[108] Dialkylphosphofluoridase

[108]

DIALKYLPHOSPHOFLUORIDASE

651

and yield misleading pictures of localization. As in the case of other histochemical techniques, conclusions require confirmation by biochemical methods. A critical and extremely lucid evaluation of this technique m a y be found in an article by Couteaux and Taxi. 4~ The author~ discuss the pitfalls as well as the presently available results and suggest several valuable improvements as to the use of pH, ionic strength, and other factors. ~ R. Couteaux and J. Taxi, Arch. anat. micrascop, et morphol, exptl. 41, 352 (1952).

[108] D i a l k y l p h o s p h o f l u o r i d a s e

I

0 (C3HTO)2~P

0 + H~O-, (CsH,O)~P

\ F

+ HF

\ OH

By A. MAZUR Assay Method

Principle. The method described is based on the fact that the hydrolysis of the phosphorus-fluorine bond in diisopropyl fluorophosphate (DFP) results in the liberation of H + as well as of F-. The reaction is carried out in Warburg vessels with a bicarbonate-carbon dioxide buffer at pH 7.4 so that the resulting liberation of carbon dioxide can be taken as an index of H + formation. Analysis for F - may be performed at the end of the reaction period. Reagents NaHCO3, 0.025 M. DFP, 0.035 millimole per 3 ml. dissolved in 0.025 M bicarbonate. Enzyme. Plasma is used per se or diluted with 0.025 M bicarbonate. Tissue extracts are prepared with 0.025 M bicarbonate (see below).

Preparation of Active Enzyme Extracts. Blood is collected with heparin to prevent clotting. Plasma is used per se or suitably diluted with 0.025 M 1Among the names used to describe this enzyme are dialkylfluorophosphataseand dialkylfluorophosphateesterase. Neither of these names is correct in the light of our knowledge concerning phosphatases and esterases. The enzyme does not split at an ester ]~nkage, nor does it form inorganic phosphate and an alcohol. Instead it splits at a phosphorus-halogen bond and must tentatively be termed a phosphofluoridase until its natural substrate~ if any exists, is determined.

652

ENZYMES OF LIPID METABOLISM

[108]

bicarbonate. R e d cells are washed twice with 5 to 10 vol. of saline and diluted four times with 0.025 M bicarbonate. Muscle and brain are freed of coagulated blood, homogenized in a Waring blendor with 9 vol. of bicarbonate, and filtered through coarse filter paper. Heart, lung, liver, and kidneys are perfused with saline in order to reduce the q u a n t i t y of blood in these tissues, homogenized, and filtered as above. (For purification see below.) Procedure. 3 ml. of D F P (0.035 millimole) is placed in the large well of a W a r b u r g flask. 0.5 ml. of e n z y m e in 0.025 M lJicarbonate is placed in the TABLE I RABm~ PLASMA ON HYDROLYSIS OF DIISOPROPYL FLUOROPHOSPHATEa The control vessels contained 0.035 millimole of diisopropyl fluorophosphate in bicarbonate buffer, pH 7.4. Others contained in addition 0.5 ml. of rabbit plasma. The data reported for plasma are not corrected for control hydrolysis in water. The values are expressed in millimoles produced per miUimole of diisopropyl fluorophosphate. EFFECT OF

C02 production in presence of

Fluoride production in presence of

Time,

min.

Water

Plasma

20 30 40 60 90 120 130 140 150 160

0.01 0.02 0.04 0.05 0.08 0.10 0.09 0.10 0.11 0.11

0.34 0.53 0.70 1.01 1.31 1.43 1.44 1.46 1.48 1.49

Water

Plasma

0.03

0.36

0.08

0.54

0.07

0.83

0.07

0.99

0.08

1.01

" A. Mazur, J. Biol. Chem. 164, 271 (1946).

first side arm. 0.5 ml. of 0.025 M bicarbonate or a solution of inhibitor or a c t i v a t o r dissolved in b i c a r b o n a t e is placed in the second side arm. A control vessel contains 0.5 ml. of b i c a r b o n a t e instead of the enzyme solution, in order to measure the extent of spontaneous hydrolysis of D F P . T h e solutions are equilibrated with shaking a t 38 ° with a gas m i x t u r e of 95 % N2 and 5 % COs. T h e reaction is s t a r t e d b y tipping the contents of the side a r m s into the m a i n chamber, of the vessel. Readings are t a k e n at 10-minute intervals, and activities are calculated from the initial reaction velocity of the straight-line portion of the reaction curve. T a b l e I lists the results obtained when the reaction is allowed to go to completion, using r a b b i t p l a s m a as a source of enzyme. T a b l e I I gives the relative activities of crude extracts of various tissues f r o m the r a b b i t and

[108]

