scholarly journals Biosynthesis in vitro of tryptophanase by polyribosomes from induced cultures of Escherichia coli

1971 ◽  
Vol 123 (3) ◽  
pp. 355-365 ◽  
Author(s):  
S. A. M. Khairul Bashar ◽  
J. H. Parish ◽  
Marjorie Brown
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Enzyme Synthesis ◽  
Supernatant Fraction ◽  
Amino Acid Residues ◽  
Radioactive Amino Acid ◽  
Tryptophanase Activity

1. Polyribosomes were isolated from Escherichia coli grown in media in which tryptophanase is induced and in which it is repressed. The polyribosomes from the induced bacteria had a small amount of tryptophanase activity associated with them. 2. A portion of the enzyme activity remained bound to polyribosomes during centrifuging in sucrose gradients. 3. Incubation of tryptophanase-containing polyribosomes with puromycin released enzyme activity. 4. The binding of the enzyme to the polyribosomes did not depend on the presence of DNA. 5. When the polyribosomes were incubated under conditions of protein synthesis with supernatant fraction obtained from repressed bacteria, a small but statistically significant increase in enzyme activity was produced. 6. When a radioactive amino acid was included in the incubation mixture for the tryptophanase system a radioactive protein was obtained whose chromatographic, electrophoretic and sedimentation properties were identical with those of tryptophanase. 7. The amount of incorporation was consistent with the amount of new enzyme synthesis predicted by the increase in enzyme activity. Both radioactive incorporation and increase in enzyme activity were shown to be energy-dependent and also negative controls were obtained by using zero-time incubations or polyribosomes isolated from either repressed cells or a mutant lacking the ability to produce tryptophanase. 8. The distribution of radioactive leucine in the carboxyl region of the newly labelled tryptophanase was examined by digesting the labelled protein with carboxypeptidases. It was shown that the radioactivity was more highly concentrated towards the carboxyl terminus when the incubation times for protein synthesis were shorter (implying that, with longer incubation times, longer lengths of polypeptide chain contained radioactive amino acid residues).

scholarly journals Cell-free synthesis of tryptophanase from Escherichia coli. Use of ribonucleic acid isolated from induced cells and a comparison of the product from a system employing ribosomes with that from one employing ribosomes and exogenous ribonucleic acid

1971 ◽  
Vol 125 (2) ◽  
pp. 643-653 ◽  
Author(s):  
J. H. Parish ◽  
S. A. M. Khairul Bashar ◽  
N. L. Brown ◽  
Marjorie Brown
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Ribonucleic Acid ◽  
Supernatant Fraction ◽  
23S Rrna ◽  
Terminal Amino Acid ◽  
Tryptic Peptides ◽  
Radioactive Amino Acid ◽  
Terminal Amino

1. Polyribosomes and RNA were isolated from cultures in which tryptophanase (EC 4.2.1.–) was induced. The polyribosomes were incubated under conditions of protein synthesis, in the presence of a radioactive amino acid and a post-ribosomal supernatant fraction obtained from repressed cells. The RNA preparations were incubated under conditions of protein synthesis in the presence of a radioactive amino acid and a supernatant fraction containing ribosomes from repressed cells. 2. The system was characterized and the synthesis of a radioactive protein with the same chromatographic properties as tryptophanase was demonstrated. This synthesis was shown to be time-dependent and required the presence of RNA from induced cultures, ribosomes and an energy supply; it was inhibited by chloramphenicol. 3. The maximum activity for the synthesis of this protein was found to be associated with 23S rRNA isolated from sucrose gradients. 4. The N-terminal amino acid of tryptophanase was labelled in the protein synthesized in this system but not in the protein synthesized by polyribosomes (without added RNA). Conversely, the C-terminal amino acid of tryptophanase was labelled in the polyribosome system but not in the RNA-containing system. 5. Tryptic digests of protein labelled in vitro were compared with those of tryptophanase. No labelled tryptic peptides were identified other than tryptophanase tryptic peptides. An analysis of the results implied that in the polyribosome system almost the complete tryptophanase subunit chain was labelled but that in the RNA-containing system these chains were incompletely synthesized. 6. Sucrose-gradient analysis of protein synthesized in the RNA-containing system suggested that it cannot be converted into structures with the same sedimentation properties as native tryptophanase. 7. The significance of these results for the assay of tryptophanase mRNA and for an understanding of the control of the translation of this mRNA in vivo is discussed.

