15.5. 1975

Specialia

543

Table II. Common and distinctive characteristics of carboxylie ester hydrolase bands A and B Carboxylic ester hydrolase bands

Acetyl esters hydrolyzed ~

ButyryI esters hydrolyzed ~

A

+

--

B

+

+

+ , Esterase activity;

Heat inactivation (60 ~ ~

+

DFP inhibition (10-4 M) ~

Approximate M. W.

--

52,000

--

63,000

, no activity. ~Previous results obtained from 25 strains of E. cell by zymogram procedure in aerylamide-agarose gel 1.

Moreover, t h e esterase b a n d s A of K~2 a n d HB10 h a v e negative charges greater than HB~ and HBls; the e s t e r a s e b a n d s B of K~2 a n d HB14 h a v e n e g a t i v e c h a r g e s g r e a t e r t h a n HB10 a n d H B l s . T h e c o m p a r i s o n of t h e slopes of t h e e s t e r a s e lines w i t h t h o s e o b t a i n e d u s i n g reference p r o t e i n s allowed a n a p p r o x i m a t e e s t i m a t e of m o l e c u l a r w e i g h t s (Figure 2). T h e v a l u e s were a b o u t 52,000 d a l t o n s (:L 5%) for esterase b a n d A a n d 63,000 d a l t o n s ( • 5%) for e s t e r a s e b a n d B. I n conclusion, t h e r e s u l t s o b t a i n e d b y m o l e c u l a r s i e v i n g effect i n p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s demonstrate that the carboxylic ester hydrolases A and B of E. coli are d i s t i n c t in m o l e c u l a r weight. T h e v a r i a t i o n s i n esterase m o b i l i t y a m o n g t h e s t r a i n s a p p e a r t o b e t h e c o n s e q u e n c e of differences in m o l e c u l a r n e t charge. T h e two molecular weight patterns supplement the characteristics o b t a i n e d p r e v i o u s l y (Table II).

Calcium and pH Homeostasis in the Snail

Rdsumd. Des @lectrophor6ses 5. diverses c o n c e n t r a t i o n s d ' a c r y l a m i d e m o n t r e n t q u e les c a r b o x y l i q u e s esters h y d r o l a s e s A e t B d'E. cell p o s s ~ d e n t d e s p o i d s moMculaires d i s t i n c t s : 52,000 et 63,000 d a l t o n s . Les v a r i a t i o n s de m o b i l i t 6 observ6es selon les souches p r o v i e n n e n t essent i e l l e m e n t de diff6rences d a n s les c h a r g e s 61ectriques. PH. GOULLET% 10

Laboratoire de Microbiologie, Facultd de 2VIddecine Xavier-Bichat, Universitd de Paris VII, 27, rue de l'Ecole de Mddecine, F - 7 5 2 7 0 Paris-Cedex 06 (France), 7d October 7974. 9 With technical assistance of Madame DANIELLE BILLOW. 10 Service de Microbiologie, H6pital Beaujon, 100, Boulevard du G6ndrai Leclerc, F-92110 Clicby, France.

