SHORT COMMUNICATIONS I63

The specificity of the interaction of adenos.;n~, trJphosphate " w i t h G - a c t i n

I n ear l ier s t u d i e s ~ wc f o u n d i n d i c a t i o n s t h a t in t h e b i n d i n g of A T P a n d I T P to G - a c t i n b o t h t i le p u r i n e r i ng a n d t h e t r J p h o s p h a t e c h a i n a re i nvo lved . Modi f ica t ions of t h e r ing s t r u c t u r e , a s in G T P , U T P , a n d C T P or .he s u b s t i t u t i o n of a d i p h o s p h a t e for t h e

t r i p h o s p h a t e g r o u p ( A D P or IDP) led to t h e loss or s u b s t a n t i a l r e d u c t i o n of t he b i n d i n g of t h e n u c l e o t i d e to G - a c t i n w i t h t h e s i m u l t a n e o u s d i s a p p e a r a n c e of t he p r o t e c t i v e effect a g a i n s t t h e loss of t h e p o i y m e r i z a b i l i t y of a c t i n d u r i n g r e p e a t e d ac id prec ip i -

t a t i on . I n th i s r e p o r t e v i d e n c e is p r e s e n t e d for t h e d i rec t or i n d i r e c ~ n v o l v e m e n t of t h e

r ibose m o i e t y of A T P in t h e b i n d i n g process , as o b t a i n e d f rom s t u d i e s on t h e in te r -

a c t i o n of d e o x y r i b o s e n u c l e o s i d e t r i p h o s p h a t e s w i t h G-ac t in .

TABLE I

THE BINDING OF VARIOUS COMPOUNDS TO THE ATe-BINDING SITE OF G-ACTIN

A G-actin solution co:.taining IO -3 .11 Tris buffer pH 7.25 (protein concentration 7-9 mg/ml) was incubated for 30 min in the presence of o.oi mg "I~C~ATP/ml (specific activity ,..2 itC/rng) at roam temperature. The free nucleotides were removed by t reatment vdth o.I g of freshly washed Dowex-I-Cl resin for each ml of the solution for 5 rain4. After centrifugation, the protein and nucleotide concentrations were measured. The investigated compounds were added in concen- trations ~ndieated in the table and after 3 ° mitt incubation at room temperature the free nucleotldes were removed by t rea tmea t with Dowex-t-Cl resin, o.2 ml of the Dowe;:-treated material was dried on filter paper and the radioactivity measured as described by LOI~-rFIELD AND EIGN~R 5

in a liquid scintillation counter.

A ddal analogtse

Moles of Radio~tivily of the bound nr.deotides

analoguglmole of aft~'r incubation with A TP bound to ac t tn

in the absence of the analogves ¢expressed as % af the original

the ana logue radioactivity)

No addition o xoo ATP 4.75 35 ITP 4.75 55 UTP 4.75 73 CTP 4.75 88 GTP 4.75 85 deATP 4.75 9o deGTP 4.75 92 deCTP 4.75 96 Ribose 5000.00 1oo Adenosine 25.oo 9z PPt* I25.oo xoo ATP x I .oo 8 ADP I I.OO 45 AMP i I .oo 93 deATP x t.oo 9 t ADP + deATP I :.oo each 47 AMP + deATP i I.OO each 8i

* Slightly polymerized.

T h e r e l a t i v e a f f in i ty e l t h e v a r i o u s d e o x y r i b o s e n u c l e o s i d e t r i p h o s p h a t e s to G- a c t i n w a s d e t e r m i n e d e s s e n t i a l l y a c c o r d i . g to STROHMAN 2 b y m e a s u r i n g the i r ab i l i t y to d i sp l ace 14C-labeled A T P f r o m t h e b i n d i n g s i te of G-ac t in . T h e m e t h o d is b a s e d

on t h e r a p i d e x c h a n g e of t h e b o u n d A T P of G-ac t i n w i t h a d d e d free A T P (ref. 3).

Bioch im. B i o p h y s . Ac ta , 57 (x962) I63-x65

I64 SHORT COMMUNICATIONS

As shown in Table I, under conditions when the exchange of the bound [14C"ATP with unlabeled ATP approaches completion, and a considerable release of the radio- active ATP from the bound form occurs on addition of ITP, no significant displace- ment of the bound nucleotide takes place in the presence of deATP and a number of structurally related compounds v i z . , deCTP, deGTP, UTP, CTP, GTP, PPt, adenosine, AMP, and ribose applied alone or in combination with deATP. The observed reduction of the bound nucleotide of G-actin in the presence of A D P is most likely due to the formation of unlabeled ATP from the added A D P through the action of myokinase and through another enzymically catalyzed A T P - A D P exchange reaction recently observed in our actin preparations.

Under the conditions of the above experiments, the interaction of various nucleoside tr iphosphates with actin was measured in the presence of ATP. The gradual replacement of the bound ATP by various nucleoside tr iphosphates in the course of repeated isoelectric precipitation 1 offers a possibility to s tudy their effect on the polymerizabil i ty of G-actin in the absence of ATP. G-actin samples were pre- cipitated four t imes in the presence of I0 -4 M ATP, deATP, deCTP, deGTP, or with- out added nucleotides. The resulting actin preparat ions were compared wi th regard to their polymefizabil i ty and nucleotide binding (Table II). Practica!ly no polymeriza tion took place in those samples which contained I0 -4 M deATP, deCTP, and deGTP.