DIALKYLPHOSPHOFLUORIDASE

653

TABLE II HYDROLYSIS OF DIISOPROPYL FLUOROPHOSPHATE BY TISSUE EXTRACTSffi

The velocity of hydrolysis of diisopropyl fluorophosphate was determined as microliters of CO~ liberated in 30 minutes from a bicarbonate buffer, p H 7.4, by 0.5 ml. of a 1 : 10 tissue extract. The plasma and red blood cell activities were calculated from the activities determined experimentally on more concentrated solutions and also expressed as those of a 1:10 dilution. All data are corrected for the hydrolysis of the diisopropyl fluorophosphate in water. Velocity of hydrolysis by

Tissue

Rabbit, #1. C02

Human, ~l. C02

274 187 129 127 55 30 20 12 9

457 319

Liver Kidney Small intestine Plasma Lung Heart Brain Muscle Red cells

--

19 50 -29 40 13

a A. Mazur, J. Biol. Chem. 164, 271 (1946).

TABLE

III

RELATIONSHIP BETWEEN ENZYME ACTIVITY AND PROTEIN TISSUES6

CONTENT OF RABBIT

Protein N is determined as the difference between total N and nonprotein N in terms of milligrams of N per 0.5 ml. of a 1 : 10 extract of tissue or dilution of plasma or red cells. Tissue

Enzyme activity ~ per rag. of protein N

Kidney Liver Plasma Lung Heart Brain Red cells Muscle " A. Mazur, J. Biol. Chem. 164, 271 (1946). b Microliters COz for 30 minutes.

370 294 22 16 12 9 2 1

654

ENZYMES OF LIPID METABOLISM

[108]

man. T a b l e I I I shows the relative activities of r a b b i t tissues on the basis of protein N content. Purification P r o c e d u r e

A partial purification of the e n z y m e f r o m r a b b i t kidney has been reported b y the author. 2 T h i s procedure yielded a p r o d u c t which represented a purification of some 13 times. A more recent s t u d y b y M o u n t e r et al. 3 outlines a m e t h o d which results in a purification of 65 to 100 times. TABLE IV DIISOPROPYLPHOSPHOFLUORIDASE ACTIVITIES IN HOG KIDNEY FRACTIONS a

Experiment Fraction 71

Kidney

33

Activity, ~1. CO~/mg. N / Recovery, Purification 30 min. % factor

Fraction A-2

6.8 5.8 4.98 4.81

60 10 12.5

230 1,600 6,500 23,000

98 87 41

7 28 100

Kidney Fraction A Fraction A-1 Fraction A-2

6.8 5.78 5.02 4.83

60 10 12.5

290 2,300 4,300 18,800

93 85 31

8 15 65

Kidney Fraction Fraction Fraction Fraction Fraction Fraction

6.8 5.8 5.05 4.7 4.8 4.8 4.8

60 10 13.5 17.5 12.5 12.5

95 85 40 27 70 34

6 12.5 32 26 37 70

Fraction A Fraction A-1

73

EtOH, pH %

A A-1 A-1 A-1 A-1 A-2

300 1,800 3,750 9,650 7,700

11,000 21,000

L. A. Mounter, C. S. Floyd, and A. Chanutin, J. Biol. Chem. 204, 221 (1953). T h e following is an outline of this m e t h o d which utilizes frozen hog kidneys: Step 1. 2500 g. of kidneys is ground in a m e a t grinder and homogenized in a Waring blendor for 3 minutes. T h e mixture is suspended in 10 1. of water, adjusted to p H 6.8 with a c e t a t e buffer, p H 4.0, and stirred for 30 minutes at 0 °. T h e p H is reduced to 5.8 with buffer, and the mixture centrifuged for 20 minutes at 45,000 )< g at 0 °. T h e clear s u p e r n a t a n t is filtered to remove lipid particles and adjusted to p H 5.8. E t h a n o l (60 %) at 5 ° is added, and the m i x t u r e is allowed to stand overnight. T h e precip= A. Mazur, J. Biol. Chem. 164, 271 (1946). 3L. A. Mounter, C. S. Floyd, and A. Chanutin, J. Biol. Chem, 204, 221 (1953).

