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Electron microscopy of multiple forms of glutamine synthetase from bacteroids and the cytosol of yellow lupin root nodules
368
Biochimica et Biophysica Acta 913 (1987) 368-376
Elsevier BBA 32875
Electron microscopy of multiple forms of glutamine synthetase from bacteroids and the cytosol of yellow lupin root nodules Vladimir L. T s u p r u n a, Olga N. Zograf a, Elena V. Orlova a, Nikolai A. Kiselev a, A l e x a n d e r V. P u s h k i n b, G u l m i r a E. Shiffelova b, N a d e z h d a A. Solovieva b, Z i n a i d a G. Evstigneeva b and W a c l a w L. K r e t o v i c h b a A. V. Shubnikov Institute of Crystallography, U.S.S.R. Academy of Sciences, and h A.IV. Bach Institute of Biochemistry, U.S.S.R. Academy of Sciences, Moscow (U.S.S.R.)
(Received 17 November 1986) (Revised manuscript received 23 March 1987)
Key words: Glutamine synthetase structure; Electron microscopy; (Lupin root nodule); (Cytosol); (Bacteroid)
The quaternary structure of multiple forms of glutamine synthetase (EC 6.3.1.2) from bacteroids and eytosol of yellow lupin (Lupinus luteus L.) root nodules has been studied by means of electron microscopy and image processing. The multiple forms of glutamine synthetase were found to possess different quaternary structures, which are, apparently, responsible for their different properties aml, possibly, for different functions. It was shown that one of the two forms of cytosol g!utamine synthetase (ghmunine synthetuse II) which is predominant in nitrogen fixation conditions has a cubic configuration with eight identical nmmm~rs arranged in two layers with point group symmetry 422 (D4). Two of the three bacterotd ~ synthetase forms, gintamine synthetase H and gintamine synthetase m , possess different quaternary s ~ . Thus, bacteroid glutamine synthetase II was found to consist of eight identical monomers arranged with point group symmetry 422 (D4) at the vertices of two squares which, unlike cytosol gimmine synthetase II are twisted about the 4-fold axis relative to each other. The quaternary structure of bacteroid glutamine synthetase II is typical for glutamine synthetases of higher plants and may indicate its plant origin. As with bacterial glutamine synthetase, bacteroid glutamine synthetase IH consists of twelve ~ nmnmners arranged with point group synmtetry 622 (D6) at the vertices of two rectilinear hexagons. Computer averaging of bacteroid gh.qamine synthetase Ill images revealed a two-donmin ~ of its ~ . Introduction A key reaction of the assimilation of ammonia products during symbiotic nitrogen fixation in legume root nodules is known to be the synthesis of glutamine [1-3], which is catalyzed by gluta-
Correspondence: A.V. Pushldn, A.N. Bach Institute of Biochemistry, U.S.S.R. Academy of Sciences, Leninsky Pr., 33, Moscow 117071, U.S.S.R.
mine synthetase (L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2). Most of the ammonia is believed to be assimilated in nodule cytosol [1-11], while bacteroids play little or no part in the process. Until recently, only one form of glutamine synthetase was known to be present in the cytosol of legume nodules [2,8,9]. In recent years, however, two molecular forms of glutamine synthetase have been reported in the cytosol of kidney beans [3,5,7] and yellow lupin [6]. One of these can be associated with the fixation of molec-
016%4838/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
369 ular nitrogen, while the other is, in many respects, close to the glutamine synthetase found in roots. The two forms of glutamine synthetase of kidney bean root nodule cytosol have equal molecular masses of about 380 kDa and consist of monomers of molecular mass of 41 kDa [7]. The molecular mass of glutamine synthetase II of the cytosol which prevails in nitrogen fixation conditions in yellow lupin root nodule cytosol was found to be 410 kDa. The enzyme consists of identical monomers with a molecular mass of 51 kDa [6]. According to other data [2], there is a form of yellow lupin root nodule cytosol glutamine synthetase with molecular mass 347 kDa consisting of identical monomers (41.5 kDa). The molecular mass of soybean root nodule cytosol glutamine synthetase was estimated to be 376 kDa, and the enzyme was found to consist of eight identical monomers with a molecular mass of 47.3 kDa [8]. White lupin root nodule cytosol glutamine synthetase [9] was shown to consist of eight identical monomers with a molecular mass of 44.7 kDa having a total molecular mass of about 360 kDa. As for the quaternary structure of legume root nodule cytosol glutamine synthetase it is only the enzyme from soybean that has been studied by electron microscopy. It was revealed [8] that it consists of eight identical monomers arranged at the vertices of a cube. Earlier, such an arrangement of monomers was found for glutamine synthetases of animals [12,13]. Unfortunately, little is known about multiple forms of bacteroid glutamine synthetase. Moreover, most of the results now available correspond not to bacteroid glutamine synthetase, but to free-living cells of rhizobia. Thus, two forms of glutamine synthetase were found in free-living cells of several Rhizobium species [14,15]. One of them was shown to comprise twelve identical monomers and had a molecular mass of about 600 kDa. The other form had a substantially lower molecular mass (about 340 kDa). The arrangement of monomers in the molecule of a larger enzyme is similar to that of glutamine synthetases from bacteria and cyanobacteria [16-21]. The authors failed to determine the quaternary structure of a smaller glutamine synthetase by electron microscopy [14]. At a high concentration of ammonia in the medium, a decrease of the activity of the dode-
cameric form of glutamine synthetase of R.
