ANALYTICAL

BIOCHEMISTRY

w,220-233

Permeabilization

of Microorganisms

G. F. MIOZZARI, Mikrobiologisches

(1978)

P. NIEDERBERGER,

Institut, ETH-Zentrum,

by Triton

X-100

AND R. HOTTER

EidgenGssische Technische Hochschule CH-8092 Ziirich, Switzerland

Ziirich.

Received March 15, 1978 A simple permeabilization procedure has been developed which allows the reliable determination of enzyme activities in situ in a variety of different microorganisms. Permeabilization is obtained by freezing cell suspensions in the presence of a low concentration of the anionic detergent Triton X-100. After thawing, the cells can be used directly in the enzyme assays. The procedure has been optimized using the yeast Saccharomyces cerevisiae. Yeast cells are completely permeabilized by Triton X-100 concentrations of 0.05% (v/v), and permeabilization is independent of cell age and cell concentration. The treatment makes the cells freely diffusible for macromolecules up to molecular weights around 70,000. Cytoplasmic and mitochondrial amino acid biosynthetic enzymes as well as aminoacyl-tRNA synthetases could be readily measured in treated cells. The method has been successfully applied to the determination of enzyme activities in other fungi as well as in gram-positive and gramnegative bacteria.

The preparation of cell-free extracts for the measurement of enzyme activities is quite laborious and time consuming, particularly when a large number of samples have to be processed at the same time. In addition, most mechanical methods used for the disruption of the cells, besides requiring relatively large amounts of cell material, completely disrupt the organizational integrity of the cells and may lead to a rapid inactivation of enzymes, a dissociation of enzyme complexes, and other artifacts. Some of these problems can be circumvented by measuring enzyme activities in situ using permeabilized cells. A number of permeabilization procedures have been reported for various organisms. In the bacterium Escherichia coli toluenization has become a standard procedure (l), and with the yeast Candida utilis cytochrome c, protamine sulfate, and bovine pancreatic ribonuclease have been used to permeabilize the cells (2). With Saccharomyces cerevisiae the application of nystatin (3), dimethylsulfoxide (DMSO)’ (4), protamine sulfate, ribonuclease or chitosan (5,6), a mixture of toluene and ethanol (7), or * Abbreviations used: DMSO, dimethylsulfoxide; DTT, dithiothreitol; PMSF, phenylmethyl sulfonyl-fluoride; TCA, trichloroacetic acid; tna-, tryptophanase-negative; trpR-, tryptophan repressor-negative. 0003-2697/78/0901-0220$02.00/O Copyright 0 1978 by Academic Ress, Inc. All rights of reproduction in any form reserved.

220

PERMEABILIZATION

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221

finally drying the cells on filters at room temperature (8) have been shown to be successful for specific purposes. The DMSO method has also been employed for the determination of tryptophan synthase in plant cells (9). We were initially interested in obtaining a method which would allow us a rapid and reproducible determination of the activities of all tryptophan enzymes in S. cerevisiae on a large number of samples which are usually harvested at different times. Among the methods described in the literature, the DMSO method was the most satisfactory as it gave consistently high enzyme activities (10). Unfortunately the method is quite laborious; it requires immediate processing of the samples after harvesting and the permeabilizing agent has to be removed prior to the enzyme assays. The anionic detergent Triton X-100 has been used successfully to render E. co/i cell preparations diffusible for macromolecules (11) and it has been shown to selectively solubilize a large proportion of the proteins of the cytoplasmic membrane in this organism (12). In this report we present a very simple and reproducible method for the determination of enzyme activities in Triton X-100 permeabilized cells. Efficient permeabilization requires only freezing and thawing of cell suspensions in the presence of a low concentration of the detergent. The cell suspensions can be frozen in the presence of the detergent and stored in a freezer until used. The suspensions are thawed just prior to the assays and used directly without further treatment. Although the method has been optimized for the tryptophan biosynthetic enzymes in S. cerevisiae, we have successfully applied it to numerous other enzymes in yeast as well as to various other eucaryotic and procaryotic microorganisms. MATERIALS

