Absolute Requirement of Ammonium Sulfate for Reconstitution of Active 70s Ribosomes from the Extreme...

Eur. J. Biochem. 233, 809-814 (1995) 0 FEBS 1995

Absolute requirement of ammonium sulfate for reconstitution of active 70s ribosomes from the extreme halophilic archaeon Halo ferax mediterranei Emma SANCHEZ and Ricardo AMILS

Centro de Biologia Molecular, CSIC-UAM, Cantoblanco, Madrid, Spain

(Received 2 August 1995) - EJB 95 1273/2

Active 70s ribosomes from the halophilic archaeon Haloferax mediterranei have been reconstituted from their isolated rRNAs and proteins. The reconstitution procedure consists of a two-step incubation ; first with 1 M ammonium sulfate and 100 mM magnesium acetate for 1 h at 42"C, followed by a 90-min incubation at 50°C after increasing the ammonium sulfate to 2 M final concentration. The total reconstitution of halophilic 70s ribosomes is a process with its own identity, which does not correspond to the conditions required for the reconstitution of the isolated subunits. Ammonium sulfate is the only salt capable of promoting the assembly of active ribosomes. The increase of ammonium sulfate salts in the second incubation step is obligatory for the isolation of functional particles.

Keywords: 70s ribosomes ; total in vitro reconstitution ; halophilic ribosomes ; protein synthesis ; ammonium sulfate.

The synthesis of ribosomes in vivo is a very complex system which requires precise coordination of the expression of many different genes and their assembly in order to allow the formation of functional ribosomes. It is well established that in vivo synthesis of ribosomal particles is the result of the simultaneous assembly of both ribosomal subunits ; a process strictly regulated within the cellular cycle (Nomura et al., 1984).

The development of in vitro reconstitution protocols for ribosomal subunits from different systems, in addition to proving that the ribosomal components have sufficient information to form fully active ribosomal particles, has made it possible to dissect the assembly processes and uncover the main rules gov- erning the structure/function relationships between the different components (Traub and Nomura, 1968; Nomura and Erdmann, 1970; Nierhaus and Dohme, 1974; Amils et al., 1979; Londei et al., 1986; Moore et al., 1986; Sanchez et al., 1990; Buck et al., 1991). From a structural point of view, there are three main variables that are considered critical in the reconstitution processes; ionic strength (mainly responsible for the selection of specific interactions), the concentration of divalent cations (mainly involved in the conformation of the rRNA and total ribosomal RNA), and the temperature (energy).

Halobacterial ribosomes have attracted the interest of riboso- mologists due to their outstanding property of requiring extreme ionic conditions, under near saturating conditions, in order to perform protein synthesis (Sanz et al., 1988). The assembly of halobacterial ribosomal particles has to be carried out at the ionic conditions required by the cell in order to overcome the osmotic pressure produced by the extreme salt concentrations of

Correspondence tu R. Amils, Centro de Biologia Molecular, Facul- tad de Ciencias, Universidad Aut6noma de Madrid Cantoblanco, E- 28049 Madrid, Spain

Fax: +34 1 3978344. Abbreviations. TP70, total ribosomal proteins; TP30, total proteins

of small ribosomal subunit; TP50, total proteins of large ribosomal subunit; SPA, subunits partial activity.

the habitats in which they develop (Bayley, 1966; Visentin et al., 1972). Furthermore, halobacterial ribosomes provide suitable material for crystallographic studies since high ionic strength favors crystallization processes (Yonath and Franceschi, 1993). The development of reconstitution procedures for halophilic ribosomal subunits, in addition to providing tools for structural and functional analysis, has been useful in determining protein- nucleic-acid interactions at high ionic strength (Sinchez et al., 1990).

All the ribosomal reconstitution processes reported up to now, with the sole exception of the system described for Esche- richia coli 70s ribosomes (Lietzke and Nierhaus, 1988), refer to isolated subunits. Since the parameters necessary for the active reconstitution of halophilic ribosomal subunits are quite different, it was considered interesting to approach the simultaneous reconstitution of both subunits to verify whether they are independent processes or whether some mutual influence exists, as suggested by Nomura and coworkers (Nashimoto and No- mura, 1970; Nomura et al., 1984).

