386

Specialia

EXPERIENTIA 2814

Table II. Cyanide (CN~ sensitive alkaline phosphatase activity in the basal and luminal area of rat renal proximal tubular segments Structure

Without CN

Basal area from PTC from PTR Luminal area from PTC from PTR

41.9• l l . 7 T 5.5 137.8J 79.2 171.0-t-70.5

(15) (7~ {12) (10)

With 10 m M CN

CN sensitive phosphatase

CN-sensitive activity total activity

20.4-t-14.8 3.82_ 1.4 3.62= 2.8 4.2z= 2.5

21.5 7.9 134.2 166.8

0.51 0.68 0.97 0.97

(7) (6) (5) (6)

PTC, proximal tubular convoluted portion: PTR, proximal tubular straight portion. ~ M K H -- inoles p-nitrophenol/kg dry weight/h at 37~ 2_ SDM. Number of analyses in brackets.

A s y e t it is n o t c l e a r w h e t h e r m i t o c h o n d r i a l m e m b r a n e c o n t a i n s a. P ' t a s e l ~ A n o t h e r i n t r a c e l l u l a r s i t e of a. P ' t a s e is t h e G o l g i a p p a r a t u s . M o r p h o m e t r i c d a t a i n r a t r e n a l t u b u l e r e v e a l a m u c h h i g h e r a m o u n t of G o l g i m e m b r a n e s i n t h e P T R t h a n in t h e P T C e p i t h e l i a 14 H o w e v e r , a.P'tase in basal area demonstrates an inverse activity p a t t e r n ( T a b l e I~ T h e c o n c l u s i o n c a n be d r a w n t h a t a . P ' t a s e a c t i v i t y ( T a b l e I a n d III in t h e b a s a l a r e a of P T C a n d P T R b e l o n g s to t h e b a s a l l a b y r i n t h m e m b r a n e . T w o t h i r d s of a. P ' t a s e in r a t r e n a l t u b u l a r e p i t h e l i a c o n s i s t of a n i n s o l u b l e d e s m o e n z v m e a n d ~/a of a s o l u b l e l y o e n z y m e ~5. T h e l y o e n z y m e is l o c a l i z e d i n t h e b a s a l l a b y rinth membrane as shown by electron microscopic t e c h n i q u e 15. T h i s m i g h t e x p l a i n t h e l a c k of a . P ' t a s e in basal labyrinth membrane fractions~6. O u r r e s u l t s s h o w t h a t a. P ' t a s e is l o c a l i z e d in t h e w h o l e s u r f a c e m e m b r a n e of t h e r e n a l p r o x i m a l t u b u l a r cell, i n c l u d i n g t h e b a s a l i n f o l d i n g s . T h i s f a c t e x c l u d e s a. P ' t a s e t o s e r v e a s a m a r k e r in d i f f e r e n t i a t i n g b r u s h b o r d e r f r o m b a s a l l a b y r i n t h . A s a p r o o f f o r t h e p u r i t y of t h e b r u s h b o r d e r f r a c t i o n , w e p r o p o s e t h e l a c k of N a K A T P a s e a c t i v i t y a s s u c h a m a r k e r . N a K A T P a s e is l o c a l i z e d o n l y i n t h e b a s a l l a b y r i n t h m e m b r a n e a n d is m i s s i n g i n t h e b r u s h b o r d e r s.

Zusammen/assung. M i t H i l f e e i n e r U l t r a m i k r o t e c h n i k konnte an isolierten gewundenen und geraden Hauptstricken der Rattenniere gezeigt werden, dass die alkalische Phosphatase nicht nur in der Brirstensaum-, sondern

R e a c t i o n of D N A w i t h P h o s p h o r i c

auch in der basalen Labyrinthmembran l o k a l i s i e r t ist. Das Enzym ist infolgedessen kein geeignetes Leit-Enzym zur Erkennung des Brirstensaumes aus einem Plasmamembrangemisch. Es wird vorgeschlagen, das Fehlen der N a K A T P a s e - A k t i v i t ~ L t a l s M a s s fiir die R e i n h e i t d e r Brirstensaumfraktion zu benutzen. U . SCHMIDT a n d U . C . DUBACH 17 with technical assistance of I. BIEDER a n d B. F U N K

Enzyme Laboratory o/ the Medical Policlinic, University of Basel, Hebelstrasse l, CH-4056 Basel (Switzerland), and Institute o/ Pathology, University of Ti~bingen (Germany), 20 December 7977.