AND METHODS

Organisms. The following microorganisms were used in the investigation: S. cerevisiae strain X2180-lA, obtained from T. Manney, Manhattan, Kansas, Schizosaccharomyces pombe strain 972 h-, obtained from U. Leupold, Bern, Switzerland, Neurospora crassa FGSC 321, obtained from K. Lerch, Zurich, Switzerland, E. co/i strain W 3110 trpRtna,-, obtained from C. Yanofsky, Stanford, California, Pseudomonas aeruginosa strain PA0 1, obtained from T. Leisinger, Zurich, Switzerland, Bacillus subtilis strain ATCC 14593, obtained from the American Type Culture Collection, Washington, D. C., Streptomyces glaucescens strain ETH 22794 a soil isolate from our own culture collection. Media and culture conditions. S. cerevisiae was grown in MV-medium (13), S. pombe in the medium described by Gutz er al. (14) and N. crassa in Vogel N medium (15). For E. cofi we used medium M9 (16), supplemented with 20 pg/ml L-tryptophan and 0.05% casamino acids, and P. aeruginosa was cultivated on medium P with 20 mM L-arginine according to Leisinger et a/. (17). B. subtilis was grown on Difco dextrose

222

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AND

HOTTER

medium containing 3 g of Bacto beef extract, 10 g of Bacto tryptose, 10 g of Bacto dextrose and 5 g/l of sodium chloride, and S. gluucescens on a medium described by Baumann and Kocher (18). All strains were grown at 30°C or 37°C (E. co/i) on a rotary shaker in 500-ml Erlenmeyer flasks with four indentations to provide adequate aeration, Growth was followed in most cases by measuring the OD,% of the cultures. With N. crassa and S. glaucescens the wet weight of cells per milliliter was measured. If not indicated separately, the cultures were harvested in the exponential growth phase. Cells of S. gfaucescens and N. crassa were separated from their culture media by filtration and all other cells by centrifugation. After washing twice with ice cold water the cells were resuspended in potassium phosphate buffer (0.1 M, pH 7.6) or Tris-HCl buffer (0.1 M, pH 7.6) as indicated in the text. Preparation of crude extracts. Crude extracts of fungi were prepared by passing cell suspensions twice through a French pressure cell at 4000 N/cm2, bacterial cells were broken by sonication with a MSE-Sonifier (150 W; 6 x 30 set with 30-set cooling intervals, amplitude setting 3, power setting “high”). For the determination of cytoplasmic enzymes, cell debris were generally removed by centrifugation (4O,OOOg, 15 min). In some cases, however, as indicated in the table and figure legends, the crude lysates were used directly without prior fractionation. If mitochondrial enzymes were to be measured, a low speed centrifugation (lOOOg, 10 min) was employed, followed by dialysis of the crude extract against potassium phosphate buffer (10 mM, pH 7, containing 1 mM EDTA). Permeabilization procedures. The following procedure was finally adopted for the permeabilization of cell suspensions with Triton X-100. The washed cell pellet is resuspended at 50 to 100 mg wet wtiml in icecold buffer containing 0.05% (v/v) Triton X-100. Depending on the enzyme to be measured we used either potassium phosphate buffer (0.1 M, pH 7.6) or Tris-HCl buffer (0.1 M, pH 7.6). The suspension is mixed by vortexing and then frozen in either liquid nitrogen, in a dry ice/acetone bath or, most conveniently, in a regular freezer at -20°C. In the latter case the suspension has to be kept frozen for at least 15 h. Prior to the enzyme measurements the frozen suspension is thawed by swirling in a 30°C water bath, avoiding any unnecessary heating, and kept in ice. Additional details are given in the text. To permeabilize cells with DMSO (4), they are washed and resuspended in potassium phosphate buffer (0.1 M, pH 7.6). DMSO is added to a final concentration of 40% and the suspension is placed in an ice bath for 10 min. After this treatment the cells are pelleted (10,OOOg, 1 min) and washed twice in phosphate buffer. Other permeabilization procedures were performed as indicated in the text. Enzyme assays and protein measurement. Glutamine-dependent