japonicum cells was observed, induced by its adenylylation, with suppressed synthesis of the other form of glutamine synthetase [14]. This fact allowed the researchers to conclude that assimilation of ammonia formed during the fixation of molecular nitrogen in legume bacteroids takes place mainly in the nodule cytosol. At the same time, as shown by experiments with bacteroids isolated from root nodules of soybean [22] and yellow lupin [23,24], ammonia does not repress the glutamine synthetase activity, and in the latter case it even stimulated it. Therefore, the evidence obtained for free-living rhizobia can hardly be applied to bacteroids, and the assimilation of ammonia produced by nitrogen fixation is likely to proceed in bacteroids as well as in the nodule cytosol. As was mentioned [25], there are three forms of glutamine synthetase in R. lupini bacteroids; two of them can also be found in culture cells, while the third one was shown to occur only in bacteroids. In nitrogen-fixing conditions the form of glutamine synthetase with a molecular mass of about 600 kDa (bacteroid glutamine synthetase III) was adenylylated, which reduced its activity [25,26]. At the same time, there was a sharp increase in the content of the other form (bacteroid glutamine synthetase II) with a molecular mass of about 340 kDa which in free-living cells of R. lupini was repressed by ammonia [24]. It can be concluded from the data available that, apart from the nodule cytosol, the assimilation of ammonia formed during symbiotic nitrogen fixation in legume nodules proceeds in the bacterial fraction of the symbiotic association as well. Since the activity of bacteroid glutamine synthetases I and III in bacteroids turned out to be insignificant [24,25], it is apparently bacteroid glutamine synthetase II that plays a key part in the assimilation of ammonia in bacteroids. The quaternary structure of glutamine synthetases from bacteroids of legume root nodules has not been determined so far. So, the aim of the present work was to study the quaternary structure of glutamine synthetase II from the plant fraction and glutamine synthetases II and III from the bacterial fraction of yellow lupin root nodules by electron microscopy.
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Materials and Methods
Purification of glutamine synthetase from yellow lupin (Lupinus luteus L.) root nodule cytosol followed the procedure which we have developed [6]. Glutamine synthetases II and III of Rhizobium lupini bacteroids were purified using our methods [24,26]. For preparations of specimens for electron microscopy a drop of protein solution in 5 mM Tris-HC1 buffer (pH 7.5) was applied to a thin carbon support grid. Then, the specimens were negatively stained with 2% phosphotungstic acid or 5% potassium silicotungstate (pH 7.0). Electron microscopy was carried out on a Philips EM 400 electron microscope at 80 kV with a magnification of 50 000. The electron micrographs were digitized with an Optronics P-1000 rotating drum microdensitometer with a sampling corresponding to 0.16 nm at specimen level. The digitized images were processed using a Nord-100 (Norsk Data) minicomputer. Images were displayed on a Tektronics graphic terminal as contour maps. The first step of processing included masking and low-pass filtering. A circular mask was used to select the part of the image containing the object, and the variance of the density was normalized. High-frequency noise was eliminated by low-pass filtering. The next step involved the alignment and calculation of the images for their averaging. The images to be averaged were aligned by a cross-correlation function with one particle selected as a reference image [27]. The quality of correlation was controlled by a correlation coefficient. Particles with a correlation coefficient higher than that of a pre-given threshold were taken for averaging. The averaged images were represented as contour maps or half-tone pictures. Rotational symmetry of particles after their centre refinement was determined using EM GROUP programs written by L. Amos [28] and adapted for an EC 1040 computer. Results and Discussion
Glutamine synthetase of yellow lupin root nodule cytosol Fig. la shows a general view of negatively stained molecules of yellow lupin root nodule
cytosol glutamine synthetase II. Several characteristic types of image of the enzyme molecule can be observed. The first type is a square projection with four protein spots whose sides are about 10 nm (Fig. lb). Two parallel protein layers separated by stain are seen in two other types (Fig. lc,d). They have square (Fig. lc) or rectangular (Fig. ld) profiles. Their lengths are about 10 nm and 13.5 nm, respectively, and their widths are the same (approx. 10 nm). The square images (Fig. lb) have rotational symmetry. The order of the rotational axis was determined by examining of the rotational power spectrum [28]. In all cases a spectrum showed the presence of 4-fold rotational symmetry. A number of such images of molecules was used for averaging and symmetrization. The selected images were aligned to a reference one by rotational and translational shift using a correlation function (see Materials and Methods). Since the averaged image showed a clearly defined 4-fold symmetry (Fig. le), it was 4-fold rotationally symmetrized. In the resulting picture four maxima of protein density are located at vertices of a square (Fig. lf,g). Analysing the projections observed (Fig.lb-d), it was inferred that the cytosol glutamine synthetase II molecule consists of eight monomers (in agreement with our preliminary findings [6]) arranged at vertices of two squares with point group symmetry 422 (D4). The molecule has a form close to a cube with sides of about 10 nm. When comparing the images of the molecule we can suggest that the first type (Fig. lb) corresponds to the frontal view projection (along the 4-fold axis), and the two other types (Fig. lc,d) are lateral views (perpendicular to this axis). The averaged image (Fig. lf, g) can then be interpreted as a projection of two eclipsed square tetramers. It should be noted that a cubic arrangement was found also for octameric glutamine synthetase of soybean root nodule cytosol [8], while the only glutamine synthetase from roots of higher plants with a known quaternary structure, the pumpkin root glutamine synthetase has a somewhat different configuration: the tetramers of one of its layers are twisted about the 4-fold axis relative to the other layers [29]. Such a quaternary structure was also observed for glutamine synthetase isoenzymes from leaves of higher plants [30-34]. A1-
371
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4
8
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i
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Fig. 1. (a) Field of glutamine synthetase from yellow lupin root nodule cytosol negatively stained with phosphotungstic acid. (b-d) Selected molecules in three characteristic orientations. Computer averaging of the images. (e) Rotational power spectrum of the average of ten aligned front view images (se~ b). The 4-fold order is shown by an arrow. (f,g) 4-fold rotationally symmetrized average. Images are represented as a half-tone picture (f) and contour map (g).
