The biosynthesis of gramicidin S in a cell-free system

1. A cell-free system prepared from Bacillus brevis cells, harvested in the late phase of growth and consisting of the 11000g supernatant, has been shown to incorporate into gramicidin S the five constituent amino acids added in labelled form. The results are consistent with complete synthesis and not merely a completion of pre-existing intermediate peptides. 2. The incorporation of ¹⁴C-labelled amino acids by the 11000g supernatant into gramicidin S requires an energy source. Omission of phosphoenolpyruvate and pyruvate kinase from the incubation mixture prevents incorporation into gramicidin S. The cell-free system incorporates [¹⁴C]-leucine, -proline and -phenylalanine over a period of 4hr. With [¹⁴C]leucine, incorporation into gramicidin S takes place in the range pH6–9 with maximum incorporation at pH7·0. High concentrations of chloramphenicol or puromycin decreased the incorporation into gramicidin S by only about 20%. 3. The 50000g supernatant exhibited no decrease in ability of incorporating [¹⁴C]valine into gramicidin S as compared with the 11000g supernatant. About 40% of the incorporating ability remained in the 105000g supernatant after 3hr. centrifugation. When recombining the 105000g sediment with the 105000g supernatant, some increase in incorporation over that obtained with the supernatant alone was obtained. The findings tend to support the view that gramicidin S is synthesized in a different manner from that of proteins.     

Kaurenolide biosynthesis in a cell-free system from Cucurbita maxima seeds

A new product obtained by incubation of [2-¹⁴C ]-mevalonic acid with a cell-free system from Cucurbita maxima endosperm was identified by GC-MS as ent-kaura-6,16-dien-19-oic acid. When this compound was reincubated with the microsomal fraction it was converted to 7β-hydroxykaurenolide and hence to 7β,12α-dihydroxykaurenolide. The dienoic acid was also obtained by incubation of ent-kaurene, ent1-kaurenol, ent-kaurenal and ent-kaurenoic acid, but not ent-7α-hydroxykaurenoic acid, with the microsomal fraction. Thus, in the C. maxima cell-free system, the kaurenolides are formed by a pathway which branches from the GA pathway at ent-kaurenoic acid and proceeds via the dienoic acid.     

The stereochemistry of biosynthesis of decaprenoxanthin in a cell-free system

Intact cells of Flavobacterium dehydrogenans grown on glucose or acetate did not incorporate mevalonic acid-[¹⁴C]. After treatment with lysozyme the protoplasts were lysed by sonication in a dilute medium containing mevalonic acid-[¹⁴C] and the cell-free system produced incorporated label into uncyclized C₄₀, monocyclic C₄₅ and bicyclic C₅₀ carotenoids of which decaprenoxanthin was the most abundant.With mevalonate-[2-¹⁴C,4R-4-³H₁] the ¹⁴C:³H ratios of the carotenoids showed that the hydrogen atoms at C-2 and C-6 of the ring and that at C-3 of the 1-hydroxy, 2-methyl but-2-ene-4-yl residues of decaprenoxanthin were derived from the 4-pro-R hydrogen atom of mevalonic acid.Mevalonate-[2-¹⁴C,2R-2-³H₁] and mevalonate-[2-¹⁴C,2S-2-³H₁] gave ratios which showed that the C-4 hydrogen atoms of decaprenoxanthin were derived from the 2-pro-S hydrogen atom of mevalonic acid.     

CRF-stimulated biosynthesis of ACTH in a cell-free system from rat anterior pituitaries

A cell-free system consisting of ribosomes, pH 5 enzymes and supernatant prepared from rat anterior pituitaries was found to be active in the incorporation of 3H-serine into ACTH. The rate of biosyntesis of ACTH, in a cell-free system as, measured by the incorporation of radioactive amino acid, and the rate of biological activity were markedly increased by the addition of CRF. The synthesis of ACTH was significantly inhibited by puromycin and RNAase but was not significantly inhibited by actinomycin D and DNAase.     

Gibberellin biosynthesis in a cell-free system from immature seeds of Pisum sativum

A cell-free system from immature pea seeds converts ¹⁴C-labelled $\text{ent}$-kaurene to $\text{ent}$-kaurenol, $\text{ent}$-kaurenal, $\text{ent}$-kaurenoic acid, $\text{ent}$-7α-hydroxykaurenoic acid, and gibberellin A₁₂-aldehyde. The latter becomes converted further to 13-hydroxygibberellin A₁₂, gibberellin A44, gibberellin A₁₂-alcohol, and several unidentified products. Thus the biosynthesis of gibberellins $\text{via ent}$-kaurene is now established for a member of the $\text{Leguminosae}$. It is the first time that 13-hydroxylation of gibberellins has been observed in a cell-free system and that gibberellin A₁₂-alcohol has been obtained in any biological system.     

Preparation and properties of a cell-free system from rat skin that catalyzes sterol biosynthesis

Homogenates of epidermis from rat skin were centrifuged at 10,000 X g for 20 min. The supernatant fraction ("whole homogenate") catalyzed the demethylation of lanosterol (C(30)) to yield C(27)-sterols. The rate of reaction was measured by the rate of release of (14)CO(2) from the 4-methyl group of lanosterol. Conditions for maximal rates of demethylation were established. Addition of increasing amounts of washed microsomes to a constant amount of substrate resulted in additional release of (14)CO(2), but the release was not proportional to the amount of microsomes. Incubation with increasing amounts of microsomes treated with Triton WR-1339 yielded a proportional rate of release of (14)CO(2). The Triton-treated microsomes were frozen and stored without loss of activity. The rate of formation of (14)CO(2) was constant up to 1 hr of incubation with both Triton-treated microsomes and whole homogenate, for which the K(m) for lanosterol was 5.0 and 3.0 X 10(-5) M, respectively. Other 4-gem-dimethyl sterols were competitive inhibitors, K(i)', 2.0 and 5.5 X 10(-5) M. The enzyme system was inhibited by arsenite. 24,25-Dihydrolanosterol, 24,25-dihydrolanostenone, and squalene were demethylated by the homogenate. The whole homogenate catalyzed the incorporation of mevalonic acid, but not acetic acid, into squalene and sterols. The enzymatic properties of the sterol synthetic system from skin resemble those of similar preparations from rat liver.     

