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 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).

C-Terminal amino acid sequence of chicken pepsinogen and its homology with sequences of other acid proteases

1980 ◽  
Vol 45 (4) ◽  
pp. 1144-1154 ◽  
Author(s):  
Miroslav Baudyš ◽  
Helena Keilová ◽  
Vladimír Kostka
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
The Other ◽  
Terminal Sequence ◽  
Terminal Part ◽  
Terminal Amino Acid ◽  
Tryptic Peptides ◽  
Terminal Amino ◽  
High Degree ◽  
Terminal Amino Acid Sequence

To determine the primary structure of the C-terminal part of the molecule of chicken pepsinogen the tryptic, chymotryptic and thermolytic digest of the protein were investigated and peptides derived from this region were sought. These peptides permitted the following 21-residue C-terminal sequence to be determined: ...Ile-Arg-Glu-Tyr-Tyr-Val-Ile-Phe-Asp-Arg-Ala-Asn-Asn-Lys-Val-Gly-Leu-Ser-Pro-Leu-Ser.COOH. A comparison of this structure with the C-terminal sequential regions of the other acid proteases shows a high degree of homology between chicken pepsinogen and these proteases (e.g., the degree of homology with respect to hog pepsinogen and calf prochymosin is about 66%). Additional tryptic peptides, derived from the N-terminal part of the zymogen molecule whose amino acid sequence has been reported before, were also obtained in this study. This sequence was extended by two residues using an overlapping peptide. An ancillary result of this study was the isolation of tryptic peptides derived from other regions of the zymogen molecule.

scholarly journals A synthetic glucagon-like peptide-1 analog with improved plasma stability

1998 ◽  
Vol 159 (1) ◽  
pp. 93-102 ◽  
Author(s):  
U Ritzel ◽  
U Leonhardt ◽  
M Ottleben ◽  
A Ruhmann ◽  
K Eckart ◽  
...  
Keyword(s):  
Amino Acid ◽  
Plasma Insulin ◽  
Rat Plasma ◽  
Plasma Stability ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1

Glucagon-like peptide-1 (GLP-1) is the most potent endogenous insulin-stimulating hormone. In the present study the plasma stability and biological activity of a GLP-1 analog, [Ser]GLP-1(7-36)amide, in which the second N-terminal amino acid alanine was replaced by serine, was evaluated in vitro and in vivo. Incubation of GLP-1 with human or rat plasma resulted in degradation of native GLP-1(7-36)amide to GLP-1(9-36)amide, while [Ser]GLP-1(7-36)amide was not significantly degraded by plasma enzymes. Using glucose-responsive HIT-T15 cells, [Ser]GLP-1(7-36)amide showed strong insulinotropic activity, which was inhibited by the specific GLP-1 receptor antagonist exendin-4(9-39)amide. Simultaneous i.v. injection of [Ser]GLP-1(7-36)amide and glucose in rats induced a twofold higher increase in plasma insulin levels than unmodified GLP-1(7-36)amide with glucose and a fivefold higher increase than glucose alone. [Ser]GLP-1(7-36)amide induced a 1.5-fold higher increase in plasma insulin than GLP-1(7-36)amide when given 1 h before i.v. application of glucose. The insulinotropic effect of [Ser]GLP-1(7-36)amide was suppressed by i.v. application of exendin-4(9-39)amide. The present data demonstrate that replacement of the second N-terminal amino acid alanine by serine improves the plasma stability of GLP-1(7-36)amide. The insulinotropic action in vitro and in vivo was not impaired significantly by this modification.

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.

Radioiodinated Human Fibrinogen. In Vitro Effects of Increasing Iodination

1975 ◽  
Author(s):  
B. E. Ly ◽  
P. Kierulf
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Clotting Time ◽  
Terminal Amino Acid ◽  
Human Fibrinogen ◽  
Terminal Amino ◽  
In Vitro Effects ◽  
Polypeptide Chains

Fibrinogen preparations with increasing contents of iodine, ranging from 0.2 to 20 atoms of iodine per molecule fibrinogen, were obtained with the ICl method. Aggregation and shortening of the thrombin clotting time occurred when the content of iodine exceeded 3 atoms per molecule.Upon the action of thrombin, the increase in N-terminal glycine, reflecting fibrin formation, was almost identical in native and iodinated fibrinogen. At visible gelation, however, decreased amounts of N-terminal glycine were found in heavily iodinated fibrinogen, thus indicating enhanced fibrin polymerization. N-terminal analysis of heavily iodinated fibrinogen demonstrated a deficiency in N-terminal tyrosine concomitantly with the apparance of a new N-terminal amino acid, identified as mono-iodo-tyrosine.Polyacrylamide gel electrophoresis at pH 8.9 revealed an increase in mobility following extensive iodination, but no shift in the isoelectric point was observed upon isofocusing.Neither clottability nor the behaviour of fibrinogen and its subunit polypeptide chains on SDS-gel electrophoresis was affected by iodination.