13 D. B. ROODYN, in The Biological Basis in Medicine (Eds. E. E. BITTAR and N. BITTAR, Academic Press, London, New York 1969), Vol. 1, p. 123. 14 TORHORST, H. P. ROHR and F. GLOOR, Verh. dt. Ges. Path. 53, 391 (1969). 16 M. M. NACHLAS, ~;. PRINN and A. M. SELmMAN, J. biophys. biochem. Cytol. 2, 487 (1956). 16 R. KINNE, J. E. ScnNiTz and E. KINNE-SAFFRAN, Pfliigers Arch. ges. Physiol. 329, 191 (1971). 1~ Grants of SNF Nr. 3.207.69 and DFG.

Acid Esters: Gasoline Additive and Insecticides

Trimethyl phosphate (TMP), a gasoline additive and the insecticides dichlorvos (DDVP, 0,0-dimethyl-2, 2-dichlo r o v i n y l p h o s p h a t e ) a n d d i p t e r e x ( d y l o x , 0, 0 - d i m e t h y l - 2 , 2, 2 - t r i c h l o r o - l - h y d r o x y e t h y l p h o s p h o n a t e ) h a , ( e r e c e n t l y been reported to exhibit 'suspicious' biologic properties suggesting that they may have adverse effects on health. 9T h u s T M P is m u t a g e n i c i n m i c e t a n d D D V P i n E. coli 3. D i p t e r e x h a s b e e n s t a t e d t o b e w e a k l y c a r c i n o g e n i c for r a t s 3 a n d D D V P t o c a u s e c h r o m o s o m e a b e r r a t i o n s in o n i o n r o o t t i p cells 4. I n t e r m s of p r e s e n t c o n c e p t s , all of t h e s e p h e n o m e n a p r e s u m a b l y r e s u l t f r o m a n e f f e c t of t h e s e a g e n t s o n c e l l u l a r D N A . I n d e e d t h e a l k y l a t i o n of D N A b y D D V P h a s b e e n r e p o r t e d 5. I n t h i s r e p o r t t h e e f f e c t of D D V P o n D N A is c o n f i r m e d , m o r e o v e r , it is shown that TMP and dipterex also alter DNA. This may t h e n p r o v i d e a c h e m i c a l b a s i s for t h e o b s e r v e d b i o l o g i c a l e f f e c t s of t h e s e s u b s t a n c e s .

E x p o s u r e of D N A t o t h e s e p h o s p h o r i c a c i d e s t e r s 6 res u l t e d i n n o t i c e a b l e d i m i n u t i o n s in t h e s e d i m e n t a t i o n coe f f i c i e n t of t h i s b i o p o l y m e r ( T a b l e ) . P r e s u m a b l y t h e s e c h a n g e s r e f l e c t a l k y l a t i o n of D N A f o l l o w e d b y s o m e d e -

1 S. S. EPSTEIN,W. BASS, E. ARNOLD and J. BISHOP, Science 168, 584 (1970). 3 G. L6FROTH, C. KIM, and S. HUSSAIN, Environ. Mutagen Soc. Newsletter 2, 21 (1969). 3 R. PREUSSMANN, Food Cosmet. Toxicol. 6, 576 (1968). a K. SAX and H. J. Shx, Jap. J. Genetics 43, 89 (1968). 5 G. L6FROTH, Naturwissensehaften 57, 393 (1970). Trimethyl phosphate was purchased from Aldrich Chemical Co., Inc., Milwaukee, Wis. Dipterex (Dylox) was received from Chemagro Corp., Kansas City, Mo., and dichlorvos (DDVP, Vapona) from Shell Chemical Co., San Ramon, Calif.

