Reassembly of the peptidyltransferase centre of larger subparticles of rabbit reticulocyte ribosomes from a core-particle and split-protein fraction.

We report the reconstruction, from a core-particle and split-protein fraction, of the larger subribosomal particle of rabbit reticulocytes. The reassembled particle was active in polyphenylalanine synthesis and in the puromycin reaction. The core-particles and split-protein fractions were obtained by treatment of the larger subparticle with salt solutions containing NH4+ and Mg2+ in the molar ratio 40:1 over the range 2.25-2.75 M-NH4Cl/56-69mM-MgCl2 at 0 degrees C. This treatment led to the loss of about eight proteins (approx. 17% of the protein moiety), which were found wholly or largely in the split-protein fraction as shown by two-dimensional gel electrophoresis. The core particle retained 5S rRNA and had much decreased (no more than 10% of control) ability to function in the puromycin reaction or in poly (U)-directed polyphenylalanine synthesis. Activity was recovered when the recombined core-particle and split-protein fractions were dialysed overnight at 4 degrees C against 0.3M-NH4Cl/15mM-MgCl2/1mM-dithiothreitol/15% (v/v) glycerol/20mM-Tris/HCl, pH 7.6, and then heated for 1 h at 37 degreesEES C. The recovery was 40-80% of the original activity. Raising the concentration of MgCL2 to 300 mM in 2.5 M-NH4CL led to the removal of seven rather than eight proteins, and the core particle remained active in the puromycin reaction. We infer that the protein retained by raising the concentration of Mg2+ is an essential component of the peptidyltransferase centre of the ribosome.     

Protein synthesis by hybrid ribosomes reconstructed from rabbit reticulocyte ribosomal core-particles and amphibian or fungal split-proteins.

It was shown that high-salt (2.75 M-NH4Cl/69mM-MgCl2) shock treatment at 0 degrees C of the larger subparticles (L-subparticles) of rabbit, Xenopus laevis and Neurospora crassa cytoplasmic ribosomes yielded split-protein fractions that were not only functionally equivalent but also interchangeable. Thus, although the remaining core-particles were inactive in both the puromycin reaction and in poly(U)-directed polyphenylalanine synthesis, activity was restored when they were combined with either homologous or heterologous split-protein fractions. This technique was used to prepare active hybrid L-subparticles, e.g. rabbit cores/N. crassa split-proteins, and also active hybrid ribosomes, e.g. rabbit smaller subparticle/X. laevis core-particle/rabbit split-proteins. Rabbit and X. laevis split-protein fractions labelled with 14C by reductive methylation with [14C]formaldehyde and sodium cyanoborohydride were both shown to bind to rabbit core-particles in approximate correlation with the degree of re-activation. The split-protein fractions of rabbit and X. laevis L-subparticles were analysed by two-dimensional and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The molecular weights (measured in sodium dodecyl sulphate gels) of the split-proteins of rabbit and X. laevis L-subparticles were found to be similar. These results demonstrate that the peptidyltransferase active centre of cytoplasmic ribosomes of eukaryotes has components that are interchangeable over a wide evolutionary range. Evidently the essential molecular architecture of the active centre is highly conserved.     

Resistance of the peptidyltransferase centre of rabbit ribosomes to attack by nucleases and proteinases.

