Determination of DFPase in rabbit and rat tissues using DF32P

Biochemical Pharmacology, 1964, Vol. 13, pp. 849-854. Pergamon Press Ltd., Printed in Great Britain.

DETERMINATION OF DFPase IN RABBIT A N D RAT TISSUES USING DF32p B. V. RAMACHANDRAN* and G. fltGREN Institute of Medical Chemistry, University of Uppsala, Uppsala, Sweden

(Received22 December1963 ; accepted26 February 1964) Abstract--The subcellular esterases of rabbit liver cells have been fractionated by DEAE-cellulose chromatography and the esterase activity of the fractions correlated with their capacity to bind radioactive diisopropyl phosphorofluoridate (DFaZP). As in the case of the rat liver enzymes the two activities are found to coincide with each other. The diisopropyl phosphorofluoridate-hydrolysing activity (DFPase) has been determined in rabbit and rat tissues using DFszP as the substrate. The values obtained are much lower than those reported in literature. The enzyme is found to be localized in the supernatant fraction and is identifiable with one of the major enzyme peaks obtained in DEAE-cellulose chromatography. WHEN radioactive diisopropyl phosphorofluoridate (DFa2p) is injected to rats large amounts o f the label are found to be incorporated in the liver. 1 A similar effect has been observed in the rabbit. 2 The liver tissue o f these species is also k n o w n to contain an active enzyme, DFPase, which hydrolyses diisopropyl phosphorofluoridate (DFP) to diisopropyl phosphate. 3, 4 This enzyme, in analogy with A-esterases 5 would be expected to act on simple substrates like p-nitrophenyl acetate ( P N P A ) 6 and not to bind DFazP since it would hydrolyse it to an inactive form. z However, in our previous study on rat liver fractions wherein the esterase activity was correlated with DFa2p-binding capacity we failed to detect any enzyme peak which could be identified as DFPase. 7 We have now extended similar studies to rabbit liver esterases, the original source of DFPase. a Using a micro-method for the determination o f D F P a s e activity it has been found that the enzyme is present only in small amounts in rat and rabbit tissues. In the liver it is found to be localized in the supernatant fraction. MATERIALS AND METHODS The materials used and the fractionation procedure adopted were the same as described in the previous papers. 7, 8 The subcellular fractions o f the rabbit liver were isolated from 32 g of the liver tissue. D F P a s e was determined as follows: 1 to 5 ml o f the enzyme fraction was diluted to 50 ml with 0.01 M Tris-acetic acid buffer, p H 7.4, and incubated with or without 10 -a M M n 2+ for 20 min at 37 °. A solution of 0.01 M DFa2P in propylene glycol (50/~1) was then added to the enzyme-buffer mixture to give a final DF32P concentration o f 10 -5 M and the incubation continued in the thermostat. The total radioactivity in 1 ml o f the digest ranged f r o m 10 to 25 × 10a cpm. Aliquots o f 5 ml were withdrawn every 5 min and extracted thrice with 10-ml portions o f ether. This was found to * Visiting Lecturer from the National Chemical Laboratory, Poona, India. 849

850

B.V. RAMACHANDRANand G. AGREN

remove the undecomposed DF32p completely. The aqueous phase which contained the protein-bound DF32P and the hydrolytic products was precipitated with an equal volume of 5 ~ trichloracetic acid (TCA) to remove the former and l-ml aliquots of the filtrate were plated in glass cups with 3 drops of 5 N N a O H for radioactivity determinations. Corrections were applied for the spontaneous hydrolysis of DF32P which under the above conditions amounted to 2.7 and 3.5 % respectively in l hr without and with Mn '~ ions. The DFa2P stock solution also contained 8.8 % of unextractable radioactive material for which a correction was applied. DFPase activity was calculated from a straight-line curve obtained by plotting the radioactivity released in TCAfiltrates against time. It was expressed in terms oftxmoles of DF~2P hydrolysed per hr, the standardization being carried out by determining the specific activity of a known weight of the stock solution of DFa2P in propylene glycol. The digest contained 0.001% of propylene glycol but the solvent even at 2.5 % concentration was found not to materially affect the results. The sensitivity of the method is approximately 0.001 t~mole of DF3'~P hydrolysed in 1 hr. Radioactivity was measured either in a Robot Scaling Equipment (LKB-produkter, Stockholm) or in a Yracerlab Superscaler 18 fitted with an end-window TGCo2 Geiger tube. RESULTS

