scholarly journals Effect of lipids, in particular cholesteryl 14-methylhexadecanoate, on the incorporation of labelled amino acids into transfer ribonucleic acid in vitro

1968 ◽  
Vol 110 (1) ◽  
pp. 1-8 ◽  
Author(s):  
J. Hradec ◽  
Z. Dušek
Keyword(s):  
Amino Acids ◽  
Ribosomal Protein ◽  
Free Cholesterol ◽  
Protein Hydrolysate ◽  
Transfer Rna ◽  
Enzymic Activity ◽  
Cholesteryl Esters ◽  
Energy Dependent ◽  
Rna Complexes

1. Rat liver pH5 enzymes and cell sap extracted with various organic solvents showed a variable decreased incorporation of labelled amino acids into s-RNA (‘soluble’ or transfer RNA) in vitro. 2. The original enzymic activity could be fully restored, though at different rates, by the addition of lipid extracts in quantities corresponding to those originally present. 3. Of the main lipid groups separated from the extract, only free cholesterol and cholesteryl esters were able to reactivate the extracted pH5 enzymes in the same way as the whole lipid extract. 4. Addition of pure cholesteryl 14-methylhexadecanoate also fully restored the enzymic activity. 5. There was no energy-dependent incorporation of labelled amino acids into ribosomal protein in the presence of extracted cell sap. Addition of cholesteryl 14-methylhexadecanoate fully restored the activity of the cell sap to incorporate labelled leucine and lysine into ribosomal protein and enhanced the incorporation of labelled protein hydrolysate and phenylalanine over the level found with the corresponding non-extracted preparations. 6. It is concluded that lipids play an important role in the synthesis of aminoacyl-s-RNA complexes and that cholesteryl 14-methylhexadecanoate may be the active lipid in this respect.

scholarly journals Effect of some polycyclic aromatic hydrocarbons on protein synthesis in vitro

1967 ◽  
Vol 105 (1) ◽  
pp. 251-259 ◽  
Author(s):  
J. Hradec
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Polycyclic Aromatic Hydrocarbons ◽  
Ribosomal Rna ◽  
Aromatic Hydrocarbons ◽  
Protein Hydrolysate ◽  
Transfer Rna ◽  
Acid Activation ◽  
Polycyclic Aromatic

1. The effect of the strongly carcinogenic polycyclic aromatic hydrocarbons benzo[a]pyrene, 3-methylcholanthrene and dibenz[a,h]anthracene and of the non-carcinogenic anthracene, pyrene and phenanthrene on protein synthesis was studied in vitro with subcellular systems from rat liver. 2. Both types of hydrocarbons affect amino acid activation and inhibit transfer of labelled amino acids from transfer RNA to ribosomes. 3. Only the carcinogenic compounds stimulate the incorporation of labelled algal-protein hydrolysate and of some individual amino acids into transfer RNA. The most active dose was 10mμg. under the conditions used. This effect is abolished by preincubation of pH5 enzymes with the carcinogens before the addition of radioactive amino acids. 4. The carcinogens stimulate the incorporation of some amino acids into ribosomal protein whereas the non-carcinogenic compounds have no such effects. 5. Polynucleotide-dependent stimulation of protein synthesis is greatly enhanced in the presence of the carcinogenic hydrocarbons when either free amino acids or transfer RNA charged with labelled amino acids are used. The non-carcinogenic compounds induce a partial inhibition of this process. 6. It is concluded, in agreement with other authors, that carcinogens may increase the number of active incorporation sites on both transfer and ribosomal RNA. Possible mechanisms of such an effect are discussed.

scholarly journals The RNA origin of transfer RNA aminoacylation and beyond

2011 ◽  
Vol 366 (1580) ◽  
pp. 2959-2964 ◽  
Author(s):  
Hiroaki Suga ◽  
Gosuke Hayashi ◽  
Naohiro Terasaka
Keyword(s):  
Amino Acids ◽  
Evolutionary History ◽  
Transfer Rna ◽  
Translation System ◽  
Rna Sequences ◽  
Rna Molecules ◽  
Rna Catalysts ◽  
Modern System ◽  
History Of