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anthranilate synthase [chorismate pyruvate-lyase (amino-accepting), EC 4.1.3.271 was tested according to Egan and Gibson (19) at pH 7.6. Anthrani[N-(5’-phosphoribosyl)-anthranilate: late phosphoribosyltransferase pyrophosphate phosphoribosyltransferase, EC 2.4.2.181 was tested by a combination of the methods of Creighton and Yanofsky (20) as described by Miozzari et al. (21). Phosphoribosylanthranilate isomerase [N-(5’phosphoribosyl)-anthranilateisomerase] was tested by a modified method of Creighton and Yanofsky (20) according to Miozzari et al. (21). Indole-3-glycerol-phosphate synthase [l-(2’-carboxyphenylamino)-ldeoxyribulose-5-phosphate carboxy-lyase (cyclizing), EC 4.1.1.481 was tested according to Wegmann and DeMoss (22) and tryptophan synthase B [L-serine hydro-lyase (adding indole), EC 4.2.1.201 according to Manney (23). For the arginine biosynthetic enzymes acetylglutamate kinase [ATP:Nacetyl-L-glutamate Sphosphotransferase, EC 2.7.2.81 and ornithine acetyltransferase [N*-acetyl-L-ornithine:L-glutamate N-acetyltransferase, EC 2.3.1.351, the assays described by Wipf and Leisinger (24), were used. For ornithine carbamoyltransferase [carbamoylphosphate:Lornithine carbamoyltransferase, EC 2.1.3.31 the method described by Theil et al. (25) were used. Histidinolphosphatase [L-histidinol-phosphate phosphohydrolase, EC 3.1.3.151 was measured according to Martin et al. (26) and threonine dehydratase [L-threonine hydro-lyase (deaminating). EC 4.2.1.161 according to Brunner ef al. (27). Arginyland tryptophanyl-tRNA synthetases [L-arginine:tRNA*‘g ligase (AMP-forming), EC 6.1.1.19; L-tryptophan:tRNAT’P ligase (AMPforming), EC 6.1.1.21 were tested according to Burkard et al. (28) with the modification that Tris-HCl buffer (1 M, pH 8.5), magnesium chloride (75 mrvr), and DL-[14C]tryptophan (20 &i/pmol) were used for tryptophanyl-tRNA synthetase. Protein was determined by the method of Herbert et al. (29), applying either the Biuret reaction (when potassium phosphate buffer was used) or the Folin-Ciocaltes reagent (when Tris-HCl buffer was used). As a standard, bovine serum albumine was used. Chemicals. All chemicals used were of analytical grade. Triton X-100 was purchased from Serva Feinbiochemica, Heidelberg, Germany. RESULTS

AND DISCUSSION

The Use of Triton X-100 as a Permeabilizing

Agent in S. cerevisiae

The permeabilization procedure was optimized using strain X2 180- 1A of S. cerevisiae grown in minimal medium (MV-medium). Cells were harvested and permeabilized under various conditions and the activities of the tryptophan enzymes determined. The results, illustrated in Fig.

224

MIOZZARI,

-2

NIEDERBERGER,

0

‘E 1% 0. 0 + f508. $k om ZE c UI 0.4 es 25 .co )

AND HiiTTER

I

. 0.2 Triton-X-100

0.4

0.6

concentration

0.8

2

1.0

4

6

e

10

(% w%)

C

w----r+ loo

200

300

Cull concentration during (mg wet weight/ml)

400

500

purmeabllizalion

FIG. 1. (A). Permeabilization as a function of Triton X-100 concentration. Strain X2180-1A was grown in MV-medium to an OD,,, of 0.7, harvested, washed, and resuspended in potassium phosphate buffer (0.1 M, pH 7.6) at 60 mg wet wt/ml. Equal volumes of Triton X-100 diluted to various extents with buffer were added to aliquots of the cell suspension, The cells were frozen at -2o”C, thawed and assayed for enzyme activity. (B). Permeabilization as a function of growth phase. Strain X2180-1A was grown in MVmedium. Aliquots of the culture were harvested and permeabilized with Triton X-100 at the OD,,, indicated. After thawing, one aliquot of each cell suspension was used directly for the determination of enzyme activities in permeabilized cells (0). whereas the remainder was lysed by passing twice through a French pressure cell at 4000 N/cm2 prior to the assays (A). The resulting lysate was not fractionated by centrifugation. The insert shows the growth curve. (C). Permeabilization as a function of cell concentration. Strain X2180-1A was grown in MV-medium to an OD,,& of 2.5. The cells were harvested by centrifugation, washed and resuspended in potassium phosphate buffer (0.1 M, pH 7.6) at a concentration of 500 mg wet wt/ml. The cell suspensions were diluted with buffer, Triton X-100 was added to a final concentration of 0.05%. the cell suspensions were frozen at -20°C. and thawed before use.

