824

Speeialia

EXPERIENTIA 26/8

t h a n n o r m a l value, w h i c h s u p p o r t s our view. The course of t h e first 2 cases was r a t h e r d i f f e r e n t as t h e y were d e t e c t e d a t t h e age oi 4 years a n d 21 m o n t h s . The diagnosis of c i t r u l l i n e m i a is h o w e v e r clearly e s t a b l i s h e d in our p a t i e n t , a n d d e a t h early in t h e n e o n a t a l period could be t h e rule r a t h e r t h a n t h e exception. T h u s citrull i n e m i a would be d i a g n o s e d m u c h m o r e r a r e l y t h a n would c o r r e s p o n d to its real o c c u r r e n c e 5.

Citrullins yon d e n n i c h t b e t r o f f e n e n F a m i l i e n m i t g l i e d e r n a b g e g r e n z t werden. H. W i c k , T. BRECHB~JHL:gR and J. GIRARD

Zusammen/assung. Die F a m i l i e n u n t e r s u c h u n g eines 3. Falles v o n Citrullinaemie s p r i c h t ffir eine a u t o s o m a l h e t e r o z y g o t e V e r e r b u n g . Die Merkmalstr~iger k h n n e n d u t c h eine einfache N t i c h t e r n s e r u m - B e s t i m m u n g des

We gratefully acknowledge the very valuable help of the family doctor of our patient, Dr. K. GkRARD, and we thank our technician, Mrs. BI~DOUCHA, for her continuous effort in pursuing promptly any suspicion of a metabolic disorder.

The

of Ovalbumin

Effect of Temperature

on the Peptic

Proteolysis

The t e m p e r a t u r e - d e p e n d e n c e of p e p t i c h y d r o l y s i s of a p r o t e i n was s t u d i e d b y BULL a n d CrJRRIE z, who e m p l o y e d ovalbumir~ as s u b s t r a t e . W e h a v e r e - e x a m i n e d t h i s prob l e m in view of t h e f i n d i n g t h a t t h e Michaelis c o n s t a n t for t h a t r e a c t i o n does n o t d e p e n d on p H 2 a n d in view of t h e e v i d e n c e t h a t Krn for t h e p e p t i c h y d r o l y s i s of s e r u m album~n 8 a n d of o v a l b u m i n 2 is t h e e q u i l i b r i u m c o n s t a n t t o t t h e dissociation of t h e e n z y m e - s u b s t r a t e c o m p l e x into enzyme and substrate. T h e k i n e t i c s of t h e a c t i o n of p e p s i n ( N u t r i t i o n a l Biochemicals Corp., t w i c e recrystallized) on o v a l b u m i n (twice recrystallized~), b o t h n a t i v e or a c i d - d e n a t u r e d 2, was followed b y d e t e r m i n i n g t h e t r i c h l o r o a c e t i c acidsoluble m a t e r i a l a b s o r b i n g at 275 nm, in aliquots r e m o v e d periodically f r o m t h e i n c u b a t i o n m e d i a ~. W h e n n a t i v e o v a l b u m i n was used as s u b s t r a t e , care was t a k e n to m e a s u r e t h e initial v e l o c i t y of p r o t e o t y s i s w i t h o u t interference of s u b s t r a t e d e n a t u r a t i o n ~,s T h e effect of t e m p e r a t u r e on t h e initial v e l o c i t y of p e p t i c h y d r o l y s i s of n a t i v e a n d d e n a t u r e d o v a l b u m i n was s t u d i e d a t t h e r e s p e c t i v e p H o p t i m a , a n d t h e results are s h o w n in T a b l e 1. A l t h o u g h we h a v e o b s e r v e d t h a t v~,~ values were c o n s i s t e n t l y lower w i t h d e n a t u r e d t h a n w i t h n a t i v e o v a l b u m i n , t h e t e m p e r a t u r e - d e p e n d e n c e of v~,~ was n o t significantly d i f f e r e n t for t h e 2 k i n d s of s u b s t r a t e , yielding a c t i v a t i o n energies (EA) of 13.7 ( ~ 1.0) kcal/mole a n d 12.7 (~- 1.0) kcal/mole for t h e n a t i v e a n d d e n a t u r e d s n b s t r a t e d , respectively. This w o u l d s e e m t o i n d i c a t e a difference in a c t i v a t i o n e n t r o p y (A S*) for t h e b r e a k d o w n of t h e e n z y m e - s u b s t r a t e c o m p l e x into p r o d u c t s . U n f o r t u n a t e l y ~chis difference b e t w e e n t h e 2 k i n d s of s u b s t r a t e could n o t be d e t e c t e d b y e s t i m a t e s of zlS* f r o m E A a n d r a t e c o n s t a n t values b e c a u s e of t h e large errors involved. W e h a v e c a l c u l a t e d f r o m our d a t a t h a t A S* for n a t i v e a n d d e n a t u r e d s u b s t r a t e s , a t 25~ was - - 8 a n d - - 1 3 ca1/ ~ respectively, b u t t h e e s t i m a t e d s t a n d a r d d e v i a t i o n for t h e s e values was 3.5 cal/~ F r o m t h e Km values a t 38 ~ we o b t a i n a n estimafie of -- 5.8 k c a l / m o l for t h e free e n e r g y of association b e t w e e n p e p s i n a n d o v a l b u m i n , b o t h n a t i v e a n d d e n a t u r e d . Alt h o u g h our s y s t e m does n o t allow m e a s u r e m e n t of t h e a c t i v a t i o n e n e r g y for t h e association of e n z y m e w i t h substrafe, t h e m a x i m u m value for this p a r a m e t e r should be 7.9 (~= 1.1) kcal/mole for t h e n a t i v e a n d 6.9 (=~ 1.4) kcal/ mote for t h e d e n a t u r e d s u b s t r a t e . T h e a b s e n c e of a signific a n t difference b e t w e e n t h e s e 2 values c o n t r a d i c t s t h e h y p o t h e s i s 5,7 t h a t a r a t e - l i m i t i n g acid d e n a t u r a t i o n , as t h e first s t e p in p e p t i c p r o t e o l y s i s w o u l d cause t h e lower p H - o p t i m a o b s e r v e d w i t h native, as c o m p a r e d w i t h de-