TABLE II POLYMERIZABILITY AND NUCLEOTIDE ~3INDING OF ACTIN SOLUTIONS PRECIPITATED

AT pn 4.75 IN THE PRESENCE OF VARIOUS NUCLEOS1DE TRIPHOSPHATES Acid precipitatit,n was carried out as described earlier x. Precipitation was repeated four times. Final concentrataon of protein o.z_z.8 mg/ml. The amount of the bound nucleotides was determined after treatment with Dowex-I-C1 resin by measuring the ultraviolet absorption of cleproteinized samples (-'.5 % HC104) in a Zeiss spectrophotometer. Viscosity measurements were carried out in an Ostwald viscometer at 26 °. Protein concentrations during ~-iscosity measurements: no nucleotide, o.77 mg/ml; ATP, o.76 mg/rnl; deATP, o.67 mg/ml; deCTP, o.73 rng/ml; deGTP,

o.78 mg/ml.

Nuclcotide added Moles of bound specific viscosity 30 rain aJter the

during a c i d nucleotide per addition of o.r M KCI precipitation 60 ooo g of actin and sro -~ M MgClt

No nucleotide 0.04 0.06 ATP i.i 5 1.72 deATP o.~56 o. IO deCTP 0.087 0.03 deGTP o.Io 0.06

The amount of the bound nucleotides and of the inorganic phosphate l iberated on ad- dition of KC1 in the presence of deoxyribosenucleoside tr iphosphates was found to be rather small compared with preparations containing ATP or ITP.

The observations presented in this paper suggest that in the specific interaction of ATP with G-actin the ribose moiety of the ATP molecule plays a rather impor tant role. The drastic reduction of the affinity of ATP to G-actin by subst i tut ing ribose with 2-deoxyribose in the molecule might indicate that the interaction of ATP with actin directly involves the OH-group in the 2 position of the ribose molecule or that the configuration of deoxyribose-adenosine t r iphosphate is al together different from that of the ATP. I t might be mentioned that myosin and H-meromyosin hydrolyse deATP zt a rate comparable to the hydrolysis of ATP.

Bioch im. B iophys . Ac ta . 57 (I962) 163-165

SHORT COMMUNICATIONS 165

This investigation has been supported by the U.S. Public Heal th Service Research Grants B-2175-C and H-5949, from the National Ins t i tu te of Neurological Diseases and Blindness and from the National Heart Inst i tute , respectively. Par t of this work was carried out during the tenure of an Established Investigatorship from the American Hear t Association, Inc.

M**scle Research Department , Ret ina Fonndat ion, ANTHONY MARTONOSI Boston, Mass . (U.S ,A . )

1 A. MARTONOSI AND. M. A.. GOUVEA, J. Biol. Chem., 236 (1961) 1345. R. C. STROHblAN, Federation Pvoc., 2o (t961) 299.

a A. MARTONOSI, M. A. GOUVEA AND J. GEkGELY, d r. Biol. Chem., 235 {I96o) I7oo. * 4 S. ASAKURA, Arch. Biochem. Biophys., 92 (x96I! t4o.

5 R. B. LOFTFIELD AND E. A.. EIGNER, Biochem. Biophys. Research Communs., z (196o) 72.

Received August I8th, 1961 Biochim. Biophys..4eta, 57 (I96~-) I63-I65

The lack of incorporation of canavanine into rabbit-musde protein in vivo

Studies by RICi~MOND 1 with StaplLvlococcns aftrens 5z4 SC and by KRUSE et al. z with Walker 256 carcinosarcoma cells cultured in vitro have derr.onstrated the incorpo- rat ion of canavanine into proteins; incorporation occurs by the replacement cf arginyl residues 2. This infc, rmation, together with the apparent requireme~at for the presence of an arginyl residue in phosphopeptides derived from muscle phosphorylase a for phosphorylase phosphatase act ivi ty 3, s t imulated the present experiments on the possible subst i ta t ion of arginyl residues by canavanine in synthesis of muscle phospho- rylase. That such a replacement might occur is indicated by the recent findings that rabbits readily incorporate p-fluorophenylalanine into various proteins in vivo'-'.

Assessment of possible canavanine incorporation required development of a suitable procedure for reliable detection of small amounts of canavanine in muscle proteins. This paper gives details of a chromatographic procedure developed for cana- vanine determination, together with results showing that rabbits fed toxic levels of canavanine do not incorporate canavanine into muscle proteins.

One-pound male rabbits were placed on three different diets; A, Nutrena rabbit pellets which contained I.O ° o arginine : B, rabbit pellets with I .o" o added canavanine; and C, rabbit pellets with 2.0 °'o added canavanine.*Crude canavanine was prepared by extract ing jack-bean meal with 50 °. o ethanol ~ and added as an aqueous concen- trate. The solution was dried evenly on ground pellets, and the ground meal repelleted for feeding. The rabbits fed i .o and 2.0°0 canavanine showed an average weekly weight gain of 16o and 4 ° g, respectively, as compared to 34 ° g for the control. Muscle from the rabbit fed 2.o o~, canavanine for 3 ° days was used for the experiments re- ported herein.

After removal of lipids, nucleic acids, amino acids and carbohydrates as described by BLOCK AND WEISS B, lZ 5 mg of the rabbit-muscle protein was hydrolyzed with IOO ml of 6 N HCI under reflux for 24 h at I iO °. The excess HC1 was removed by

Biochim. Biophys..-Icta, 57 (I96:') I65-z67