Stereochemical specificity of the biosynthesis of the alkyl ether bond in alkyl ether lipids.

The stereochemical course of the formation of the alkyl ether bond in alkyl ether lipids was investigated through the synthesis of stereospecifically labeled acyl R- or S-[1-3H]dihydroxyacetone 3-phosphate (DHAP) starting from L-glyceraldehyde. It was demonstrated directly that the formation of the alkyl ether bond results in the stereospecific exchange of the pro-R C-1 hydrogen of DHAP with a proton of water. The configuration of the hydrogen that is retained on C-1 after formation of the alkyl ether bond was also investigated. The alkyl ether lipid was degraded, and the DHAP backbone isolated as glycerol, converted to DHAP via glycerol 3-phosphate and treated with either aldolase or triose phosphate isomerase. The results demonstrated that the retained hydrogen on C-1, which was pro-S in the starting substrate, was pro-S in the product alkyl ether.     

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.     

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.     

Regularity of isoprenoid biosynthesis in the ether lipids of archaebacteria

The location of paired and unpaired ¹³C atoms in the 16,16′-biphytanyl components of the lipids of Caldariella acidiophila following incorporation of acetate-[1,2-¹³C₂] shows that the overall process of isoprenoid biosynthesis in this archaebacterial species follows a normal pattern and that the head-to-head linkage of the two tetraprenyl chains occurs stereoselectively.     

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 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.     

酵母微生物油脂的生物合成研究

摘要：目的 微生物油脂可作为制备绿色能源生物柴油的原料。对酵母微生物油脂的生物合成方法进行研究。方法 以斯达油脂酵母Lipomyces starkeyi AS 2.1560为菌种进行微生物油脂生物合成。首先获得大量细胞,将细胞收集后,转移至葡萄糖溶液中进行油脂合成。结果 斯达油脂酵母可在不含有其他营养成分的葡萄糖溶液中快速合成油脂,细胞油脂含量可达到细胞干重的60%以上。菌龄对油脂生成影响不明显,糖浓度过高抑制油脂生成,40 g/L葡萄糖溶液中60 h合成油脂最多,达到65.2%,并有进一步积累的可能,在（0.5～6）×108个/mL,接种细胞的密度越大,油脂合成能力越低。合成油脂成分主要为棕榈酸和油酸。结论 斯达油脂酵母细胞增殖与油脂生物合成可分开进行,其油脂成分与普通动植物油脂成分相似。     

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.     

The biosynthesis of C55 polyprenols by a cell-free preparation of Lactobacillus plantarum

A cell-free supernatant of lysates of Lactobacillus plantarum catalyses the synthesis of lipids from [2-¹⁴C]mevalonate. Of the added mevalonate, 7.5% is incorporated into lipids, which were fractionated into five components. About 4% of the radioactivity in these lipids co-chromatographs with compounds shown by mass spectrometry, n.m.r. and i.r. spectroscopy to be C₅₅ polyprenols, and about 2% co-chromatographs with a hexamer. The rest of the radioactivity is in more complex fractions. Analysis by mass spectrometry, n.m.r. and i.r. spectroscopy shows that the major C₅₅ polyprenol is undecaprenol, accompanied by an isomer containing one reduced isoprene unit. A Kuhn–Roth degradation of [¹⁴C]polyprenols indicates that the supernatant catalyses synthesis of these compounds de novo.     

The biosynthesis of a choline nucleotide by a cell-free extract from Streptococcus pneumoniae.

Choline, a component of the wall teichoic acid of Streptococcus pneumoniae, was converted to cytidine diphosphocholine via choline phosphate by enzymes which were identified in cell-free extracts of the pneumococcus. The first enzyme, choline kinase, was investigated in some detail. It appeared to have a pH optimum of 7.3 to 7.4 and was stimulated by Mg2+. Kinetic studies gave an apparent Michaelis constant (Km) for ATP of I mM, and for choline of 0.19 mM, with Vmax values of 3 nmol min-1 (mg protein)-1 and 0.5 nmol min-1 (mg protein)-1 respectively. The second enzyme, CDPcholine pyrophosphorylase was specific for CTP and had a requirement for Mg2+ with an optimum at 7 mM.     

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.