A general method for the localization of enzymes that produce phosphate, pyrophosphate, or CO2 after polyacrylamide gel electrophoresis

ANALYTICAL

121, 17-22 (1982)

BIOCHEMISTRY

A General Method for the Localization of Enzymes That Produce Phosphate, Pyrophosphate, or CO, after Polyacrylamide Gel Electrophoresis H. G. NIMMO Department

of Biochemistry,

AND G. A. NIMMO

Glasgow

University,

Glasgow

Cl 2 8QQ,

Scotland

Received June 2, 198 1 Previous workers have stained gels for enzymes that produce inorganic phosphate by using the insolubility of calcium phosphate. This method can also be applied to enzymes that produce pyrophosphate or C02. The white bands of the precipitated calcium salt are clearly visible when viewed against a dark background and can be photographed or scanned. The method can be used at pH 6 and above; the level of Ca*’ required is reduced at higher pH values. The sensitivity of the method is tested by injecting the various anions into presoaked gels; as little as 10 nmol of phosphate or pyrophosphate and 100 nmol of CO* produce clearly visible precipitates.

It is very useful to be able to stain for enzyme activity after polyacrylamide gel electrophoresis. A number of authors have stained for enzymes that catalyze the release of phosphate by utilizing the insolubility of calcium phosphate (see, e.g., Ref. (1)). The precipitated calcium phosphate can be converted to lead sulfide (the lead conversion method) (2-5), photographed directly (6-8), or subsequently stained with Alizarin red S (3,9). These methods have been used only for a limited range of enzymes, in conditions of high pH or high Ca*+ ion concentration. In this paper we show that the calcium salt precipitation method can be applied to enzymes that produce pyrophosphate or CO2 in addition to those releasing phosphate. The appearance of the precipitated calcium salt affords a very sensitive method of detection. We present results illustrating that the method can be used for a wide variety of enzymes under different conditions of pH and Ca*+ ion concentration. MATERIALS

AND

(EC 4.1.1.1), and yeast uridine-5’-diphosphoglucose (UDPG)’ pyrophosphorylase (EC 2.7.7.9) were obtained from Sigma (London) Chemical Company, Poole, Dorset, United Kingdom. Alkaline phosphatase (EC 3.1.3.1) from calf intestine was obtained from Boehringer Corporation (London), Lewes, Sussex, United Kingdom. Fructose 1,6-bisphosphatase (EC 3.1.3.11) was purified from ox liver (8). The arom multienzyme complex containing 5-dehydroquinate synthase (EC 4.6.1.3) and 3-enolpyruvylshikimate-5-phosphate (EPSP) synthase (EC 2.5.1.19) was purified from Neurospora crassa (10). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (EC 1.2.1.12) was purified from rabbit muscle (11). RNA polymerase (EC 2.7.7.6) purified from Escherichia co/i (12), and chorismate synthase (EC 4.6.1.4) partially purified from N, crassa were gifts from Dr. J. R. Coggins. The three 3-deoxy-D-arabino-heptulo’ Abbreviations used: UDPG, uridine-S-diphosphoglucose; EPSP, 3-enolpyruvylsbikimate-5-phosphate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DAHP, 3-deoxy-D-arabino-heptulosonate-7-phosphate.

METHODS

Materials. Wheat germ acid phosphatase (EC 3.1.3.2), yeast pyruvate decarboxylase 17

0003-2697/82/050017-06$02.00/O Copyright Q 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.