C h i l d r e n ' s H o s p i t a l o/ the U n i v e r s i t y o/ Basel, C H - 4 0 0 0 B a s e l 5 (Switzerla~r 9 F e b r u a r y 1970.

n a t u r e d , p r o t e i n s u b s t r a t e s . If this h y p o t h e s i s were valid, t h e a c t i v a t i o n e n e r g y for t h e association of e n z y m e w i t h n a t i v e s u b s t r a t e w o u l d include t h a t of t h e acid d e n a t u r a t i o n s . W e h a v e f o u n d t h a t acid d e n a t u r a t i o n of ovalb u m i n , in t h e c o n d i t i o n s p r e v a i l i n g in our e n z y m e reactions, has an a c t i v a t i o n e n e r g y of 33.9 (~= 3.6) kcal/mole

Table I. Effect of temperature on K m and Vmax for peptic proteolysis of native and denatured ovaibumin Temperature (~

K m • 10~ (M)

Vmax (AA/rain)

Native ovalbumin 20.0 25.0 30.0 35.0 38.0

2.00 (:k 0.19) 1.43 (=c 0.25) 1.00 (~ 0.11) 0.88 {:~ 0.23) 0.80 ( i 0.12)

0.340 0.530 0.730 1.130 1.180

{4- 0.026) (:~ 0.035) (-- 0.028) ( i 0.015) (~= 0.020)

Denatured ovalbumin 19.0 29.0 33.3 38.0 40.0

1.47 0.97 0.83 0.77 0.74

0.173 0.416 0.476 0.640 0.675

(• 0.007) (~ 0.018) (-[- 0.006) (~=0.010) (~- 0.006)