[lO~]

DIALKYLPHOSPHOFLUORIDASE

655

itate is recovered b y centrifugation at 3000 X g. This represents fraction A, which is 6 to 8 times purified. Step 2. T h e precipitate is suspended in 2 1. of 0.002 M NaHCOs and stirred for 1 hour at 0 °. The insoluble material is discarded. The solution is adjusted to p H 5.0 with buffer, and a small precipitate removed b y centrifugation. T h e solution is then adjusted to 10 % ethanol at - 5 ° and allowed to stand for 30 minutes. The precipitate is removed and dissolved in 0.025 M N a H C 0 s . This is fraction A-1 and represents a purification of 15 to 37 times. Step 3. T h e solution is adjusted to p H 5.0 at room temperature and clarified b y centrifugation. The supernatant is adjusted to 12.5 % ethanol, and the mixture allowed to stand for 40 minutes at 25 ° . T h e precipitate which forms is removed, and the supernatant fluid allowed to stand at - 5 ° for 2 hours. The precipitate is recovered and brought into solution as before. This is fraction A-2 and represents an over-all purification of 65 to 100 times. Table IV illustrates the activities of the various fractions and the yields obtained. Properties

Specificity. The enzyme is active in the splitting of the P-F linkage in a variety of fluorophosphates. Table V illustrates the activity of plasma and TABLE V HYDROLYSIS OF RELATED FLUOROPHOSPHATESa

The activities are expressed as microliters of CO2 liberated per minute when 0.5 ml. of 1 : 1 rabbit plasma or 1:10 rabbit liver extract was used in the standard hydrolysis mixture containing 0.035 millimole of the dialkyl fluorophosphate in a bicarbonate buffer, pH 7.4. The hydrolysis of the fluorophosphate in water was determined by substituting bicarbonate solution for the enzyme. Enzyme activity of Fluorophosphate Dimethyl fluorophosphate Diethyl fluorophosphate Diisopropyl fluorophosphate Ethylmethyl fluorophosphate

Hydrolysis in water 10 2 1 2

Plasma Liver extract 114 77 15 14

73 97 22 51

A. Mazur, J. Biol. Chem. 164, 271 (1946).

of a liver extract on the dimethyl, diethyl, ethyl methyl, and diisopropyl fluorophosphates. However, no normal substrate was found which could be split b y the enzyme. Table VI shows the lack of identity of the enzyme with acetylcholinesterase, glycerophosphatase, or lipase. When the enzyme splits D F P , no inorganic phosphate is liberated.

656

[108]

ENZYMES OF LIPID METABOLISM

Activators. T h e partially purified enzyme from rabbit kidney as well as t h a t from hog kidney is inhibited b y dialysis. Addition to the dialyzed partially purified enzyme of the dialyzate, Ca ++, or Mg ++ restored the enzyme activity. M o u n t e r et al. 3 report a striking activation b y Mn++ and to a slighter extent b y Co++. Histidine was also found to be a p o t e n t a c t i v a t o r of the enzyme in the presence of M n ++. Other amino acids which potentiated the activity of the enzyme are cysteine, thiolhistidine, and serine. T h e greatest enzyme activity was obtained b y these workers in the presence of purified enzyme, Mn++, and 2,2'-dipyridyl. TABLE VI REI~TIONSmP BETWEEN FLUOROPHOSPHATE-HYDROLYZINO ENZYME AND PHOSPHATASE, CHOLINESTERASE, AND ESTERASE ACTIVITIES6

The phosphatase activities are measured by the production of inorganic phosphate from ~-glycerophosphate in 1 hour. The activities of esterase and cholinesterase are expressed as microliters of C02 liberated by the action of 0.5 ml. of the enzyme solution in a bicarbonate buffer, pH 7.4, containing 0.015 mole per liter of acetylcholine, triacetin, tripropionin, or methyl butyrate. Enzyme activity in Substrate Dilsopropyl fluorophosphate, ~d. COz Acetylcholine, ~1. CO~ Glycerophosphate, rag. P04 Triacetin, id. CO~ Tripropionin, ~1. C02 Methyl butyrate, iA. C02

Fraction A, Table IV

Fraction H, Table IV

388 0 0.144b 288 510 277

3640 0 0.080 14 49 7

a A. Mazur, J. Biol. Chem. 164, 271 (1946). v This quantity of inorganic phosphate accounts for but 4.6% of the available phosphate from glyeerophosphate. In a similar experiment with rat intestinal phosphatase, 61% of the available phosphate from glycerophosphate was hydro° lyzed in 1 hour.

Inhibitors. T h e enzyme is inhibited b y h e a v y metal ions such as H g ++ and Cu ++. I t is only slightly inhibited b y iodoacetate, and not at all b y iodosobenzoate or fluoride. M a r k e d inhibition of the purified enzyme b y p-chloromercuribenzoate and b y phenyl mercuric nitrate is reported. Effect of Temperature and pH. Exposure of the partially purified enzyme solution from liver to 60 ° for 5 minutes results in an almost complete inactivation (6 % of its original activity). T h e enzyme is stable at icebox temperatures for 18 hours at a p H of 7.0 to 9.8. Below 7.0 its activity falls, and at p H 4.6 it is reduced to less t h a n 2 % of the activity of the enzyme a t p H 7.0. T h e purified enzyme has its optimum activity from p H 75. to 8.0.