Resistance of uridine 5′-fluorophosphate to alkaline phosphatase and its sensitivity to 5′-nucleotidase

194

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 96985

RESISTANCE OF U R I D I N E 5'-FLUOROPHOSPHATE TO A L K A L I N E PHOSPHATASE AND ITS SENSITIVITY TO 5'-NUCLEOTIDASE Z. K U ~ E R O V ~ A.'~I) J. ~KODA

Department o/ Molecular Biology, Institute o/ Organic Chemistry and Biochemistry, Czechoslovak Academy o/ Sciences, Prague 6 (Czechoslovakia) (Received May 25th, 1971)

SUMMARY

Uridine 5'-fluorophosphate is fully resistant towards alkaline phosphatase (EC 3.1.3.1) and acid phosphatase (EC 3.1.3.2) but hydrolysed by 5'-nucleotidase (EC 3.1.3.5) to uridine. Uridine 5'-fluorophosphate is dephosphorylated by 5'-nucleotidase more slowly than uridine 5'-phosphate. Russell's viper venom splits uridine 5'-fluorophosphate to a mixture of uridine 5'-phosphate and uridine.

Nucleoside 5'-fluorophosphates have not been studied to any great extent. The first nucleoside fluorophosphates were prepared by WITTMANN1 who found that the analogues are readily split by snake venom phosphodiesterase whereas spleen phosphodiesterase does not attack them. NICHOL and co-workers ~ observed that inosine 5'-fluorophosphate is not a substrate of inosine 5'-phosphate dehydrogenase from Aerobacter aerogenes, or of adenylate synthetase from Escherichia coli but that it acts as a weak competitive inhibitor of both enzymes. They showed further that inosine 5'-fluorophosphate neither inhibits nor activates spleen or snake venom phosphodiesterase, pancreatic ribonuclease or adenosine monophosphate deaminase; snake venom phosphodiesterase will split inosine 5'-fluorophosphate to inosine 5'-phosphate. We decided within a wider investigation of anomalous nucleotides with an altered "inorganic" part of the molecule to study the behaviour of nucleoside 5'fluorophosphate of the pyrimidine type in some enzyme systems. The following enzymes not yet tested with nucleoside 5'-fluorophosphate were selected for this purpose: alkaline phosphatase (EC 3.I.3.I), acid phosphatase (EC 3.I.3.2), 5'-nucleotidase (EC 3.I.3.5), a mixture of phosphodiesterase (EC 3.1.4.1) and 5'-nucleotidase, and finally Russell's viper venom. Uridine 5'-fluorophosphate was prepared from uridine 5'-phosphate (preparation of Calbiochem) according to WITTMANN1 with slight modifications. Alkaline phosphatase was a preparation of Worthington. Acid phosphatase was prepared from prostatic glands by Dr. V. Pa~es from our Institute. Phosphodiesterase from Crotalus terr. terr. was a preparation of Boehringer. 5'-Nucleotidase from Crotalus adamanteus was a preparation of British Drug House Ltd; this preparation was found to be free of any detectable activity of either non-specific phosphatase or phosphodiesterase. Biochim. Biophys. Acta, 247 (1971) 194-196

195

URIDINE 5'-FLUOROPHOSPHATE

We have found that alkaline phosphatase and acid phosphatase did not split uridine 5'-fluorophosphate. Whereas under the same conditions, uridine 5'-phosphate was split completely after i h of incubation, no cleavage products were found from uridine 5'-fluorophosphate even after 5 h of incubation. Further, a tenfold excess of uridine 5'-fluorophosphate would not even partially inhibit the cleavage of uridine 5'-phosphate by alkaline phosphatase or acid phosphatase. Snake venom phosphodiesterase split uridine 5'-fluorophosphate giving rise to uridine 5'-phosphate. Uridine 5'-fluorophosphate was split five times more rapidly than uridylyl-3'-5'-uridine under the same conditions. Russell's viper venom split 5'-fluorophosphate giving rise to uridine 5'-phosphate and uridine. When a mixture of phosphodiesterase and 5'-nucleotidase was used, more uridine was formed than with 5'-nucleotidase alone since 5'-nucleotidase split not only uridine 5'-fluorophosphate but also the uridine 5'-phosphate formed by phosphodiesterase from uridine 5'-fluorophosphate. 5'-Nucleotidase split uridine 5'-phosphate more rapidly than uridine 5'-fluorophosphate (Fig. i); the optimal pH for both substrates was practically identical (Fig. 3). The same degree of cleavage of uridine 5'-fluorophosphate was achieved by a 4-fold greater concentration of the enzyme than was required for the cleavage of uridine 5'-phosphate (Fig. 2).