Larger ribosomal subparticles (L-subparticles) of rabbit ribosomes were treated with either ribonucleases (I or T1) or proteinases (trypsin or chymotrypsin), and their capacity to function in poly(U)-directed polyphenylalanine synthesis and in the puromycin reaction was investigated. The effects of pretreatment of L-subparticles on the reconstruction of active subparticles from core-particles derived by treatment with 2.75 M-NH4Cl/69 mM-MgCl2 and split-protein fractions were also examined. The protein moiety of proteinase-treated L-subparticles was analysed by one-dimensional sodium dodecyl sulphate/polyacrylamide- and two-dimensional polyacrylamide-gel electrophoresis. The introduction of 16--100 scissions in the RNA moiety had no effect on the activity of the L-subparticles in polyphenylalanine synthesis, and there was no effect on the stability of L-subparticles to high-salt shock treatment and a marginal effect on the reconstruction of L-subparticles from high-salt-shock core-particles and split-protein fractions. In contrast, L-subparticles treated with low amounts of trypsin (0.56 ng of trypsin/microgram of L-subparticle) were inactive in polyphenylalanine synthesis, and their capacity to function in partial-reconstruction experiments was diminished. Activity in the puromycin reaction was increased by 70% as a result of trypsin treatment (280 ng of trypsin/microgram of L-subparticle). At least two of the acidic proteins implicated in the translocation function were not affected by trypsin treatment. Trypsin-treated L-subparticles had lost their capacity to bind the smaller ribosomal subparticle (S-subparticle). The protein(s) needed for S-subparticle binding were shown to be present in high-salt-shock cores. At least six proteins associated with the core-particles were attack during trypsin treatment of L-subparticles. An examination of L-subparticles isolated from trypsin-treated polyribosomes showed that the amount of trypsin necessary to decrease the activity of the subparticle by 50% was about twice that needed in the treatment of L-subparticles alone. The largest protein of rabbit L-subparticles (approx. 51 000 daltons) was cleaved in a stepwise fashion by trypsin to fragments of approx. 40 000 daltons. This protein was also cleaved by chymotrypsin.     

Re-activation of the peptidyltransferase centre of rabbit reticulocyte ribosomes after inactivation by exposure to low concentrations of magnesium ion.

1. The larger subrivosomal particles of rabbit reticulocytes retained full activity in the puromycin reaction and in poly(U)-directed polyphenylalanine synthesis after 4h at 0 degrees C when buffered 0.5M-NH4Cl/10-30mM-MgCl2 was the solvent. 2. Activity in the puromycin reaction was diminished to approx 10% after 15-30 min at 0 degrees C when the concentration of MgCl2 was lowered to 2mM. 3. Activity was not restored when the concentration of MgCl2 was raised from 2mM to 10-30 mM at 0 degrees C. However, activity was recovered as measured by both assay systems when the ribosome fraction was heated to 37 degrees C at the higher concentrations of MgCl2. 4. Recovery of activity was noted during the course of the polyphenylalanine synthesis in 50 mM-KCl/5mM-MgCl2/25mM-Tris/HCl, pH 7.6, at 37 degrees C. Re-activation was slow at 20 degrees C and below. 5. No more than about 5% of the protein moiety of the subparticle was lost in 0.5M-NH4Cl on decreasing MgCl2 concentration from 10mM to 2mM. No proteins were detected in the supernatant fractions by gel electrophoresis after ribosomes were separated by differential centrifugation. The supernatant fraction was not essential for the recovery of activity. However, at higher (e.g. 1M) concentrations of NH4Cl, proteins were split from the subparticle. 6. The loss and regain of activity found on lowering and restoring the concentration of MgCl2 at 0.5M-NH4Cl appears to arise from a conformational change that does not seem to be associated with a loss and regain of particular proteins. 7. A 2% decrease in E260 was noticed when the concentration of Mg2+ was restored, and the change in the spectrum indicated a net increase of approx. 100A-U base-pairs per subribosomal particle. 8. When the concentration of Mg2+ was restored, S20,W of the subparticle remained at 52+/- 1S until the sample was incubated at 37 degrees C when S20,W increased to 56 +/- 1S compared with the value of 58 +/- 1S for the subparticle as originally isolated.     

Location of the SH group of the alkali light chain on the myosin head as revealed by electron microscopy

The location of the single cysteinyl residue of the alkali light chain on the myosin head was determined by electron microscopy. The cysteinyl residue of isolated alkali light chain 2 was biotinylated and the light chain was exchanged with that of heavy meromyosin in 4.7 M-NH4Cl. Avidin was attached to the biotin in the heavy meromyosin and the complex was rotary shadowed and observed in the electron microscope. The distance from the head-rod junction to the centre of avidin was 8(+/- 3) nm (mean value +/- standard deviation: n = 105).     