Figure 1 gives the pattern obtained in the DEAE-cellulose chromatography of rabbit liver subcellular enzymes. The esterase activity as determined by the PNPAmethod 9 has been correlated with the capacity of each fraction to bind DF32P and the curves indicate that as in the case of the rat enzymes the two activities run parallel to each other. The nuclear fraction has two major peaks and the mitochondria are found to contain numerous enzyme peaks of low esterase activity. It is likely that some of these are derived as impurities from other fractions. The microsomal fraction has the maximum esterase and DF32P-binding activity. The esterase activity of the particulate fractions towards PNPA is completely inhibited by 10 -~ M DF32P. The supernatant fraction has 7 esterase peaks and all of them are found to incorporate DFS2p. However, the esterase activity of peaks 3 and 4 (Fig. 1) is not completely suppressed by 10 6 M DF~"P, about 32 and 14% respectively of the original activity being found to be DFP-resistant. The amount of DFP-insensitive esterase in other enzyme peaks is too small to be determined accurately. The esterase peaks of the particulate as well as the supernatant fraction are in general more active towards phenyl butyrate and propionate than the acetate. Since in these studies there is no esterase peak which can be identified as DFPase, this activity was determined in tissue homogenates of the rabbit and the rat to obtain an idea of the approximate amounts present. The results are given in Table 1. The values are expressed as t~moles of DF~2P hydrolysed by 1 g of tissue, 1 ml of plasma or subcellular material derived from 1 g of liver tissue. Nitrogen values to which the DFPase activity refers are also given. The results are only indicative and not absolute since they are based only on a few experiments and it is well known that individual animals may show large variations, it It is observed that the DFPase activity as determined by this method is very low in tissues, only of the order of a fraction of a /~mole DF32P hydrolysed in 1 hr. The liver and kidney DFPases are activated by Mn 2~ while the plasma enzyme is inhibited. The results are in broad qualitative agreement

Determination of DFPase in rabbit and rat tissues using D P ~ P 2.0

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F1G. 1. Esterase and DP2P-binding activity of subcellular enzymes of rabbit liver cells. The microsomes and mitochondria were derived from 32 g and the nuclear and supernatant fractions from 8 g of liver tissue. The particulate fractions were solubilized with 0-5 % sodium deoxycholate in 0.01 M Tris-acetic acid buffer, pH 7.4. After filtering through Sephadex G-25 the material was applied to a column of DEAE-cellulose (volume 350 ml; dimensions 2"3 x 84 cm). Solvent system: 0'0l M Trisacetic acid buffer, pH 7'4 up to 10 tubes after which gradient elution was started with the above buffer in the mixing chamber and 0'1 M sodium acetate in buffer in the reservoir. At about tube No. 100 the mixing chamber contained 0.1 M sodium acetate in buffer and the reservoir the same buffer containing 1"0 M sodium acetate. Fractions of 10-12 ml each were collected at 15-min intervals. All operations were carried out between 0-4 °. Esterase activity was determined according to Huggins and Lapides g and DFazP incorporation by the procedure described previously3 Optical readings were taken in a Zeiss Model PMQ II spectrophotometer.

852

B . V . RAMACHANDRANand G. AGREN

with those o f M o u n t e r et aL z° I n no case was histidine f o u n d to potentiate the activation by M n 2+. W i t h the exception of the heterogenous rat nuclear material, b o t h in the r a b b i t a n d in the rat liver, D F P a s e is concentrated in the s u p e r n a t a n t fraction. The degree o f activation o f r a b b i t liver s u p e r n a t a n t by M n 2+ is m u c h higher t h a n that o f the rat liver s u p e r n a t a n t . The localization of D F P a s e in the s u p e r n a t a n t fraction indicates that the hydrolysis o f D F P is b r o u g h t a b o u t by a distinct enzyme a n d is n o t due to the unspecific hydrolytic activity exhibited by esterases in general towards various substrates since the microsomes which have the highest esterase activity towards P N P A are f o u n d to exhibit a comparatively low D F P a s e activity. Adie a n d T u b a zz f o u n d that sarinase is localized in the s u p e r n a t a n t fraction. I n our own TABLE 1. DFPASE CONTENT OF RABBIT AND RAT TISSUE MATERIAL DFPase activity in t~moles of DF~2P hydrolysed in 1 hr Material