(Helix pomatia): Effects of C02 and CaCl~ Infusion

W h e n snails (Helix aspersa 1 a n d H. pomatia 2) are e x p o s e d to 5 - 1 0 % COs, t h e c o n c e n t r a t i o n s of b i c a r b o n a t e a n d c a l c i u m in t h e h a e m o l y m p h rise in a p p r o x i m a t e l y 2 : 1 r a t i o a n d r e a c h n e w s t e a d y levels in 2 - 6 h, w i t h negligible c h a n g e in sodium, p o t a s s i u m a n d m a g n e s i u m . W i t h t h e i m p o r t a n c e of ionic b a l a n c e to cellular f u n c t i o n in m i n d , we h a v e s t u d i e d t h e r e s u l t i n g r e l a t i o n s h i p b e t w e e n p H a n d free calcium. T h e r e s u l t s i n d i c a t e a n u n e x p e c t e d l y slow rise in c a r b o n d i o x i d e t e n s i o n a n d also a n a p p a r e n t h o m e o stasis of t h e ionic p r o d u c t [Ca++]. [COg] a t v a l u e s well a b o v e t h e s o l u b i l i t y p r o d u c t s for c a l c i u m c a r b o n a t e . Materials and methods. H y d r a t e d , f a s t e d snails (H. pomatia) were e x p o s e d t o 5 - 1 0 % CO 2 in O 3 or i n f u s e d w i t h 150 m M CaCI=. t t a e m o l y m p h was d r a w n f r o m c a n n u l a e t i e d i n t o t h e o p t i c t e n t a c l e s ~ for t h e d e t e r m i n a t i o n of p H 1 a n d t o t a l c a l c i u m =. C o n c e n t r a t i o n s of free c a l c i u m were c a l c u l a t e d o n t h e a s s u m p t i o n t h a t 1 m M of t h e t o t a l was b o u n d t o h a e m o c y a n i n (Figure 2) a n d t h a t t h e o n l y o t h e r b o u n d c a l c i u m was t h a t of t h e ion p a i r C a l i C O +. I n t h e e x p e r i m e n t s o n t h e effects of c a r b o n dioxide, it was a s s u m e d , for t h e c a l c u l a t i o n of c o n c e n t r a t i o n s of C a l i C O + a n d also CO i , t h a t t h e t o t a l b i c a r b o n a t e c o n c e n t r a t i o n , i n c l u d i n g free I-ICe;-, C a l i C O ~ a n d M g H C O +, a w a s i n i t i a l l y a t y p i c a l 30 m M a n d t h a t it rose w i t h t o t a l c a l c i u m in 2 : 1 ratio. T h e d i s s o c i a t i o n c o n s t a n t s of C a l i C O + a n d M g H C O ~ were b o t h t a k e n ~ as 160 m M a n d t h e c o n c e n t r a t i o n of m a g n e s i u m was t a k e n as 10 r a M . I n t h e i n f u s i o n e x p e r i m e n t s t h e c o n c e n t r a t i o n s of ionized b i c a r b o n a t e were c a l c u l a t e d f r o m t h e p H of s a m p l e s e q u i l i b r a t e d a t 20~ w i t h 2~o CO~e; t h e c a r b o n d i o x i d e t e n s i o n s in v i v o were c a l c u l a t e d f r o m t h i s a n d t h e in r i v e pH.

Results and discussion. F i g u r e 1 shows, as a repres e n t a t i v e e x a m p l e , t h e t i m e course of t h e c h a n g e s in t o t a l a n d free c a l c i u m a n d in C a H C O g a n d p H in t h e h a e m o l y m p h of a snail exposed t o 8.7% CO S. F i g u r e 2 a shows tile r e l a t i o n s h i p b e t w e e n p H a n d ionized c a l c i u m for 7 snails e q u i l i b r a t i n g w i t h 5 - 1 0 % CO 2. D u r i n g t h e s e changes, t h e p H m o s t l y falls as t h e c o n c e n t r a t i o n of c a l c i u m rises and, since t h e level of b i c a r b o n a t e rises a l o n g w i t h t h a t of calcium, it follows t h a t t h e t e n s i o n of c a r b o n dioxide increases w i t h s i m i l a r t i m e course - r a t h e r t h a n , say, s t a b i l i z i n g in 10-20 rain as in Man. T h e d e l a y of several h o u r s i n v o l v e d in t h e a t t a i n m e n t of t h e n e w s t e a d y s t a t e is t h e r e f o r e largely d u e t o slow e n t r y of c a r b o n d i o x i d e (most of w h i c h b e c o m e s b i c a r b o n a t e ) r a t h e r t h a n t o a n i n h e r e n t slowness in b i c a r b o n a t e g e n e r a t i o n . A t n o r m a l r a t e s of m e t a b o l i s m ~, m o s t of t h e a c c u m u l a t i n g c a r b o n d i o x i d e could in a n y case b e metabolic. T h e a v e r a g e d i n i t i a l a n d f i n a l v a l u e s of p H a n d ionized c a l c i u m for t h e s a m e 7 snails are s h o w n in F i g u r e 2b. T h e c o n t i n u o u s c u r v e c o r r e s p o n d s t o a c o n s t a n t ionic p r o d u c t [Ca++].ECOg], of 3.6 m M 2, c h o s e n t o b e t h e a v e r a g e p e r t a i n i n g in t h e snails w h i l e still in air. T h e n e a r c o n s t a n c y of t h e ionic p r o d u c t in e a c h snail suggests t h a t

1 R. F. BURTON, Comp. Biochem. Physiol. 37, 193 (1970). R. F. BURTON, Comp. Biochem. Physiol., in press. 3 I. GREENWALD,J. biol. Chem. 741, 789 (1941). 5 H. WESEMEIER, Z. vergl. Physiol. 43, 1 (1960).