15.4. 1972

387

Specialia

p u r i n a t i o n ; this in t u r n causes scission of t h e p h o s p h o d i ester b a c k b o n e 7 leading to lower s e d i m e n t a t i o n coefficients. H e a t i n g of t h e m o d i f i e d D N A results 1. in f u r t h e r d e p u r i n a t i o n a n d h e n c e f u r t h e r scission a n d 2. in separat i o n of t h e s t r a n d s a n d t h e r e f o r e t h e u n m a s k i n g of addit i o n a l ' h i d d e n ' (single-chain) b r e a k s t h a t p r e v i o u s l y were stabilized (i. e. hidden) b y t h e d o u b l e - s t r a n d e d s t r u c t u r e . T h e s e effects cause a f u r t h e r r e d u c t i o n in s e d i m e n t a t i o n coefficient (Table). The c o n t r o l D N A , on t h e o t h e r h a n d , does n o t s h o w this p r o n o u n c e d decrease in s e d i m e n t a t i o n coefficient u p o n t h e r m a l d e n a t u r a t i o n . A d d i t i o n a l e x p e r i m e n t s r e v e a l e d t h a t e x p o s u r e of deo x y g u a n o s i n e t o t h e s e s u b s t a n c e s r e s u l t e d in t h e f o r m a t i o n of a p r o d u c t w i t h c h r o m a t o g r a p h i c a n d s p e c t r a l p r o p e r t i e s similar to t h o s e of 7 - m e t h y l d e o x y g u a n o s i n e . These find-

Effects of phosphoric acid esters on the properties of DNA Additions

Sedimentation coefficients (s) Before heating After heating

None Dipterex DDVP Trimethylphosphate

18.4 11.2 15.7 15.7

17.5 7.4 9.1 10.3

To 1 ml of calf thymus DNA (1 mg per ml of 0.015 M NaC1 in 0,01 M phosphate buffer, pH 7.0) either 1 ~xl of DDVP, 1 [xl of trimethylphosphate or 100 tzg dipterex were added. The mixtures were incubated at 56~ for 42 h whereupon DNA was precipitated with ethanol, the insoluble fibers washed extensively with ethanol and the DNA redissolved in 0.15M NaC1. Portions of the specimens were placed in a boiling water bath for 10 rain. and then immersed into an ice-bath. Sedimentation coefficients were determined in a Spinco Model E analytical ultracentifuge equipped with an ultraviolet optical system.

ings s u p p o r t t h e a b o v e p o s t u l a t e d m e c h a n i s m of action in w h i c h a l k y l a t i o n of D N A i s t h e first s t e p in t h e d e g r a d a t i v e process, t h e y are also in accord w i t h t h e k n o w n alkyls t i n g p o t e n t i a l of t h e s e s u b s t a n c e s (see refs. 1 andS). I n view of t h e e s t a b l i s h e d r e l a t i o n s h i p b e t w e e n t h e a b i l i t y of a s u b s t a n c e to r e a c t w i t h D N A a n d its p o t e n t i a l t o i n d u c e d e t r i m e n t a l effects ( m u t a t i o n s , carcinogenesis a n d t e r a t o g e n e s i s ) a n d because of w i d e s p r e a d h u m a n exp o s u r e t o t h e a g e n t s studied, w a y s to e l i m i n a t e h u m a n c o n t a c t w i t h t h e s e s u b s t a n c e s should be s o u g h t s.

Rdsumd. L ' a d d i t i o n de p h o s p h a t e t r i m 6 t h y l , < (0, 0-dimethyl-2, 2, 2-trichloro- 1 - h y d r o x y e t h y l p h o s p h o n a t e ) h du D N A p r o v o q u e la d 6 g r a d a t i o n de c e t t e macromol~cule. L a r6action semble ~tre due ~ une alkyla t i o n d u r6sidu de g u a n i n e du DNA. Ces o b s e r v a t i o n s p e u v e n t fournir une e x p l i c a t i o n c h i m i q u e a u x effets biologiques de ces s u b s t a n c e s . H. S. ROSENKRANZ a n d S. ROSENKRANZ

Department o/Microbiology, College o/ Physicians and Surgeons, Columbia University, N e w York (New York 70032, USA), 15 November 1972.

P. D. LAWLEV,in Progress in Nucleic Acid Research and Molecular Biology (J. N. DAVlOSONand W. E. COHN; Academic Press, New York 1966), vol. 5, p. 89. s This investigation was supported by a grant from the Damon Runyon Memorial Fund for Cancer Research and by the Annie R. Masch Memorial Grant for Cancer Research from the American Cancer Society. One of the authors (H. S. R.) is a Research Career Development Awardee of the U.S. Public Health Service No. 2-K3-GM-29, 024.