The development of the total reconstitution system of active halophilic 70s particles from their isolated components should facilitate the availability of homogeneous particles suitable for crystallographic analysis. Additionally, ribosomes with modified components may be suitable for structural and functional analysis, thus avoiding the tedious preparation of active subunits and subsequent damage to some ribosomal components.

MATERIALS AND METHODS

Preparation of rRNA, ribosomal proteins, ribosomes and ribosomal subunits. H. mediterranei ribosomes and ribosomal subunits were prepared as described previously (Sinchez et al., 1990). Total ribosomal proteins (TP70) and rRNA or purified rRNA species (16s rRNA, 23s rRNA and 5s rRNA) were prepared following procedures described in Sdnchez et al. (1990).

810 Sgnchez and Amils (EM J . Biochem. 233)

Reconstitution procedure. In a final volume of 1.50 pl, 1 A,h(, unit total rRNA was incubated with a 2.5-M excess of TP70 for 60 min at 42°C in the presence of 1 M (NH,),SO,, 30 mM Tris/HCI, pH 8.4, 100 mM MgAc2 and 5 mM 2-mercaptoethanol. After increasing the concentration of (NH,J,SO, to 2 M, the reconstitution mixture was incubated at 50°C for 90 min. The stepwise procedure starts with the preparation of the saline solution. Ammonium sulfate was added in solid form and heated briefly at 42°C until fully dissolved. Then, TP70 was gently added prior to the addition of rRNA. After the first incubation, the solid ammonium sulfate was added and dissolved very gently, with the help of a micropipette in order to avoid the formation of bubbles that may affect the assembly process.

Analysis of reconstituted particles. The sedimentation be- haviour of reassembled particles was determined by centrifugation in sucrose density gradients (2-32'10) in the presence of 3.2 M KCI, 60 mM MgAc, and 20 mM Tris/HCI, pH 8.4. The gradients were run for 2.5 h at 15"C, 35000 rpm in a Beckman SW41 rotor. If necessary, the reconstituted particles were purified from the corresponding sucrose gradient fractions and sedi- mented after 15 h centrifugation at 40000 rpm and 10°C with a Beckman T86.5 rotor. Pellets were resuspended in 3 M KCI, 60 mM MgAc,, 20 mM Tris/HCl, pH 7.4, and 6 mM 2-mercaptoethanol, or in a buffer similar to the reconstitution buffer described above and stored at -80°C.

The polypeptide-synthesizing activity of reconstituted particles was measured by direct sampling of aliquots from the reconstitution mixtures or by using purified reconstituted particles, in a cell-free synthesis of poly(Phe), directed by poly(U), described in Shnchez et al. (1990). To estimate the partial activity of each subunit in the reconstitution process as a measure of the extension of their assembly, equivalent amounts of native subunits, 30s or SOS, were added to the correspondent protein synthesis mixtures. The activities obtained, which will be referred to as subunits partial activity (SPA), monitor the maximal functional activity obtained for the subunits during different stages of the assembly process.

RESULTS Reconstitution procedure. Ribosomal components of Halo- ferax tnediterrmei were isolated following standard protocols (Sinchez et al., 1990). Both components, rRNA and proteins (TP70), maintain their ability to reconstitute active particles after storage for several months without requiring high salt in the medium.