Purification and properties of glutamate synthase and glutamate dehydrogenase from Bacillus megaterium.

Bacillus megaterium N.C.T.C. no. 10342 exhibits glutamate synthetase (EC 2.6.1.53) and glutamate dehydrogenase (EC 1.4.1.4) activities. Concentrations of glutamate synthase were high when the bacteria were grown on 3mM-NH4Cl and low when they were grown on 100mM-NH4Cl, whereas glutamate dehydrogenase concentrations were higher when the bacteria were grown on 100mM-NH4Cl than on 3mM-NH4Cl. Glutamate synthase and glutamate dehydrogenase were purified to homogeneity from B. megaterium grown in 10mM-glucose/10mM-NH4Cl. The purified enzymes had mol.wts. 840000 and 270000 for glutamate synthase and glutamate dehydrogenase respectively. The Km values for substrates with NADPH and coenzyme were (glutamate synthase activity shown first) 9 micron and 360 micron for 2-oxoglutarate, 7.1 micron and 8.7 micron for NADPH, and 0.2 mM for glutamine and 22 mM for NH4Cl, similar values to those of enzymes from Escherichia coli. Glutamate synthase contained NH3-dependent activity (different from authentic glutamate dehydrogenase), which was enhanced 4-fold during treatment at pH 4.6 NH3-dependent activity was generally about 2% of the glutamine-dependent activity. Amidination of glutamate synthase by the bi-functional cross-linking reagent dimethyl suberimidate inactivated glutamine-dependent glutamate synthase activity, but increased NH3-dependent activity. A cross-linked structure of mol.wt. approx 200000 was the main product formed.     

The partial purification of sodium-plus-potassium ion-dependent adenosine triphosphatase from the gills of Anguilla anguilla and its inhibition by orthovanadate.

1. (Na+ +K+)-dependent ATPase was partially purified from eel gills by a procedure in which the microsomal fraction of crude preparations of chloride cells was selectively extracted with sodium dodecyl sulphate. 2. The microsomal specific activity was increased 2-fold during optimal treatment with detergent. 3. The final preparation (56% pure) had a specific activity of 341 mumol of ATP hydrolysed/h per mg of protein and a turnover number of 3560 min-1. The number of ouabain-binding sties equalled the number of sites phosphorylated by ATP. 4. Both sodium orthovanadate and ouabain inhibited the purified preparation more than the microsomal fraction, vanadate being more effective on an equimolar basis than ouabain. 5. Inhibition by orthovanadate was not enhanced at 28 mM-as compared with 1mM-MgCl2 and was not reversed by beta-adrenergic agonists (cf. Josephson & Cantley (1977) Biochemistry 16, 4572--4578). 6. Of various other metallic oxyanions tested only niobate proved an effective inhibitor of the enzyme although this anion was less effective than orthovanadate. 7. Orthovanadate partially inhibited phosphorylation of the enzyme by ATP in the presence of 28 mM-MgCl2.     

Stoichiometry of GTP hydrolysis during peptide synthesis on the ribosome. I. Factor-independent GTPase and ATPase of ribosomal preparations

It has been found that preparations of Escherichia coli (MRE-600) ribosomes can display GTPase and ATPase activities independent of elongation factors EF-Tu and EF-G. The GTPase and ATPase are localized on ribosomal 50S subparticles, whereas 30S subparticles are free of the activities and do not stimulate them upon association with the 50S subparticles to form complete ribosomes. The GTPase and ATPase can be removed from the ribosomes and their 50S subparticles by treatment with 1 M NH4Cl or 50% ethanol in the cold. Ribosomal preparations freed from the factor-independent GTPase and ATPase retain their basic functional features. The data obtained do not permit to solve finally whether the factor-independent GTPase and ATPase revealed are components of ribosomes or represent a contamination rather firmly bound to the ribosomes. However, in any case this finding can contribute to an uncoupled hydrolysis of GTP and should be considered when studying the stoichiometry of triphosphate expenditure in the process of ribosomal protein synthesis.     