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though there are still no reliable data about the quaternary structure of glutamine synthetase in roots of legumes, and specifically of lupin, one may expect it not to be much different from that of glutamine synthetase of pumpkin roots [29]. The octameric glutamine synthetases with cubic arrangement were also found in animals [12,13,35-37] and Neurospora crassa [38].
Glutamine synthetase H of Rhizobium lupini bacteroids Different types of molecule images can also be observed in micrographs of bacteroid glutamine synthetase II (Fig. 2a-e), which depend on the respective orientation of the molecule when adsorbed onto the support film. The first type is represented by annular projections with a diameter of 9-10 nm. Their central space is often completely filled with stain (Fig. 2b). Rotational power spectrum of such images very often showed the presence of a rotational symmetry. To amplify the main details, these images were computer averaged. The result of averaging clearly shows the predominance of the 4-fold symmetry (Fig. 2f). Therefore, it was additionally 4-fold rotationally symmetrized. In the resulting picture (Fig. 2g,h) one can see a circular region with eight maxima of protein density grouped in pairs, and the internal cavity filled with stain. In the images of the three other types (Fig. 2c-e), two strips of protein separated by stain are seen. All these projections have the same width (about 7 nm), but differ in length and details of image. The images of the second (Fig. 2c) and the third (Fig. 2d) types are rectangular. They are characterized by a 2m symmetry and have a length of 9-10 and 8-9 nm, respectively. The projections of the fourth type (Fig. 2e) are trapezium shaped. Comparing all these images one can suppose that the first type is a front view (along the 4-fold axis) of molecules, while all the others are side views (perpendicular to the 4-fold axis). The projection of the first type is most frequently observed and apparently corresponds to the most stable position of the molecule on the support film. The above data can be summarized by the following three-dimensional model of bacteroid glutamine synthetase II molecule: eight elongated monomers are arranged with point group symme-
try 422 (D4) at vertices of two squares twisted about the 4-fold axis relative to each other at an angle of about 40 ° . Therefore, the projections shown in Fig. 2c,d correspond to the orientation of particles observed along the two 2-fold axes, lying at 45 ° . The projection of the fourth type (Fig. 2e) occurs when the molecule is observed along the bisector between these axes. Also, an angular shift of tetramers of two layers leads to protein density having four paired maxima in the first type of images and to the appearance of trapezium-shaped projections (the fourth type). In some images of the first type (Fig. 2b) one can see four dark spots of stain in the centre of the ring, separated by white regions, which most probably correspond to protein. The presence of protein inside the annular region is also confirmed by the fact that there is no visible 'central channel' in the lateral projections of the molecule. These observations indicate that the enzyme molecule has no annular form, but contains an internal cavity at the expense of hollows in monomers, as shown in the model (Fig. 2b). Therefore, the ring-shaped projections of the molecule (Fig. 2b) may be explained by penetration of the stain into the internal cavity. According to this model, the monomer volume is 30-35 nm3, corresponding to a molecular mass of 45 + 5 kDa. These data are in good agreement with parameters of the bacteroid glutamine synthetase II molecule determined by the gel filtration method [24]. Thus, the bacteroid glutamine synthetase II molecule consists of eight identical monomers arranged in accordance with point group symmetry 422 (I)4). Up until now, the octameric structure has been found only for glutamine synthetases of eucaryotic organisms which, as opposed to procaryotic ones (from bacteria and cyanobacteria), contain twelve identical monomers. The arrangement of monomers in bacteroid glutamine synthetase II molecule at vertices of two squares twisted about the 4-fold axis appears to be very similar to that observed for glutamine synthetases of higher plants [29,30-34]. The greatest similarity is observed with glutamine synthetase of pea seeds [34], which is immunochemically indistinguishable from a glutamine synthetase of lupin seeds [38]. Our results indicate that glutamine synthetase
373
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Fig. 2. (a) Field of glutamine synthetase II from bacteroids of R. lupini negatively stained with phosphotungstic acid. (b-e) Images of the first, second, third and fourth types, respectively. Different appearances of the model are shown on the right, corresponding to the different projections of the molecule. Computer averaging of the images: (f) rotational power spectrum of the average of ten aligned front view images (see b). The 4-fold order is shown by an arrow. (g,h) 4-fold symmetrized average. Images are represented as a half-tone picture (g) and a contour map (h).
374
II of R. lupini bacteroids may have a plant origin and that the structural gene of the enzyme might, in one way or another, have been acquired by rhizobia from the genome of higher plants.
Glutamine synthetase I l l of Rhizobium lupini bacteroids Electron micrographs of bacteroid glutamine synthetase III also show individual molecules in different positions (Fig. 3a). The majority of the enzyme molecules are predominantly shown form the front and have a hexagonal profile with a diameter of 13-14 nm (Fig. 3b). The molecules are often seen in two side views, which reveal two protein layers either as four spots (Fig. 3c), or as two strips (Fig. 3d). These projections have the same width of 8 + 1 nm. Some of the molecules in the side-view projections form stacks with a 4-4.5 nm spacing between protein layers (Fig. 3a). Projections of the bacteroid glutamine synthetase III molecule are very similar to those of Escherichia coli glutamine synthetase, described by Valentine et al. [16] and also to projections of glutamine synthetase I (with a molecular mass of 600 kDa) from free-living cells of R. japonicum, described by Darrow [14]. It can be concluded from the images obtained that the enzyme is composed of twelve identical monomers arranged at vertices of two eclipsed hexagons with point group symmetry 622 (D6). The dimensions of the molecule are 13-14 nm in diameter and 8 + 1 nm high. The quaternary structure of bacteroid glutamine synthetase III molecule is similar to the quaternary structure of glutamine synthetases of several bacteria and cyanobacteria studied both by electron microscopy [14,16-21,26] and X-ray analysis [40-42]. All these molecules have D 6 symmetry and are of a similar size. The result of the three-dimensional reconstruction from images of 6 + 1 helical chains of glutamine synthetase molecules from E. coli [43] is consistent with this symmetry. Image analysis of micrographs of single glutamine synthetase particles, including images obtained with the low-dose technique has been performed by Frank et al. [44,45] and Kunath and coworkers [46]. Computer averaging of particles revealed a division of monomers into two domains.