18

NIMMO

AND

sonate-7-phosphate (DAHP) synthases (EC 4.1.2.15) were partially purified from N. CRZSSQ(13). Fatty acid synthase was purified from rabbit mammary gland (14). Isocitrate dehydrogenase (EC 1.1.1.42) was purified from E. co/i ( 15). Polyacrylamide gel electrophoresis was carried out in 3, 3.5, or 7% polyacrylamide, 7-cm tube gels in Tris-glycine (16) or Trisacetate (17) buffers. Gels were preelectrophoresed for 20 min and the tracking dye (bromphenol blue) was electrophoresed into the gels before the enzyme samples were loaded. Electrophoresis was carried out at 5°C with a current of 3 to 4 ma/tube. Gels to be stained for activity were soaked in the

NIMMO

buffer to be used for staining (see Table 1) at 37°C for 20 to 30 min. They were then transferred to staining mixture containing Ca*+ ions and all necessary substrates and cofactors. Staining was carried out at room temperature or 37°C. Gels were stained for protein in Coomassie brilliant blue G-250. One unit of enzyme activity is defined as the amount that causes the conversion of 1 pmol of substrate to product per minute. RESULTS

We have stained polyacrylamide gels for several different enzyme activities by incubating them in the presence of a suitable

TABLE ENZYMES

THAT Activity loaded (mu)

Phosphate-producing enzymes Fructose l&bisphosphatase

1

BEEN STAINED

PH

Cd+ (mM)

BY Ca’+

PRECIPITATION

Other assay conditions

10

10

8.8

20

50 IIIM glycine-KOH. 10 rn~ Mg’+, 5 rn~ fructose 1.6bisphasphatc, 100 IIIM KCI 100 rn~ Tris-HCI, 150 NM NADH, 1 IIIM ATP, 5 IIIM 3. phosphoglycerate, I tn~ dithiothnitol, IO @g/ml phosphoglycerate kinase. 20 IIIM Mg’+

10

10

50 rn~ glycine-KOH, 0.1 mu phosphc-enolpyruvate, 0.1 IIIM crythrose 4-phosphate

10 10

10 10

Chmismate synthase

8

10

Alkaline phospbatase

IO

IO

4

5

200

20.

9

2

a

20

7.5

100

Pyruvate decarboxylasc

8.5

2

Isecitrate dcbydrogcnasc

7.5

100

GAPDH DAHP synthase (Phc sensitive) DAHP synthasc (Tyr wtsitive) DAHP syathase (Tfp sensitive) Lkbydroquinatc synthase EPSP synthase

Acid phosphatase Pymphosphate-producing enzymes UDPG pyropbospborylase RNA pelymerasc

CO,-producing enzymes Fatty acid syntbasc

4’

HAVE

AND DISCUSSION

1600 18 II 160 I 70 600

50 rn~ glycinc-KOH, 50 PM NAD+, 1 mu DAHP 50 IIIM glycim-KOH, I IIIM shikimate 5-phosphate, 2 HIM phosphcenolpyruvate 100 IIIM Tris-HCI, 50 PM EPSP, 0.5 rn~ NADPH, IO PM FMN 50 IIIM glycine-KOH, 1.5 IIIM pnitrophcnyl phosphate, 0.1 nv.t Zn*+, 1 IIIM Mg*+ 100 III&I sodium acetate, 0.6 mhi p-nitrophcnyl phosphate 50 IIIM glycine-KOH, 500 FM glucose I-phosphate, 8 rn~ Mg’+, 2 rntd UTP 100 rn~ Tris-HCI, 12 IIIM M$i, 0.1 rn~ EDTA, I mu dithiothreitol. 0.2 M KCI. 1.6 q/ml calf thymus DNA (in gel), 0.8 rn~ GTP, 0.8 mht CTP, 0.8 rn~ UTP. 0.8 IIIM ATP 100 nm Tri.-HCI. 100 FM amtyl CoA, 100 PM malonyl CoA, 100 ,IM NADPH 100 nw Tri-HCI, 5 rn~ sodium pyruvate, 67 PM thiamin pyrephosphate 100 rn~ Tris-HCl, I rn~ isocitrate, 1 rn~ NADP+, 100 rind Mn’+

Note. The mncunts of activity loaded do cot repre.se.at the minimum activity that could be detected, except where indicated by an asterisk. These activities were measured under optimal assay conditions. It should be emphasized that the activity of an enzyme after electrophoresis and in the staining mixture may be considerably lower. This is discussed in detail for fatty acid synthase in the text.