Optimal reconstitution of H. mediterrunei 70s ribosomes requires a two-step procedure. 1 A,,,, total rRNA and a 2.5-M excess of total ribosomal proteins (TP70) are incubated in 1 M (NH,)2S0,, 100 mM MgAc,, 30 mM Tris/HCl. pH 8.4, and 5 mM 2-mercaptoethanol for 60 min at 42°C. This is followed by a second incubation at 50°C for 90 min after increasing the ammonium sulfate concentration, added it as a solid to a final concentration of 2 M.

sedimentation analysis of the reconstitution products showed the presence of two discrete particles with sedimentation coefficients similar to 30s and 50s native subunits, respectively (Fig. l a ) . 70s ribosomal particles of H. mediterrunei are not observed in sucrose gradients even at concentrations of magnesium acetate as high as 1.50 mM. This property was described earlier for Halobacterium saliriariuin ribosomes (Rauser and Bayley, 1968). It is not unusual that halophilic ribosomes go unnoticed, since ribosomes froin the thermophilic archaeon Sul-

, fdobi i .~ .sii/fiitm*iciis display a similar behavior (Cammarano et al., 1982). In this case. the presence of mRNAs is necessary for the formation of 70s particles in a sucrobe gradient.

Q 8 N

migrated distance (cm)

Fig. 1. Sedimentation profiles of reconstitution mixture during the assembly process of H. mediterrunei ribosomes under different conditions. A 2.5 M excess of TP70 and 1 ALb,, unit rRNA in a buffer containing 100 mM MgAc,, 30 mM Tris/HCI, pH 8.4, and 5 mM 2-mercaptoethanol were incubated under optimal conditions of the 70s reconstitution process described in Materials and Methods. (a) At the end of the process; (c) after the first step. The reconstitution process was carried out using the one-step procedure at 42°C for 60 min in the presence of (b) 3 M KCl and (d) 2 M KCI. Native 70s ribosomes of H . mediterrunei were used for sedimentation control of active particles (e). High-salt- containing sucrose gradients were prepared and run as described in Ma- terials and Methods.

Table 1. Poly(phenyla1anine) synthesizing activity of Huloferux mediterrunei reconstituted 70s ribosomes. Reconstituted 70s particles were direct sampled on activity reaction mixture. Background of activity (all components minus 70s particles, 350 cpm) has been subtracted.

~ ~ ~~

Radio- Asso- Activity activity ciation

C P d mol Phe/ % assay 70s mix particles

Native 70s 10 830 6.5 100 Reconstituted 70s 9 530 5.71 88

Under the optimal conditions described above, the protein synthesis activity of the reconstituted 70s particles was 885G when compared to the activity of native ribosomes (Table 1).

Optimization of the reconstitution process. Three variables play a major role in the ribosomal reconstitution processes; (a) ionic strength, (b) concentration of divalent cations. (c) temper-

Sinchez and Amils ( E m J , Biochem. 233) 81 1

\

0 I I I I 1 - I I k l I

0 25 1 1 5 2 2 5 3

amonium sulfate concentration (M)

Fig. 2. Influence of ammoniun sulfate salts on the reconstitution process of H. mediterrunei 70s ribosomes. The polymerization activity of the reconstituted particles was measured as a function of increasing concentrations of the ammonium sulfate salts under different conditions of the process. 2 M TP70 and 1 A,,,, unit rRNA in 1 O O m M MgAc,, 30 mM TridHCl, pH 8.4, and 5 mM 2-mercaptoethanol were incubated (- V -) in the one-step procedure at 42°C for 60 min, (-A-) at different concentrations of ammonium sulfate at 42°C for 60 min followed by subsequent additions of ammonium sulfate to a final concentration of 2 M and further incubation at 50°C for 90 min, (-Q-) in 1 M ammonium sulfate at 42°C for 60 min with subsequent additions of ammonium sulfate at different concentrations and further incubation at 50°C for 90 min. 4-, dependence of the 50s reconstitution process on the ammoniun sulfate concentration (Sinchez et al., 1990); -=-, dependence of the 30s reconstitution process on the ammonium sulfate salt concentration (unpublished data).

ature. Optimal conditions for each parameter of the reconstitution of 7 0 s particles of H. mediterranei were obtained. The yield of reconstitution and the activity of the 70s particles were obtained as described above. The SPA of the correspondent subunits was determined after complementation of the protein synthesis mixture with either one of the native subunits.