1 for anthranilate synthase, are typical for the response obtained with the other enzymes of the pathway. While Triton X-100 concentrations in excess of 1% were used to render E. cofi cells permeable to macromolecules (1 l), only very small concentrations of the detergent are required for an efficient permeabilization of yeast cells (Fig. 1A). The addi-

PERMEABILIZATION

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tion of 0.05% (v/v) Triton X-100 to freshly harvested cell suspensions prior to freezing gives maximum activity for all five tryptophan biosynthetic enzymes (only anthranilate synthase shown in Fig. IA). The uv-absorption spectrum of Triton X-100 has a peak at 274 nm and a shoulder at 282 nm (not shown). While the detergent does not interfere with the determination of the other tryptophan enzymes, it can cause a concentration dependent background absorption in the indole-3-glycerolphosphate synthase assay, which is read at 290 nm. However, if the Triton X-100 concentration in the cell suspension is kept below 0. I%, background absorption of the detergent in the final assay is negligible. For this reason a Triton X-100 concentration of 0.05% (v/v), the lowest concentration required to give complete permeabilization, was used in all further experiments. The results presented in Fig. 1B show that the permeabilization procedure is equally effective at all growth stages of a batch culture grown in MV-medium. The activity of anthranilate synthase measured in permeabilized cells is about 30% higher than the activity in an unfractionated crude extract of the same cell suspension throughout the growth curve. Moreover the efficiency of permeabilization does not depend on the concentration (in mg wet cells/ml) of the cell suspension during permeabilization over a wide range (14-500 mg wet cells/ml; Fig. IC). For convenience we routinely resuspend our washed pellets at a concentration of 50-100 mg wet cells/ml; this gives us predictable enzyme and protein contents. The exact amount of cell suspension to be used per assay obviously depends on the sensitivity of the enzyme assay and on the specific activity of the enzyme in that particular culture. In many cases the permeabilized cell suspension has to be diluted severalfold prior to use in the enzyme assay. In Table 1 the activities of two tryptophan biosynthetic enzymes obtained by the Triton X-100 method are compared to the activities measured in the same cell suspension using other methods. While freezing alone or addition of Triton X-100 alone uncovers only a small fraction of the enzyme activities, freezing in the presence of the detergent yields enzyme activities that are comparable to or higher (e.g., anthranilate synthase) than the activities measured in the same cell suspension after breaking the cells by repeated passages through a French pressure cell. This means that the permeabilization procedure makes the enzymes completely accessible to exogenous substrates. The Triton X-100 method is also superior to both DMSO (4) and toluene/ethanol treatment (7), even when these methods are used in combination with a freeze/thaw step, while protamine sulfate treatment (5,6) gave only very poor permeabilization in our hands (Table 1). An additional advantage of the Triton X-100 method is that the detergent does not affect enzyme activities or interfere with the enzyme assays when used at low concentrations (see above), and thus, in contrast to DMSO and toluene/ethanol, does

226

MIOZZARI,

NIEDERBERGER, TABLE

AND Hi.JTTER

1

ACTIVITIES OF TRYFTOPHAN ENZYMES IN SACCHAROMYCES CEREVISIAE UNDER VARIOUS PERMEABILIZATION CONDITIONS Treatment of freshly harvested cell suspension”

Anthranilate synthase (nmoUmg protein. min)

None Freeze/thaw6 Triton X-100 alone’ Triton X-100 + freeze/thaw Crude extractd DMSO DMSO + freeze/thaw’ Tolueneiethanol + freeze/thaw’ Protamine sulfate”