Aminoacylation of tRNA is an essential event in the translation system. Although in the modern system protein enzymes play the sole role in tRNA aminoacylation, in the primitive translation system RNA molecules could have catalysed aminoacylation onto tRNA or tRNA-like molecules. Even though such RNA enzymes so far are not identified from known organisms, in vitro selection has generated such RNA catalysts from a pool of random RNA sequences. Among them, a set of RNA sequences, referred to as flexizymes (Fxs), discovered in our laboratory are able to charge amino acids onto tRNAs. Significantly, Fxs allow us to charge a wide variety of amino acids, including those that are non-proteinogenic, onto tRNAs bearing any desired anticodons, and thus enable us to reprogramme the genetic code at our will. This article summarizes the evolutionary history of Fxs and also the most recent advances in manipulating a translation system by integration with Fxs.

scholarly journals Ribosomal protein L7a is encoded by a gene (Surf-3) within the tightly clustered mouse surfeit locus.

1989 ◽  
Vol 9 (1) ◽  
pp. 224-231 ◽  
Author(s):  
A Giallongo ◽  
J Yon ◽  
M Fried
Keyword(s):  
Amino Acids ◽  
Ribosomal Protein ◽  
Ribosomal Subunit ◽  
Ribosomal Protein Gene ◽  
Mouse Cell ◽  
Nucleic Acid Hybridization ◽  
Peptide Sequence ◽  
In Vitro Translation ◽  
Cell Components

The mouse Surfeit locus, which contains a cluster of at least four genes (Surf-1 to Surf-4), is unusual in that adjacent genes are separated by no more than 73 base pairs (bp). The heterogeneous 5' ends of Surf-1 and Surf-2 are separated by only 15 to 73 bp, the 3' ends of Surf-1 and Surf-3 are only 70 bp apart, and the 3' ends of Surf-2 and Surf-4 overlap by 133 bp. This very tight clustering suggests a cis interaction between adjacent Surfeit genes. The Surf-3 gene (which could code for a basic polypeptide of 266 amino acids) is a highly expressed member of a pseudogene-containing multigene family. By use of an anti-peptide serum (against the C-terminal nine amino acids of the putative Surf-3 protein) for immunofluorescence and immunoblotting of mouse cell components and by in vitro translation of Surf-3 cDNA hybrid-selected mRNA, the Surf-3 gene product was identified as a 32-kilodalton ribosomal protein located in the 60S ribosomal subunit. From its subunit location, gel migration, and homology with a limited rat ribosomal peptide sequence, the Surf-3 gene was shown to encode the mouse L7a ribosomal protein. The Surf-3 gene is highly conserved through evolution and was detected by nucleic acid hybridization as existing in multiple copies (multigene families) in other mammals and as one or a few copies in birds, Xenopus, Drosophila, and Schizosaccharomyces pombe. The Surf-3 C-terminal anti-peptide serum detects a 32-kilodalton protein in other mammals, birds, and Xenopus but not in Drosophila and S. pombe. The possible effect of interaction of the Surf-3 ribosomal protein gene with adjacent genes in the Surfeit locus at the transcriptional or posttranscriptional level or both levels is discussed.

scholarly journals Cloning of the Schizosaccharomyces pombe gene encoding diadenosine 5′,5′′′-P1,P4-tetraphosphate (Ap4A) asymmetrical hydrolase: sequence similarity with the histidine triad (HIT) protein family

1995 ◽  
Vol 312 (3) ◽  
pp. 925-932 ◽  
Author(s):  
Y Huang ◽  
P N Garrison ◽  
L D Barnes
Keyword(s):  
Amino Acids ◽  
Schizosaccharomyces Pombe ◽  
Sequence Similarity ◽  
Enzymic Activity ◽  
Protein Family ◽  
Partial Purification ◽  
Binding Motif ◽  
Local Similarity ◽  
Multicopy Plasmid