Dependence on Ca2+ of the activities of phosphatidylinositol 4,5-bisphosphate phosphodiesterase and inositol 1,4,5-trisphosphate phosphatase in smooth muscles of the porcine coronary artery.

The activities of phosphatidylinositol 4,5-bisphosphate (PIP2) phosphodiesterase (PDE) and inositol 1,4,5,-trisphosphate (IP3) phosphatase in the particulate and cytosol fractions prepared from porcine coronary artery smooth muscles were examined using 32P-labelled PIP2 and IP3 as substrates, respectively. The activity of PIP2 PDE, as assessed from the production of IP3, in the cytosol fraction was about 10-fold higher than that in the particulate fraction. In the absence of MgCl2, the activity of PIP2 PDE in both fractions showed no causal relation to the free Ca2+ concentration in the physiological range of 10(-7)-10(-5) M, but was enhanced remarkably by 10(-4) M free Ca2+. The addition of 1 mM-MgCl2 to the assay medium markedly inhibited the activity of PIP2 PDE in both fractions in the presence of free Ca2+ (10(-8)-10(-5) M). In the absence of MgCl2, 10(-5)M-acetylcholine (ACh) produced IP3, and this action was blocked by 3 X 10(-6) M-atropine. The ACh-induced activation of PIP2 PDE ceased in the presence of 1 mM-MgCl2; however, the reactivation occurring on the addition of 10 microM-guanosine 5'-[gamma-thio]triphosphate did not depend on the free Ca2+ concentrations (10(-7)-10(-5)M). The activities of IP3 phosphatase, determined from decrease in the amount of IP3 in the particulate and cytosol fractions, had much the same potency in both fractions. The activity of IP3 phosphatase in the cytosol fraction was enhanced by MgCl2 in a concentration-dependent manner, the maximal value occurring at 1 mM-MgCl2, and was also enhanced in the presence of physiological concentrations of free Ca2+ (10(-7)-10(-6) M). These findings suggest that the activation of PIP2 PDE which occurs with application of ACh in the presence of guanine nucleotides and 1 mM-MgCl2 is independent of the free Ca2+ concentration, and that the hydrolysis of IP3 by phosphatase increases, depending on the concentration of free Ca2+.     

pH effects on cystine transport in lysosome-rich leucocyte granular fractions.

Inhibitors of lysosomal acidification (4,4'-di-isothiocyanostilbene-2,2'-disulphonate, NN'-dicyclohexylcarbodi-imide, carbonyl cyanide m-chlorophenylhydrazone, NH4Cl and methylamine hydrochloride) did not alter cystine egress or countertransport in polymorphonuclear-leucocyte lysosome-rich granular fractions at pH 7.0. Together, 2 mM-MgCl2/MgATP and 90 mM-KCl stimulated cystine egress 2-fold, but this effect also was not influenced by inhibitors of ATP-dependent lysosomal acidification. MgCl2/MgATP stimulated cystine transport at pH 5.5, but the effect also occurred with MgCl2, MgSO4 or MnCl2 alone, was prevented by chelation, and was not seen with NaATP; therefore, it was considered a bivalent-cation, not an ATP, effect. Proton-pump-mediated acidification of lysosomes does not appear to be required for cystine transport in normal polymorphonuclear-leucocyte granular fractions, as reported for lymphoblast lysosomes.     

The acidic ribosomal phosphoprotein of eukaryotes and its relationship to ribosomal proteins L7 and L12 of Escherichia coli.

The acidic ribosomal phosphoprotein, Lgamma, of Krebs II ascites cells was further characterized and compared with proteins L7 and L12 of Escherichia coli. Ribosomal protein Lgamma was selectively removed from 60S sibosomal subunits by 50% ethanol and 1M-NH4Cl, and antibodies raised against protein Lgamma cross-reacted with E. coli protein L12 in immunodiffusion experiments. These and other, previously reported, data support the proposal that the uekaryotic counterpart of E. coli proteins L7 and L12 is phosphorylated.     

