Eur. J. Biochem. 201.257-263 (1991) 0 FEBS 1991 0014295691006307

N-Myristoyl-transferase activity in cancer cells Solubilization, specificity and enzymatic inhibition of a N-Myristoyl transferase from L1210 microsomes Jean Albert BOUTIN ', Jean-Pierre CLARENC ', Gilles FERRY ',Anne-Pascale ERNOULD', Georges REMOND', Michel VINCENT' and Ghanem ATASSI Laboratoire de Biochimie Moltculaire, Departement de Cancerologie Experimentale Division de Chimie D., Institut de Recherches SERVIER, Suresnes, France (Received April 3/May 31, 1991) - EJB 91 0433

The activity catalyzed by N-myristoyl tranferase (NMT) is described for the first time in microsome-rich fractions from the murine leukemia cell line L1210, rat brain and mouse liver as biological sources. The enzyme from each source can accomodate various types of proteins (protein kinase A, virus structural gag protein or pp60src) as modelized by the use of their N-terminal derived peptides (GNAAAARR, GQTVTTPL and GSSKSKPKDP, respectively). As for some other types of membrane-bound enzymes, NMT activity can be enhanced by pretreatment with various types of detergents, amongst which Triton 770 and deoxycholate were the most potent. Further experiments on the L1210 microsome-rich fractions demonstrate that these two detergents were able to solubilize the microsomal enzyme, without modifying its substrate specificy. Finally, three compounds described in the literature to be inhibitors of NMT activity from other sources were tested for L1210 microsome-associated activity. None of them show any significant potency in inhibiting this activity . A new compound, myristoylphenylalanine, shows a slightly better inhibitory effect on the L1210 microsomal activity than the reference compounds with a median inhibitory concentration (I(&) of 0.2 mM.

Myristoylation is a post-translational acylation of proteins catalyzed by NMT [ l , 21. From the literature, a growing number of proteins undergo this modification, provided that as an absolute requirement, these proteins bear a glycine at their Nterminus (see [3, 41 for reviews). Amongst the proteins so acylated, three categories can be defined : endogenous proteins (such as protein kinase A, guanyl-nucleotide-bindingprotein, NADHlcytochrome-b, reductase); oncogene products like pp60v-src, p561ck, p59fyn or p62yes; virus structural proteins such as gag from various types of retrovirus, for example Moloney murine leukemia virus and human immunodefficient virus (see [5, 61 for reviews). Several lines of evidence showed both in virology and cancerology that the suppression by directed mutagenesis of the site of myristoylation leads to (a) the suppression of the transforming potency of pp6Ov-src [7 - 91; (b) the assembly of non-infectious viral particles for VPO [lo, 111 and VP1 [12], and (c) the inhibition of viral-particle formation and budding for gag [13-151. Furthermore, numerous viral oncogenes cfes, abl, ras) are expressed in cancerous cell lines as gag-onc fusion proteins which are the active transforming species of the corresponding Correspondence to J. A. Boutin, Dtpartement de Canctrologie Experimentale, Institut de recherches Servier, 11 rue des Moulineaux, F-92150 Suresnes, France Abbreviations. NMT, N-myristoyl transferase; ICs0, median inhibitory concentration. Enzymes. N-myristoyl transferase (EC 2.3.1.97); Acyl-CoA synthetase (EC 6.2.1.3).