Diadenosine 5′,5‴-P1,P4-tetraphosphate (Ap4A) asymmetric hydrolase (EC 3.6.1.17) is a specific catabolic enzyme of Ap4A found in Schizosaccharomyces pombe. We have previously described the partial purification of Ap4A hydrolase from S. pombe [Robinson, de la Peña and Barnes (1993) Biochim. Biophys. Acta 1161, 139-148]. We determined the sequence of the N-terminal 20 amino acids of Ap4A hydrolase and designed two degenerate PCR primers based on the sequence. The 60 bp DNA fragment obtained by PCR, which is specific to Ap4A hydrolase, was used to isolate the Ap4A hydrolase gene, aph1, from S. pombe by screening a genomic DNA library in a multicopy plasmid. Ap4A hydrolase activity from the crude supernatant of a positive S. pombe transformant was about 25-fold higher than the control. There was no detectable stimulation of enzymic activity by phosphate. The aph1 gene from S. pombe contains three introns. The intron boundaries were confirmed by sequencing the cDNA of the aph1 gene from a S. pombe cDNA library. The deduced open reading frame of the aph1 gene codes for 182 amino acids. Two regions of significant local similarity were identified between the Ap4A hydrolase and the histidine triad (HIT) protein family [Séraphin (1992) DNA Sequence 3, 177-179]. HIT proteins are present in prokaryotes, yeast, plants and mammals. Their functions are unknown, except that the bovine protein inhibits protein kinase C in vitro. All four histidine residues which are conserved among the HIT proteins, including the HxHxH putative Zn(2+)-binding motif, are conserved in the Ap4A hydrolase. In addition, there are two regions of similarity between the Ap4A phosphorylases I and II from Saccharomyces cerevisiae and Ap4A hydrolase from S. pombe. These regions overlap with the HIT protein similarity regions. The aph1 gene from S. pombe is the first asymmetrical Ap4A hydrolase gene to be cloned and sequenced.

Process Optimization and Composition Analysis of Antioxidant Enzymatic Hydrolysate from Squid By-Products by Papain

2011 ◽  
Vol 79 ◽  
pp. 198-203 ◽  
Author(s):  
Bin Wang ◽  
Jia Hui Ma ◽  
You Le Qu ◽  
Xian Wang ◽  
Li Li
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protein Hydrolysate ◽  
Essential Amino Acids ◽  
Single Factor ◽  
Scavenging Ratio ◽  
Dpph Scavenging ◽  
By Products ◽  
Total Amino Acids

In order to evaluate the high-value application of squid by-products yielded hydrolysate, the process of preparation and purification technology, chemical composition and in vitro antioxidant activity of the hydrolysate were investigated. The optimal conditions of papain hydrolysis were obtained by single-factor experiments and orthogonal test with the DPPH• scavenging ratio as index, amino acid composition was analysed by automatic amino acid analyzer, the hydrolysate was isolated with a Sephadex G-25 column. Based on single-factor experiments, the hydrolysate with the DPPH• scavenging ratio being 53.96 % was gained under the optimal condition of enzymolysis temperature of 45 °C, enzymolysis time of 3 h, total enzyme dose of 1.2 %, and the pH value of 7. The protein content of the hydrolysate reached up to 17.53 %, and the essential amino acids were accounted for 51.06 % of total amino acids. The largest content amino acid was glutamic acid, which accounted for 10.74 % of total amino acids. Compared with the amino acid profiles recommended by FAO/WHO, the quality of the protein hydrolysate was high, as it was rich in essential amino acids, including isoleucine, leucine, lysine, methionine and cystine, threonine, tryptophan, and valine, which covered 88 %-100 % of the FAO/WHO recommended. The hydrolysate was divided into three fractions (F1-F3) using a Sephadex G-25 column, the F1 possessed the highest antioxidation activity with the reducing power, •OH and DPPH• scavenging ratio being 0.236, 18.13 % and 63.85 % at the concentration of 5 mg/mL. Compared with the retention time of the reduced glutathione chromatomap, the relative molecular mass of F1 was higher than 307, F2 and F3 was lower than 307. The result revealed that the protein hydrolysate from squid by-products by papain had strongly antioxidant capacity in vitro and high nutrition, and this finding provided a new way of advanced exploitation of squid scrap resources.

Ribosomal protein L7a is encoded by a gene (Surf-3) within the tightly clustered mouse surfeit locus

1989 ◽  
Vol 9 (1) ◽  
pp. 224-231
Author(s):  
A Giallongo ◽  
J Yon ◽  
M Fried
Keyword(s):  
Amino Acids ◽  
Ribosomal Protein ◽  
Ribosomal Subunit ◽  
Ribosomal Protein Gene ◽  
Mouse Cell ◽  
Nucleic Acid Hybridization ◽  
Peptide Sequence ◽  
In Vitro Translation ◽  
Cell Components