We also obtained averaged hexagonal projection of the bacteroid glutamine synthetase III molecule. Thirty images were studied, ten of which (with the best correlation coefficients) were used for averaging. A power spectrum of the average of ten aligned particles displayed 6-fold symmetry (Fig. 3e). Half-tone and contour map representation of the 6-fold rotationally symmetrized average is shown in Fig. 3f,g. One can see six protein units containing one major outer and one minor inner density peak, separated by a lower density region where the stain can penetrate. The averaged image (Fig. 3f,g) has slight handedness which can be explained by differences in staining of one of two apposing hexamers. Recently, the X-ray structure of bacterial glutamine synthetase from Salmonella typhimurium at 3.5 A resolution has been reported [42]. The atomic model obtained shows the localization of the active sites at the subunit interfaces and suggests a mechanism for regulation of the rate of synthesis of glutamine in response to covalent modification and feedback inhibition. The protein is composed of 12 identical subunits arranged with 622 point group symmetry. The molecule has an outer diameter of 143 A and a height of 103 ~,. The projected structure of the single hexamer down the 6-fold axis of symmetry shows distinct handedness arising from the skew appearance of the subunits. The subunits have a region of low protein density which can be penetrated by stain, leading to two density peaks in subunit projection observed by electron microscopy. Thus, the structures of the glutamine synthetase III molecule from R. lupini bacteroids and other bacterial glutamine synthetases are very similar as seen with negative staining resolution, and appear to be compatible with the X-ray diffraction study [42]. In summary, it can be concluded that the multiple forms of glutamine synthetase of bacterial and plant fractions of the yellow lupin and R. lupini symbiotic association have appreciably different quaternary structures. Apparently, such distinctions are responsible for different properties and, perhaps, different functions of these glutamine synthetases.
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2 1
e
i
i
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4
6
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Fig. 3. (a) Field of glutamine synthetase llI from bacteroids of R. lupini negatively stained with potassium silicotungstate. (b) Selected molecules showing the front view, and two side views (c,d). Computer averaging of the images: (e) rotational power spectrum of the average of ten aligned front view images (see Fig. 3b). The 6-fold order is shown by an arrow. (f,g) 6-fold rotationally symmetrized average. Images are represented as a half-tone picture (f) and contour map (g). The two-domain structure of monomers is visible.
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Acknowledgments The authors express their deep gratitude to Dr. L. Amos for providing the computation programs. References 1 Evstigneeva, Z.G. and Pushkin, A.V. (1983) in Molecular Mechanisms of Nitrogen Assimilation in Plants (Kretovich, W.L., ed.), pp. 198-234, Nauka, Moscow 2 Chen, J. and Kennedy, I.R. (1985) Phytochemistry 24, 2167-2172 3 Cullimore, J.V. and Miflin, B.J. (1984) J. Exp. Bot. 153, 581-587 4 Boland, M.J., Fordyce, A.M. and Greenwood, R.M. (1978) Aust. J. Plant Physiol. 5, 533-539 5 Lara, M., Cullimore, J.V., Lea, PJ., Miflin, B.J., Johnston, A.W.B. and Lamb, J.W. (1983) Pianta 157, 254-258 6 Sadikova, G.E., Solovieva, N.A., Pushkin, A.V., Tsuprun, V.L., Shubin, V.V., Evstigneeva, Z.G. and Kretovich, W.L. (1985) Biokhimiya 50, 936-940 7 Cullimore, J.V., Lara, M., Lea, PJ. and Miflin, B.J. (1983) Planta 157, 245-253 : " ~i 8 McParland, R.H,, Guevara J.G, Becker, R.R. and Eyans, H.J. (1976) Biochem. J. 153, 597-606 9 McCormack, D.K., Farnden, K.J.F. and Boland, M.J. (1982) Arch. Biochem. Biophys. 218, 561-571 10 O'Gara, F. and Shanmugam, K.T. (1976) Biochim. Biophys. Acta 437, 313-321 11 Howitt, S.M. and Gresshoff, P.M. (1985) J. Gen. Microbiol. 131, 1433-1440 12 Haschemeyer, R.H. (1968) Trans. N.Y. Acad. Sci. 30, 875-891 13 Tate, S.S. and Meister, A. (1973) in The Enzymes of Glutamine Metabolism (Prusiner, S. and Stadtman, E.R., eds.), pp. 76-128, Academic Press, New York 14 Darrow, R.A. (1980) in Glutamine: Metabolism, Enzymology, and Regulation (Mora, J. and Palacios, R., eds.), pp. 139-166, Academic Press, New York 15 Fuchs, R.L. and Keister, D.L. (1980) J. Bacteriol. 144, 641-648 16 Valentine, R.C., Shapiro, B.M. and Stadtman, E.R. (1968) Biochemistry 7, 2143-2152 17 Deuel, T.F., Ginsburg, A., Yeh, J., Shelton, E. and Stadtman, E.R. (1970) J. Biol. Chem. 245, 5195-5205 18 Alef, K., Burkardt, H.-J., Horstmann, H.-J. and Zumft, W.G. (1981) Z. Naturforsch. 36c, 246-254 19 Siedel, J. and Shelton, E. (1979) Arch. Biochem. Biophys. 192, 214-224 20 Sampaio, M.J.A.M., Rowell, P. and Stewart, W.D.P. (1979) J. Gen. Microbiol. 111, 181-191 21 Orr, J., Keefer, L.M., Keim, P., Nguen, T.D., Wellems, T., Heinrikson, R.L. and Haselkorn, R. (1981) J. Biol. Chem. 256, 13091-13098
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Biochimica et Biophysica Acta 913 (1987) 368-376
Elsevier BBA 32875
Electron microscopy of multiple forms of glutamine synthetase from bacteroids and the cytosol of yellow lupin root nodules Vladimir L. T s u p r u n a, Olga N. Zograf a, Elena V. Orlova a, Nikolai A. Kiselev a, A l e x a n d e r V. P u s h k i n b, G u l m i r a E. Shiffelova b, N a d e z h d a A. Solovieva b, Z i n a i d a G. Evstigneeva b and W a c l a w L. K r e t o v i c h b a A. V. Shubnikov Institute of Crystallography, U.S.S.R. Academy of Sciences, and h A.IV. Bach Institute of Biochemistry, U.S.S.R. Academy of Sciences, Moscow (U.S.S.R.)
