97, 43-47 (1979)
A Simplified EDWARD L. KRUG, Department
Assay for Phospholipase NANCY
Received November 13, 1979 A new assay for phospholipase C activity that uses alkaline phosphatase to convert phosphorylchohne to inorganic phosphate is described. The determination of inorganic phosphate is performed in the presence of phosphatidylcholine and protein after the addition of sodium dodecyl sulfate. Phospholipase C activity determined by this coupled enzyme assay agrees well with data obtained by extracting and measuring phosphoryl[W]choline produced from phosphatidyl[ methyl-Wlcholine. The assay is sensitive to 1 nmol of phosphate, requires no removal of protein or phospholipid, and will work with a variety of phospholipid substrates. The assay is faster and more sensitive than previously published procedures. Stimulation of phospholipase C from Closfridium perfringens by ammonium sulfate is also reported.
Phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 184.108.40.206) from Clostridium perfringens catalyzes the reaction phosphatidylcholine
which otherwise interferes with phosphate determination (10). We also report stimulation by ammonium ions of the phospholipase C from C. perfringens.
+ phosphorylcholine METHODS
Materials. C. perfringens (strain ATCC 13124, American Type Culture Collection, Rockville, Md.) was grown as reported by Cassidy et al. (11) and harvested in lateexponential growth phase by centrifugation at 6000g for 30 min. Crude phospholipase C was obtained by ammonium sulfate precipitation of the supernatant. The 40-60% saturated ammonium sulfate fraction was dialyzed against distilled water at 4°C with water changes every 6 h, until the conductivity was the same as that of glassdistilled water. The dialysate was then lyophilized and stored at -20°C. Alkaline phosphatase (Escherichia cofi, type III-S), bovine serum albumin (fraction V, fatty acid free), phosphorylcholine chloride, and SDS were obtained from Sigma Chemical Company, St. Louis, Missouri. Egg yolk phosphatidylcholine was prepared using the procedure of Pangborn (12) and stored in
Phosphatidylcholine is the preferred substrate but phosphatidylethanolamine and sphingomyelin can also serve as substrates (1). Phospholipase C can be used to probe the association of membrane-associated enzymes and receptors with the phospholipid milieu of the membrane (2-8). This enzyme is, therefore, a powerful tool for studying membrane structure and function. Unfortunately, the assays for phospholipase C activity are tedious, insensitive, and often cannot be used with a variety of substrates (9). We have developed an improved assay in which alkaline phosphatase is used to liberate inorganic phosphate from phosphorylcholine. The phosphate is determined calorimetrically in the presence of SDS,’ precluding acid-precipitation of the protein r Abbreviation (lauryl sulfate).
used: SDS, sodium dodecyl sulfate
0003-2697/79/110043-05$02.00/O Copyright 0 1979 by Academic Press. Inc. All rights of reproduction in any form reserved.
absolute ethanol at -20°C. Radioactive phosphatidyl[methyl-14C]choline, 50 mCi/ mmol, was purchased from ICN Chemical and Radioisotope Division, Irvine, California. Phospholipase C assay. Assay I. Sequential phospholipase C-alkaline phosphatase incubations were performed in disposable glass 6 x 50-mm test tubes containing the following concentrations of reagents in a total volume of 40 ~1: 50 mM Tris-HCl (pH 7.3 at 37”C), 6.3 mM CaCI,, 0.13 mg/ml bovine serum albumin, 2.5 mM phosphatidylcholine, 25% ethanol, 0.15 M (NHJ2S04, and 0.1 to 1 x 10e3 unit of phospholipase C. The best dispersion of phospholipid was obtained by adding the reagents in the above order, followed by agitation with a Vortex mixer after each addition. The phosphatidylcholine was added as a 10 mM solution in absolute ethanol. All components except the phospholipase C were usually combined in one large suspension, then aliquots removed for each assay to be performed. The assay mixtures were incubated at 37°C for 15 min in a shaking water bath. The reaction was terminated by the addition of 10 ~1 of 0.2 M EDTA (pH 7.3). Phosphorylcholine was converted to choline and inorganic phosphate by adding 0.15 unit of alkaline phosphatase to the assay mixture and incubating at 37°C for 45 min in a shaking water bath. The alkaline phosphatase was purchased as a suspension in ammonium sulfate and diluted to 15 units/ml in 10 mM Tris-HCl + 1 mM MgCl, (pH 7.3 at 37°C). The final concentration of ammonium sulfate in the assay mixture was 0.026 M. Inorganic phosphate was determined by the method of Ames (13) in the presence of SDS (10). Each assay received 40 ~1 of 20% SDS and 200 ~1 of freshly prepared ascorbatemolybdate reagent (1 part 10% ascorbic acid:6 parts 0.42% ammonium molybdate in 1 N H,SO,), and was then incubated at 45°C for 20 min or 37°C for 60 min. The absorbance was determined at 820 nm. One nanomole of phosphate corresponds to an optical density of about 0.085. When