retroviruses [16]. More recently, two sets of experiments showed that the addition of the myristoylation signal to the proto-oncogene p2lras [17] or to v-erb-B [18] greatly enhanced their transforming potential. These observations demonstrate the major role played by myristoylation and thus by the NMT, in these pathological processes. As stressed recently by Mitsuya and coworkers [19], myristoylation is potentially a major target for antiretroviral agents. Although the process (i.e. myristoylation) involved in the maturation of the viral precursor proteins is an attractive target for antiviral chemotherapy, it may prove difficult, according to De Clercq [20], to design drugs that selectively interfere with this or these enzyme(s). Despite this statement, inhibitors of NMT could also lead to anticancer compounds as briefly presented above. NMT from Saccharomyces cerivisiae has been explored, purified, cloned and expressed in Escherichia coli [21- 271. Its specificity towards substrates [23, 251 and cosubstrates [28311 has been extensively documented. Nevertheless, attention must be drawn to the fact that the yeast enzyme might be in nature very different from the enzyme(s) isolated from other more complex sources. Concerning the NMT from other sources, such as mammalian organs or camercdl lines, rather little is known so far. In particular, only a few experiments have been conducted using cytosols from the murine-muscle BC3H1 cell line [3], rat liver [25] or rat brain E32, 331 as source of the activity. Because of the possible involvement of NMT in one stage of cellular transformation, it became primarily important to describe and study such NMT activity from a cancer cell

258 line such as L1210, a murine leukemia cell line, in order to document the nature and compartmentalization of this activity in one such source. In the present work we studied microsome-associated NMT activity from L1210, its activation pattern by various types of detergent and subsequent solubilization, its specificity towards three model peptides and the effect of some compounds described by others on other models as potential inhibitors. Some of these observations were compared with sources obtained from rat brain and mouse liver microsomes. The microsomal NMT was found to behave like a typical microsomal enzyme and to potently accomodate endogenousderived, oncogene-product-derived and virus structural-protein-derived peptides. Furthermore, the inhibitory potency of three potentially active inhibitors (GLAAAARR described by Towler et al. [24], myristoylglycinal diethylacetal described by Shoji et al. [34] and cerulenin described by Saermark and Bex [35]) was measured. These compounds failed to inhibit microsomal NMT activity at concentrations that could be of some pharmacological use. A new compound, N-myristoylphenylalanine, was synthetized that shows slight inhibitory potency to microsomal NMT.

Frozen rat brains and mouse livers were obtained from EUROMEDEX (Strasbourg, France). They were carefully thawed at 4°C in 10 vol. buffer A, minced with scissors and homogenized with a glasslglass dounce. The homogenate was submitted to the same differential centrifugation preparation as described above. The final pellet was resuspended in buffer A supplemented with 0.5 sucrose. Activation and solubilization of N-myristoyl transferuse Activation and solubilization tests were run classically [37, 381 as described for other membrane-bound enzymes such as UDP-glucuronosyltransferase [39]. Briefly, 10% (niass/vol.) aqueous solutions of detergents were added to the microsomal preparation in order to give a final detergent concentration of 2%. The solution was tested as such before the preparation underwent 105000 x g centrifugation in an AIRFUGE centrifuge (Beckman). The supernatants were collected and tested for their NMT activities using GNAAAARR as substrate. Protein contents were measured by the method of Bradford [40]. N-Myristoyl-transferase assay