(Received 17 November 1986) (Revised manuscript received 23 March 1987)
Key words: Glutamine synthetase structure; Electron microscopy; (Lupin root nodule); (Cytosol); (Bacteroid)
The quaternary structure of multiple forms of glutamine synthetase (EC 6.3.1.2) from bacteroids and eytosol of yellow lupin (Lupinus luteus L.) root nodules has been studied by means of electron microscopy and image processing. The multiple forms of glutamine synthetase were found to possess different quaternary structures, which are, apparently, responsible for their different properties aml, possibly, for different functions. It was shown that one of the two forms of cytosol g!utamine synthetase (ghmunine synthetuse II) which is predominant in nitrogen fixation conditions has a cubic configuration with eight identical nmmm~rs arranged in two layers with point group symmetry 422 (D4). Two of the three bacterotd ~ synthetase forms, gintamine synthetase H and gintamine synthetase m , possess different quaternary s ~ . Thus, bacteroid glutamine synthetase II was found to consist of eight identical monomers arranged with point group symmetry 422 (D4) at the vertices of two squares which, unlike cytosol gimmine synthetase II are twisted about the 4-fold axis relative to each other. The quaternary structure of bacteroid glutamine synthetase II is typical for glutamine synthetases of higher plants and may indicate its plant origin. As with bacterial glutamine synthetase, bacteroid glutamine synthetase IH consists of twelve ~ nmnmners arranged with point group synmtetry 622 (D6) at the vertices of two rectilinear hexagons. Computer averaging of bacteroid gh.qamine synthetase Ill images revealed a two-donmin ~ of its ~ . Introduction A key reaction of the assimilation of ammonia products during symbiotic nitrogen fixation in legume root nodules is known to be the synthesis of glutamine [1-3], which is catalyzed by gluta-
Correspondence: A.V. Pushldn, A.N. Bach Institute of Biochemistry, U.S.S.R. Academy of Sciences, Leninsky Pr., 33, Moscow 117071, U.S.S.R.
mine synthetase (L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2). Most of the ammonia is believed to be assimilated in nodule cytosol [1-11], while bacteroids play little or no part in the process. Until recently, only one form of glutamine synthetase was known to be present in the cytosol of legume nodules [2,8,9]. In recent years, however, two molecular forms of glutamine synthetase have been reported in the cytosol of kidney beans [3,5,7] and yellow lupin [6]. One of these can be associated with the fixation of molec-
016%4838/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
369 ular nitrogen, while the other is, in many respects, close to the glutamine synthetase found in roots. The two forms of glutamine synthetase of kidney bean root nodule cytosol have equal molecular masses of about 380 kDa and consist of monomers of molecular mass of 41 kDa [7]. The molecular mass of glutamine synthetase II of the cytosol which prevails in nitrogen fixation conditions in yellow lupin root nodule cytosol was found to be 410 kDa. The enzyme consists of identical monomers with a molecular mass of 51 kDa [6]. According to other data [2], there is a form of yellow lupin root nodule cytosol glutamine synthetase with molecular mass 347 kDa consisting of identical monomers (41.5 kDa). The molecular mass of soybean root nodule cytosol glutamine synthetase was estimated to be 376 kDa, and the enzyme was found to consist of eight identical monomers with a molecular mass of 47.3 kDa [8]. White lupin root nodule cytosol glutamine synthetase [9] was shown to consist of eight identical monomers with a molecular mass of 44.7 kDa having a total molecular mass of about 360 kDa. As for the quaternary structure of legume root nodule cytosol glutamine synthetase it is only the enzyme from soybean that has been studied by electron microscopy. It was revealed [8] that it consists of eight identical monomers arranged at the vertices of a cube. Earlier, such an arrangement of monomers was found for glutamine synthetases of animals [12,13]. Unfortunately, little is known about multiple forms of bacteroid glutamine synthetase. Moreover, most of the results now available correspond not to bacteroid glutamine synthetase, but to free-living cells of rhizobia. Thus, two forms of glutamine synthetase were found in free-living cells of several Rhizobium species [14,15]. One of them was shown to comprise twelve identical monomers and had a molecular mass of about 600 kDa. The other form had a substantially lower molecular mass (about 340 kDa). The arrangement of monomers in the molecule of a larger enzyme is similar to that of glutamine synthetases from bacteria and cyanobacteria [16-21]. The authors failed to determine the quaternary structure of a smaller glutamine synthetase by electron microscopy [14]. At a high concentration of ammonia in the medium, a decrease of the activity of the dode-
cameric form of glutamine synthetase of R.