N (mg)

Without activator

With Mn2+ (10-3 M)

Values from literature*

18.2 11'9 17-5 5.5 0'4 2.2 6.9

0-958 0-443 0.261 0'021 0-008 0'028 0.072

5-124 0' 125 1-617 0.041 0-004 0.040 0.656

167"0 113'4 244'6

24.3 5.8 19.0 7"2 2.3 4'5 8.7

0.315 0.125 0-483 0.146 0.025 0.091 0.177

2' 175 0-049 1"620 0'328 0"089 0'070 0'332

113-9 (489.6)

Rabbit

Kidney Plasma Liver Liver Nucleus Liver Mitochondria Liver Microsomes Liver Supernatant Rat

Kidney Plasma Liver Liver Nucleus Liver Mitochondria Liver Microsomes Liver Supernatant

187'5 (356.3)

The values are expressed as tzmoles of DF32P hydrolysed by 1 g of tissue, 1 ml of plasma or subcellular material derived from 1 g of liver tissue in 1 hr at 37 ° and at pH 7.4 (0"01 M Tris-acetic acid buffer). Nitrogen values (micro-Kjeldahl) given refer to this quantity of material. Tissue homogenates and liver fractions were dispersed in Tris-acetic buffer in a dilution of l : 10 or I : 5 and suitable aliquots were used for determinations. The procedure used is given under the methods. The sensitivity of the method is approximately 0.001/zmole DF32P hydrolysed in 1 hr. * The rabbit and rat values were calculated from figures given by Mazur3 and Mounter~a respectively. in-vivo studies z with rats it was observed that that the DFazP c o n t e n t of the s u p e r n a t a n t

fraction decreased progressively with time p r o b a b l y due to the D F P a s e activity of this fraction. As D F P a s e seems to be concentrated m a i n l y in the supernatant, the c h r o m a t o graphic fractions obtained by DEAE-cellulose c h r o m a t o g r a p h y (Fig. 1) were analysed for this activity by the m e t h o d described. One ml o f each fraction was i n c u b a t e d with M n 2+ for 20 rain at 37 ° a n d then with 10 -5 M DFa2P for 1 hr. The radioactivity

Determination of DFPase in rabbit and rat tissues using DF==P

853

released in the TCA-filtrate was d e t e r m i n e d for each fraction a n d corrected for hydrolysis. F i g u r e 2 gives the D F P a s e activity curve o f t h e s u p e r n a t a n t fraction s u p e r i m p o s e d o n the curve for esterase activity t o w a r d s P N P A . I t is o b s e r v e d t h a t D F P a s ¢ is c o n c e n t r a t e d in p e a k 3 which, incidentally, is also n o t c o m p l e t e l y inhibited b y D F ~ P . T h e a m o u n t o f D F P a s e in other enzyme p e a k s o f the s u p e r n a t a n t as well as o f the particulate fractions is t o o small for accurate assay. A

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FiG. 2. Correlation of DFPase and B-esterase in the supernatant fraction of rabbit liver cells. The fractions used were the same as indicated in Fig. 1 (cell-sap). For DFPase, 1 ml of each fraction was incubated with 0.05 ml of 2 × 10-8 M Mn 2+ for 20 rain at 37 °. One ml of 2 × 10-5 M DI~aP in 0.01 M Tris-acetic acid buffer, pH 7"4, was then added and the incubation continued for 1 hr. The digest was extracted thrice with 5-ml portions of ether and 1 ml of the aqueous phase was precipitated with an equal volume of 5 ~ TCA. Other details are given under Methods. DISCUSSION Interest in enzymes which hydrolyse o r g a n o p h o s p h o r u s anticholinesterases centres on their possible role in the detoxicating m e c h a n i s m o f the a n i m a l a n d the e v a l u a t i o n o f their use in the t h e r a p y o f o r g a n o p h o s p h a t e poisoning. 12 H o w e v e r , little is k n o w n a b o u t the identity o f the various enzymes hydrolysing o r g a n o p h o s p h a t e s or their a m o u n t s present in tissues to assess the extent to which they m a y c o u n t e r toxicity. The m e t h o d used b y m o s t w o r k e r s for the assay o f these enzymes is the m a n o m e t r i c technique in which the acid liberated is t a k e n as a measure o f the hydrolysis o f the o r g a n o p h o s p h a t e . In the case o f D F P , 2 molecules o f acid will be generated for each molecule o f D F P hydrolysed. It is also k n o w n t h a t when D F P acts as an i n h i b i t o r