(=c 0.13) (-~-0.09) (:k 0.08) (• (~ 0.05)

Initial velocities at several substrate concentrations, ranging from 5 • 10-~ to 3 • 10 ~M, were measured at pH 0.8 with the native and pH 1.65 with the denatured ovaIbumin. K m and Vr~axvalues were estimated from the least-square equation for the 1]v versus 1/[S] plots. Vmax is expressed by the increase of absorbance of the trichloraeetic acid filtrate at 275 nm/miu. Pepsin concentration was 2.8 • 10 "M in all the experiments. Figures in parentheses indicate the standard deviations calculated from the error of the estimates of the intercepts and slopes of the least-square lines.

1 H. B. BULLand B. T. CURRIs J. Am. chem. Sot. 77, 2758 (1949). 2 Q. S. TAHIN and A. C. M. PAIv.~, Enzymologia 37, 153 (1969). a j. R. CANNand J. A. Ih~LAPpERJR., J. biol. Chem. 236, 2446 (1961). 4 R, A. KEKWmKand R. K. CANNAX,Biochem. J. 30, 227 (1936). 5 L. K. CI-fRISTENSEN,Arch. Biochem. Biophys. 57, 163 (1955). 6 K. J. LAIDLER, The Chemical Kinetics o[ Enzyme Action (Oxford University Press, Oxford 1958), p. 201. 7 M. SCHLAMOWlTZand L. U. PETERSO~-, J. biol. Chem. 234, 3137 (1959). 8 K. LINDERSTROM-LANG, R. D. HOTCHKISS and G. JOHANSI~N, Nature 742, 996 (1938).

16. 8. 1970

Specialia

(Table II), in good a g r e e m e n t w i t h p u b l i s h e d d a t a obt a i n e d a t h i g h e r p H v a l u e s 9, ~0 T h i s is s i g n i f i c a n t l y larger t h a n t h e m a x i m u m v a l u e for t h e a s s o c i a t i o n of p e p s i n with native ovalbumin. T h e t e m p e r a t u r e - d e p e n d e n c e of Krn (Table I) yields v a l u e s for t h e c h a n g e s in n o r m a l e n t h a l p y a n d e n t r o p y t h a t are n o t s i g n i f i c a n t l y d i f f e r e n t for t h e 2 f o r m s of t h e s u b s t r a t e . T h e large errors i n h e r e n t in t h e m e t h o d used for o b t a i n i n g t h o s e v a l u e s do n o t allow a fine i n t e r p r e t a tion, b u t i t is s i g n i f i c a n t t h a t t h e a s s o c i a t i o n of p e p s i n a n d

825

o v a l b u m i n is all e n d o t h e r m i c process w i t h a large increase in e n t r o p y . T h i s also occurs in t h e p e p t i c h y d r o l y s i s of s m a l l s y n t h e t i c s u b s t r a t e s n, a n d w o u l d i n d i c a t e t h a t h y d r o p h o b i c b o n d i n g p l a y s a p r e p o n d e r a n t role in t h e a s s o c i a t i o n of p e p s i n w i t h its s u b s t r a t e .

Rdsumd. P a r t a n t de l'effet de la t e m p 6 r a t u r e sur la cin~tique de la p r o % o l y s e p e p t i q u e de l ' o v a l b u m i n e n a t i v e ou denatur~e, on a estim6 l%nergie d ' a c t i v a t i o n , l ' e n t h a l p i e et ! ' e n t r o p i e de l ' a s s o c i a t i o n e n z y m e - s u b s t r a t . Q. s. TAHIN a n d A. C. M. PAIVA

Table II. Effect of temperature on the rate of acid denaturation of ovalbumin Temperature (~ hI • 108 (mid -1)

25.0 7

30.0 20

35.0 55

40.0 130

Department o/ Biophysics and Physiology, Escola Paulista de Medicina, Sgo Paulo, S.P. (Brazil), 28 January 7970.

