Stimulation of gastric mucosal protein synthesis by different molecular forms of gastrin.

Protein synthesis in gastric mucosa was studied by measuring the incorporation of labeled amino acids into protein by isolated gastric mucosal ribosomes in a cell-free system. In 48-hour fasted rats, administration of the synthetic analogues pentagastrin, tetragastrin and gastrin-17 or naturally occurring molecular forms of human gastrin (G-14, G-34) markedly enhanced (23-123%) the capacity of the gastric mucosal ribosomes to synthesize endogenous mRNA-directed protein in a cell-free system. In the presence of exogenous mRNA (poly-U), the gastric mucosal ribosomes from the saline-treated controls showed a higher poly(U)-directed protein synthesis, compared to each fo the gastrin-treated groups. The protein/polyphenylalanine ratio which represents a ratio of polysomes to monosomes was found increased in ribosomes from the gastrin-treated groups.     

Methylglyoxal bis(guanylhydrazone) elimination of polyamine effects on protein synthesis

The effect of methylglyoxal bis(guanylhydrazone) (MGBG), a structural analog of polyamines, on protein synthesis has been studied in the presence and absence of spermidine. The spermidine stimulation of polyphenylalanine- and MS2 RNA-directed RNA replicase synthesis in an Escherichia coli cell-free system and of globin synthesis in a rabbit reticulocyte cell-free system disappeared with the addition of MGBG. The spermidine reduction of misincorporation of leucine during polyphenylalanine synthesis in both E. coli and wheat germ cell-free systems was also disturbed by MGBG. MGBG noncompetitively interfered with polyamine stimulation of polyphenylalanine and globin synthesis, suggesting that MGBG could bind to both RNA and the complex of RNA and polyamine. MGBG was preferentially bound to ribosomal RNA among ribosomal RNA, poly(U), and calf thymus DNA, and strongly inhibited the amount of polyamine bound to ribosomal RNA. These results suggest that MGBG elimination of polyamine effects on protein synthesis may occur through the disturbance of polyamine binding to ribosomal RNA.     

Involvement of 30S ribosomal protein S1 in poly(U)-directed polyphenylalanine synthesis.

The effect of 30S ribosomal protein S1 on poly(U)-directed polyphenylalanine synthesis was studied using a highly purified cell-free system which was devoid of endogenous S1. The system consisted of homogeneous preparations of EF-Tu, EF-Ts, and EF-G, and 70S ribosomes from which protein S1 had been removed by poly(U)-cellulose column chromatography. It was found that protein S1 was indispensable for translation of poly(U) by an S1-depleted system at low concentrations of poly(U). On the other hand, at higher concentrations of poly(U), a considerable amount of polyphenylalanine was synthesized in the absence of added S1. The stimulatory effect of S1 was observed at all Mg2+ concentrations examined but was most pronounced at 10 mM Mg2+. Some physicochemical properties of the protein were also studied. It was demonstrated that the protein has an elongated shape with an axial ratio of approximately 8.5.     

Effects of retinoic acid on protein synthesis in cultured melanoma cells

Retinoic acid reduces the growth rate of mouse S91 melanoma cells in culture and increases the proportion of cells in the G₁ phase of the cell cycle. Because of the integral role protein synthesis has been shown to play in growth control we studied the effect of retinoic acid on the protein synthesis machinery with a cell-free system developed from the melanoma cells. This system was capable of translating endogenous mRNA, exogenous globin mRNA, and the synthetic template poly(U). Of the above activities of the protein synthesis system only the translation of endogenous mRNA was reduced significantly in the cell-free system prepared from retinoic acid-treated cells. Analyses of the amount and function of RNA revealed that treatment with retinoic acid leads to reductions in total RNA content, in the proportion of ribosomes in polysomes, in the amount of poly(A)RNA, and in the amount of polysome-associated mRNA. All these effects of retinoic acid contribute to the decrease in protein synthesis activity of treated cells. Two-dimensional electrophoresis anlaysis of L-[³⁵S]methionine-labeled proteins produced by untreated and treated cells revealed only a few quantitative differences. We suggest that retinoic acid-induced suppression of protein synthesis activity may be the cause for growth inhibition.     