The reconstitution of 70s ribosomes implies the simultaneous formation of both the 30s and the 50s subunits. Since we had previously developed optimal reconstitution for either one of the halophilic ribosomal subunits (Sjnchez et al., 1990; Sinchez, unpublished results) the first attempts were based on the extrapolation of the optimal conditions obtained from the isolated subunits.

Ionic strength of the medium. We explored the influence on the reconstitution process of four salts; (NH4),S04, KCI, NH,CI and NaCI. A wide range of concentrations of each salt (0.5- 3.5 M) were assayed. A 2-M excess of TP70 over the corresponding rRNA was added to a reaction mixture containing 60 mM MgAc,, 30 mM Tris/HCl, pH 8.4, and 5 mM 2-mercaptoethanol, and incubated in the presence of increasing concentrations of (NH,),SO, (0.75-2 M) in a one-step reaction at 42°C for 2 h (Fig. 2). Analysis of sedimentation profiles of the reconstitution products shows the presence of two broad peaks with sedimentation coefficients similar to those of 30s and S0S ribosomal subunits at 1 M (NH,),SO,. Thus, the existence of heterogeneous assembled particles for either ribosomal subunit (Fig. 1 c) was revealed. No significant activity of 70s ribosomes was achieved under these conditions. with 20% being the high-

Table 2. 70s activity and SPA under different reconstitution conditions. All the assays were carried out in the presence of 100 mM MgAc,, 30 mM Tris/HCl, pH 8.4, and 5 mM 2-mercaptoethanol by incubation at 42°C for 2 h. The assay with 2 M KCl (1st step) and 3 M KC1 (2nd step) was carried out under similar ionic conditions referred to before (for the first four conditions) using a two-step incubation procedure at 42°C and 50°C for 1 h. The assay with 1 M (NH,),SO, (1st step) and 2 M (NH,),SO, (2nd step) was carried out under optimal 70s reconstitution conditions as described in Materials and Methods. The SPA were determined as described in Materials and Methods. Reconstitution effi- ciencies were measured for at least three independent experiments and two different preparations of ribosomes.

Ionic conditions Final 30s 50s of the reconstitution assay for activity partial partial

2"" step ribosomes 1" step as 70s activity activity ____

9% ____ __

1 M (NH,),SO, - 14-20 59-64 52-57 2 M (NH,),SO, - 0 27-30 0

0 26-30 32-39 2 M KCL - 0 29-34 42-49 3 M KCl -

2 M KCI 3 M KCI 0 27-32 41-45 1 M (NH,),SO, 2 M (NH,),SO, 77-88 84-90 68-75

est polymerization activity obtained at 1 M (NH,),SO, (Table 2). The determination of SPA for each subunit revealed the existence of higher partial activity for each subunit than the total activity of the reconstituted 70s particles (Table 2).

Due to the low level of activity detected under these conditions and keeping in mind that the lack of activity could be related to the need for a conformational change (Dohme and Nierhaus, 1976), we tried a two-step reconstitution procedure involving an increase in temperature at the second step, providing additional energy to the system. The incubation of a 2-M excess of TP70 and 1 A,,,, unit rRNA in the presence of 1 M (NH,),SO,, 60 mM MgAc,, 30 mM Tris/HCl, pH 8.4, and 5 mM 2-mercaptoethanol, incubated at 42 "C for 60 min, followed by a second incubation for 90 min at different temperatures (48, S O , 55 or 60"C, respectively) did not affect the reconstitution values.

Similar results were obtained when increasing concentrations of KCI were assayed while keeping the rest of the variables constant. Analysis of sedimentation profiles of reconstitution products shows the presence of two discrete peaks with smaller sedimentation coefficients than those expected for 30s and 50s components (Fig. 1 b, d). No polymerization activity as 70s ribosomes was detected when the reconstitution products were assayed. The addition of complementary native subunits revealed the existence of 30-40% partial activity for each of the ribosomal subunits (Table 2). Similar to the results obtained for (NH,),SO,, no improvement of the activity was obtained at different KC1 concentrations when a second step with increased temperature was carried out.

