Magnesium replacement by polyamines in higher plant cell-free polyphenylalanine synthesis

The characteristics and specific requirements for the formation of polyphenylalanine from Phe-[¹⁴C] in a barley ribosome cell-free incorporation system were detailed. The polyamines spermine, spermidine and putrescine, and the inorganic cations Ca²⁺, Ba²⁺ and Mn²⁺ demonstrated different capabilities for replacing the Mg²⁺ requirement in the incorporation system. Spermine was extremely efficient in this respect, followed by spermidine; all of the cations tested showed discrete concentration ranges of effectiveness. The data supported the suggestion that, at least to a certain extent, the cation requirement for protein syntheis may be non-specific.     

On the cell-free system of protein synthesis from mammalian mitochondria

Summary Some properties of a submitochondrial cell-free system for protein synthesis are described. The system was prepared from rat liver mitochondria lysed with Triton X-100, and the lysate was characterized by a linear rate of [¹⁴C]amino acid incorporation for 15–20 min with subsequent decline in activity. The incorporation reaction was inhibited by chloramphenicol and was in-sensitive to cycloheximide. Poly(U) addition stimulated [¹⁴C]phenylalanine incorporation by the preincubated submitochondrial system. Upon the addition of 7.5S mRNA that was iso-lated from mitochondria the major translation product was identified as a hydrophobic poly-peptide which in some properties (solubility in chloroform-methanol mixture) was similar to one of polypeptides synthesized by the sub-mitochondrial system on endogeneous mRNAs.     

Effect of polyamines on prostaglandin synthesis in various cell-free systems

Synthesis of PGF_2α by bovine uterus and guinea pig lung microsomes and that of TXB₂ by human platelet and rat spleen microsomes were stimulated by spermine. PGE₂ synthesis by bovine seminal vesicle and porcine lung microsomes, and 6-keto-PGF_1α synthesis by bovine seminal vesicle and uterus microsomes were inhibited by spermine. When phospholipid-free prostaglandin synthetase from bovine seminal vesicle was used instead of microsomes, the inhibition of PGE₂ synthesis by spermine disappeared. The inhibition of PGE₂ synthesis by spermine gradually appeared with an increase of phospholipid added. Among phospholipids tested, phosphatidylcholine was the most effective for the inhibition of PGE₂ synthesis by spermine.     

Concentration-dependent effects of natural polyamines on peptide chain initiation and elongation in a cell-free system of protein synthesis

Spermidine and spermine at submillimolar concentrations stimulate the rate of incorporation of amino acid into protein in a cell-free system, directed either by endogenous or exogenous mRNA (TMV, globin). The stimulatory effects of these polyamines are exerted at both the stages of initiation and elogation and are more pronounced in the case of TMV or globin mRNA, amounting to approximately 2.3-fold stimulation over the polyamine-free system. The number of polysomes and the polysome-associated radioactivity increase approximately 2-fold in the presence of spermine. Synthesis of large polypeptides is a characteristic feature of the stimulatory event. However, elevated concentrations of spermidine and spermine strongly inhibit amino acid incorporation into protein. Inhibition is manifest at the stage of peptide elongation. In the case of endogenous mRNA the addition of an excess of polyamines results in a non uniform inhibition of amino acid incorporation. A most interesting finding is that, with increasing concentrations of polyamines, the intensity of four bands with Mr values of 63000, 44000, 15500 and 12500 respectively, increases or leastwise remains constant while others fade, indicading differential translation of proteins in the presence of polyamines.     

Rapid production of milligram quantities of proteins in a batch cell-free protein synthesis system

We developed a cell-free protein synthesis system that produces more than 1mg/ml of recombinant proteins in two hours. A basal system that supports the stable maintenance of ATP and amino acids was constructed by using high concentrations of CP (100 mM) and amino acids (3 mM). Approximately 0.6 mg/ml of protein was produced during the batch incubation of the basal system. We found that the accumulation of inorganic phosphate reduces the concentration of free magnesium ions and that there exists a critical concentration of magnesium at which the protein synthesis is halted. Based on this finding, we attempted to extend the duration of the protein synthesis by keeping the magnesium concentration sufficiently high throughout the reaction period. The protein synthesis reaction continued for at least 2 h when the reaction was repeatedly supplemented with magnesium, and approximately 1.2 mg/ml of active CAT or GFP was produced. The simple, fast, and highly productive cell-free protein synthesis system described herein should offer a versatile platform for the preparation of protein molecules in various post-genomic efforts.     

Synaptosomal protein synthesis in a cell-free system

—Synaptosomes were isolated from cerebral cortex of young rats and incubated with ¹⁴C-labelled l -leucine in vitro. Amino acid incorporation into proteins of the synaptosomal cytoplasm, mitochondria and membrane components was observed. There was no incorporation into proteins of the vesicles. The protein-synthesizing system was not stimulated by the addition of either ATP or an ATP-generating system. ATP at all concentrations was inhibitory. Two different protein-synthesizing systems operate in the synaptosome. One, sensitive to inhibition by chloramphenicol and related antibiotics, is found in the mitochondrial subfraction and the other, inhibited by cycloheximide, is located either in the membrane components or the synaptosomal cytoplasm. This second system resembles the eukaryotic ribosomal system in its sensitivity to cycloheximide. Both the synaptosomal soluble fraction and the synaptosomal membrane fractions were shown previously to contain RNA. This RNA could function in protein-synthesizing mechanisms in the synaptosome. These results deomonstrate that protein is synthesized in axonal components and show that it is unnecessary to postulate that all axonal protein is supplied by somato-axonal flow.     