Calcium stimulation of polyphenylalanine synthesis in a bovine heart cell-free system

A bovine myocardial cell-free system, active in polyphenylalanine synthesis, has been studied. When Ca²⁺ was present under suboptimal Mg²⁺ concentrations (2 and 5 mM) a marked stimulation of the poly(U) directed macromolecular synthesis was obtained. Calcium did not stimulate the aminoacylation of bovine heart tRNA^Phe The evidence suggests that calcium is required, in conjunction with other cations, for an efficient translation of synthetic polynucleotides.     

Inhibition of protein synthesis in mouse myeloma cells by Ricinus communis toxin.

The mechanism of inhibition of protein synthesis in mouse myeloma cells by Ricinus communis toxin was studied. No significant disaggregation of polysomes into monosomes was detected in the toxin-treated cells. The activity of the polysomes isolated from the cells treated with the toxin in protein synthesis was remarkably lower than that of the untreated cells, while the activity of the supernatant enzyme fraction was retained. The ribosomes derived from the polysomes of the toxin-treated cells were inactive in poly(U)-dependent polyphenylalanine synthesis. The activity of ribosomes reconstituted by hybridizing subunits derived from the ribosomes of normal and toxin-treated cells were measured in poly(U)-dependent polyphenylalanine synthesis, and the 60 S subunit was revealed to be inactive. These results indicate that the target of action of the toxin towards intact cells is the 60 S ribosomal subunit.     

Inhibition of polypeptide synthesis initiation by a change of Mg++ concentration in wheat germ cell-free systems

Polypeptide synthesis programmed by poly(U) and globin mRNA has been studied in cell-free extracts from wheat germ. A two-step reaction with a preincubation at high Mg⁺⁺ levels followed by a second step carried out after a shift to a low Mg⁺⁺ concentration and the addition of labeled amino acids is described. Under these conditions the initiation of polyphenylalanine synthesis can be blocked without affecting the elongation of polypeptide chains. This procedure allows the selective inhibition of polypeptide synthesis initiation without using any drug or antibiotic.     

Inhibition of Cell-Free Protein Synthesis by Hydrostatic Pressure

Pressure inhibition of cell-free polypeptide synthesis is manifested in the same manner as that observed in the intact cell: (i) starting at approximately 200 atm, there is a progressive inhibition with increasing pressures; (ii) there is complete inhibition at 680 atm; (iii) incorporation into polypeptide is instantaneously reversible after pressure release and proceeds at a rate parallel to an atmospheric control; and (iv) the volume change of activation (DeltaV*) is 100 cm(3)/mole. Peptide bond formation per se can occur at a pressure level which is totally inhibitory to polypeptide synthesis. The one investigated step in translation that is inhibited in an identical manner is the binding of aminoacyl-transfer ribonucleic acid (AA-tRNA) to the ribosome-messenger RNA (mRNA) complex. The volume change of activation (DeltaV*) calculated for the binding reaction is also 100 cm(3)/mole. Thus, the inability of AA-tRNA to bind to ribosomes and mRNA under pressure, possibly in conjunction with translocation, appears to be responsible for the observed inhibition of the translational mechanism.     

Inhibition of protein synthesis by ricin: experiments with rat liver mitochondria and nuclei and with ribosomes from Escherichia coli (Short Communication)

1. Ricin, a toxic protein from the seeds of Ricinus communis which inhibits poly(U)-directed polyphenylalanine synthesis by rat liver ribosomes (Montanaro et al., 1973), does not affect protein synthesis by isolated rat liver mitochondria. 2. The toxin is ineffective also on poly(U)-directed polyphenylalanine synthesis in reconstituted systems with ribosomes isolated from rat liver mitochondria or from Escherichia coli. 3. Ricin inhibits protein synthesis by isolated rat liver nuclei, but at concentrations much higher than those affecting rat liver ribosomes.     

