r a c t e r i s t i c s of mammalian histidine decarboxylating e n z y m e s z Biochim. Biophys. Acta 50, 177. Werle, E., 1936, Uber die Bildung von Histamin aus Histidin dutch t i e r i s c h e s Gewebe, Bioehem. Z. 288, 292. Werle, E. and W. Lorenz, 1966, Histamin und Histidindecarboxylasen in Schilddrilse und Thymus, Biochem. ]?harmacol. 15, 1059.