Autogenous regulation of histone mRNA decay by histone proteins in a cell-free system.

We tested the hypothesis that histone mRNA turnover is accelerated in the presence of free histone proteins. In an in vitro mRNA decay system, histone mRNA was degraded four- to sixfold faster in reaction mixtures containing core histones and a cytoplasmic S130 fraction than in reaction mixtures lacking these components. The decay rate did not change significantly when histones or S130 was added separately, suggesting either that the histones were modified and thereby activated by S130 or that additional factors besides histones were required. RecA, SSB (single-stranded binding), and histone proteins all formed complexes with histone mRNA, but only histones induced accelerated histone mRNA turnover. Therefore, the effect was not the result of random RNA-protein interactions. Moreover, histone proteins did not induce increased degradation of gamma globin mRNA, c-myc mRNA, or total poly(A)- or poly(A)+ polysomal mRNAs. This autoregulatory mechanism is consistent with the observed accumulation of cytoplasmic histone proteins in cells after DNA synthesis stops, and it can account, in part, for the rapid disappearance of histone mRNA at the end of S phase.     

Characterization of cell-free synthesis of collagen by lung polysomes in a heterologous system.

In normal lung growth, post-pneumonectomy lung growth, and in possibly several lung disorders, there are marked alterations in the density of collagen and changes in the rate of synthesis of collagen relative to the synthesis of other lung proteins. To provide a technology to begin to understand these changes at the molecular level, polysomes were prepared from rabbit lung and translated in a heterologous cell-free system including rabbit reticulocyte 0.5 M KCl ribosomal wash fraction and liver tRNA. Collagen was shown in the cell-free product by collagenase sensitivity, hydroxylation of incorporated proline by peptidyl prolyl hydroxylase, agarose gel chromatography, and sodium dodecyl sulfate acrylamide gel electrophoresis. The cell-free system was optimized with respect to K+, Mg2+, amino acids, and ribosomal wash fraction and used under conditions where total protein synthesis and collagen synthesis are linear with respect to time and amount of polysomes. Under these conditions, collagen synthesis was directed almost entirely by polysomes derived from the endoplasmic reticulum. Polysomes isolated from late fetal lung directed collagen synthesis at twice the rate (per polysome) as those polysomes isolated from adult lung. Similar changes were seen if lung tRNA replaced liver tRNA and if lung ribosomal wash fraction replaced reticulocyte wash fraction. Although these changes in cell-free lung collagen synthesis with tissue explants, further studies will have to be carried out to determine whether, in fact, age-related alterations in control of lung collagen synthesis are truly explained by these findings.     

Effect of polyamines on yeast cell-free protein synthesizing system. II. Increase stability of cell-free system in the presence of spermine.

The addition of spermine, at concentration which stimulates protein synthesis, to the yeast cell-free system significantly increases the thermal stability of the latter. Similar stabilizing effect of polyamine is observed for ribosome-poly U-ac-phe-tRNA complexes. These results suggest that the stimulatory effect of polyamines on the in vitro protein synthesis might be partly due to the increased stability of ribosomes and ribosome-peptydyl-tRNA complexes.     

Stimulation of collagen synthesis in a cell-free system by mRNA from chick embryos.

Embryonic chick RNA was translated in a cell-free system derived from wheat germ. One of the products synthesized in vitro under the direction of this RNA could be identified as collagen on the basis of collagenase digestion experiments and sodium dodecylsulfate-acrylamide gel electrophoresis. By submitting the RNA to chromatography on oligo(dT)-cellulose, a 26-30-fold enrichment of the mRNA coding for collagen was achieved.     

Necessity of polyamines for maximum isoleucyl-tRNA formation in a rat liver cell-free system

From a study of the effect of polyamines on aminoacyl-tRNA formation of nine amino acids in a rat liver cell-free system, it is shown that isoleucyl-tRNA formation in the presence of polyamines is much greater than that in presence of Mg²⁺. The data suggest that polyamines may play an important role in protein synthesis by regulating aminoacyl-tRNA formation.     

Differential stimulation by polyamines of phage RNA-directed synthesis of proteins

The effect of polyamines on Q beta and MS2 phage RNA-directed synthesis of three kinds of protein in an Escherichia coli cell-free system has been studied. With both phage RNAs, the degree of stimulation of protein synthesis by spermidine was in the order RNA replicase greater than A protein, while the synthesis of coat protein was not stimulated significantly by spermidine. The synthesis of RNA replicase was stimulated by 1 mM spermidine approx. 8-fold. From the results of Q beta RNA direct alanyl-tRNA and seryl-tRNA binding to ribosomes and initiation dipeptide synthesis, it is suggested that the preferential stimulation of the synthesis of RNA replicase by spermidine is due at least partially to the stimulation of the initiation of RNA replicase synthesis.