The mouse Surfeit locus, which contains a cluster of at least four genes (Surf-1 to Surf-4), is unusual in that adjacent genes are separated by no more than 73 base pairs (bp). The heterogeneous 5' ends of Surf-1 and Surf-2 are separated by only 15 to 73 bp, the 3' ends of Surf-1 and Surf-3 are only 70 bp apart, and the 3' ends of Surf-2 and Surf-4 overlap by 133 bp. This very tight clustering suggests a cis interaction between adjacent Surfeit genes. The Surf-3 gene (which could code for a basic polypeptide of 266 amino acids) is a highly expressed member of a pseudogene-containing multigene family. By use of an anti-peptide serum (against the C-terminal nine amino acids of the putative Surf-3 protein) for immunofluorescence and immunoblotting of mouse cell components and by in vitro translation of Surf-3 cDNA hybrid-selected mRNA, the Surf-3 gene product was identified as a 32-kilodalton ribosomal protein located in the 60S ribosomal subunit. From its subunit location, gel migration, and homology with a limited rat ribosomal peptide sequence, the Surf-3 gene was shown to encode the mouse L7a ribosomal protein. The Surf-3 gene is highly conserved through evolution and was detected by nucleic acid hybridization as existing in multiple copies (multigene families) in other mammals and as one or a few copies in birds, Xenopus, Drosophila, and Schizosaccharomyces pombe. The Surf-3 C-terminal anti-peptide serum detects a 32-kilodalton protein in other mammals, birds, and Xenopus but not in Drosophila and S. pombe. The possible effect of interaction of the Surf-3 ribosomal protein gene with adjacent genes in the Surfeit locus at the transcriptional or posttranscriptional level or both levels is discussed.

scholarly journals Kinetics of Hyaluronidase Inhibition by Rice (Oryza sativa L.) Protein Hydrolysate

2020 ◽  
Vol 10 (24) ◽  
pp. 9087
Author(s):  
Hui-Ju Chen ◽  
Fan-Jhen Dai ◽  
Siao-Ling Fan ◽  
Yu-Chun Huang ◽  
Chi-Fai Chau ◽  
...  
Keyword(s):  
Amino Acids ◽  
Inhibitory Activity ◽  
Protein Hydrolysate ◽  
Oryza Sativa L ◽  
Skin Aging ◽  
Protein Hydrolysates ◽  
Total Amino Acid ◽  
Competitive Reaction ◽  
Rice Protein

Research on the skin’s maintenance and protection against aging has gradually progressed toward phytocosmetics. This study investigated the in vitro hyaluronidase inhibitory activity of rice protein hydrolysate obtained by using bacterial amylase and protease against skin aging-related enzymes. Here, the molecular weights of rice protein hydrolysates were in the range 5–63 kDa. Every 100 g of a rice protein hydrolysate contains approximately 2960 mg of total amino acid, including essential amino acids (893 mg) and branched-chain amino acids (591 mg). A kinetic study showed that hyaluronidase inhibition by the rice protein hydrolysate occurs through a competitive reaction mechanism. Achieving effective hyaluronidase inhibitory activity, the rice protein hydrolysate had a half maximal inhibitory concentration of 7.61 mg/mL. Because hyaluronidase activity inhibition is crucial for treating skin aging, rice protein hydrolysates should be considered as cosmeceutical ingredients.

A basic region neighboring the lysine-rich C-terminus of protein SRP19 is required for binding to signal recognition particle RNA

1991 ◽  
Vol 69 (9) ◽  
pp. 649-654 ◽  
Author(s):  
Christian Zwieb
Keyword(s):  
Amino Acids ◽  
Rna Binding ◽  
Signal Recognition Particle ◽  
Site Directed Mutagenesis ◽  
Signal Recognition ◽  
C Terminus ◽  
Terminal Amino ◽  
Rna Complexes ◽  
Mutant Polypeptides

To identify some of the determinants in the 19-kilodalton protein of signal recognition particle (SRP19) for binding to signal recognition particle RNA, two mutant derivatives of the SRP19 were constructed, lacking 14 and 24 C-terminal amino acids. Polypeptides were transcribed and translated in vitro and tested for their ability to bind to signal recognition particle RNA by retention of protein–RNA complexes on DEAE–Sepharose. Both mutant polypeptides form complexes with the RNA, demonstrating that the 24 C-terminal amino acids, which include a lysine-rich sequence at positions 136–144, are dispensable. A third mutant polypeptide, in which eight additional amino acids were removed by oligonucleotide-directed digestion of the mRNA, was unable to bind. The amino acids in the sequence PKLKTRTQ correspond to positions 113–120; they are suggested to be involved in interaction with signal recognition particle RNA.Key words: signal recognition particle, site-directed mutagenesis, protein–RNA binding.