The first-order rate constants (/~i) were estimated by following the decrease of solubility of 2.5 • 10-4M ovalbumin solutions kept at pH 0.8. The solubility was determined by periodically removing aliquots that were diluted 40-folt with 2M acetate buffer (pH 4.75} containing 0,5 M MgSO~, add reading the absorbanee of the filtrate at 275 rim.

9 H. K. CUBIN, Biochem. J. 23, 25 (1929). 10 R. J. GIBBS, M. BInR and F. F. NORD, Arch. Biochem. Biophys. 33, 345 (1951). n E. J. CASE'Zand K. J. LAIDLER, J. Am. chem. Soe. 72, 2159 (1950). a2 We are indebted to the Brazilian National Research Council (CNPq) for support.

Norepinephrine-Sensitive

in Brown

Na+/K+ ATPase

Activity

N o r e p i n e p h r i n e (NE) released f r o m s y m p a t h e t i c neur o n a l t e r m i n a l s a p p e a r s to m e d i a t e t h e increased t h e r m o genesis in b r o w n adipose tissue d u r i n g cold stress 2, a. T h i s response, w h i c h is b l o c k e d b y fl-adrenergic a n t a g o n i s t s , a p p a r e n t l y i n v o l v e s a c t i v a t i o n of lipolysis v i a t h e a d e n y l cyclase, Y, 5'-cyclic A M P system4, 5. H o w e v e r , t h e bioc h e m i c a l m e c h a n i s m s u n d e r l y i n g t h e N E - s t i m u l a t i o n of r e s p i r a t i o n in b r o w n f a t are still c o n t r o v e r s i a l a. T h e u n c o u p l i n g agent, 2 , 4 - d i n i t r o p h e n o l (DNP), i n j e c t e d i.v. i n t o c o l d - a c c l i m a t e d rats, e n h a n c e s t h e t h e r m o g e n i c r e s p o n s e of t h e b r o w n f a t d u r i n g cold stress as well as d u r i n g N E a d m i n i s t r a t i o n ; t h i s implies t h a t in a c t i v a t e d b r o w n fat, r e s p i r a t i o n is coupled t o o x i d a t i v e p h o s p h o r y l a t i o n G, W e t h u s p r o p o s e d a) t h a t t h e N E i n d u c e d r e s p i r a t o r y e l e v a t i o n of t h e b r o w n f a t i n i t i a l l y reflects i n c r e a s e d a v a i l a b i l i t y of s u b s t r a t e r a t h e r t h a n of A D P , a n d b) t h a t t h i s is a c c o m p a n i e d b y a n i n c r e a s e d cellular A T P r e q u i r e m e n t 6. T h e f i n d i n g t h a t N E ( w h e t h e r of e x o g e n o u s or n e u r o n a l origin) d e p o l a r i z e d t h e m e m b r a n e s of t h e s e cells in v i v o 7, s u g g e s t e d t h a t in t h e N a + / K + d i s t r i b u t i o n a n d p e r h a p s also t h e N a + / K + p u m p , a l t e r a t i o n s m i g h t occur d u r i n g s t i m u l a t i o n of t h e n n o g e n e s i s b y t h e c a t e c h o l a m i n e . H e n c e we e x a m i n e d t h e effect of N E o n t h e N a + / K + A T P a s e s y s t e m associated w i t h t h e m e m b r a n e ion p u m p . T h e m e t h o d o l o g y e n t a i l e d r e m o v a l of b r o w n adipose tissue f r o m d e c a p i t a t e d male, L o n g - E v a n s r a t s t h a t h a d b e e n cold a c c l i m a t e d (exposure t o 5 ~ 50% R . H . , w i t h 12 h h i g h / l o w l i g h t cycle for 4 - 8 weeks). T h e b r o w n f a t was cleared of e x t r a n e o u s tissues a n d t h e n h o m o g e n i z e d in a m i x t u r e (9/1, v o l u m e / w e i g h t ) c o n t a i n i n g 250 m M sucrose, 2 m M E D T A , a n d 2 m M T E S (N Tris ( h y d r o x y m e t h y l ) m e t h y l - 2 - a m i n o m e t h a n e sulfonic acid), p H 7.0 a t 0~ T h e h o m o g e n a t e was c e n t r i f u g e d 10 m i d a t 14,000 • g, t h e o v e r l y i n g f a t r e m o v e d a n d t h e supern a t a n t d e c a n t e d for assay. A T P a s e a c t i v i t y was determ i n e d in 2 m e d i a (final v o l u m e 1.5 ml): i.e., (A) in millimoles p e r liter: 20 T E S (pH 7.2), 27 sucrose, 8.7 KC1, 70 NaC1, 2.7 E D T A , 5 MgC12, 4 NaCN, 6.7 N a 2 A T P