Fluorescence of the Soluble 'Sweet-Sensitive' Protein Complexes with Sugars The i n t e r a c t i o n of a ' s w e e t - s e n s i t i v e ' p r o t e i n w i t h sugars a n d s a c c h a r i n was originally s t u d i e d b y t h e m e t h o d of difference s p e c t r o s c o p y 1. C h a n g e s in o p t i c a l d e n s i t y u p o n t i t r a t i o n w i t h various c o n c e n t r a t i o n s of d i f f e r e n t sugars were r e c o r d e d a t 277 nm, t h e u l t r a v i o l e t m a x i m u m of t h e p r o t e i n using a t a n d e m d o u b l e c o m p a r t e m e n t techn i q u e 3. The U V - a b s o r p t i o n s p e c t r a of p r o t e i n s is m a i n l y due t o t h e i r c o n t e n t of t h e a r o m a t i c a m i n o acid tyrosine, t r y p t o p h a n and, to a lesser e x t e n t , p h e n y l a l a n i n e a. The region 278-281 n m is c h a r a c t e r i s t i c a l l y t h e m a x i m u m p e a k area 4. The fluorescence of p r o t e i n s originates a l m o s t e n t i r e l y f r o m t h e t y r o s y l a n d t r y p t o p h e n y l residues. C o n s e q u e n t l y , c o n f o r m a t i o n a l m o d i f i c a t i o n s can be followed b y o b s e r v i n g c h a n g e s in e i t h e r t y r o s i n e or t r y p t o p h a n fluorescence int e n s i t y . This s t u d y of t h e fluorescence of t h e 'sweet-sensit i v e ' p r o t e i n a n d its sugar c o m p l e x e s was u n d e r t a k e n because of t h e k n o w n s e n s i t i v i t y of t h e emission of t h e aromatic chromophores. Materials and methods. T h e ' s w e e t - s e n s i t i v e ' p r o t e i n was e x t r a c t e d f r o m e p i t h e l i u m of cow a n d dog t o n g u e s b y t h e m e t h o d p r e v i o u s l y described 1, 5 w i t h t h e following except i o n s : 1. t h e 75,000 • g or 105,000 • g s u p e r n a t a n t was n o t s u b j e c t e d to a m m o n i u m sulfate b u t was f r a c t i o n a t e d b y

t h e u l t r a f i l t r a t i o n in a n A m i c o n cell w i t h a ])iaflow m e m .brane of r e t e n t i o n of molecular w e i g h t s 100,000 a n d u p : 2. t h e r e t e n t a t e f r o m u l t r a f i l t r a t i o n was s u b j e c t e d to c a t i o n e x c h a n g e on B i o - R e x 63 resin (control No. 7429) a n d t h e p r o t e i n e l u t e d w i t h 1.0 M s o d i u m b i c a r b o n a t e - s o d i u m carb o n a t e b u f f e r p H 10.0 plus 1M NaC1. T h e p r o t e i n w a s d i a l y z e d in t h e u l t r a f i l t r a t i o n cell u n t i l e q u i l i b r i u m to 0.1 N s o d i u m p h o s p h a t e buffer p H 7.0 was achieved. E m i s s i o n s p e c t r a were m e a s u r e d w i t h a n i n s t r u m e n t built in t h e l a b o r a t o r y 6. Optical d e n s i t y (OD) r e c o r d i n g s were m a d e on a Cary 15 r e c o r d i n g s p e c t r o p h o t o m e t e r . I n

1 F. R. DASTOLI and S. PRICE, Science 75,1 (3750), 905 (1966). T. T. HERSKOVlTS and M. LASKOWSKIJR., J. biol. Chem. 237, 2481 (1962). a D. B. WETLAUFER,Adv. Protein Chem. 17, 303 (1962). 4 T. T. HERSKOVlTS,in Methods in Enzymology (Ed. C. W. H. HIRS; Academic Press, New York 1967), vol. ll, p. 750. 5 F. R. DASTOLI,D. V. LOPIEKESand S. PRICE, Biochemistry 7, 1160 (1968). 6 M. J. KRONMANand L. G. HOLMES,Photochem. Pbotobiol., in press (1971).