Cell-free Biosynthesis Research Articles

Cell-free biosynthesis of multiple preprosomatostatins: characterization by hybrid selection and amino-terminal sequencing.

In vitro translation of mRNA isolated from islets of Langerhans results in the synthesis of three major preprosomatostatins of Mr 19 000, 18 000, and 16 000, each of which can be resolved into several isoelectric forms [Warren, T. G., & Shields, D. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3729-3733]. Here we present further characterization of the somatostatin precursors by (i) hybrid selection translation of specific preprosomatostatin mRNAs, (ii) in vitro proteolytic processing of the nascent preprosomatostatins synthesized from hybrid-selected mRNAs, (iii) comparison of their tryptic peptides, and (iv) partial amino-terminal sequence analysis of the signal peptide regions. Hybrid selection experiments using specific cDNA clones demonstrated which preprosomatostatin species corresponded to previously characterized precursor cDNAs [Hobart, P., Crawford, R., Shen, L. P., Picket, R., & Rutter, W. J. (1980) Nature (London) 288, 137-141]; thus, the polypeptide encoded by plasmid pLaS1 corresponds to one form of the Mr 18 000 preprosomatostatins while one form of the Mr 16 000 preprosomatostatins is encoded by pLaS2. Analysis of the tryptic peptides demonstrated that the Mr 16 000 molecule possessed the mature hormone sequence at the carboxyl terminus, as had been shown for the Mr 19 000 and 18 000 precursors. Partial NH2-terminal sequence analysis (a) confirmed the data from hybrid selection and (b) demonstrated that the Mr 18 000 precursor contained a signal peptide manifesting amino acid heterogeneity at certain positions in the signal peptides of each preprosomatostatin. It is suggested that this heterogeneity might account, in part, for variants of the preprosomatostatin molecules.

The alpha subunit of pituitary glycoprotein hormones. Formation of three-dimensional structure during cell-free biosynthesis.

The alpha subunit of pituitary glycoprotein hormones. Formation of three-dimensional structure during cell-free biosynthesis.

Studies on the cell-free biosynthesis of CNS membrane proteins.

The biosynthesis of CNS membrane proteins was studied in cell-free systems containing membrane-bound polysomes (rough endoplasmic reticulum; RER) or free polysomes from rat forebrain. In previous studies of CNS membrane proteins using two-dimensional gel electrophoretic analysis, five proteins (mol. wt. -pI: 75K, 5.4, 68K 5.6, 61K 5.1, 58K 5.1, and 36K 5.6) were found in cell membrane fractions including preparations enriched in RER, smooth endoplasmic reticulum, and plasma membranes. One of these proteins, 68K 5.6, was also present in cytosol and comigrated with a microtubule-associated protein. In our present study, cell-free systems containing RER were found to synthesize the 75K, 5.4, 61K 5.1, and 58K 5.1 proteins. A protein, 34K 5.65, similar (but not identical) to the 36K 5.6 protein was also synthesized. After cell-free synthesis, 75K 5.4 and 58K 5.1 proteins could be purified by concanavalin A affinity chromatography. Of the five common membrane proteins previously identified, only 68K 5.6 protein was synthesized by the free polysome population. The free polysomes were also found to synthesize cyclic AMP binding proteins at 48K and 54K, known from previous studies to be present in both cytosol and plasma membrane fractions in mammalian brain tissue. In conclusion, RER synthesized proteins found exclusively in CNS membrane fractions, whereas free polysomes synthesized those proteins found in both soluble and membrane compartments.

Vellosimine reductase. A specific enzyme involved in the cell-free biosynthesis of sarpagine type alkaloids

Vellosimine reductase is a substrate and cofactor specific enzyme which catalyzes the NADPH-dependent reduction of vellosimine ( 4 ) forming 10-deoxy-sarpagine ( 5 ) in the cell-free biosynthesis of sarpagine type alkaloids.

Cell-free biosynthesis of erythroglycan in a microsomal fraction from K-562 cells.

Particulate membrane preparations from K-562 [human CML (chronic-myelogenous-leukaemia)-derived] cells catalyse the transfer of [3H]galactose from UDP-[3H]-galactose and [3H]N-acetylglucosamine from UDP-[3H]N-acetylglucosamine into an endogenous product that on digestion with Pronase yields long-chain glycopeptides (mol.wt. 7000--10 000) called 'erythroglycan'. Incorporation of either labelled sugar increased up to 60 min of incubation time. The labelled erythroglycan was isolated by chromatography on Sephadex G-50 and characterized by digestion with endo-beta-galactosidase from Escherichia freundii, followed by analysis on Bio-Gel P-2 and paper chromatography. This digestion gave the following four products: (1) a disaccharide with the sequence beta GlcNAc-beta Gal; (2) a trisaccharide with the sequence betaGal-betaGlcNAc-beta Gal; (3) a larger oligosaccharide containing galactose and N-acetylglucosamine; and (4) a putative protein-linkage region.