0.01 0.18 0.02 1.33 1.02 0.66 0.86 0.77 0.10

Indole-3-glycerolphosphate synthase (nmoVmg protein.min) 0.14 0.38 0.36 2.16 1.95 1.16 1.76 1.67 0.19

(1Strain X2180-IA was grown in MV-medium to an OD,,, of 0.7, harvested and washed twice with distilled water and once with potassium phosphate buffer (0.1 M, pH 7.6). The cells were resuspended in buffer at 50 mg wet wt/ml, divided into several portions, and subjected to further treatment as indicated. All treatments were performed in phosphate buffer. b Cells were frozen in liquid nitrogen and thawed by swirling in a water bath at 3O”C, avoiding any unnecessary heating. c Triton X-100 was added to a final concentration of 0.05% and the suspension was kept in ice for 60 min prior to the enzyme assays. d The cells were disrupted by passing the cell suspensions twice through a French pressure cell at 4000 N/cm2. The resulting lysate was not fractionated by centrifugation; thus, the activities obtained in this “extract” are directly comparable to the values measured in permeabilized cells. p The DMSO treatment was preceded and followed by afreeze/thaw cycle in liquid nitrogen. f As with DMSO, the toluenelethanol treatment was preceded and followed by a freeze/thaw cycle in liquid nitrogen. Toluene and ethanol were added to the thawed cell suspension to 0.5 and 4% final concentration respectively. The suspension was shaken 5 min by hand and refrozen. g Protamine sulfate was added to I mg/ml final concentration. The suspension was mixed by vortexing and kept at 30°C for 30 min. Protamine sulfate was removed by centrifugation and the suspension was washed twice with buffer. The cells were finally resuspended in the original volume of buffer. Cells kept at +4”C in the presence of protamine sulfate (instead of 30°C) showed no activity at all.

not have to be removed prior to the assays. In other experiments we found that additional freeze/thaw cycles either preceding or following the permeabilization step do not affect enzyme activities. Similarly, enzyme activities are independent of the speed at which the cell suspensions have been frozen. We find identical enzyme activities for samples frozen in liquid nitrogen, in a dry ice/acetone bath, or at -20°C in a regular freezer. For convenience we routinely freeze our cells by trans-

PERMEABILIZATION

BY TRITON TABLE

PROTEIN

AND

PERMEABILIZED

Fraction Supematant Sediment

Protein 23 77

ENZYME

ACTIVITIES

CELLS

2 IN THE SUPERNATANT

OF

OF SACCHAROMYCESCEREVISIAE" Phosphoribosyl anthranilate isomerase

Anthranilate synthase

Anthranilate phosribosyl transrerase

(132.WO)

(7O.ow

(35.ow

62.5 37.5

97.5 2.5

3.5 96.5

227

X-100

Indole glycerolphosphate synthase (132.000) 7.5 92.5

Tryptophan synthase (16o.ow 3.5 %.5

” Two parallel cultures of strain XZIWIA were harvested at an OD, of 1.5 and pemteabilized separately. After thawing. the cell suspensions were divided into wo pans. One part was used for the deterntination of rotol enzyme activities and protein. The other part was centrifuged (20,OLWg. IO min at 4°C). the supernatant was separated from the pellet and used for the determination of enzyme activities and protein (supemotanr). The pellet was washed once with potassium phosphate buffer (0.1 M, pH 7.6) and resuspended in the original volume before measurement of enzyme activities and protein (sedimmt). In both parallel experiments the supetnatant and the sediment taken together contained more than 90% of the original (total) protein and of the enzyme activities. The values given in the table show the distribution of protein and enzyme activities (in %) in the supernatant and the washed sediment (average of two experiments). Below the enzyme designations the corresponding molecular weights are given in parentheses. The estimated approximate molecular weights of anthranilate synthase and indole-3-glycerolphosphate synthase are taken from Schiirch-Rathgeb (30), for anthranilate phosphoribosyl transferax from Hiitter and DeMoss (31). for phosphoribosylanthrailate isomerase from DeMoss (32) and finally for tryptophan synthase from Manney (33).