scholarly journals STUDIES ON PROTEIN SYNTHESIS IN VITRO

1957 ◽  
Vol 41 (1) ◽  
pp. 219-231 ◽  
Author(s):  
A. Korner ◽  
H. Tarver
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Incubation Medium ◽  
Free Amino ◽  
Opposite Effect ◽  
Specific Activity ◽  
Rapid Rate ◽  
Radioactive Amino Acid ◽  
Release Data

The release of radioactive amino acid from the particulate fractions separated from the prelabelled livers of rats by centrifugation has been studied under various conditions. Although pure fractions may not have been obtained, great differences in behavior were observed. In the mitochondria and nuclei dinitrophenol (10–4 M) causes an inhibition of release, but in microsomes the opposite effect is observed. When the incubation medium is fortified with ATP and phosphocreatine, release is inhibited. In microsomes and nuclei the inhibition proceeds to the extent that the incorporation of preformed radioactive amino acid occurs. Protein is synthesized at a rapid rate. In incubations longer than 1 hour there is always a release of radioactive amino acid. It is concluded from these results that the interpretation of release data from slices or systems such as those studied is impossible without further information concerning some of the unknown variables. The most important unknown is the specific activity of the "free" amino acid in the particulates and the effect of carrier amino acid in the medium of this specific activity.

scholarly journals Amino Acid Residues Involved in the Functional Integrity of Escherichia coli Methionine Aminopeptidase

1999 ◽  
Vol 181 (15) ◽  
pp. 4686-4689 ◽  
Author(s):  
Chen-Hsiang Chiu ◽  
Chao-Zong Lee ◽  
Kung-Shih Lin ◽  
Ming F. Tam ◽  
Lih-Yuan Lin
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Binding Site ◽  
Metal Binding ◽  
Substrate Binding ◽  
Type I ◽  
Amino Acid Residues ◽  
Methionine Aminopeptidase ◽  
Cobalt Ions

ABSTRACT Amino acid residues in the metal-binding and putative substrate-binding sites of Escherichia coli methionine aminopeptidase (MAP) were mutated, and their effects on the function of the enzyme were investigated. Substitution of any amino acid residue at the metal-binding site resulted in complete loss of the two cobalt ions bound to the protein and diminished the enzyme activity. However, only Cys70 and Trp221 at the putative substrate-binding site are involved in the catalytic activity of MAP. Changing either of them caused partial loss of enzyme activity, while mutations at both positions abolished MAP function. Both residues are found to be conserved in type I but not type II MAPs.

scholarly journals Restoration of a Defective Lactococcus lactis Xylose Isomerase

2004 ◽  
Vol 70 (7) ◽  
pp. 4318-4325 ◽  
Author(s):  
Joo-Heon Park ◽  
Carl A. Batt
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Lactococcus Lactis ◽  
Xylose Isomerase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Triple Mutant ◽  
Sequence Homologies

ABSTRACT The genes (xylA) encoding xylose isomerase (XI) from two Lactococcus lactis subsp. lactis strains, 210 (Xyl−) and IO-1 (Xyl+), were cloned, and the activities of their expressed proteins in recombinant strains of Escherichia coli were investigated. The nucleotide and amino acid sequence homologies between the xylA genes were 98.4 and 98.6%, respectively, and only six amino acid residues differed between the two XIs. The purified IO-1 XI was soluble with K m and k cat being 2.25 mM and 184/s, respectively, while the 210 XI was insoluble and inactive. Site-directed mutagenesis on 210 xylA showed that a triple mutant possessing R202M/Y218D/V275A mutations regained XI activity and was soluble. The K m and k cat of this mutant were 4.15 mM and 141/s, respectively. One of the IO-1 XI mutants, S388T, was insoluble and showed negligible activity similar to that of 210 XI. The introduction of a K407E mutation to the IO-1 S388T XI mutant restored its activity and solubility. The dissolution of XI activity in L. lactis subsp. lactis involves a series of mutations that collectively eliminate enzyme activity by reducing the solubility of the enzyme.