Due to the lack of positive results i n the formation of active 70s halophilic ribosomes by overlapping the optimal conditions for the reconstitution of each subunit, we tested a two-step procedure in which different salt concentrations were used in each step. A 2-M excess of protein fraction over 1 A,,,, unit rRNA in 1 M (NH,),SO,, 100 mM MgAc,, 30 mM Tris/HCl, pH 8.4, and 5 mM 2-mercaptoethanol, was incubated at 42°C for 60 min. After increasing the ammonium sulfate concentration from 1.25 M to 3 M, a second incubation was performed at 42°C for 90 min. The greatest activity, 78%, was obtained after increasing the ammonium sulfate in the reconstitution mixture to 2 M final concentration (Fig. 2).

81 2 Sinchez and Amils (Eur J. Biochem. 233)

% activity loo

80

60

40

2 0

n d I 1 I I , I

0 20 40 BO 80 100 120 1 4 0 160 180 200

MgAc, (mW

Fig. 3. Effect of magnesium concentration on the reconstitution of halophilic ribosomes and ribosomal subunits. (-A-) 70s particles ; (-D-) 30s particles (unpublished data), (-0-) 50s particles (Sanchez et al., 1990). 70s particles were reconstituted under optimal conditions as described in Materials and Methods. 50s and 30s particles were reconstituted under optimal conditions as described in SBnchez et al. (1990) and unpublished data, respectively. The activity of the reconstituted ribosomes is given relative to that of native 70s particles, corresponding to the amount of rRNA present in the aliquot of reconstitution mixtures sampled during the assay.

Once the active 70s reconstituted ribosomes had been obtained, optimization of the different parameters was analyzed. First, the influence of different concentrations of (NH,),SO, in the first step of the reconstitution was examined. Maximum efficiency of reconstitution was obtained at 1 M salt concentration (Fig. 2).

Similar experiments to those described for ammonium sulfate using different combinations of KCI concentrations did not produce active 70s particles. No improvement of the SPA values was detected under these conditions when compared with those obtained under the one-step reaction (Table 2) and the sucrose gradient analysis showed a similar pattern to those obtained when the process was carried out under a one-step reaction (Fig. 1 b, d).

In agreement with the results obtained for the reconstitution of the halophilic subunits, neither NH,CI nor NaCl were capable of promoting the assembly of active ribosomes.

Influence of divalent cations. To evaluate the influence of divalent cations on the reconstitution of halophilic 70s ribosomes, increasing concentrations of MgAc, over 10-200 mM, were added at a 2-M excess of TP70 and 1 A,,,, unit rRNA. This mixture was incubated at 42°C for 60min in the presence of 1 M (NH,),SO,, 30 mM Tris/HCI, pH 8.4, and 5 mM 2-mercaptoethanol. After increasing the ammonium sulfate concentration, and keeping the other conditions constant, the reaction mixture was incubated at 42°C for 90 min. At low Mg2' concentration there is no reconstitution of ribosomal activity, which reflects the need for divalent cations in the assembly of 50s subunits (Fig. 3). There is a small but constant increase in the efficiency of reconstitution of 70s subunits over 80-200mM MgAc,, which somehow reflects the unusual insensitivity of the 30s subunit reconstitution to the Mg" concentration. Moreover, the high concentration of Mg" cations required for the 70s reconstitution process of H. mediterranei is probably needed to com- pete with the high concentration of monovalent cations, 4 M equivalents NH,', present in the reconstitution mixture.

Although the optimal concentration of MgAc, observed for the 70s ribosomal reconstitution process of H. mediterranei was 200 mM, 100 mM MgAcz was used in all reconstitution experiments. This lower concentration was preferred, given that the

slight increase in efficiency at 200 mM was counteracted by the inhibition produced at high concentrations of Mg2+ in the protein synthesis assays.

rRNMprotein ratio. Similar to the results obtained from reconstitution experiments of individual halophilic subunits, a 2.5- M excess of proteins (TP70) over rRNA was required for maximal efficiency of 70s ribosome reconstitution (data not shown).