The effect of high hydrostatic pressure on eukaryotic protein synthesis

The pressure response of two eukaryotic protein synthesizing systems has been characterized. The rabbit reticulocyte system has been tested, both in vivo and in vitro, using endogenous polysomes and polyuridylic acid (poly U). In addition, the poly U-directed polyphenylalanine synthesizing system obtained from wheat germ was utilized. The effect of pressure on eukaryotic protein synthesis has been found to be basically similar to that observed in prokaryotic systems, although the response of the eukaryotic protein synthesizing system is somewhat more complex signifying a greater influence of overlapping reactions. Magnesium was found to affect eukaryotic systems in much the same way as has been reported for prokaryotic systems, i.e., increasing the Mg²⁺ concentration in a protein synthesizing system increases the barotolerance exhibited by that system. Under conditions of high Mg²⁺ concentration, however, extreme (up to 160%) stimulation of protein synthesis at lower pressure levels was observed in the eukaryotic systems. Such high stimulation is not apparent in prokaryotic systems. The poly U-directed wheat germ system exhibited the most barotolerant polypeptide synthesis ever seen in our laboratory. This extreme barotolerance was only slightly decreased when the system was tested at reduced concentrations of magnesium.     

Effect of norspermidine and its related triamines on the cell-free polyphenylalanine synthesizing system from Vibrio parahaemolyticus

The effect of norspermidine and its structurally related triamines on the cell-free polyphenylalanine synthesizing system from Vibrio parahaemolyticus was examined in connection with the requirement of the system for monovalent cation. In the absence of norspermidine, the maximal incorporation of [14C]phenylalanine into hot trichloroacetic acid insoluble material was observed under ionic conditions of 12 mM Mg2+ and 50 mM NH4+. K+ could partially substitute for NH4+, but Na+ could not. The addition of norspermidine to the polyphenylalanine synthetic reaction mixture not only lowered the optimal Mg2+ concentration, but it also stimulated the polyphenylalanine synthesis up to 2-fold with no significant increase in misincorporation of [14C]leucine. Other triamines having one or two methylene chains more than norspermidine were also effective in eliciting these effects. Furthermore, Na+ could not support the polyphenylalanine synthesis even in the presence of norspermidine and, on the contrary, inhibited the polyphenylalanine synthesis induced by NH4+ regardless of whether norspermidine was present or not. These findings are discussed in comparison with the properties of other bacterial cell-free systems.     

Initiation factors in protein synthesis by free and membrane-bound polyribosomes of liver and hepatoma.

The activity of initiation factors obtained from free and membrane-bound polyribosomes of liver and of transplantable H5123 hepatoma of rats was investigated by using an assay of protein synthesis in vitro in which poly (U)-directed polyphenylalanine synthesis was measured. Initiation factors of membrane-bound polyribosomes prepared by using the anionic detergent deoxycholate exhibited less activity in incorporating [14C]phenylalanyltRNA into polypetides than did initiation factors of free polyribosomes. However, when membrane-bound polyribosomes were prepared after using the non-ionic detergent Triton X-100, no significant differences in activities in polyphenylalanine synthesis were observed between the initiation factors of free and membrane-bound polyribosomes. These results suggest that Triton X-100 is preferable to deoxycholate in the isolation of of initiation factors from polyribosomes. Initiation factors, prepared by using Triton X-100, of free polyribosomes of hepatoma exhibited greater activity in the stimulation of polyphenylalanine synthesis than did the initiation factors of free or membrane-bound polyribosomes of host livers or of membrane-bound polyribosomes of hepatomas.     