The assay used in this work is the assay described by Towler et al. [22] with minor modifications. Briefly, for about Peptides and chemicals 30 assays, 80 pl [3H]myristic acid (0.9 Ci/l, Amersham) in ethanol was dissolved in 0.8 ml buffer B (20mM Hepes, GNAAAAK was synthesized by Novabiochem, Switzerland. GNAAAARR, GLAAAARR, GQTVTTPL and GSS- pH 7.4, 10 mM MgCI2, 0.2 mM EGTA, 2 mM dithiothreitol) KSKPKDP were synthesized by NeoSystem Laboratories to which were added: 50 p1 20 mM lithium CoA and 0.1 ml (Strasbourg, France). GY, GQ, GYG, GYA, GGAG and 50 mM ATP (both from Sigma, St Louis, Mo.). The reaction GDDDDK-P-naphthylamide were purchased from Bachem was started by adding 0.2 ml acyl-CoA synthetase (Sigma, (Bubendorf, Switzerland) and GVT from Sigma (St Louis, St Louis, Mo.) at 1 mU/pI in 50 mM Hepes, pH 7.3. After Mo.). Myristoylphenylalanine, myristoylglycinal and 30 min at 37 "C,aliquots of 45 p1 were added to conical plastic myristoylglycinal diacetal were prepared by a general syn- tubes with 50 pl buffer C (10 mM Hepes, pH 7.4, 5 mM thesis for N-acyl amino acids as described by Lapidot et al. MgCI2, 0.1 mM EGTA, 1 mM dithiothreitol), 20 p1 enzyme source and 20 p1 peptide substrate (I mgjml in water). The [36]. Myristic acid (ethanol solution) was from Amersham. reaction ran for 30 min at 37°C and was stopped by adding 0.2 ml methanoljtrichloroacetic acid (10: 1, by vol.). Tubes Biological sources were kept in ice for 30 min and centrifuged at 13000 x g for L1210, a murine leukemia cell line, was obtained from the 5 min. 70 p1 supernatant was injected in a WATERS HPLC American Tissue Culture Collection. Cells were grown in 750- station equipped with a Nucleosyl CIS, 7-pm pore size ml rollers in RPMI 1640 supplemented with penicillin (50 U / (0.46 cm x 10 cm) column. The radioactivity was monitored ml), streptomycin (50 pg/ml), glutamine (2 mM), Hepes on-line with a Berthold HPLC monitor LB 507 A in presence (10 mM) and 10% (by vol.) fetal calf serum (all from GIBCO of 4 ml/min HPLC scintillator fluid (Zinsser 303). The analysis France). The rollers were gased with C02/air, (5:95, mol YO) took 30 min and was run at 1.5 ml/min gradient-wise from at 37°C. Batches of about 10'" cells were obtained weekly. water/0.05% (by vol.) triethylamine/O.l % (by vol.) trifluoroCells were collected by low-speed centrifugation (600 x g) in a acetic acid in acetonitrile/O.l '30(by vol.) trifluoroacetic acid (6:4) to 100% acetonitrile/O.l% (by vol.) trifluoroacetic acid. Jouan GR-411 centrifuge, washed three times with NaCIIP, (CaC12, 0.1 g/l; KC1,0.2 g/l; KH2P04, 0.2 g/l; MgCI2, 0.047 g/l; NaCI, 8 g/l; Na2HP04, 1.15 g/l), counted and checked RESULTS for viability by the trypan-blue exclusion method. The final cell pellet was suspended in 3 vol. buffer A (SO mM Hepes/ HPLC analysis NaOH, pH 7.4, 2 mM EGTA, 1 mM dithiothreitol, 1 mM Three typical chromatograms are presented in Fig. 1 for phenylmethylsulfonyl fluoride, 5 pg/ml aprotinin, 10 pg/ml GNAAAARR (Fig. 1A), GSSKSPKDP (Fig. 1B) and trypsin inhibitor, 10 pg/ml leupeptin) and submitted to a series GQTVTTPL (Fig. 1C). For all the sources used, the myrisof 10-s sonication bursts until all cells were lysed as checked toylated peptide retention times were constant. under a microscope. The homogenate was centrifuged Traces of [3H]myristoyl-CoA can be observed on the (15 000 x g for 10 min) and the supernatant further centrifuged chromatograms at a retention time of about 10 rnin (Fig. 1A). at 105000 x g for 60 min. The final pellet was suspended in buffer A supplemented with 0.5 M sucrose and referred to as Detergent activation and solubilization the microsomal suspension. Lysed yeasts were obtained from Springler Fould, and ofthe L1210 microsomal N M T artivitv were submitted to clarification and a DEAE-chromatographic NMT activity was found in microsomes prepared from step as described by Towler and Glaser [24]. log-phase growing L1210. We tested the capacity of a large MATERIALS AND METHODS