Cell-free biosynthesis of cytokinins in cultured tobacco cells

Cell-free biosynthesis of free cytokinins not related to the degradation of t-RNA was demonstrated by using enzyme preparation extracted from cultured tobacco (Nicotiana tabacum cv. Xanthi) cells. The reaction mixture for cytokinin biosynthesis contained 20 mM Tris-HCl buffer (pH 7.6), 5 mM magnesium acetate, 0.5 mM isopentenylpyrophosphate, 50 μM adenosine as a substrate and dialyzed enzyme extract. After 2.5 hours of reaction at 27°C, N6-(Δ2-isopentenyl)adenosine, N6-(Δ2-isopentenyl)adenine and trans-zeatin were biosynthesized. The radioactivity of [U-14C]adenosine was incorporated into these three cytokinins after 30 minutes of reaction in the cell-free system. From the time course of biosynthesis of cytokinins and the incorporation of [U-14C]adenosine into cytokinins, we concluded that adenosine accepts the isopentenyl group directly to form N6-(Δ2-isopentenyl) adenosine. This is converted to trans-zeatin via N6-(Δ2-isopentenyl)adenine by several enzymes.

Cell-free biosynthesis of different high molecular weight forms of bovine neurophysins I and II coded by hypothalamic mRNA.

Cell-free biosynthesis of different high molecular weight forms of bovine neurophysins I and II coded by hypothalamic mRNA.

Cell-free biosynthesis of discadenine, a spore germination inhibitor of Dictyostelium discoideum

Cell-free biosynthesis of discadenine, a spore germination inhibitor of Dictyostelium discoideum

Cell-free biosynthesis of the O-acetylated N-acetylneuraminic acid capsular polysaccharide of group C meningococci.

A cell-free system was established to study the biosynthesis of group C meningococcal capsular polysaccharide, an alpha-2 leads to 9-linked N-acetylneuraminic acid (NeuAc) homopolymer containing O-acetyl groups at either C7 or C8. Sialyltransferase activity, isolated from group C meningococcus strain C-11, catalyzed incorporation of [14C]NeuAc from CMP (CMP--[14C]NeuAc) into polymeric form. This sialyltransferase was stimulated by addition of meningococcus group C and Escherichia coli K92 capsular polysaccharides, the latter being an alpha-2 leads to 8- and alpha-2 leads to 9-linked NeuAc heteropolymer. Group C meningococcal sialyltransferase did not require divalent ions but was stimulated by Mn2+. Attempts to demonstrate a lipid-soluble intermediate in the biosynthesis of this NeuAc polymer were unsuccessful. Meningococcal group C sialyltransferase incorporated NeuAc into a membrane-associated product. The polysaccharide can be extracted from the membrane-bound fraction with Triton X-100. The newly synthesized polysaccharide coprecipitates with authentic group C antigen in meningococcal group C antiserum and is degraded by sodium metaperiodate, indicating that the NeuAc polymer synthesized by the cell-free system consists of alpha-2 leads to 9 linkage. Meningococcal group C spheroplast membranes contain an O-acetylase that can catalyze the transfer of acetyl groups from acetyl coenzyme A to the in vitro-synthesized polysaccharide.

5'-AMP is a direct precursor of cytokinin in Dictyostelium discoideum.

ISOPENTENYLADENINE (i6Ade) and closely related derivatives occur naturally either as free bases or as constituents of tRNA1,2. Although these cytokinins have been detected in various organisms, it is still not clear whether they are produced by the degradation of tRNA or whether they are synthesised by some pathway not involving tRNA. A spore germination inhibitor, discadenine, isolated from Dictyostelium discoideum was found to be an i6Ade derivative with a 3-amino-3-carboxypropyl group in the 3 position3—the cells contained a large quantity of free i6Ade, presumably a precursor of discadenine4. These results led us to investigate the cell-free biosynthesis of i6Ade in this organism. We report here the existence of a cytokinin biosynthetic pathway not related to tRNA degradation, showing that 5′-AMP is the actual acceptor molecule for the isopentenyl group. Our findings suggest that the biosynthesis of cytokinin in higher plants may proceed in a similar fashion.