Comparison of cell-free protein synthesis by different regions of chicken brain

The cell-free protein synthesis by the postmitochondrial supernatant from chicken cerebrum was twofold greater than protein synthesis by the cerebellum or optic lobes. Ribosomal aggregation of mRNA and ribonuclease activity of the postmitochondrial supernatant from the three brain regions was not statistically different. The higher protein synthetic activity of the cerebral postmitochondrial supernatant was associated with both the postribosomal supernatant (cell sap) and microsomal fractions. Cerebral monomeric ribosomes were more active in polyuridylic acid directed polyphenylalanine synthesis than monomeric ribosomes from either the cerebellum or optic lobes. The ability of cerebral cell sap to support polyuridylic acid directed polyphenylalanine synthesis was 1.6 to 2 times greater than cell sap from the other two regions. Cell sap factors other than tRNA^phe or phenylalanyl-tRNA synthetases appear to be responsible for the higher protein synthetic activity of the cbr cell sap.     

Effects of hyperoxia on composition and rate of synthesis of fatty acids in Escherichia coli

Growth of and fatty acid synthesis in Escherichia coli were inhibited by oxygen at partial pressures above 1 atm and were prevented by exposure to oxygen at 4.2 atm on membranes incubated on a minimal medium. Growth and fatty acid synthesis returned to control rates when cells were removed from hyperoxia to air. The spectrum of fatty acids produced was unchanged by oxygen at pressures which reduced the rate of synthesis. In situ fatty acids were stable to oxygen at pressures which prevented growth and synthesis. Reinitiation of synthesis after complete inhibition in hyperoxia occurred without production of aberrant fatty acids. Fatty acid synthetase specific activity was virtually unchanged, compared with air controls, in cells exposed either to 3.2 or to 15.2 atm of oxygen. The spectrum of fatty acids synthesized by cell-free extracts during incubation in 4.2 atm of oxygen was not different from air-incubated controls. Synthetase assays included added NADPH, acyl carrier protein, mercaptoethanol, and malonyl coenzyme A; hence, damage, other than reversible sulfhydryl oxidation, to the apoenzymes of synthetase was ruled out.     

CHARACTERISTICS AND PRODUCTS OF A CELL-FREE POLYPEPTIDE SYNTHESIZING SYSTEM FROM NEONATAL AND ADULT MOUSE BRAIN

—The regulation of protein synthesis by ribosomes isolated from mouse brain tissue was studied using a cell-free polyphenylalanine synthesizing system. Polypeptide synthesis was followed by assaying translocation and analysing the reaction products by BD-cellulose chromatography. The brain ribosomal activity could be divided by these methods into two distinct steps : binding of aminoacyl-tRNA to the ribosome and active translocation leading to subsequent polyphenylalanine synthesis. In comparison to initial binding of aminoacyl-tRNA, translocation in the cell-free system increased the incorporation of labelled phenylalanine by 10-fold. An analysis of the reaction products clearly showed active ribosomal synthesis of oligophenylalanine from [³H]phe-tRNA. Ribosomes isolated from neonatal brain tissue were 2–4 times as active as those obtained from adult brain tissue in polypeptide synthesis. In addition, polypeptides synthesized on the more active ribosomes from neonates tended to be of greater chain length than those from adult. Therefore, the maturation-dependent decrease in ribosomal protein synthetic activity during neural development was shown to be directly associated with the ribosome particles.     

Isolation of ribosomal subunits containing intact rRNA from human placenta: estimation of functional activity of 80S ribosomes.

We have elaborated a method for the isolation of ribosomal subunits from fresh unfrozen human placenta containing intact rRNA and a complete set of ribosomal proteins. Activity of 80S ribosomes obtained by reassociation of 40S and 60S subunits in nonenzymatic poly(U)-dependent binding of Phe-tRNA(Phe) was equal to 80% (above 1.5 mol [14C]Phe-tRNA(Phe) is coupled to 1 mol of ribosomes). The activity of 80S ribosomes in poly(U)-directed synthesis of polyphenylalanine was tested in a polysome-free protein-synthesizing system from rabbit reticulocytes. About 100 mol of phenylalanine residue was polymerized by a mole of ribosomes at a rate of 0.83 residues per minute in this system (2 h, 37 degrees C).     