The biosynthesis of fibroin: II. The synthesis of fibroin in a crude cell-free system

The properties of a crude cell-free system able to incorporate amino acids into fibroin are described. Fibroin is characterized by its composition and its sedimentation properties on sucrose gradients.Two populations of membrane-bound ribosomes can be characterized by the utilization of puromycin and detergents for releasing the newly made chains. One population produces fibroin, which remains associated with the membrane fraction, the other produces different proteins which are released into the cell sap.     

Kaurene and kaurenol biosynthesis in cell-free system of Phaseolus coccineus suspensor

It is shown that suspensor tissue of Phaseolus coccineus can biosynthesize ent-kaur-16-ene and ent-kaur-16-en-19β-ol, two key precursors in the biosynthesis of gibberellins.     

The biosynthesis of 12α-hydroxylated gibberellins in a cell-free system from cucurbita maxima endosperm

A previously unknown pathway for the biosynthesis of 12α-hydroxylated gibberellins was found in a cell-free system from Cucurbita maxima endosperm. The microsome fraction converts the gibberellin precursor GA_12-aldehyde simultaneously to GA₁₂ and 12α-hydroxy-GA₁₂-aldehyde. The ratio of these products is pH-dependent: above pH 6.5, the production of GA₁₂ is favoured, whilst below pH 6.5, 12α-hydroxy-GA₁₂-aldehyde is the predominant product. 12α-Hydroxy-GA₁₂-aldehyde is converted further by soluble enzymes to 12α-hydroxy-GA₁₄, 12α-hydroxy-GA₁₅, 12α-hydroxy-GA₃₇ and several unidentified products. This conversion is optimal between pH 6.0 and 6.5 in contrast to the previously known conversion of GA₁₂-aldehyde to GA₄₃ by soluble enzymes, which is optimal at pH 7.5. GA₅₈, a major 12α-hydroxylated endogenous constituent of C. maxima endosperm, was not obtained when 12α-hydroxy-GA₁₂-aldehyde was used as a substrate, but it was obtained together with GA₄ when GA₉ was incubated with a preparation containing both microsomal and soluble enzymes.     

In vitro biosynthesis of plasminogen in a cell-free system directed by mRNA fractions isolated from monkey liver

mRNA was isolated from total RNA of monkey liver by oligo(dT)-cellulose chromatography and was translated in a rabbit reticulocyte cell-free system. Analysis of the translation products immunoprecipitated with specific antibodies to monkey plasma plasminogen revealed a molecule with characteristics similar to those of native plasminogen. The purification of the mRNA by centrifugation on sucrose gradients indicated the presence of plasminogen mRNAs in both the 23S and 18S RNA fractions. Both plasminogen mRNAs can be further purified by chromatography on Sepharose 4B. Affinity chromatography of the proteins synthesized in vitro by total mRNA from liver, as well as by the purified mRNAs, on L-lysine-substituted Sepharose revealed that both major plasma plasminogen forms (1 and 2) are synthesized, as precursors, in the system. The in vitro synthesized plasminogen is similar in its physical and chemical properties to native plasma plasminogen as determined by its ability to bind to L-lysine-substituted Sepharose and its molecular interaction with streptokinase. The purified mRNAs were also translated in the presence of dog pancreas microsomal membranes, and and fractionated on concanavalin A-Sepharose. The 23S mRNA directed the synthesis of a plasminogen molecule similar to the circulating plasma plasminogen form 1, whereas the 18S mRNA directed the synthesis of a molecule similar to the circulating plasma plasminogen form 2. Our evidence indicates that the synthesis of the two major circulating plasma plasminogen forms is directed in the liver by separate mRNAs.     

Further studies on the biosynthesis of gramicidin S and proteins in a cell-free system from Bacillus brevis

1. An improved 11000g cell-free system for the incorporation of [(14)C]valine into gramicidin S has been obtained. The cell-free extract used was the supernatant obtained by treating Bacillus brevis with ultrasonics for 1min. followed by centrifugation at 11000g. The optimum pH for the incorporation was 8.2-8.4 and the optimum Mg(2+) concentration 0.05m. The presence of ammonium sulphate (0.1m) and K(+) (0.01m) increased the incorporation. 2. Cell-free extracts prepared from cells harvested in the early phase of growth (extinction value 0.1) incorporated negligible amounts of [(14)C]valine into gramicidin S compared with that incorporated by cell-free extracts prepared from cells harvested in the late phase of growth (extinction value 0.5). This was not due to the presence of inhibitors in the cell-free extracts prepared from cells harvested early, since there was no marked decrease in gramicidin S synthesis in a mixture of extracts prepared from cells harvested early and late in the growth phase. 3. The small incorporation of [(14)C]valine into protein, which took place in cell-free extracts from cells harvested in the late growth phase, was not inhibited by puromycin, chloramphenicol and ribonuclease. However, the substantial incorporation that took place in cell-free extracts prepared from cells harvested in the early phase of growth was completely inhibited by puromycin, chloramphenicol and ribonuclease. On mixing cell-free extracts prepared from cells harvested early and late in the growth phase, it appeared that the small incorporation that occurs in extracts from cells harvested in the late phase of growth was not due to cellular inhibitors.