Factors controlling the activities of phosphatidate phosphohydrolase and phosphatidate cytidylyltransferase. The effects of chlorpromazine, demethylimipramine, cinchocaine, norfenfluramine, mepyramine and magnesium ions.

1. Microsomal membranes from rat liver were incubated with ATP, CoA, Mg2+, [14C]palmitate, F- and sn-glycerol 3-phosphate in order to label them with [14C]phosphatidate. These membranes were isolated and used in a second incubation in which [3H]CTP was present, and the simultaneous synthesis of [14C]diacylglycerol and [3H]CDP-diacylglycerol was measured. 2. The addition of phosphatidate phosphohydrolase, which had been partially purified from the particle-free supernatant, supplemented the activity of the endogenous phosphohydrolase, but it did not alter the rate of CDP-diacylglycerol formation. 3. Adding EDTA inhibited phosphatidate cytidylyl-transferase activity and stimulated the activity of the phosphohydrolases by removing excess of Mg2+. 4. Increasing the concentration of Mg2+, norfenfluramine or chlorpromazine in the assay system stimulated cytidylyltransferase activity, but decreased the activities of both phosphohydrolases. 5. The mechanism for the stimulation of cytidylyl=transferase activity by the cationic drugs and Mg2+ was investigated with emulsions of phosphatidate and the microsomal fraction of rat liver. 6. There was a threshold concentration of about 5mM-MgCl2 below which no cytidylyltransferase activity was detected in the presence or absence of norfenfluramine. Just above this threshold concentration norfenfluramine stimulated cytidylyltransferase activity, but this stimulation disappeared as the Mg2+ concentration was raised to its optimum of 20mM. Norfenfluramine therefore partially replaced the bivalent-cation requirement. 7. At 30 mM-MgCl2 amphiphilic cationic drugs inhibited cytidylyltransferase activity at relatively high concentrations in a non-competitive manner with respect to phosphatidate. 8. The implications of these results are discussed with respect to the regulation of the synthesis of the acidic phospholipids compared with the synthesis of phosphatidylcholine, phosphatidylethanolamine and triacylglycerol.     

ADP-ribosyltransferase in isolated nuclei from sea-urchin embryos.

The activity of ADP-ribosyltransferase in nuclei isolated from sea-urchin embryos was estimated by the incorporation of [adenosine-14C]NAD+ into the acid-insoluble fraction. Hydrolysis of this acid-insoluble product by snake venom phosphodiesterase yielded radioactive 5'-AMP and phosphoribosyl-AMP. The incorporation of [14C]-NAD+ was inhibited by 3-aminobenzamide and nicotinamide, potent inhibitors of ADP-ribosyltransferase. [14C]NAD+ incorporation into the acid-insoluble fraction results from the reaction of ADP-ribosyltransferase. The optimum pH for the enzyme in isolated nuclei was 7.5. The enzyme, in 50 mM-Tris/HCl buffer, pH 7.5, containing 0.5 mM-NAD+ and 0.5 mM-dithiothreitol, exhibited the highest activity at 18 degrees C in the presence of 14 mM-MgCl2. The apparent Km value for NAD+ was 25 microM. The activity of the enzyme was measured in nuclei isolated from the embryos at several stages during early development. The activity was maximum at the 16-32-cell stage and then decreased to a minimum at the mesenchyme blastula stage. Thereafter its activity slightly increased at the onset of gastrulation and decreased again at the prism stage.     

Association of ribosomal subunits. IV. Polyamines as active components of the association factor from Bacillus stearothermophilus.

The association of ribosomal subparticles induced by several associating agents has been studied under different conditions. The following observations were made: 1. Spermidine was able to produce the association of subunits, and the concentration and temperature curves of this reaction were similar to those obtained with association factor. The product formed in the latter case was more stable. 2. The association at low Mg2+ concentration was higher with association factor than with polyamines. 3. The temperature-dependent binding of spermidine to 30-S subunits formed an active complex, which was converted into the 30S-50S couples by the addition of 50-S subparticles at low temperature. A similar behaviour has been previously shown for the complete association factor and its low molecular weight fraction. 4. The same unstable form of 30S-50S couples has been obtained either with spermidine or with the low molecular weight component (AFII) of the association factor. In both cases the protein fraction AFI was able to complete the reaction by stabilizing the subunit couple. 5. After glutaraldehyde fixation the products of the reactions with spermidine or association factor behaved in a similar way when they were submitted to long sucrose-gradient centrifugations. 6. The analysis of association factor preparations has shown that they contain spermidine as well as spermine. The polyamine levels in association factor could account for part of the total associating activity.     