Oxytocin analogues with non-coded amino acid residues in position 8: [8-Neopentylglycine]oxytocin and [8-cycloleucine]oxytocin

1987 ◽  
Vol 52 (9) ◽  
pp. 2317-2325 ◽  
Author(s):  
Jan Hlaváček ◽  
Jan Pospíšek ◽  
Jiřina Slaninová ◽  
Walter Y. Chan ◽  
Victor J. Hruby
Keyword(s):  
Amino Acid ◽  
Solid Phase Synthesis ◽  
Solid Phase ◽  
The Other ◽  
Amino Acid Residues ◽  
Phase Synthesis ◽  
Other Hand ◽  
Fragment Condensation

[8-Neopentylglycine]oxytocin (II) and [8-cycloleucine]oxytocin (III) were prepared by a combination of solid-phase synthesis and fragment condensation. Both analogues exhibited decreased uterotonic potency in vitro, each being about 15-30% that of oxytocin. Analogue II also displayed similarly decreased uterotonic potency in vivo and galactogogic potency. On the other hand, analogue III exhibited almost the same potency as oxytocin in the uterotonic assay in vivo and in the galactogogic assay.

Synthesis of Fragments of the Vasoactive Intestinal Peptide (VIP) and Analysis of Their Residual Biological Activities

1995 ◽  
Vol 60 (7) ◽  
pp. 1229-1235 ◽  
Author(s):  
Ivana Zoulíková ◽  
Ivan Svoboda ◽  
Jiří Velek ◽  
Václav Kašička ◽  
Jiřina Slaninová ◽  
...  
Keyword(s):  
Amino Acid ◽  
Biological Activities ◽  
Residual Activity ◽  
Detailed Examination ◽  
Amino Acid Residues ◽  
Linear Peptide ◽  
Zone Electrophoresis ◽  
Capillary Zone

The vasoactive intestinal (poly)peptide (VIP) is a linear peptide containing 28 amino acid residues, whose primary structure indicates a low metabolic stability. The following VIP fragments, as potential metabolites, and their analogues were prepared by synthesis on a solid: [His(Dnp)1]VIP(1-10), VIP(11-14), [D-Arg12]VIP(11-14), [Lys(Pac)15,21,Arg20]VIP(15-22), and VIP(23-28). After purification, the peptides were characterized by amino acid analysis, mass spectrometry, RP HPLC, and capillary zone electrophoresis. In some tests, detailed examination of the biological activity of the substances in vivo and in vitro gave evidence of a low, residual activity of some fragments, viz. a depressoric activity in vivo for [His(Dnp)1]VIP(1-10) and a stimulating activity for the release of α-amylase in vitro and in vivo for [Lys(Pac)15,21,Arg20]VIP(15-22) and VIP(23-28).

Engineering two mutants of cDNA-encoding G2 subunit of soybean glycinin capable of self-assembly in vitro and rich in methionine

Biologia ◽  
2007 ◽  
Vol 62 (4) ◽  
Author(s):  
Reda Sammour
Keyword(s):  
Amino Acid ◽  
Self Assembly ◽  
Seed Quality ◽  
Amino Acid Residues ◽  
Methionine Residues

AbstractThe main goal of this work was to make the cDNA-encoding subunit G2 of soybean glycinin, capable of self-assembly in vitro and rich in methionine residues. Two mutants (pSP65/G4SacG2 and pSP65/G4SacG2HG4) were therefore constructed. The constructed mutants were successfully assembled in vitro into oligomers similar to those occurred in the seed. The successful self-assembly was due to the introduction of Sac fragment of Gy4 (the codons of the first 21 amino acid residues), which reported to be the key element in self-assembly into trimers. The mutant pSP65/G4SacG2HG4 included the acidic chain of Gy4 (HG4), which was previously molecularly modified to have three methionine residues. This mutant will be useful in the efforts to improve the seed quality.

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