phospholipase C was omitted from the assay, the phosphate blank absorbance was about 0.040. Assay II. Conditions for assay II were the same as those for assay I except that the initial incubation mixture included 0.15 unit of alkaline phosphatase and ammonium sulfate at a final concentration of 0.07 M. The reaction was terminated after 15 min at 37°C by the addition of EDTA, and inorganic phosphate was determined as described above. Specific activity was expressed as micromoles of inorganic phosphate produced per minute per milligram of phospholipase C protein. Protein was determined by the method of Lowry et al. (14) with bovine serum albumin as the standard. RESULTS
If alkaline phosphatase is to be used in an assay for phospholipase C activity, the phosphatase must be able to convert all of the phosphorylcholine produced by phospholipase C to inorganic phosphate. When phosphorylcholine was added to a complete incubation mixture minus phospholipase C, the alkaline phosphatase converted at least 95% of the added phosphorylcholine to inorganic phosphate (Fig. 1). Production of inorganic phosphate was linear with respect to the concentration of phosphorylcholine up to at least 0.6 mM. The ability of alkaline phosphatase to completely hydrolyze enzymatically produced phosphorylcholine was demonstrated by an experiment in which phosphatidyl[merhyl-14C]choline was used as substrate. The amount of inorganic phosphate produced in the presence of alkaline phosphatase was the same as the amount of phosphoryl[‘*C]choline produced in the absence of the phosphatase (Fig. 2). The data in Fig. 2 were obtained by a procedure in which the incubation with alkaline phosphatase followed that with phospholipase C (assay I). The feasibility of including the alkaline phosphatase in
C ASSAY TABLE
Specific activity’ 4.13 3.78 4.00 6.14 7.92
Control NaCl Na$O, NH&l (NH&SO,
FIG. 1. Production of inorganic phosphate from the alkaline phosphatase hydrolysis of phosphorylcholine. Incubation mixtures contained all of the components for assay I except phospholipase C. Release of inorganic phosphate was determined as outlined under Methods. The slope of the line is 0.95 with a correlation coefficient of 1.00.
the phospholipase C incubation (assay II) was also examined. Surprisingly, inclusion of alkaline phosphatase in the phospholipase C incubation caused a stimulation of phospholipase C activity of about 2.5-fold (Fig. 3A). Heat-inactivated alkaline phosphatase caused the same level of stimulation, suggesting that the ammonium sulfate in which the alkaline phosphatase had been suspended was the cause of the stimulation. Including an equivalent amount of ammonium sulfate in the phospholipase C incubation in assay I resulted in the same activity as assay II (Fig. 3). With either assay phospholipase C activity was linear with time to 30 min (Figs. 2 and 3A) and protein to 36 pg (Fig. 3B). Other salts of sulfate and ammonium were tested for their effects on phospholipase C activity. The stimulation was apparently specific for the ammonium ion since ammonium chloride stimulated and sodium sulfate and sodium chloride had no effect (Table 1). Ammonium ion could not be replaced by K+ or Cs+ (data not shown). The optimal stimulation of phospholipase C occurred at 0.15 M ammonium sulfate in assay I and 0.07 M in assay II (Fig. 4). Alka-
OF SALTS ON PHOSPHOLIPASE ACTIVITY IN ASSAY I"
+- 0.27 k 0.02 -r- 0.01 + 0.26 + 0.14
u Experimental conditions were as outlined under Methods except that the phospholipase C incubation was for 30 min. * SdtS were present at a Concentration Of 0.054 M in each assay. The control received distilled water. c Units per milligram of phospholipase C protein 2 range for duplicate assays.