Extraction of proteins from Saccharomyces cerevisiae ribosomes under nondenaturing conditions

The differential sensitivity of ribosomal proteins to removal by salts has been studied. Proteins were extracted from the large and small subunits of cytoplasmic ribosomes from Saccharomyces cerevisiae by washing the individual subunits with a series of solutions containing increasing concentrations of NH4Cl (0.74-3.56 M) for a defined time (20 min) at 0 degrees C. The molar ratio of magnesium to ammonium ions of 1:40 was maintained to protect the ribosomal subparticles from complete disassembly. Proteins extracted under each salt condition were analyzed for composition by two-dimensional polyacrylamide gel electrophoresis. The relative quantity of each protein was determined. Most proteins were not removed from the ribosomal particle completely by any one condition, but were preferentially enriched in a single fraction. Whereas most proteins could be solubilized, several proteins remained predominantly or exclusively with the final core particle. The kinetics of protein release from both subunits at a single NH4Cl concentration (0.74 M) were also studied. Release of protein was time dependent, i.e., longer extraction generally removed more of the same proteins. However, prolonged treatment (240 min) of subunits, even at the same salt concentration, resulted in removal of additional species of proteins in varying amounts. Among the ribosomal RNA species, only the 5 S RNA species was released from the ribosomal particles upon treatment.     

Polyribosomes, isolated from rat liver in a low ionic strength medium, capable of autonomic translation in a cell-free system without the addition of cellular fluid

Polyribosomes isolated from the liver in the presence of 10 mM KCl and purified by centrifugation through 2 M sucrose were shown to incorporate [3H]leucine both into aminoacyl-tRNA and polypeptides in a cell-free system without cell sap. The incorporation of [3H]leucine showed a linear increase within 80-100 min and was then levelled off. The system was sensitive to cycloheximide, puromycin and ethionine and needed ATP, GTP and unlabeled amino acids. The quantitation of tRNA in polyribosomes (the fraction which did not sediment with the subparticles after polyribosome dissociation) revealed more than two tRNA molecules per 80S monosome. It is likely that this tRNA excess as well as the earlier established presence of aminoacyl-tRNA synthetases and elongation factors promote the autonomic translation of polyribosomes.     

Study of the internal structure of Escherichia coli ribosomes by neutron and x-ray scattering.

Neutron scattering curves of the small and large subparticles of Escherichia coli ribosomes are presented over a wide range of scattering angles and for several contrasts. It was verified that the native ribosome structure was not affected by ²H₂O in the buffer. The reliability of the neutron scattering curves, obtained in H₂O buffer, was established by X-ray scattering experiments on the same material.The non-homogeneous distribution of RNA and protein in the subparticles of E. coli ribosomes is confirmed, with RNA predominantly within the particle and protein predominantly on its periphery. The distances between the centres of gravity of the RNA and protein components do not exceed 25 Å and 30 Å, in the large and small subparticles, respectively.The volume occupied by the RNA within the large and small subparticles is determined. The ratio of the “dry” volume of the RNA to the occupied volume is found to be 0.56; it is the same in both subparticles. Such packing of RNA is characteristic of single helices of ribosomal RNA at their crystallization and of the helices in transfer RNA crystals. A conclusion is drawn that RNA in ribosomes is in a similar state.Experimental scattering curves for the small subparticle depend significantly on the contrast in the angular region in which the scattering is mainly determined by the particle shape. The scattering curve, as infinite contrast is approached, is similar to that calculated for the particle as observed by electron microscopy. Thus, the long-existing contradiction between electron microscopy data (an elonggated particle with an axial ratio 2:1) and X-ray data (an oblate particle with an axial ratio 1:3.5), concerning the overall shape of the 30 S subparticle, is settled in favour of electron microscopy. The experimental neutron scattering curve of RNA within the small subparticle is well-described by the V-like RNA model proposed recently by Vasiliev et al. (1978).Experimental data are given to support the hypothesis that the maxima in the X-ray scattering curves, in the region of scattering angles corresponding to Bragg distances of 90 to 20 Å, arise from the ribosomal RNA component alone. It is shown that the prominence of the peaks in this region of the scattering curve depends only on the scattering fraction of the RNA component. The scattering fraction can be changed both by using the “native contrast” (ribosomal particles containing different amounts of protein) and by varying the solvent composition. The maxima are most pronounced where the RNA scattering fraction is highest or in solvents where the protein density is matched by the solvent. The scattering vectors of the maxima in the X-ray and neutron scattering curves, however, remain unchanged. This allows us to propose the tight packing of RNA as a common principle for the structural arrangement of RNA in ribosomes.     