scholarly journals Mutations in 23S rRNA and Ribosomal Protein L4 Account for Resistance in Pneumococcal Strains Selected In Vitro by Macrolide Passage

2000 ◽  
Vol 44 (8) ◽  
pp. 2118-2125 ◽  
Author(s):  
A. Tait-Kamradt ◽  
T. Davies ◽  
M. Cronan ◽  
M. R. Jacobs ◽  
P. C. Appelbaum ◽  
...  
Keyword(s):  
Amino Acids ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Serial Passage ◽  
Single Amino Acid ◽  
Rrna Genes ◽  
23S Rrna ◽  
Erythromycin Resistance ◽  
Single Amino Acid Change

ABSTRACT The mechanisms responsible for macrolide resistance inStreptococcus pneumoniae mutants, selected from susceptible strains by serial passage in azithromycin, were investigated. These mutants were resistant to 14- and 15-membered macrolides, but resistance could not be explained by any clinically relevant resistance determinant [mef(A),erm(A), erm(B), erm(C),erm(TR), msr(A), mph(A),mph(B), mph(C), ere(A),ere(B)]. An investigation into the sequences of 23S rRNAs in the mutant and parental strains revealed individual changes of C2611A, C2611G, A2058G, and A2059G (Escherichia colinumbering) in four mutants. Mutations at these residues in domain V of 23S rRNA have been noted to confer erythromycin resistance in other species. Not all four 23S rRNA alleles have to contain the mutation to confer resistance. Some of the mutations also confer coresistance to streptogramin B (C2611A, C2611G, and A2058G), 16-membered macrolides (all changes), and clindamycin (A2058G and A2059G). Interestingly, none of these mutations confer high-level resistance to telithromycin (HMR-3647). Further, two of the mutants which had no changes in their 23S rRNA sequences had changes in a highly conserved stretch of amino acids (63KPWRQKGTGRAR74) in ribosomal protein L4. One mutant contained a single amino acid change (G69C), while the other mutant had a 6-base insert, resulting in two amino acids (S and Q) being inserted between amino acids Q67 and K68. To our knowledge, this is the first description of mutations in 23S rRNA genes or ribosomal proteins in macrolide-resistant S. pneumoniae strains.

Binding of Tissue Plasminogen Activator to Vascular Grafts

1989 ◽  
Vol 61 (01) ◽  
pp. 131-136 ◽  
Author(s):  
Richard A Harvey ◽  
Hugh C Kim ◽  
Jonathan Pincus ◽  
Stanley Z Trooskin ◽  
Josiah N Wilcox ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Polymer Surface ◽  
Enzymic Activity ◽  
Vascular Prostheses ◽  
Chemically Modified ◽  
Insoluble Surfactant ◽  
Tissue Plasminogen

SummaryTissue plasminogen activator labeled with radioactive iodine (125I-tPA) was immobilized on vascular prostheses chemically modified with a thin coating of water-insoluble surfactant, tridodecylmethylammonium chloride (TDM AC). Surfactant- treated Dacron, polytetrafluoroethylene (PTFE), silastic, polyethylene and polyurethane bound appreciable amounts of 125I- tPA (5-30 μg 125I-tPA/cm2). Upon exposure to human plasma, the amount of 125I-tPA bound to the surface shows an initial drop during the first hour of incubation, followed by a slower, roughly exponential release with a t½ of appoximately 75 hours. Prostheses containing bound tPA show fibrinolytic activity as measured both by lysis of clots formed in vitro, and by hydrolysis of a synthetic polypeptide substrate. Prior to incubation in plasma, tPA bound to a polymer surface has an enzymic activity similar, if not identical to that of the native enzyme in buffered solution. However, exposure to plasma causes a decrease in the fibrinolytic activity of both bound tPA and enzyme released from the surface of the polymer. These data demonstrate that surfactant-treated prostheses can bind tPA, and that these chemically modified devices can act as a slow-release drug delivery system with the potential for reducing prosthesis-induced thromboembolism.

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