544

Speeialia

Mean values ( • standard deviations) of total calcium, ionized bicarbonate, pH and PCO2 in the haemolymph of 4 snails just before and just after infusion with 1 ml of 150 mM CaCI2, and also 3.4-6.3 h later (final samples)

Total calcium (raM) Ionized bicarbonate (raM) pH PCO2 (mm Hg)

Before infusion

After infusion

Final sample

10.5 ~z 3.4

20.7 ~ 1.8

14.7 • 1.5

26.0 -L 4.9 7.77 ~ 0.05 14.1 -t- 2.0

20.8 • 4.2 16.5 ~ 2.6 7.65 zL 0.06 7.60 ~z 0.02 14.3 -4- 1.1 13.1 • 1.8

Total Ca

mF'/ 10 7.9 pH 7.7 5 7.5

pH_ 9 =

j~-'---"

:

0

r

i

-2

r

0

i

r

i

2

9 7.5

CaliCO3+ i

I

I

4

T

6

8h

Time in 8.7% CO 2

Fig. 1. Changes in total and free calcium and in CaliCO + and pH in haemolymph of a hydrated snail placed in 8.7% CO2 in O2 at time zero,

a)

pH

b)

,\

7.9

7.7

X

7.5

73

8

10

12

14

6

8

10

EXPENIENTIN 31/5

t h e h a e m o l y m p h could b e in e q u i l i b r i u m w i t h solid calcium c a r b o n a t e , b u t in f a c t it is h i g h l y s u p e r s a t u r a t e d w i t h r e s p e c t to b o t h t h e c o m m o n forms, a r a g o n i t e a n d calcite, a n d can r e m a i n so because of t h e p r e s e n c e of p h o s p h a t e a n d p e r h a p s o t h e r c r y s t a l poisons t h a t p r e v e n t nucleation. (The solubility p r o d u c t for calcite a t 20 ~ a n d t h e ionic s t r e n g t h of snail h a e m o l y m p h is a p p r o x i m a t e l y 0.1 m M 2, while t h a t of a r a g o n i t e is a b o u t 60 ~o higher6). T h e r e is so m u c h calcium c a r b o n a t e a b o u t t h e b o d y t h a t t h e r e p r e s u m a b l y exists some b o d y fluid t h a t is in e q u i l i b r i u m w i t h it. If b i c a r b o n a t e a n d calcium ions could r e a d i l y m o v e f r o m t h e h a e m o l y m p h i n t o t h i s fluid a n d t h e r e p r e c i p i t a t e , t h e n t h e c o n c e n t r a t i o n s in t h e h a e m o l y m p h w o u l d quickly fall. This could be p r e v e n t e d b y a n o p p o s i n g active t r a n s p o r t of one or m o r e of t h e following ions: Ca++, H+, O H - , HCO~ a n d CO~. To e x p l a i n t h e c o n s t a n c y of ECa++] 9[CO ~) in t h e h a e m o l y m p h , one n e e d n o w only p o s t u l a t e t h a t t h e m e c h a n i s m of ion t r a n s p o r t o p e r a t e s to m a i n t a i n a c o n s t a n t e l e c t r o c h e m i c a l p o t e n t i a l g r a d i e n t for t h e t r a n s p o r t e d ion(s) - as can t h e s o d i u m p u m p in r a t d i a p h r a g m 7 - a n d t h a t t h e o t h e r ions are in equilibrium. Calcium a n d h y d r o g e n ions h a v e o p p o s i t e effects on t h e m e m b r a n e p o t e n t i a l of a p a r t i c u l a r n e u r o n e in t h e b r a i n of H. pomatla a n d CHRISTOFFERSEN8 has s u g g e s t e d t h a t t h e s e effects m i g h t p r o v i d e a stabilizing m e c h a n i s m ill acidosis. Figure 2 b s h o w s t h a t ionized calcium rises on a v e r a g e b y 11.5 m M p e r p H u n i t a n d t h i s c o m p a r e s well w i t h t h e v a l u e t h a t is n e e d e d t o p r e v e n t c h a n g e s in r e s t i n g p o t e n t i a l ; t o j u d g e f r o m CHRISTOFFERSEN'S Figures l c a n d 6b, t h i s is 6-13 m M p e r p H unit. F o u r snails were each infused t h r o u g h t h e i r cannulae, w i t h o u t a p p a r e n t ill effect, w i t h 1 m l of 150 m M CaC18 over 9 rain. H a e m o l y m p h was s a m p l e d j u s t before this a n d again 11-16 rain a f t e r t h e infusion a n d 2-4 m o r e t i m e s over t h e n e x t 3.4-6.3 h. H a e m o l y m p h b i c a r b o n a t e was d i l u t e d b y t h e i n f u s a t e and, since FCO 2 was little c h a n g e d , t h e p H fell (Table). B i c a r b o n a t e c o n c e n t r a t i o n s t h e n fell f u r t h e r t o n e w s t e a d y values, while calcium c o n c e n t r a t i o n s fell w i t h o u t stabilizing. As p r e v i o u s l y f o u n d S, b i c a r b o n a t e (total) a n d calcium left t h e h a e m o l y m p h in less t h a n 2:1 r a t i o ( m e a n r a t i o = 0 . 9 3 : 1 ) . Values of [Ca++] 9ICOn] rose as a result of t h e infusions, b u t a l w a y s eventually fell below- p r e - i n f u s i o n values, b e i n g lower on average t h e n b y 31 • s.d. 16~o. If t h e s a m e m e c h a n i s m o p e r a t e s here, in reverse, as in r e s p i r a t o r y acidosis, it seems t h a t calcium m u s t also leave t h e h a e m o l y m p h b y another mechanism not involving bicarbonate.