The influence of temperature in the two-step system. The effect of temperature on the ribosomal reconstitution processes is related to the energy required for ribosomal assembly. To test the effect of temperature in the two-step system, 1 A,,,, unit rRNA and a 2.5-M excess of TP70 were incubated at 36 ,42 and 50°C, in 1 M (NH,),SO,, l 0 0 m M MgAc,, 30 mM Tris/HCl, pH 8.4, and 5 mM 2-mercaptoethanol for 60 min, followed by a second incubation for 90 min at the same temperature after increasing the ammonium sulfate concentration to 2 M. Optimal activity was achieved when the reconstitution process was carried out at 42°C (Fig. 4). The optimum temperature is similar to that observed for the reconstitulion of 50s ribosomal subunits of H. mediterranei (SBnchez et al., 1990) and is within the optimal temperature range for growth of this microorganism.

In order to ascertain the effect of a temperature change in the two-step reconstitution system, a 2.5-M excess of TP70 and the correspondent rRNA were incubated in a buffer containing 1 M (NH,),SO,, 100 mM MgAc,, 30 mM Tris/HCI, pH 8.4, and 5 mM 2-mercaptoethanol for 60 min at 42°C. After increasing the ammonium sulfate to 2 M, a second incubation was performed for 90 min at different temperatures. The maximum reconstitution efficiency, 88 %, was obtained when the second step was carried out at 50°C (Fig. 4).

Kinetics. As the reconstitution process consists of a two-step procedure, we had to study the kinetics of each step separately. 1 rRNA was incubated with a 2.5-M excess of TP70 in 1 M (NH,),SO,, 100 mM MgAc,, 30 mM Tris/HCl, pH 8.4, and 5 mM 2-mercaptoethanol at 42°C for different time periods. Af- ter increasing the ammonium sulfate concentration, a second incubation was carried out at 50°C for 90min. During the first step, a 60-min incubation was sufficient to obtain maximum reconstitution efficiency. When the incubation period of the second step was varied while keeping the other conditions constant,

SBnchez and Amils ( E m J. Biochem. 233) 813

% activity 100 I

" I b

30 35 40 45 50 55 60 65

O C Tenperatare

Fig. 4. Effect of temperature on the reconstitution process of 70s ribosomes. (-A-) Both steps of the process were carried out at the same temperature; (-0-) two-step reconstitution performed at 42 "C for the first step and at different temperatures for the second step The 70s reconstitution process was carried out under optimal ionic conditions, as described in Materials and Methods

optimal reconstitution values were obtained after 90 min. Longer periods of incubation did not damage the activity of the reconstituted particles.

DISCUSSION

This paper presents an efficient system for reconstituting active 70s ribosomes of H. mediterranei from their isolated components, riboproteins and rRNA. The studies carried out re- veal that the simultaneous in vitro assembly of both the 30s and 50s ribosomal subunits is not related to the conditions required for the reconstitution processes of the individual ribosomal subunits (Sinchez et al., 1990; unpublished results), as is the case for E. coli 70s ribosomes (Lietzke and Nierhaus, 1988) but a process with its own identity.

Two main issues arise in relation to the conditions required for total reconstitution of 70s ribosomes of H. mediterranei when compared with those needed for the assembly of individual ribosomal subunits; its strict dependence on ammonium sulfate salts and the need for a two-step incubation system. Comparing the salt-concentration dependence of the reconstitution processes for the 30s and 50s halophilic ribosomal subunits (Fig. 2), it was expected that active 70s particles would be obtained within a range 1 - 1.2 M ammonium sulfate as well as 3 - 3.4 M KCI. Nevertheless, a very low level of activity of reconstituted 70s particles, around 20%, resulted when the process was carried out using one-step in the presence of 1 M (NH,),SO,. The analysis of the sedimentation profiles of the reconstitution products under these conditions (Fig. 1 c) revealed the existence of heterogeneous assembled particles in each peak. The mixture contained an assortment of aborted particles resulting from non- specific interactions with active reconstituted particles as the product of specific interactions of the different ribosomal components. When the results are compared with those obtained for the reconstitution of isolated subunits, it appears that the simultaneous presence of all the ribosomal components, riboproteins (TP30, total proteins of small ribosomal subunit + TP50, total proteins of large ribosomal subunit) and rRNAs (16s + 23s + 5 S rRNAs), in the reconstitution mixture favors the appearance of a strong background of non-specific interactions among these components. The introduction of more restrictive ionic condi-