854

B.V. RAMACHANDRANand G. fikGREN

o n e molecule o f H F is liberated. The error f r o m this source in the assay o f D F P a s e is assumed to be negligible but it m a y be significant if the a m o u n t o f D F P - s e n s i t i v e enzymes is very large c o m p a r e d to D F P a s e . F r o m the present series o f studies 7, s it a p p e a r s likely that the B-esterases (DFP-sensitive esterases) o f the m i c r o s o m a l fraction m a y affect the d e t e r m i n a t i o n o f D F P a s e in u n f r a c t i o n a t e d tissues by the m a n o m e t r i c method. The values o b t a i n e d by the present method are much lower t h a n those r e p o r t e d by M a z u r 3 a n d M o u n t e r et al. 4 for r a b b i t a n d rat tissues The reason m a y be the one stated a b o v e or due to the nature o f the substrate used by us whose purity c a n n o t be so effectively c o n t r o l l e d as that o f un-labelled D F P . It is observed that D F P a s e occurs in the same enzyme p e a k which also i n c o r p o r a t e s r a d i o a c t i v i t y (Fig. 2). This m a y m e a n t h a t the enzyme p e a k is still heterogenous o r that the enzyme has m o r e than one active centre, one inhibited by DF32P (B-esterase)~ a n d therefore i n c o r p o r a t i n g r a d i o a c t i v i t y a n d a n o t h e r concerned with the hydrolysis o f D F P . J a n d o r f a n d M c N a m a r a 2 drew attention to the fact that the tissues which are k n o w n to be rich in D F P a s e are also those which actively i n c o r p o r a t e r a d i o a c t i v i t y f r o m DFa2P. A n o t h e r possibility is t h a t the D F P - D F P a s e i n t e r m e d i a t e which m a y be f o r m e d is h y d r o l y s e d so slowly t h a t the radioactive label can be detected as proteinb o u n d in the present technique. M n 2÷ ions m a y p l a y a role in accelerating this rated e t e r m i n i n g second step. The finding t h a t D F P - s e n s i t i v e a n d insensitive esterases are localized in the m i c r o s o m e s a n d s u p e r n a t a n t respectively is o f interest in the purification a n d further study o f these enzymes. Acknowledgements--The authors are thankful to the Medical Defence Committee of the Swedish

Medical Research Council for generous financial support. The skilful technical assistance rendered by Miss Gunilla Bengtsson, Mr. Roman Bijan and Mrs. lnga H~iggl6v is gratefully acknowledged.

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 1I. 12. 13.

REFERENCES G. flt,GREN and B. V. RAMACHANDRAN, Acta physiol, scand. (in press). B. J. JANDORFand P. D. MCNAMARA,J. Pharmacol. 98, 77 (1950). A. MAZUR,J. biol. Chem. 164, 271 (1946). L. A. MOUNTER, L. T. H. DIEN and A. CHANUTtN,J. biol. Chem. 215, 691 ([955). W. N. ALDRIDGE,Biochem. J. 53, ll0, 117 (1953). L. A. MOUNTER,J. biol. Chem. 209, 813 (1954). B. V. RAMACHANDRANand G. /~GREN, Bioehem. Pharmacol. 12, 981 (1963). B. V. RAMACHANDRAN,L. ENGSTR6Mand G. /~GREN, Bioehem. Pharmacol. 12, 167 (1963). C. HUGGINSand J. LAPIDES,J. biol. Chem. 170, 467 (1947). L. A. MOUNTER,J. biol. Chem. 215, 705 (1955). P. A. ADIEand J. TUBA, Canad. J. Bioehem. Physiol. 36, 21 (1958). J. A. COHEN and M. G. P. J. WARRINGA,Bioehim. Biophys. Aeta, 26, 29 (1957). L. A. MOUNTER in Handbuch der Experimentellen Pharmakologie, Ergiinztmgswerk, XV, p. 486. Springer-Verlag, Berlin (1963).