ferring them from an ice bath to a -20°C overnight or longer. Effect of Triton X-100 on Morphology, Integrity of S. cerevisiae

freezer and letting them sit

Viability,

and Structural

The gross morphology of Triton X-100 permeabilized yeast cells as observed with a phase contrast microscope is essentially unaltered except that the cells look smaller and show less contrast than untreated cells. The viability of permeabilized cell suspensions is reduced to about 5 x 10-4-10-5 depending on the permeabilization conditions used, slow freezing and short exposures to -20°C increase the number of survivors without a measurable effect on the efficiency of permeabilization (results not shown). Between 20 and 25% of the total protein is released into the supernatant by the Triton X-100 treatment (Table 2). In analogy to E. cofi, most of the protein may selectively be released from the cytoplasmic membrane (12). However, as reported for E. co& (ll), treatment with the detergent also leads to the release of freely diffusible small proteins from the cytoplasm into the supernatant. As shown in Table 2, two of the tryptophan enzymes, (anthranilate phosphoribosyl transferase and phosphoribosylanthranilate isomerase) are mainly found in the supernatant, whereas the three other enzymes remain associated with the cells and can be sedimented by centrifugation. The molecular weights of the different enzymes indicate that proteins with molecular weights below about 70,000 are readily released from permeabilized cells. This suggests

228

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AND

HijTTER

that enzymes retained within the cells must be accessible for very large exogenous substrates. Since centrifugation and washing essentially removes small molecules from permeabilized cell suspensions, it provides an easy means of “desalting”; this is particularly useful for the removal of residual amounts of analogs or amino acids which would otherwise interfere with the determination of certain enzyme activities (e.g., allosteric enzymes). It is also conceivable that the method could be used as a first step in the purification of any of the small enzymes (e.g., phosphoribosylanthranilate isomerase) that are quantitatively released into the supernatant by the detergent. No loss in enzyme activity could be detected for any of the tryptophan enzymes after storage of permeabilized cell suspensions for several weeks at -20°C. However, anthranilate synthase, unlike the other enzymes of the pathway, is rapidly inactivated when permeabilized cells are thawed and stored at 0°C (Fig. 2). Under these conditions the stability of the enzyme is only slightly better than in crude extracts, but clearly inferior to DMSO permeabilized cells. The stability of the enzyme can be increased considerably (to about the same level in all cases) by the addition of glycerol and the protease inhibitor PMSF to the cell suspensions (shown only for Triton X-100 permeabilized cells in Fig. 2). The relatively low stability of anthranilate synthase in Triton X-100 permeabilized cells compared to cells treated with DMSO suggests that Triton X-100 affects the structural integrity of the cells more extensively than DMSO. In particular, it may lead to the release of proteases from vacuoles. Thus, treatment of yeast cells with Triton X-100 does not represent a particularly mild permeabilization method. Application of the Method to Cytoplasmic Enzymes in S. cerevisiae

and Mitochondrial

Figure 3 shows an example of the application of the permeabilization method. Derepression of the tryptophan enzymes was induced by addition of DL-5-methyl-tryptophan to an exponentially growing culture of S. cerevisiae (13). Samples were harvested and permeabilized at various times. After all the samples had been collected, enzyme activities were determined for all samples in parallel. As has been reported elsewhere (21) the activities of the tryptophan enzymes determined in permeabilized cells closely reflect the activities of the same enzymes in vivo. Although the method has been optimized for the determination of the tryptophan enzymes, we have successfully applied it to a number of additional yeast enzymes (Table 3). In all cases, the activities measured in Triton X-100 permeabilized cells are higher than, or at least comparable to the activities observed in crude extracts. Among the enzymes tested are cytoplasmic amino acid biosynthetic enzymes, aminoacyl-tRNA syn-