Rdsumd. D a n s l ' h O m o l y m p h e des escargots (Helix pomatia) traitOs avec le CO2 le c a l c i u m ionique a u g m e n t e et le p H d i m i n u e de telle fa~on que le p r o d u i t [Ca++]- [CO ~ ] ainsi que l ' a s s o c i a t i o n du calcium avec l'hOmocyanine r e s t e n t k peu pros c o n s t a n t . Le t e m p s requis p o u r a t t e i n d r e une s i t u a t i o n s t a b l e est regl6 p a r l'entrOe lento du CO2. AprOs i n j e c t i o n de CaC1 v [Ca ++] et [HCO~] d i m i n u e n t , ainsi que le p r o d u i t [Ca++3 9[CO~].

~

R. F. BURTON a n d IR. T. MATHIE

12

Ionized calcium (mM)

Fig. 2 (a). Relationships between pill and ionized calcium for 7 snails equilibrating with 5-10% CO2 in 02. (b) Average initial (O) and final (0) values of pl-I and ionized calcium for the same snails. Initial concentrations of total calcium averaged 8.3 ~ s.d. 2.3 mM. The continuous curve represents the relationship between pH and [Ca++] corresponding to a constant value of [Ca++] 9[CO~]. This is 3.6 mM ~ if pK( for carbonic acid is taken as 9.50. The broken curve represents the relationship between pH and [Ca++] that is needed if the amount of calcium bound by haemocyanin* in a 2% solution (containing also 10 mM magnesium) is to remain constant at 1 raM.

Institute o/ Physiology, University o] Glasgow, Glasgow G12 8QQ (Scotland), 78 Novembe~ 1974.

~tL EDMOND and J. 1K. T. M. GIESKES, Geochim. cosmochim. Acta 3d, 1261 (1970). 7 H. A. FOZZAt~Dand D. 1K. KIPNIS, Science 156, 1257 (1967). s G. R. J. CHRISTO~FERSEN, Comp. Biochem. Physiol. 46A, 371 (1973). 4 R. F. BURTON, Comp. Biochem. Physiol. 41A, 555 (1972). 6 J.