259

cE

-

Table 1. Activation and solubilization of N-myristoyltrunsferase activity f r o m L1210 leukemia cell microsomes Microsomal protein concentration was 17.5 mg/ml throughout the experiment. The ratio mg detergent/mg microsomal protein was 1 :1.14. The activities have not been corrected for the solubilized protein content which was not determined. Activation was measured after treatment of microsomes with 2% (mass/vol.) detergent with GNAAAARR as the substrate. Activity in supernatant after 105000 x g centrifugation of 2% (mass/vol.) detergent-treated microsomes measured with GNAAAAR as substrate. The activity associated with the microsomes before any treatment was taken as 100%. Frozen/thawed microsomes were used in these experiments. sol/activated, percentage activity after centrifugation versus percentage activity before centrifugation; sol/control, percentage activity after solubilization versus no detergent; N, nonionic; A, anionic; Z, zwitterionic

h

28ee-1

1

2

P 3

0

Time (min)

],,

My=-GQTVTTPL

n

2

-E 1

a

Detergents

,e

Enzyme activity

0

activation by 2% detergent

sol/activated sol/control

Time (min)

mo{

nJ

Myr-CSSKSKPKDP

solubilization by 2% detergent

I

Timelmin)

Fig. 1. HPLC pro$les of peptide myristoylation by the microsome. Measurements were as described in Materials and Methods using a (10 cm x 4.6 cm) Nucleosyl C18, 7-pm pore size column. GNA4R2, GNAAAARR; Myr, myristate. Y-axis is labelled ‘output (mv)’ as recorded by the on-line radioactivity detector

spectrum of detergents, either of an anionic or non-ionic nature to activate microsomal-associated NMT activity and to solubilize this activity in an activated form. These experiments, unlikc some other ones throughout the present work were conducted with frozen/thawed microsomal preparations. Table 1 compares the activity before and after centrifugation of the enzyme mixed with various detergents. It is clear that the best detergents were Triton 770 and deoxycholate which gave more than 100% of the untreated microsomal activity solubilized (162% and 132%, respectively). In these experiments, the microsomal protein concentration was 17.5 mg/ ml. The detergent/protein ratio (by mass) is thus 1: 1.14. This figure is close to the one required for the solubilization of membranes by deoxycholate [37]. The activation potency of these two detergents was studied at various concentrations from 0.1% up to 5% (massivol.). Fig. 2 depicts the results of this experiment. It shows clearly that NMT is activated by these two detergents. The profiles of activation show broad maxima for 2% (mass/vol.) deoxycholate and 1YO(mass/vol.) triton 770, while 3% (mass/ vol.) of either detergent suppresses the activity almost entirely. Furthermore, by comparing frozen/thawed microsoinal NMT activity with native microsomal activity, it can be seen that freezing/thawing partially removes latency. Considering the results obtained for the solubilization of the NMT activity, we scaled up the system, using a complete batch of microsomes and 2% (mass/vol.) of these two deter-

Microsomes (control) Buffer A (control) Triton X-100 (N) Brij 56 (N) Brij 35 (N) Lubrol PX (N) Triton 770 (N) Poe W-1 (N) Chaps (Z) Mega-9 (N) Triton XQS-20 (N) Tween 85 (N) Nonipet P40 (N) Deoxycholate (A) Triton N-101 (N) Triton N-42 (N)

100 97 36 27 24 66 i 98 44 64 100 0 36 74 171 104 102

0 0 8 0 6 162 13 12 15 0 14

14 163 99 97

0 0 29 0 9 81 30 19 15 0 39 19 95 95 95

gents. With both detergents and GNAAAARR as substrate, some NMT activity remains associated with the pellet (about 20% of the total activity, with 12 - 14 mg/ml protein, giving specific activities of 0.4 and 1.1 for triton 770 and deoxycholate treatments, respectively) but a great deal of activity was solubilized and active in the supernatant in which 5-6 mg/ ml protein was found, giving specific activities of 6.7 and 5.8 pmol . min-’ . mg protein-’ for triton 770 and deoxycholate extractions, respectively. Specificity of the microsomal enzymes