PERMEABILIZATION

BY TRITON

229

X-100

l

25

50

75

100

125

time in hat 0°C FIG. 2. Stability of anthranilate synthase in Triton X-100 permeabilized cell suspensions of Sacchuromyces cerevisinr at 0°C. Triton X-IOO-containing samples: Strain X2180-1A was grown to an OD,,, of 2.4 in MV-medium, harvested, washed, and permeabilized with Triton X-100. After thawing, the cell suspensions were kept in ice: one aliquot was supplemented with glycerol [ 15% (v/v)] and PMSF (lo-” M). The crude extract was prepared by passing an aliquot of the permeabilized cell suspension through a French pressure cell twice at 4000 N/cm*. followed by centrifugation (40,OOOg. 20 min at 4°C). DMSO treated samples: DMSO permeabilized cells were prepared by subjecting freshly harvested cells of strain X2180-1A to the standard DMSO permeabilization procedure, including removal of the permeabilizing agent (4). The initial anthranilate synthase specific activities of the different samples were set at 100%.

thetases and mitochondrial enzymes. It has been evidenced by others (24,34,35) that the mitochondrial enzymes tested are located in the mitochondrial matrix. It is not known, whether the permeabilization procedure solubilizes the enzymes or just makes the mitochondrial membrane permeable to the substrates. The successful in situ measurement of aminoacyl-tRNA synthetases (Table 3) further demonstrates that Triton X100 permeabilized cells are freely accessible to large molecules like tRNAs, which have molecular weights in the range of 30,000. In control experiments it was shown that the reaction is fully dependent on exogenously supplied tRNA and Mg2+ ions, while omitting ATP reduces the activity to 25% (P. Staheli, unpublished results from this laboratory). Although we have tested only a limited number of enzymes, the results obtained with various cytoplasmic and two mitochondrial enzymes sug-

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AND HUTTER

FIG. 3. Kinetics of derepression of the tryptophan enzymes in Saccharomyces cerevisiae. DL-S-Methyltryptophan (5 x 10m4M final concentration) was added to an exponentially growing culture of strain X2180-1A at an OD J48 of 0.25 (r = 0). At the indicated time points aliquots of the cultures were harvested and permeabilized in potassium phosphate buffer (0.1 M, pH 7.6) containing 0.05% Triton X-100. After all the samples had been collected, the cell suspensions were thawed and the enzyme activities determined. The basal enzyme levels in MV-medium were set arbitrarily at 1.0 for all enzymes. Growth inhibition by the analog was 32%. Uninhibited control culture, A; Smethyltryptophan, 0.

gest, that many if not most yeast enzymes are made accessible by Triton X-100 permeabilization. One exception has been observed, however, with acetyl glutamate synthase, a particulate enzyme (24,35) which could be measured in crude extracts but not in Triton X-100 permeabilized cells (B. Wipf, unpublished results from this laboratory). Application

of the Method to Other Microorganisms

In an effort to determine whether permeabilization with Triton X-100 could be applied to other microorganisms besides S. cerevisiae, we subjected two other fungi as well as various gram-positive and gram-negative bacteria to the standard permeabilization procedure as it was developed for yeast enzymes. Table 4 shows the levels of indoleglycerol phosphate synthase obtained with permeabilized cell sus-

PERMEABILIZATION TABLE

BY TRITON

231

X-100

3

ACTIVITIESOF VARIOUS CYTOPLASMIC AND MITOCHONDRIAL ENZYMES IN PERMEABILIZED CELLS OF SACCHAROMYCESCEREVISIAE Specific enzyme activity (nmolimg protein .min) Crude extracts”

Cells permeabilized with Triton X-100 and freeze/thaw

Cytoplasmic enzymes Histidinolphosphatase” Ornithine carbamoyltransferase” Threonine dehydratase’ Arginyl-tRNA synthetased Tryptophanyl-tRNA synthetase”