Adipose

Tissue 1

(Sigma) ; (B) differed f r o m (A) o n l y in c o n t a i n i n g 190 m M sucrose a n d no KC1 or NaC1. E a c h r e a c t i o n s y s t e m cont a i n e d 0.477 ~ 0.013 m g s u p e r n a t a n t n i t r o g e n K After i n c u b a t i o n a t 30 ~ for 20 miD, t h e r e a c t i o n was t e r m i n a t e d w i t h 015 m l 1 . 0 N HC104, t h e t u b e s c e n t r i f u g e d a t 0~ for 10 rain a t 1000 • g a n d t h e i n o r g a n i c p h o s p h a t e in t h e s u p e r n a t a n t a s s a y e d 9 a g a i n s t a p p r o p r i a t e controls. T h e N a + / K + A T P a s e a c t i v i t y is here defined as t h e i n o r g a n i c p h o s p h a t e released in t h e p r e s e n c e of Na+, K + a n d Mg++ ( m e d i u m A) m i n u s t h a t in t h e a b s e n c e of KCI a n d NaC1 ( m e d i u m B)10. I n t h e p r e s e n c e of N E , t h e N a + / K + A T P a s e was m a r k e d l y s t i m u l a t e d . T h e increase was dose d e p e n d e n t (Figure) a n d m a x i m a l w i t h 6 m M N E . T h e fact t h a t t h i s e n h a n c e m e n t was a b o l i s h e d b y o u a b a i n (Table) suggests t h a t t h i s A T P a s e is p a r t of t h e N a + / N + p u m p a s s o c i a t e d w i t h t h e cell m e m b r a n e .

i Supported in part by research grallts NASA No. NGR-05-004-035 and USPHS No. HD-03268. P.A.H. is a PHS predoctoral fellow {No. 1-GM-33, 505-04). A preliminary report of this work was presented at the 1969 Fall meeting of the Arn. Physiol. Society; an abstract of this talk is contained in The Physiologist 72, 257 (1969). We thank J. F. DETREK for his technical assistance. 2 D. HULL and M. M. SEO*LL, J. Physiol. 187,458 {1965). R. E. SMITH and B. A. HORWlTZ, Physiol. Rev. 49, 330 (1969). 4 A. BEVIZ, L. LUNDHOLIVi and t~. MOHME-LUNDHOLI% Br. J.

Pharmac. 3d, 198P (1968). 5 N. REED and J. N. FAIN, J. biol. Chem. 243, 2843 (1968). 6 B. A. HORWITZ,P. A. HERD and R. E. SMITH,Can. J. Physiol. Pharmae. 46, 897 (1968). 7 B. A. HOR'vVlTZ, J. ~VL HOROWITZ JR. and R. E. SMITH, Proe.

natn. Acad. Sci., USA 64, 113 (1969). s T. S. MA and G. ZtJAZACA,Ind. Eng. Chem. Analyt. Edn. 14, 280 (1942). 9 G. A. Go~oei, J. Lab. elin. Med. 27, 955 (1942). 10 R. L. POST and A. K. SEN, in Methods in Enzymology, Oxidation and Phosphorylation (Eds. R. W. ESTABROOKand M. E. PULLMAN; Academic Press, NewYork, 1967), p. 762.