SOME ACTIVITY

a

b

c

STAINS

d

FIG. I. Typical gels stained for protein and enzyme activity. Electrophoresis was carried out as described under Methods (16) in 3% polyacrylamide gels for UDPG pyrophosphorylase and in 7% gels for GAPDH. The conditions used for the activity stains are described in Table I. (a) I6 gg GAPDH stained for activity for 10 min at 37”C, (b) 3 pg GAPDH stained for protein, (c) 20 pg UDPG pyrophosphorylase stained for activity for 20 min at 37”C, and (d) IO rg UDPG pyrophosphorylase stained for protein.

mixture of substrates and Ca’+ ions. Enzymes that have been stained successfully are listed in Table 1 and typical gels and conditions are shown in Figs. 1 and 2 and Table 1. Depending on the activity and stability of the enzyme, bands of calcium phosphate, pyrophosphate, or carbonate required 10 min to 20 h to precipitate. In some cases, a band of precipitate was formed slightly ahead of the tracking dye (Figs. 1 and 2); this was caused by the presence of phosphate in the enzyme sample. Although the optimal conditions for staining any particular enzyme must be determined individually, some general recommendations can be made. Because enzyme activity is very sensitive to pH, we decided that the best way to determine general conditions suitable for staining was to inject samples of phosphate, pyrophosphate, and carbonate ions into polyacrylamide gels that had been preincubated in buffers of various

19

FOR ENZYMES

pHs and containing various concentrations of Ca2+ ions. The minimum amounts of each anion detected under various conditions are shown in Table 2. No precipitates were obtained below pH 4 or with less than 10 PM Ca2+ ions. The stain was more sensitive and there was less diffusion of the injected ions at higher pHs and higher concentrations of Ca2+ ions. These conclusions have been largely confirmed in our attempts to stain for enzyme activities. For example, gels were stained for fructose 1,&bisphosphatase, acid phosphatase, UDP glucose pyrophosphorylase, and fatty acid synthase activity at a wide range of pH values (pH 4- 10) and Ca2+ ion concentrations (200 mM-10 PM). In general, the bands of precipitate were sharper at higher pHs and concentrations of Ca2+ and no bands were obtained below pH 5 or 16 PM ca’+. In order to obtain sharp bands of precipitate in the presence of less than 5 mM Cazt‘, it is necessary to include Ca*’ in the presoaking buffer to ensure that the concentration of CaZf in the gel is high enough to

a

bc

d

e

f!3

FIG. 2. Polyacrylamide gels loaded with a range of amounts of fatty acid synthase. Electropboresis was carried out as described under Methods (16) in 3.5% gels. Gels a-f were stained for activity for 45 min at 37°C as described in Table I and gel g was stained for protein. The amounts of fatty acid synthase used in pg were (a) 3.125, (b) 6.25, (c) 12.5, (d) 25, (e) 37.5, (f) 50, and (9) 3.125.

20

NIMMO

AND TABLE

NIMMO 2

MINIMUM AMOVNTS (nmol) OF PHOSPHATE, PYROPHOSPHATE, AND CABONATE IONS NECESSARY TO GIVE A VISIBLE PRECIPITATE IN POLYACRYLAMIDE GELS UNDER VARIOUS CONDITIONS OF PH, AND Ca2+ PH Anion pi

Ca*+ (M) 10-l 10-2 1o-3 lO-4 lO-5 10+

co:-

10-l 10-* 10-’ IO+ lo-5 lo+

PPi

10-l lo-2 lo-) lO-4 lO-5 lO-6

3 71000

>I000

4

5

6

1000 >lOOO

1000 >lOOO

100 100 >iOOO

1000 >I000

7

8 10 10

>I000

>I000

1000 >I000

100 1000 >lOOO

100 >lOOO

100 100 >I000

10 10 ZlOOO

9

10

10 10 10 10 100 >I000

10 10 10 10 10 >I000

10 10 10 10 10 >lOOO

100 100 100 100 1000 71000

100 100 100 100 1000 71000

100 100 100 100 1000 71000

10 10 10 10 10

10 10 10 10 10 7-1000

10 10 10 10 10 71000

71000

Nofe. Polyacrylamide gels were equilibrated in buffers of various pHs and containing various concentrations of Cazc as indicated. They were then injected with 1, 10, 100, and 1000 nmol (52 ~1) of phosphate, pyrophosphate, and carbonate. In some cases, this caused a local rise in pH and the gels were soaked for a further 4 h to reequilibrate the pH. This table indicates the smallest amounts of each ion which gave a Permanent, clearly visible precipitate.