line phosphatase activity was not affected by ammonium sulfate over the range of concentrations tested (Fig. 4). Since some phospholipid substrates are not completely soluble in ethanol, experiI
FIG. 2. Comparison of alkaline phosphatase assay 1 with production of phosphoryl[W]choline. Phosphatidyl[merhyl-Wlcholine, 2.8 mCi/mmol, was used as substrate. At the end of the first incubation all reactions were stopped with EDTA. One set of incubations was extracted by the method of Bligh and Dyer (15) and the amount of phosphoryl[i4C]choline present in the aqueous phase was determined. The other set received alkaline phosphatase and was carried through the standard protocol for assay I. This figure shows the data obtained from measuring either phosphoryl[‘4C]choline (A) or inorganic phosphate (0) production. The deviation of duplicate incubations was less than 6% for both procedures.
FIG. 3. (A) Time course of phospholipase C activity in assays I and II. Data represent assay I without ammonium sulfate (0) or with 0.1 M ammonium sulfate (A), and assay II with 0.1 M ammonium sulfate (0). Experimental conditions were as outlined under Methods. The deviation of duplicate incubations was less than 10%. (B) Linearity of phospholipase C activity vs protein concentration in assays I and II. Both assays I (A) and II (0) were performed as outlined under Methods, and contained 0.1 M ammonium sulfate. The deviation of duplicate incubations was less than 5%.
- 100 D
ments were performed to examine the effect of detergents on phospholipase C activity. With a final concentration of 0.08% (w/v) sodium deoxycholate or Triton X-100, phospholipase C activities were, respectively, 68 and 58% of that determined with ethanol. These detergents did not inhibit alkaline phosphatase activity at this concentration. Increasing the detergent concentration to 1.4% (w/v) decreased the observed phospholipase C activity to less than 10% of the control value. DISCUSSION
A number of procedures have been developed for assaying phospholipase C activity (9), but none of these assays is suitably (NH&SO., IM) sensitive and rapid. A major drawback of the egg yolk turbidimetric procedure is the FIG. 4. Ammonium sulfate stimulation of phosphoundefined nature of the substrate and the lipase C. Data represent the assay I (A) and assay 11 inability to use other defined phospholipid (@) using If-min incubations as outlined under Methods. The deviation of duplicate incubations was less substrates. Meas~ement of acid liberation than 1.5%. The effect of ammonium sulfate on alkaline is unsuitable for impure enzyme preparaphosphatase is also shown (0). Experimental conditions since activity of other hydrolytic entions were as outlined for assay I except for the omiszymes can also result in acid production. sion of phospholipase C and the addition of 15 nmol Depletion of a radioactive substrate from of phosphorylcholine. The deviation of duplicate incubations was less than 0.4%. an ether phase (6) is rapid but lacks sensi-
tivity and requires the synthesis of a variety of radioactive phospholipids in order to study phospholipase activity with various substrates. The most direct assay procedures employ measurement of phosphorylcholine production, usually by acid hydrolysis of an acid-soluble portion of the incubation mixture. Phosphatase digestion of phosphorylcholine has been reported (16- 18), but these methods usually involve lengthy incubations with the phosphatase (4-8 h) and arid-precipitation of the protein and phospholipid before determination of inorganic phosphate. The procedures described in this report either use a short incubation with alkaline phosphatase (assay I) or the alkaline phosphatase is present in the phospholipase incubation (assay II), and in either case inorganic phosphate is determined without prior removal of protein and phospholipid. The assay is quick, quantitative, inexpensive, and sensitive to 1 nmol phosphate. It is adaptable to a variety of defined phospholipid substrates and also to other phospholipase C-type enzymes. In this laboratory we have used it to assay sphingomyelinase C activities from C. perfringens and S~~~~yl~c~~cus aureu.r , and phospholipase C activity from Bacillus cereus.
ACKNOWLEDGMENTS This work was supported by the National Institutes of Health Grant HD 10580, and a grant from the Muscular Dystrophy Association. This is Journal Paper No. 7397 from the Purdue Agriculture Experimental Station.
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