japonicum cells was observed, induced by its adenylylation, with suppressed synthesis of the other form of glutamine synthetase [14]. This fact allowed the researchers to conclude that assimilation of ammonia formed during the fixation of molecular nitrogen in legume bacteroids takes place mainly in the nodule cytosol. At the same time, as shown by experiments with bacteroids isolated from root nodules of soybean [22] and yellow lupin [23,24], ammonia does not repress the glutamine synthetase activity, and in the latter case it even stimulated it. Therefore, the evidence obtained for free-living rhizobia can hardly be applied to bacteroids, and the assimilation of ammonia produced by nitrogen fixation is likely to proceed in bacteroids as well as in the nodule cytosol. As was mentioned [25], there are three forms of glutamine synthetase in R. lupini bacteroids; two of them can also be found in culture cells, while the third one was shown to occur only in bacteroids. In nitrogen-fixing conditions the form of glutamine synthetase with a molecular mass of about 600 kDa (bacteroid glutamine synthetase III) was adenylylated, which reduced its activity [25,26]. At the same time, there was a sharp increase in the content of the other form (bacteroid glutamine synthetase II) with a molecular mass of about 340 kDa which in free-living cells of R. lupini was repressed by ammonia [24]. It can be concluded from the data available that, apart from the nodule cytosol, the assimilation of ammonia formed during symbiotic nitrogen fixation in legume nodules proceeds in the bacterial fraction of the symbiotic association as well. Since the activity of bacteroid glutamine synthetases I and III in bacteroids turned out to be insignificant [24,25], it is apparently bacteroid glutamine synthetase II that plays a key part in the assimilation of ammonia in bacteroids. The quaternary structure of glutamine synthetases from bacteroids of legume root nodules has not been determined so far. So, the aim of the present work was to study the quaternary structure of glutamine synthetase II from the plant fraction and glutamine synthetases II and III from the bacterial fraction of yellow lupin root nodules by electron microscopy.
370
Materials and Methods
Purification of glutamine synthetase from yellow lupin (Lupinus luteus L.) root nodule cytosol followed the procedure which we have developed [6]. Glutamine synthetases II and III of Rhizobium lupini bacteroids were purified using our methods [24,26]. For preparations of specimens for electron microscopy a drop of protein solution in 5 mM Tris-HC1 buffer (pH 7.5) was applied to a thin carbon support grid. Then, the specimens were negatively stained with 2% phosphotungstic acid or 5% potassium silicotungstate (pH 7.0). Electron microscopy was carried out on a Philips EM 400 electron microscope at 80 kV with a magnification of 50 000. The electron micrographs were digitized with an Optronics P-1000 rotating drum microdensitometer with a sampling corresponding to 0.16 nm at specimen level. The digitized images were processed using a Nord-100 (Norsk Data) minicomputer. Images were displayed on a Tektronics graphic terminal as contour maps. The first step of processing included masking and low-pass filtering. A circular mask was used to select the part of the image containing the object, and the variance of the density was normalized. High-frequency noise was eliminated by low-pass filtering. The next step involved the alignment and calculation of the images for their averaging. The images to be averaged were aligned by a cross-correlation function with one particle selected as a reference image [27]. The quality of correlation was controlled by a correlation coefficient. Particles with a correlation coefficient higher than that of a pre-given threshold were taken for averaging. The averaged images were represented as contour maps or half-tone pictures. Rotational symmetry of particles after their centre refinement was determined using EM GROUP programs written by L. Amos [28] and adapted for an EC 1040 computer. Results and Discussion
Glutamine synthetase of yellow lupin root nodule cytosol Fig. la shows a general view of negatively stained molecules of yellow lupin root nodule
cytosol glutamine synthetase II. Several characteristic types of image of the enzyme molecule can be observed. The first type is a square projection with four protein spots whose sides are about 10 nm (Fig. lb). Two parallel protein layers separated by stain are seen in two other types (Fig. lc,d). They have square (Fig. lc) or rectangular (Fig. ld) profiles. Their lengths are about 10 nm and 13.5 nm, respectively, and their widths are the same (approx. 10 nm). The square images (Fig. lb) have rotational symmetry. The order of the rotational axis was determined by examining of the rotational power spectrum [28]. In all cases a spectrum showed the presence of 4-fold rotational symmetry. A number of such images of molecules was used for averaging and symmetrization. The selected images were aligned to a reference one by rotational and translational shift using a correlation function (see Materials and Methods). Since the averaged image showed a clearly defined 4-fold symmetry (Fig. le), it was 4-fold rotationally symmetrized. In the resulting picture four maxima of protein density are located at vertices of a square (Fig. lf,g). Analysing the projections observed (Fig.lb-d), it was inferred that the cytosol glutamine synthetase II molecule consists of eight monomers (in agreement with our preliminary findings [6]) arranged at vertices of two squares with point group symmetry 422 (D4). The molecule has a form close to a cube with sides of about 10 nm. When comparing the images of the molecule we can suggest that the first type (Fig. lb) corresponds to the frontal view projection (along the 4-fold axis), and the two other types (Fig. lc,d) are lateral views (perpendicular to this axis). The averaged image (Fig. lf, g) can then be interpreted as a projection of two eclipsed square tetramers. It should be noted that a cubic arrangement was found also for octameric glutamine synthetase of soybean root nodule cytosol [8], while the only glutamine synthetase from roots of higher plants with a known quaternary structure, the pumpkin root glutamine synthetase has a somewhat different configuration: the tetramers of one of its layers are twisted about the 4-fold axis relative to the other layers [29]. Such a quaternary structure was also observed for glutamine synthetase isoenzymes from leaves of higher plants [30-34]. A1-
371
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1 "
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e
4
8
I
i
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f
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Fig. 1. (a) Field of glutamine synthetase from yellow lupin root nodule cytosol negatively stained with phosphotungstic acid. (b-d) Selected molecules in three characteristic orientations. Computer averaging of the images. (e) Rotational power spectrum of the average of ten aligned front view images (se~ b). The 4-fold order is shown by an arrow. (f,g) 4-fold rotationally symmetrized average. Images are represented as a half-tone picture (f) and contour map (g).
372
though there are still no reliable data about the quaternary structure of glutamine synthetase in roots of legumes, and specifically of lupin, one may expect it not to be much different from that of glutamine synthetase of pumpkin roots [29]. The octameric glutamine synthetases with cubic arrangement were also found in animals [12,13,35-37] and Neurospora crassa [38].