cause precipitation as soon as the enzyme reaction is initiated. The gels were viewed against a dark background and, when an activity stain of sufficient intensity had been obtained, the gels were removed from the staining mixture and were stored in 50 mM glycine-KOH, pH 10, 5 mM Ca2+ either at 5’C or in the presence of an antibacterial agent. Under these conditions, gels can be stored for several months with little deterioration. Gels can be photographed by reflected light against a dark background (Figs. 1 and 2) and can be scanned (Fig. 3). This method of staining is quite sensitive. When a range of amounts of fatty acid synthase was electrophoresed and stained for

activity, as little as 3 pg of enzyme was easily detected (Fig. 2). By eluting the fatty acid synthase from an unstained gel and assaying it, we found that only 7% of the activity was recovered after electrophoresis. The specific activity of the fatty acid synthase was 0.16 U/mg when assayed under the conditions used for staining (Table l), and it was unaffected by the presence of the Ca2+ ions. Therefore, as little as 35 PLJ of enzyme were detected. Similar experiments were carried out on other enzymes, and as little as 0.1 pg of UDP glucose pyrophosphorylase and 0.2 pg of fructose 1,6-bisphosphatase were detected; however, it is difficult to assess the actual activities of these two enzymes in the staining mixture because they are both

SOME ACTIVITY

28I dye

STAINS

top

front

‘L

1 cm

FIG. 3. Spectrophotometric scans of gels containing UDPG pyrophosphorylase which have been stained for protein and enzyme activity. Photographs of the gels are shown in Fig. 1. Both gels were scanned at 600 nm. (a) Activity stain, (b) protein stain.

strongly inhibited by Ca’+ ions. To assess further the sensitivity of the stain for phosphate-producing enzymes, alkaline phosphatase was subjected to electrophoresis and was stained for activity as described in Table 1. As soon as a band of precipitated calcium phosphate was clearly visible, the enzyme reaction was stopped by removing the gel and adding EDTA to a final concentration of 50 mM. Thep-nitrophenol which had been released by the enzyme was eluted from the gel and measured at 4 10 nm. Forty nanomoles of p-nitrophenol was released; therefore, less than 40 nmol of calcium phosphate can form a visible precipitate, because the EDTA will not have instantly stopped the alkaline phosphatase reaction. Similar results were obtained in the experiments described in Table 2, in which gels were presoaked in various buffers and then injected with samples of phosphate, pyrophosphate, and carbonate. No clearly visible precipitate was observed for samples of less than 10

21

FOR ENZYMES

nmol phosphate or pyrophosphate or for samples of less than 100 nmol carbonate (Table 2). Thus, even under optimum conditions, the staining method appears to be about 10 times less sensitive for carbonate than it is for phosphate and pyrophosphate. Clearly, the amount of precipitate formed will depend upon the amount of product formed by the enzyme, but it will also depend on the relative rates at which the product is formed and can diffuse away from the enzyme. Therefore, in practice, the stain may be slightly less sensitive than the results shown in Table 2 would indicate and it may not always be advantageous to warm the gels during staining, particularly if the enzyme has a low QlO. In our hands, the lead conversion stain (2-5) and Alizarin red S stain (3,9) do not increase the sensitivity of the staining method for photographs or for scanning, although they would be of advantage for more opaque gel systems such as starch. ACKNOWLEDGMENTS We would like to thank many colleagues, particularly Dr. C. A. Fewson and Dr. J. R. Coggins, for their helpful suggestions and gifts of enzymes. This work was supported by a grant from the Medical Research Council, London.

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NIMMO

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