The CAMP-dependent protein kinase N-terminal-derived heptapeptide (GNAAAAK) and its derivative (GNAAAARR) with ArgArg replacing Lys were used as substrates for the NMT. Surprisingly, the true protein-kinase-A-derived peptide (GNAAAAK) is not a substrate for NMT, possibly through cyclization of the peptide or aspecific retention onto the analytical column material.. GNAAAARR is a good substrate for the enzyme [24]. We tested three other peptides as substrates: GQTVTTPL, the p l Sgag-derived peptide [41], GSSKSKPKDP, the p60src-derived peptide [42] and GLAAAARR, one of the yeast NMT-specific inhibitors [24]. The two former are good substrates for L1210 microsomal NMT, while

260 Table 3. Comparison of the specificity of the microsomal and the 2% (mass/vol.) deoxycholate-solubilized N-myristoyl transferase from L1210 Experiments were clasically conducted by incubating the microsomal fractions with detergent for 30min a t 4"C, then centrifuged at 105000 x g for 1 h. The subsequent supernatant was used as the source for the assay of NMT activity. The source for native microsomeassociated NMT activity was freshly prepared microsomes. Peptide concentration was 0.3 pM. n.d., not detectable

*.-.-

o k . . 0

7 1

. %

I

2

.

I

3

.

I

4

7 5

Peptides

NMT activity

DOC (rnass/voL)

native microsomes

DOC-solubilized microsomes

pmol myristoylated peptide . min-' . mg protein- ' GNAAAARR GSSKSKPKDP GQTVTTPL Proteins (mgiml)

%

Ttlion 710

(rnasrlvol.)

Fig. 2. Activation profiles of L1210 microsomal N-myristoyl-transferm e by two detergents: Triton 770 and deoxycholate. Experiments were conducted with detergent concentrations ranging from 0 to 5% (by mass). Freshly prepared microsomal fractions (dashed lines) and frozen/thawed microsomal fractions (full lines) were used in the second set of measurements. Microsomal fractions were prepared from L1210 as described in Materials and Methods. Activities are expressed as area under the curve (AUC) corresponding to myristoylated peptide . 30 min-' and were conducted with GNAAAARR as the substrate. DOC, deoxycholate

GLAAAARR is not myristoylated by any of the enzymatic preparations (Table 2). As an internal standard, we introduced an experiment using the yeast cytosolic partially purified NMT preparation [24]. The difference in specificity of the NMT obtained from this yeast source is outstanding, since the best substrate for

n.d.

6.2

1.4

16.3

1.1

17.4

18.4

6.6

yeast-derived enzyme (GNAAAARR) is the worst accomodated by the L1210-derived source. For the other sources (i.e. mouse liver and rat brain microsomal fractions), the same observation occurs, since the best substrates are the src-derived and gag-derived peptides, of similar accomodation, while the protein-kinase-A-derived peptide is poorly recognised (Table 2). The treatment by deoxycholate 2% (massivol.) of all the microsomal sources enhances the velocity towards all the substrates, without changing their relative rates when compared to each other with GNAAAARR < GQTVTTPL < GSSKSKPKDP, although the difference between the two former is more attenuated in L1210-derived microsomes than with brain microsomes and liver microsomes. The activity of the yeast enzyme is unchanged by detergent treatment. The deoxycholate solubilization of L1210 microsomal NMT does not modify the order of myristoylation of those three peptides: GNAAAARR < GSSKPKDKDP d GQTVTTPL (Table 3). Mouse liver microsomes were treated with various concentrations of three detergents (Triton 770, Triton X-100 and

Table 2. Influence of deoxycholate activation on the specificities of microsomal N-myristoyl transferuse from various origins Peptides were used at a final concentration of 0.3 mM. Yeast enzyme was purified according to [24]. All the microsomal sources used in these experiments were stored several weeks at -80°C. n.d., not detectable; DOC, deoxycholate Peptide

Yeast

Microsome

L1210 -DOC

mouse liver +DOC

-DOC

rat brain +DOC

-DOC

+DOC

pmol myristoylated peptide . min- ' . mg protein-' GNAAAARR GSSKSKPKDP

145.5

2.05

18.1

4.6

4.02 10.5

7.6

15.5

1.9

4.8

23.6

42.6

8.6

16.2

GQTVTTPL

58.3

5.8

9.6

19.8

31.1

3.6

8.1

GLAAAARR

n.d.

n.d.

ad.

n.d.

n.d.

n.d.

n.d.