Separation of myocardial cytoplasmic acidic inhibitor proteins which inhibit hepatic protein biosynthesis in vitro

Cytoplasmic inhibitor proteins of rabbit and pig myocardium have been separated and their in-vitro effects on hepatic cell-free protein biosynthesis reported. Three protein peaks could be separated from myocardial cytoplasmic pH 5 fraction by DEAE and CM cellulose column chromatography. There was a difference in the elution pattern of the acidic inhibitor proteins obtained from pig and rabbit myocardium. The myocardial acidic inhibitor proteins inhibited a liver cell-free protein biosynthesizing system, but not a myocardial cell-free system.

Acute effects of exposure to orthochlorobenzylidene malononitrile (CS) and the development of tolerance.

1<h3>Abstract</h3> Many natural metabolites have antibacterial, antiviral, or anticancer effects and can be developed into new drugs. However, working with the microorganisms that produce these products can be challenging since they are not as well characterized as a model organism like <i>Escherichia coli</i>. In this paper, we investigate the potential for a cell-free transcription-translation (TX-TL) system to provide a rapid prototyping platform for characterizing new genetic pathways. We use the valinomycin biosynthesis pathway as a test case, and we show successful heterologous expression of the heterodimeric valinomycin synthetase (VlmSyn, Vlm1: 374 kDa and Vlm2: 284 kDa) from <i>Strep-tomyces tsusimaensis</i> within the TX-TL system. Using LC-MS analysis, we find that valinomycin is produced at low but detectable levels, even when only one out of the three basic precursors is fed into the system. Our work represents another step towards applying cell-free biosynthesis to the discovery and characterization of new natural products.

Cell-free biosynthesis of precursors of polyprenyl derivatives of p-hydroxybenzoate in mammalian and bacterial systems.

Research Article| June 01 1972 Cell-free biosynthesis of precursors of polyprenyl derivatives of p-hydroxybenzoate in mammalian and bacterial systems H Rudney H Rudney Search for other works by this author on: This Site PubMed Google Scholar Biochem J (1972) 128 (1): 13P–14P. https://doi.org/10.1042/bj1280013P Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation H Rudney; Cell-free biosynthesis of precursors of polyprenyl derivatives of p-hydroxybenzoate in mammalian and bacterial systems. Biochem J 1 June 1972; 128 (1): 13P–14P. doi: https://doi.org/10.1042/bj1280013P Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Journal Search Advanced Search This content is only available as a PDF. © 1972 London: The Biochemical Society1972 Article PDF first page preview Close Modal You do not currently have access to this content.

The conversion of mevalonic acid into gibberellin A12-aldehyde in a cell-free system from Cucurbita pepo

A cell-free system prepared from immature seed of Cucurbita pepo incorporates the label from mevalonate-2-(14)C into ent-kaur-16-en-19-oic acid (I), ent-7α-hydroxy-kaur-16-en-19-oic acid (II), and ent-gibberell-16-en-7-al-19-oic acid (III) (gibberellin A12-aldehyde). The products were identified by gas liquid chromatography and by combined gas chromatography-mass spectrometry of the methylated and trimethylsilylated fractions. The radioactivity of the compounds was established by recrystallisation to constant specific radioactivity. Gibberellin A12 (IV), also detected in the system after incubation by combined gas chromatographymass spectrometry may be an artefact, derived from gibberellin A12-aldehyde by a non-enzymatic conversion.With gibberellin A12-aldehyde, the cell-free biosynthesis of an ent-gibberellane has been achieved for the first time.

Cell-free biosynthesis of wool keratin proteins.

Cell-free biosynthesis of wool keratin proteins.

The cell-free biosynthesis of clavine alkaloids

The cell-free biosynthesis of clavine alkaloids

The cell-free biosynthesis of the glycoprotein of membranes from Ehrlich ascites carcinoma cells.

The cell-free biosynthesis of the glycoprotein of membranes from Ehrlich ascites carcinoma cells.

Localization of the site of proline hydroxylation during the cell-free biosynthesis of collagen

We have previously reported a cell-free system derived from chick embryo homogenates in which proline-C 14 is incorporated into protein-bound hydroxyproline associated with the microsomal fraction (Peterkofsky and Udenfriend, 1961). No significant amounts of protein-bouund hydroxyproline-C 14 were found in the soluble protein fraction. The present communication describes studies carried out with this system which give some information concerning the site of proline hydroxylation.

Cell-free biosynthesis of the tropolone ring

Cell-free biosynthesis of the tropolone ring