Glutamine synthetase H of Rhizobium lupini bacteroids Different types of molecule images can also be observed in micrographs of bacteroid glutamine synthetase II (Fig. 2a-e), which depend on the respective orientation of the molecule when adsorbed onto the support film. The first type is represented by annular projections with a diameter of 9-10 nm. Their central space is often completely filled with stain (Fig. 2b). Rotational power spectrum of such images very often showed the presence of a rotational symmetry. To amplify the main details, these images were computer averaged. The result of averaging clearly shows the predominance of the 4-fold symmetry (Fig. 2f). Therefore, it was additionally 4-fold rotationally symmetrized. In the resulting picture (Fig. 2g,h) one can see a circular region with eight maxima of protein density grouped in pairs, and the internal cavity filled with stain. In the images of the three other types (Fig. 2c-e), two strips of protein separated by stain are seen. All these projections have the same width (about 7 nm), but differ in length and details of image. The images of the second (Fig. 2c) and the third (Fig. 2d) types are rectangular. They are characterized by a 2m symmetry and have a length of 9-10 and 8-9 nm, respectively. The projections of the fourth type (Fig. 2e) are trapezium shaped. Comparing all these images one can suppose that the first type is a front view (along the 4-fold axis) of molecules, while all the others are side views (perpendicular to the 4-fold axis). The projection of the first type is most frequently observed and apparently corresponds to the most stable position of the molecule on the support film. The above data can be summarized by the following three-dimensional model of bacteroid glutamine synthetase II molecule: eight elongated monomers are arranged with point group symme-
try 422 (D4) at vertices of two squares twisted about the 4-fold axis relative to each other at an angle of about 40 ° . Therefore, the projections shown in Fig. 2c,d correspond to the orientation of particles observed along the two 2-fold axes, lying at 45 ° . The projection of the fourth type (Fig. 2e) occurs when the molecule is observed along the bisector between these axes. Also, an angular shift of tetramers of two layers leads to protein density having four paired maxima in the first type of images and to the appearance of trapezium-shaped projections (the fourth type). In some images of the first type (Fig. 2b) one can see four dark spots of stain in the centre of the ring, separated by white regions, which most probably correspond to protein. The presence of protein inside the annular region is also confirmed by the fact that there is no visible 'central channel' in the lateral projections of the molecule. These observations indicate that the enzyme molecule has no annular form, but contains an internal cavity at the expense of hollows in monomers, as shown in the model (Fig. 2b). Therefore, the ring-shaped projections of the molecule (Fig. 2b) may be explained by penetration of the stain into the internal cavity. According to this model, the monomer volume is 30-35 nm3, corresponding to a molecular mass of 45 + 5 kDa. These data are in good agreement with parameters of the bacteroid glutamine synthetase II molecule determined by the gel filtration method [24]. Thus, the bacteroid glutamine synthetase II molecule consists of eight identical monomers arranged in accordance with point group symmetry 422 (I)4). Up until now, the octameric structure has been found only for glutamine synthetases of eucaryotic organisms which, as opposed to procaryotic ones (from bacteria and cyanobacteria), contain twelve identical monomers. The arrangement of monomers in bacteroid glutamine synthetase II molecule at vertices of two squares twisted about the 4-fold axis appears to be very similar to that observed for glutamine synthetases of higher plants [29,30-34]. The greatest similarity is observed with glutamine synthetase of pea seeds [34], which is immunochemically indistinguishable from a glutamine synthetase of lupin seeds [38]. Our results indicate that glutamine synthetase
373
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f
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Fig. 2. (a) Field of glutamine synthetase II from bacteroids of R. lupini negatively stained with phosphotungstic acid. (b-e) Images of the first, second, third and fourth types, respectively. Different appearances of the model are shown on the right, corresponding to the different projections of the molecule. Computer averaging of the images: (f) rotational power spectrum of the average of ten aligned front view images (see b). The 4-fold order is shown by an arrow. (g,h) 4-fold symmetrized average. Images are represented as a half-tone picture (g) and a contour map (h).
374
II of R. lupini bacteroids may have a plant origin and that the structural gene of the enzyme might, in one way or another, have been acquired by rhizobia from the genome of higher plants.
Glutamine synthetase I l l of Rhizobium lupini bacteroids Electron micrographs of bacteroid glutamine synthetase III also show individual molecules in different positions (Fig. 3a). The majority of the enzyme molecules are predominantly shown form the front and have a hexagonal profile with a diameter of 13-14 nm (Fig. 3b). The molecules are often seen in two side views, which reveal two protein layers either as four spots (Fig. 3c), or as two strips (Fig. 3d). These projections have the same width of 8 + 1 nm. Some of the molecules in the side-view projections form stacks with a 4-4.5 nm spacing between protein layers (Fig. 3a). Projections of the bacteroid glutamine synthetase III molecule are very similar to those of Escherichia coli glutamine synthetase, described by Valentine et al. [16] and also to projections of glutamine synthetase I (with a molecular mass of 600 kDa) from free-living cells of R. japonicum, described by Darrow [14]. It can be concluded from the images obtained that the enzyme is composed of twelve identical monomers arranged at vertices of two eclipsed hexagons with point group symmetry 622 (D6). The dimensions of the molecule are 13-14 nm in diameter and 8 + 1 nm high. The quaternary structure of bacteroid glutamine synthetase III molecule is similar to the quaternary structure of glutamine synthetases of several bacteria and cyanobacteria studied both by electron microscopy [14,16-21,26] and X-ray analysis [40-42]. All these molecules have D 6 symmetry and are of a similar size. The result of the three-dimensional reconstruction from images of 6 + 1 helical chains of glutamine synthetase molecules from E. coli [43] is consistent with this symmetry. Image analysis of micrographs of single glutamine synthetase particles, including images obtained with the low-dose technique has been performed by Frank et al. [44,45] and Kunath and coworkers [46]. Computer averaging of particles revealed a division of monomers into two domains.