7.4

11

Protein content (mgiml)

2.2

18.4

261 Table 4.Detergent activation of mouse liver microsomul N-myristoyl transferase Experiments were classically conducted by incubating the microsomal fractions with detergent for 30 min a t 4°C. The mixture was then used as a source for the NMT assay Conditions

Peptide GNAAAARR

GSSKSKPKDP

G QT V TT P L

pmol myristoylated peptide . 30 min-' Control

7.8

23.6

19.9

Triton 770 2% (mass/vol.)

8.6

59.7

30.5

Deoxycholate 1% (massivol.)

8.4

25.5

17.3

Deoxycholate 2% (mass/vol.)

15.5

42.6

30.3

Triton XlOO 0.5% (massjvol.)

7.3

17.1

13.8

Triton XlOO 1% (massivol.)

4.9

15.7

6.4

deoxycholate) and the enzyme activity was subsequently measured with our three reference peptides. Table 4 shows striking differences in the activation of the individual peptide myristoylations as a function of the detergent used. The most surprising observation is that GNAAAARR myristoylation is poorly enhanced by the 2% (mass/vol.) Triton 770 treatment. The K , and V,,, of the L1210 deoxycholate-activated microsomal NMT for the three peptides were measured. Since [3H]myristoyl-CoA is generated in situ, it is impossible to know its concentration, and therefore difficult to know if the transferase is saturated by its cosubstrate. To partly avoid this problem we generated batches of [3H]myristoyl-CoAand used the same batch for a given set of experiments. Thus, the concentration of [3H]myristoyl-CoA for a given K , determination is constant. Fig. 3 shows a linear relationship in doubleinverse construction (l/v = f(l/[S]). The K , were 0.33, 0.17 and 0.033 mM for protein kinase A, gag and src, respectively, (Fig. 3A, B and C). Furthermore, the following short peptides were tried as substrates of the microsomal preparation from L1210: GY, GQ, GYG, GYA, GVT, GGAG, GDDDDK. None showed detectable levels of activity (results not shown). These results are in agreement with those described by Towler and Glaser [22] for other sequences. These authors concluded that the minimum size for a peptide substrate to be accomodated by NMT is 7 - 8 amino acids.

0.05

7

0.mJ

'

'

'

10

.

'

20

'

30

11s ( mw" ) ,

030,

::y ./

0.20 1

.-op'E

0.10

i

z

4

-

0.05

>

Inhibition of microsomal NMT activity

It is obvious that research for specific inhibitors of this particular enzyme is of major importance from a pharmacological point of view. Because rather little is known about NMT issued from a mammalian source, we tested the three compounds known to inhibit the myristoylation of either proteins or peptides in various situations. Myristoylglycinal, cerulenin and the protein-kinase-A-derived octapeptide inhibitor were tested. Cerulenin is a rather poor inhibitor of the enzyme, with an ICso of about 0.5 mM. The octapeptide (GLAAAARR) shows no activity, Myristoylglycinal the Shoji's patented inhibitor (either under its free or its diacetal forms [34]) is active at concentrations equal to or higher than 1 mM. The newly synthetised compound myristoylphenylalanine shows a better inhibitory potency on the enzyme with an IC,o of 0.2 mM.

0.w

0

100

200 300

400

500

600

700 BOO

Fig. 3. Double-reciprocal plots of N-myristoyl-transferase measured with three different peptides. The assays were conducted with 2 % (massivol.) deoxycholate-activated L1210-derived rnicrosomal preparations as described in Materials and Methods. For each determination the same batch of in situ prepared [3H]myristoyl-CoA was used, hence, the [3H]myristoyl-CoA concentration is not known. (A) peptide GNAAAARR; (B) peptide GQTVTTPL; (C) peptide was GSSKPKDKDP