tions by increasing the ammonium salt concentration in the second incubation step might facilitate the destruction of formed abortive particles and simultaneously preclude their formation while specific interactions are favoured.

Accordingly, the lack of efficiency of KCI in promoting the in vitro formation of active 70s particles could be related to this effect. Analysis of the conditions required for the reconstitution of individual subunits shows that they can be efficiently assembled in a 3-3.4-M concentration range of KCl. (Sinchez et al., 1990; unpublished results). The analysis of the sedimentation profiles of reconstituted 70s particles obtained in the presence of KC1 salts (Fig. 1 b, d) revealed the presence of two peaks with smaller sedimentation coefficients than the expected 30s and 50S, which evidence the formation of abortive particles as well as incomplete and non-stable particles. In this case, it is impos- sible to further constrain the specific interactions by increasing the ionic strength, since we are working near the solubility limit of KCI salts in the reaction mixture. It is also interesting to note that, like functional protein synthesis, the activity of halophilic ribosomes is not regulated by the compatible solute of the cell (KCI). This probably reflects the in vivo situation in which the intracellular ionic concentration is dependent upon the ionic strength of the habitat in which they develop. It is reasonable to postulate that the regulation of specific interactions in the cell is controlled by components which do not vary in concentration according to ecological conditions. We already know that ammonium sulfate is not an intracellular component of extreme halophilic microorganisms (Sanz et al., 1988). Thus, an intracellular component with physicochemical properties similar to this macromolecule-ordering salt is likely to have physiological im- plications. It must also be mentioned that the observed effects on halophilic protein synthesis and reconstitution of isolated halophilic subunits, are not only dependent on cations but also on the corresponding counteranions, which are critical to the total reconstitution of 70s ribosomes. These observations make the analysis of the effects produced by the different components much more complicated.

The existence of heterogeneous particles in functionally in- active reconstitution products, many with structural features closely resembling those of active particles, can interfere with the analysis of activity by interacting with complementary active reconstituted subunits and sequestering activity measurements. The detection of partial activity for the reconstituted subunits should rely on the use of two different effects; (a) the full functional expression of the active reconstituted particles present in the reconstitution mixture rescued by the presence of an excess of Complementary external native subunits, and (b) the possible functional rescue of particles, partially active particles, with the help of conformational changes introduced by their interaction with complementary native subunits. From the results obtained with KCI salts, we can assume that up to 40% of the activity of the reconstituted subunits can be rescued from the reconstitution mixture when native subunits are added. However, these reconstituted subunits are not capable of protein synthesis as are 70s ribosomes in the absence of added native subunits. This result correlates with the differences found between the low activity of 70s ribosomes of the reconstituted particles at 1 M ammonium sulfate and the high partial activity exhibited by the reconstituted subunits detected by the addition of external native subunits (Table 2) . This differential effect disappears when reconstituted particles reach a fully active conformation (Table 2). These results underline the complexity of the assembly model under study. The practical applications of the reconstituted halophilic ribosomes described recommend their disclosure. The structural changes affecting the assembled particles during the reconstitution process will be the challenge of further studies.

814 Sanchez and Amils (Eur: J . Biochcm. 233)

We thank Dr Brian Duval and Rosemary Amils for the critical read- ing of the manuscript. This work was supported by the Spanish Intermin- isterial Commission for Science and Technology grant PB 92-0129 (DGICYT), an E. C. grant BIO-2-CT93-0274, and an institutional grant from the Fundacidn Ramcin Arrces.

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