i

i

[

i

F1

I

N260

1

0.5~ E o

20

40

t,min 60

0 I 10-~ 10"3 10-2 ~1-1 10° E 101

Fig. I. Rate•fc•eavage•furidine5'-ph•sphateanduridine5'-f•••r•ph•sphateby5'-nuc•e•tidase. T h e i n c u b a t i o n m i x t u r e c o n t a i n e d 2. 5 ffmoles s u b s t r a t e a n d i o o # g e n z y m e per ml, o.i M T r i s HC1 (pH 8) a n d o.i IV[ MgSO,. I n c u b a t i o n w a s carried o u t a t 37 °. T h e c o n c e n t r a t i o n of t h e indiv i d u a l c o m p o u n d s was e s t i m a t e d after s e p a r a t i o n of aliquots of t h e m i x t u r e b y p a p e r electrophoresis ( W h a t m a n 3, 15oo V, 60 min, o.o 5 M t r i e t h y l a m m o n i u m b i c a r b o n a t e buffer, p H 8.5). A f t e r d e t e c t i o n u n d e r u l t r a v i o l e t irradiation t h e e l e c t r o p h o r e t o g r a m was c u t into sections w h i c h were eluted w i t h water. T h e c o n c e n t r a t i o n s of c o m p o u n d s in t h e eluates were calculated f r o m t h e a b s o r b a n c e at 260 n m . I, uridine split f r o m uridine 5 ' - p h o s p h a t e ; 2, u r i d i n e split f r o m uridine 5 ' - f l u o r o p h o s p h a t e ; 3, uridine 5 ' - p h o s p h a t e ; 4, u r i d i n e 5 ' - f l u o r o p h o s p h a t e . Fig. 2. C o m p a r i s o n of e n z y m i c d e g r a d a t i o n of uridine 5 ' - p h o s p h a t e a n d u r i d i n e 5'-fluorophosp h a t e at different c o n c e n t r a t i o n s of 5'-nucleotidase. T h e conditions were t h e s a m e as described for Fig. i, e x c e p t for t h e 3o-min i n c u b a t i o n used here a n d t h e c o n c e n t r a t i o n of e n z y m e w h i c h is as i n d i c a t e d in t h e figure, i, uridine 5 - p h o s p h a t e ; 2, uridine 5 ' - f l u o r o p h o s p h a t e .

It follows from the results shown that different types of phosphatase behave quite differently toward uridine 5'-fluorophosphate. Our findings demonstrated a pronounced difference in the mechanism of action between non-specific phosphatases and specific phosphatase, 5'-nucleotidase. It is assumed that nucleoside 5'-fluorophosphates might serve as novel and useful models for study of mechanism of action of these enzymes as well as for specific enzyme assays. Biochim. Biophys. Acta, 247 (1971) 194-196

196

z. KUCEROV2k, J. ~KODA

70 Uridine %

"7.15 7.50

&00

8.50 9.00 9.50 pH

Fig. 3. Effect of pH on the cleavage of uridine 5'-phosphate and uridine 5'-fluorophosphate. The conditions were the same as described for Fig. I, except for the 6o-min incubation used here. Striped bars, uridine sprit from uridine 5'-fluorophosphate; blank bars, uridine split from uridine 5'-phosphate.

REFERENCES i R. WITTraA~N, Chem. Ber., 96 (1963) 771. 2 Q. W. NICHOL, A. NOMURA AND A. HAMPTON, Biochemistry, 6 (1967) lOO8.

Biochim. Biophys. Acta, 247 (i97 I) 194-196