DISCUSSION A great deal of studies have been conducted on the cytosolic enzyme extracted from yeast. Only a few experiments have been published on NMT activities from wheat germ [29],

262 rabbit reticulocytes [2], rat brain and liver. These works were actually performed on cytosolic fractions and therefore, the present report is the first one describing microsomal NMT activity. The data gathered in the present study clearly indicate that the microsomal NMT is a membrane-bound enzyme comparable to other membrane-bound enzymes such as UDP glucuronosyl transferase(s) with which it has the common feature of being capable of accomodating substrates of various chemical structures. The enzyme is a latent enzyme and can be easily solubilized in an active form by Triton 770 or by the anionic detergent, deoxycholate. Due to this latency, it may be thought that the microsomes are deprived of any NMT activity, against the observations of Wilcox et al. [l] for whom myristoylation is an early post-translational event, therefore, most probably, associated with the endoplasmic reticulum. Furthermore, the solubilization step is not lethal for the enzyme activity which keeps its specificity intact, probably through favorable interactions with the detergent/phospholipid mixture. Yeast NMT has been shown by Rudnick et al. [27] to act using a preferred-ordered or ping-pong reaction mechanism. Similarly, from our primary data, the L1210 microsomal enzyme seems to obey a Michaelis-Menten mode of action, showing a linear double-inverse relationship. The K,,, globaly compare well with those published for similar peptides with the yeast purified enzyme [24], although comparison is made difficult by the non-saturated conditions used in the present paper and possibly in the work of Towler et al. [24]. Interspecies or interorgan comparisons of the behaviour of an enzyme can document indirectly the heterogeneity of a family of isoenzymes, as Boutin [43] pointed out for an important group of microsomal enzymes (namely, UDPglucuronosyl transferases). We therefore tested several tissue-derived microsomes and noted their behaviour during activation with our three substrates. Discrepancies between tissues, together with variations in latency restricted to one of those substrates (Tables 2, 3 and 4) lead us to speculate that there are several isoforms associated with the microsomal fractions. This will be demonstrated with partially purified preparations. On brain, the present work also demonstrates the presence of two subcellular localizations of NMT activities, since we also found a great deal of activity in brain cytosol (results not shown), as described also by McIlhinney and McGlone [32, 331 as it was for all the other sources (L3210 and liver). The most striking observation is that L1210 cytosol does not catalyse the myristoylation of GNAAAARR as also observed with cytosol from HL60 (human promyelocytic leukemia cell line), CEM (immortalized T lymphocytes), rat brain and mouse liver cytosols (J. A. Boutin, J. P. Clarenc and M. F. Burbridge, unpublished results). Except for the octapeptide GLAAAARR and myristoylCoA analogues [44, 451 no data exist in the literature on inhibitors tested on NMT activity. The result obtained with myristoylphenylalanine is somewhat encouraging. It consists of a leader compound from which better inhibitors of NMT activity will be derived, in the near future. The present work was intended to open the way for solubilization and purification of the different enzymatic entities suggested in the various studies conducted here. Furthermore, the role of the microsomal enzyme(s) in myristoylation of pp60src, p561ck or pl4gag have not been shown to date. The possibility that a cytosolic entity was responsible for the v i h s structural protein myristoyla&on is

strongly suggested by the cytosolic site of synthesis of the structural proteins of some retroviruses. It is obviously of major importance that cells susceptible either to infection by virus or to express, for any reason, high levels of myristoylated oncogene products should be screened for their levels of NMT activities as for some colon carcinomas overexpressing a myristoylated pp60c-src 1461. The peptide specificities and the succeptibility of the various subcellular compartment-associated NMT(s) to the different classes of inhibitors will then be studied. Such a work is currently in progress in our laboratory with microsomal versus cytosolic NMT of various origins. The authors are indebted to Dr J. F. Prost for hisconstant interest for this project, to Dr A. M. Pierre for his comments, to Mrs A. Genton and Mr M. F. Burbridge for their assistance and to Mr J. M. Barret and Mrs G. Guillaume-Dechartre for their help in preparing this manuscript.

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