We also obtained averaged hexagonal projection of the bacteroid glutamine synthetase III molecule. Thirty images were studied, ten of which (with the best correlation coefficients) were used for averaging. A power spectrum of the average of ten aligned particles displayed 6-fold symmetry (Fig. 3e). Half-tone and contour map representation of the 6-fold rotationally symmetrized average is shown in Fig. 3f,g. One can see six protein units containing one major outer and one minor inner density peak, separated by a lower density region where the stain can penetrate. The averaged image (Fig. 3f,g) has slight handedness which can be explained by differences in staining of one of two apposing hexamers. Recently, the X-ray structure of bacterial glutamine synthetase from Salmonella typhimurium at 3.5 A resolution has been reported [42]. The atomic model obtained shows the localization of the active sites at the subunit interfaces and suggests a mechanism for regulation of the rate of synthesis of glutamine in response to covalent modification and feedback inhibition. The protein is composed of 12 identical subunits arranged with 622 point group symmetry. The molecule has an outer diameter of 143 A and a height of 103 ~,. The projected structure of the single hexamer down the 6-fold axis of symmetry shows distinct handedness arising from the skew appearance of the subunits. The subunits have a region of low protein density which can be penetrated by stain, leading to two density peaks in subunit projection observed by electron microscopy. Thus, the structures of the glutamine synthetase III molecule from R. lupini bacteroids and other bacterial glutamine synthetases are very similar as seen with negative staining resolution, and appear to be compatible with the X-ray diffraction study [42]. In summary, it can be concluded that the multiple forms of glutamine synthetase of bacterial and plant fractions of the yellow lupin and R. lupini symbiotic association have appreciably different quaternary structures. Apparently, such distinctions are responsible for different properties and, perhaps, different functions of these glutamine synthetases.
375
2 1
e
i
i
2
4
6
0
10 12 I
g
~m
Fig. 3. (a) Field of glutamine synthetase llI from bacteroids of R. lupini negatively stained with potassium silicotungstate. (b) Selected molecules showing the front view, and two side views (c,d). Computer averaging of the images: (e) rotational power spectrum of the average of ten aligned front view images (see Fig. 3b). The 6-fold order is shown by an arrow. (f,g) 6-fold rotationally symmetrized average. Images are represented as a half-tone picture (f) and contour map (g). The two-domain structure of monomers is visible.
376
Acknowledgments The authors express their deep gratitude to Dr. L. Amos for providing the computation programs. References 1 Evstigneeva, Z.G. and Pushkin, A.V. (1983) in Molecular Mechanisms of Nitrogen Assimilation in Plants (Kretovich, W.L., ed.), pp. 198-234, Nauka, Moscow 2 Chen, J. and Kennedy, I.R. (1985) Phytochemistry 24, 2167-2172 3 Cullimore, J.V. and Miflin, B.J. (1984) J. Exp. Bot. 153, 581-587 4 Boland, M.J., Fordyce, A.M. and Greenwood, R.M. (1978) Aust. J. Plant Physiol. 5, 533-539 5 Lara, M., Cullimore, J.V., Lea, PJ., Miflin, B.J., Johnston, A.W.B. and Lamb, J.W. (1983) Pianta 157, 254-258 6 Sadikova, G.E., Solovieva, N.A., Pushkin, A.V., Tsuprun, V.L., Shubin, V.V., Evstigneeva, Z.G. and Kretovich, W.L. (1985) Biokhimiya 50, 936-940 7 Cullimore, J.V., Lara, M., Lea, PJ. and Miflin, B.J. (1983) Planta 157, 245-253 : " ~i 8 McParland, R.H,, Guevara J.G, Becker, R.R. and Eyans, H.J. (1976) Biochem. J. 153, 597-606 9 McCormack, D.K., Farnden, K.J.F. and Boland, M.J. (1982) Arch. Biochem. Biophys. 218, 561-571 10 O'Gara, F. and Shanmugam, K.T. (1976) Biochim. Biophys. Acta 437, 313-321 11 Howitt, S.M. and Gresshoff, P.M. (1985) J. Gen. Microbiol. 131, 1433-1440 12 Haschemeyer, R.H. (1968) Trans. N.Y. Acad. Sci. 30, 875-891 13 Tate, S.S. and Meister, A. (1973) in The Enzymes of Glutamine Metabolism (Prusiner, S. and Stadtman, E.R., eds.), pp. 76-128, Academic Press, New York 14 Darrow, R.A. (1980) in Glutamine: Metabolism, Enzymology, and Regulation (Mora, J. and Palacios, R., eds.), pp. 139-166, Academic Press, New York 15 Fuchs, R.L. and Keister, D.L. (1980) J. Bacteriol. 144, 641-648 16 Valentine, R.C., Shapiro, B.M. and Stadtman, E.R. (1968) Biochemistry 7, 2143-2152 17 Deuel, T.F., Ginsburg, A., Yeh, J., Shelton, E. and Stadtman, E.R. (1970) J. Biol. Chem. 245, 5195-5205 18 Alef, K., Burkardt, H.-J., Horstmann, H.-J. and Zumft, W.G. (1981) Z. Naturforsch. 36c, 246-254 19 Siedel, J. and Shelton, E. (1979) Arch. Biochem. Biophys. 192, 214-224 20 Sampaio, M.J.A.M., Rowell, P. and Stewart, W.D.P. (1979) J. Gen. Microbiol. 111, 181-191 21 Orr, J., Keefer, L.M., Keim, P., Nguen, T.D., Wellems, T., Heinrikson, R.L. and Haselkorn, R. (1981) J. Biol. Chem. 256, 13091-13098
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