Subunit interactions of rabbit muscle aldolase. Conformational change of aldolase in magnesium chloride and its relationship to the dissociation of subunits.

The effect of Escherichia coli ribosomal protein S1 on translation has been studied in S1-depleted systems programmed with poly(U), poly(A) and MS2 RNA³. The translation of the phage RNA depends strictly on the presence of S1. Optimum poly(U)-directed polyphenylalanine synthesis and poly(A)-programmed polylysine synthesis also require S1. Excess S1 relative to ribosomes and messenger RNA results in inhibition of translation of MS2 RNA and poly(U), but not of poly (A). In the case of phage RNA translation, this inhibition can be counteracted by increasing the amount of messenger RNA. Three other 30 S ribosomal proteins (S3, S14 and S21) are also shown to inhibit MS2 RNA translation. The effects of S1 on poly(U) translation were studied in detail and shown to be very complex. The concentration of Mg²⁺ in the assay mixtures and the ratio of S1 relative to ribosomes and poly(U) are crucial factors determining the response of this translational system towards the addition of S1. The results of this study are discussed in relation to recent developments concerning the function of this protein.     

The regulation of protein synthesis in animal cells by serum factors.

We have investigated the regulation of protein synthesis in animal cells by serum factors. Withdrawal of serum from the medium of actively dividing Vero cells resulted in an immediate decline in the rate of peptide chain elongation (Hassell and Engelhardt, 1973). Assay of elongation factor I (EFI) activity in the post-ribosomal supernatant as well as that associated with the ribosomes revealed that serum deprivation resulted also in reduction in the activity of this factor. The decline in the activity of EFI after serum deprivation occurred to the same extent and at the same time as the decline in the in vivo rate of protein synthesis and the in vitro peptide synthetic capacity of cell-free extracts. A temporal correlation therefore exists among the in vivo rate of protein synthesis, the peptide synthetic activity of cell-free extracts, and the activity of EFI. The activity of peptidyl transferase was not altered by serum deprivation. The loss of extract peptide synthetic activity resulting from serum deprivation was reversible since serum addition to previously serum-starved cultures resulted in full restoration of activity for polyphenylalanine (polyPhe) synthesis within 3 h. Moreover, RNA synthesis was not required for this turn-on of polyPhe synthesis. Vased on these data we conclude that a translational control mechanism is operative in Vero cells deprived of serum.     

Ribosomes of the extremely thermophilic eubacterium Thermotoga maritima are uniquely insensitive to the miscoding-inducing action of aminoglycoside antibiotics.

Poly(U)- and poly(UG)-programmed cell-free systems were developed from the extreme thermophilic, anaerobic eubacterium Thermotoga maritima, and their susceptibility to aminoglycoside and other antibiotics was assayed at a temperature (75 degrees C) close to the physiological optimum (80 degrees C) for cell growth and in vitro polypeptide synthesis, using a Bacillus stearothermophilus system as the reference. The synthetic capacity of the Thermotoga assay mixture was abolished by the eubacterium-targeted drugs chloramphenicol, thiostrepton, and kirromycin. However, streptomycin, the disubstituted 2-deoxystreptamines (kanamycin, gentamicin, neomycin, and paromomycin), and the monosubstituted 2-deoxystreptamine (hygromycin) all failed to promote translational misreading of poly(U) on Thermotoga ribosomes; they also failed to block polyphenylalanine synthesis at a low (less than 10(-4) M) concentration and did not inhibit Thermotoga cell growth at a high (10 micrograms/ml) concentration even though Thermotoga ribosomes possess the 16S rRNA sequences required for aminoglycoside action. In contrast to the other eubacteria, Thermotoga elongation factor G was also refractory to the steroid inhibitor of peptidyl-tRNA translocation fusidic acid.