57.0 250 III 0.20 0.03

65.1 34s 300 0.25 0.05

Mitochondrial enzymes” Acetylglutamate kinase Ornithine acetyltransferase

0.13 4.62

0.16 3.42

(’ The values for crude extracts obtained after centrifugation are corrected for their lower content of protein compared to permeabilized cells (68% for crude extracts for cytoplasmic, 79% for mitochondrial enzymes). b Histidinol phosphatase and ornithine carbamoyltransferase were measured in cells permeabilized in Tris-HCI buffer (0.1 M, pH 7.6), containing 0.05% Triton X-100. Crude extracts were prepared in the same buffer. c The data were supplied by D. Schulthess from our institute (unpublished data). DMSOpermeabihzed cells gave a value of 220. d The data were supplied by P. Staheli from our institute (unpublished data). DMSOpermeabilized cells gave values of 0.17 and 0.015 respectively. Crude extracts as well as Triton X-100 permeabilized cells were dialyzed against Tris-HCI buffer (0.1 M, pH 7.4, containing 0.06 M KCI. 0.01 M MgCI,, 0.01 M mercaptoethanol, and 50% glycerol). ’ The data were supplied by B. Wipf from our institute (unpublished data). Crude extracts as well as Triton X-100 permeabilized cells were dialyzed against potassium phosphate buffer ( 10 mM, pH 7, containing 1 mM EDTA).

pensions of the different strains and compares them to either crude extracts or to partially permeabilized suspensions of the same strain. In all cases the activities obtained in permeabilized cells were higher than those in crude extracts. The comparison of the combined effects of freezing and thawing and Triton X-100 with the individual treatments alone reveals substantial differences between the various microorganisms. In some organisms freezing and thawing alone will uncover a large proportion of the enzyme activity, while in others Triton X-100 seems to have the predominant effect. In most cases, however, full permeabilization depends on the combined effects of both Triton X-100 and freezing and thawing. Using this method we have been able to measure the activity

232

MIOZZARI,

NIEDERBERGER, TABLE

AND HijTTER

4

ACTIVITY OF IND~LE-~-G~~~ER~LE~H~~PH,~~~ PROCARYOTK

AND EUCARYOTIC

SYNTHA~E IN VARIOUS

MICROORGANISMS

Specific enzyme activity (nmol/mg protein. min) Organism

Whole cells

Triton X-100”

Freeze/ thawb

Freeze/thaw + Triton X-100

Crude extracts’

0.14

0.36

0.38

2.16

1.95

0.05

0.09

1.29

1.69

1.27

0.24

0.19

0.67

0.62

0.56

0.32

1.59

1.02

1.94

1.51

0.64

I.46

0.76

1.06

0.88

1.90

5.58

3.38

6.80

6.31

0.25

0.78

0.65

0.94

0.92

Fungi Saccharomyces

cerevisiae

strain X2180-IA Schizosaccharomyces

pombe

strain 972 hNeurospora

crassa

strain FGSC 321 Bacteria Gram-positive Bacillus

subtilis

strain ATCC 14593 Streptomyces

glaucescens

strain ETH 22794 Gram-negative Escherichia

co/i

strain W 3110 Pseudomonas

strain PA0 1

aeruginosa

a Triton X-100 was added to a final concentration of 0.05% and the cell suspension was kept in ice for 60 min prior to the enzyme assays. b The cells were frozen at -20°C for 24 h and then thawed prior to the enzyme assays. c Crude extracts of fungi were prepared with the French pressure cell and bacteria by sonication (see Materials and Methods). Values are corrected for lower protein content in centrifuged crude extracts compared to permeabilized cells. Centrifuged crude extracts contained between 60 and 70% of the total protein.

of anthranilate synthase in Schizosaccharomyces pombe, an enzyme that is not detectable in crude extracts (data not shown). The results demonstrate the usefulness of the method for permeabilizing a broad range of microorganisms, including both eucaryotes and procaryotes. Since our method has originally been developed for permeabilizing S. cerevisiae, it is conceivable that the minimum detergent concentration required for complete permeabilization of a given microorganism may differ from the values used in this study. The wide range of application, its simplicity, and reliability make permeabilization with Triton X-100 a useful tool in most cases in which enzyme activities have to be determined. It seems particularly useful for the determination of enzyme activities from small amounts of cell material, and in all cases involving simultaneous processing of a large number of samples .

PERMEABILIZATION

BY TRITON

X-100

233

ACKNOWLEDGMENTS We are grateful to our colleagues T. Leisinger, for performing some of the enzyme assays. technical assistance. This work was supported Scientific Research, project no. 3.053.73.

D. Schulthess, P. Staheli. and B. Wipf We thank Miss B. Gilnter for expert by the Swiss National Foundation for

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19. 20. 21. 22. 23. 24. 25. 26.

27. 28. 29. 30. 31. 32. 33. 34. 35.

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