Isolation and study of the properties of Streptococcus pyogenes ribosomes

Ribosomes from Streptococcus pyogenes, group A, strain 29 were studied. A comparison of different methods of ribosomal isolations has shown that the homogenous ribosomal samples can be obtained by the method of differential ultracentrifugation using tris-HCl buffer. The ribosomes of S. pyogenes had the sedimentation coefficient of 70S and consisted of 65% of protein and 35% of nucleic acids; the ribosomes dissociated into subparticles with the sedimentation coefficients of 50S and 30S under a low magnesium concentration. Thus the S. pyogenes ribosomes do not differ from the ribosomes of procaryotes. It was shown that the ratios of 70S, 50S and 30S ribosomal subparticles in the cells depend on the growth phase of S. pyogenes. The cells of the middle and the late logarithmic phase contained 50S and 30S particles in a stoichiometric ratio. In the cells of the late stationary growth phase there was a deficiency of 30S ribosomal subparticles which does not result from a loss during the isolation procedure, as it was already observed in the initial 30S fraction.     

Characterization of L-Glutamate Binding Sites in Rat Spinal Cord Synaptic Membranes: Evidence for Multiple Chloride Ion-Dependent Sites

The effects of various ions on L-glutamate (L-Glu) binding sites (Na+-dependent, Cl(-)-dependent, and Cl(-)-independent) in synaptic plasma membranes (SPM) isolated from rat spinal cord and forebrain were examined. Cl(-)-dependent binding sites were over twofold higher in spinal cord (Bmax = 152 +/- 34 pmol/mg protein) as compared to forebrain SPM (Bmax = 64 +/- 12 pmol/mg protein). Na+-dependent binding, on the other hand, was nearly sixfold less in spinal cord (Bmax = 74 +/- 10 pmol/mg protein) compared to forebrain SPM (408 +/- 26 pmol/mg protein). Uptake of L-Glu (Na+-dependent) was also eightfold less in the P2 fraction from spinal cord relative to forebrain (Vmax of 2.89 and 22.3 pmol/mg protein/min, respectively). The effects of Na+, K+, NH4+, and Ca2+ on L-Glu binding sites were similar in both regions of the CNS. In addition, in spinal cord membranes, Br-, I-, and NO3- were equivalent to Cl- in their capacity to stimulate L-Glu binding, whereas F- and CO3- were less effective. Cl(-)-dependent L-Glu binding in spinal cord membranes consisted of two distinct sites. The predominant site (74% of the total) had characteristics similar to the Cl(-)-dependent binding site in forebrain membranes [i.e., Ki values of 5.7 +/- 1.4 microM and 119 +/- 38 nM for 2-amino-4-phosphonobutyric acid (AP4) and quisqualic acid, (QUIS), respectively]. The other Cl(-)-dependent site was unaffected by AP4 but was blocked by QUIS (Ki = 14.2 +/- 4.8 microM).