A major Litomosoides carinii microfilarial sheath glycoprotein (gp22): amino terminal sequence and immunological studies with corresponding synthetic peptides

Parasitology ◽  
1991 ◽  
Vol 103 (3) ◽  
pp. 387-394 ◽  
Author(s):  
G. Bardehle ◽  
F. J. Conraths ◽  
F. Fahrenholz ◽  
M. Hintz ◽  
D. Linder ◽  
...  
Keyword(s):  
Amino Acid ◽  
Synthetic Peptides ◽  
General Pattern ◽  
Amino Sugar ◽  
Amino Acid Sequences ◽  
Terminal Amino Acid ◽  
Amino Terminal ◽  
Terminal Amino ◽  
Amino Terminal Sequence

The major glycoprotein of the sheath of Litomosoides carinii microfilariae (gp22) was analysed for its amino acid and amino sugar composition. It is rich in proline, glutamine/glutamic acid and glycine and contains (N-acetyl)galactosamine. The N-terminal amino acid sequence was determined up to position 37. It consists of a group of 6 repeats of the pentapeptide sequence methionine-glycine-proline-glutamine-proline with two minor modifications in repeats 3–6, while the first two repeats follow the general pattern more loosely. Identical N-terminal amino acid sequences were found in at least two other sheath polypeptides (33 kDa, 39 kDa). Antisera prepared against 3 overlapping synthetic peptides corresponding to the amino terminus of gp22 recognized different epitopes. They all reacted with identical patterns of sheath polypeptides. The antisera failed to recognize antigens of 4th-stage larvae of L. carinii. In contrast, cross-reacting epitopes were detected in other parasite stages. Antisera reacted with material surrounding embryos and microfilariae in the uterus of females, and caused patchy fluorescence on the sheath of blood-derived and in vitro-released microfilariae.

scholarly journals A C-terminal amino acid substitution in the γ-chain caused by a novel heterozygous frameshift mutation (Fibrinogen Matsumoto VII) results in hypofibrinogenaemia

2010 ◽  
Vol 104 (08) ◽  
pp. 213-233 ◽  
Author(s):  
Ayumi Haneishi ◽  
Kazuyoshi Yamauchi ◽  
Fumiko Terasawa ◽  
Toshiro Ito ◽  
Fumihiro Ishida ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Frameshift Mutation ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Glycosylation Site ◽  
Terminal Amino Acid ◽  
Ovary Cells ◽  
Terminal Amino

SummaryWe found a novel hypofibrinogenemia designated as Matsumoto VII (M-VII), which is caused by a heterozygous nucleotide deletion at position g.7651 in FGG and a subsequent frameshift mutation in codon 387 of the γ-chain. This frameshift results in 25 amino acid substitutions, late termination of translation with elongation by 15 amino acids, and the introduction of a canonical glycosylation site. Western blot analysis of the patient’s plasma fibrinogen visualised with anti-γ-chain antibody revealed the presence of two extra bands. To identify the extra bands and determine which of the above-mentioned alterations caused the assembly and/or secretion defects in the patient, 11 variant vectors that introduced mutations into the cDNA of the γ-chain or γ′-chain were transfected into Chinese hamster ovary cells. In vitro expression of transfectants containing γΔ7651A and γΔ7651A/399T (γΔ7651A with an amino acid substitution of 399Asn by Thr and a variant lacking the canonical glycosylation site) demonstrated a reduction in secretion to approximately 20% of the level seen in the transfectants carrying the normal γ-chain. Furthermore, results from other transfectants demonstrated that eight aberrant residues between 391 and 398 of the M-VII variant, rather than the 15 amino acid extension or the additional glycosylation, are responsible for the reduced levels of assembly and secretion of M-VII variant fibrinogen. Finally, the results of this study and our previous reports demonstrate that the fibrinogen γ-chain C-terminal tail (388–411) is not necessary for protein assembly or secretion, but the aberrant amino acid sequence observed in the M-VII variant (especially 391–398) disturbs these functions.

scholarly journals Energy-dependent protein folding: modeling how a protein folding machine may work

F1000Research ◽  
2021 ◽  
Vol 10 ◽  
pp. 3
Author(s):  
Harutyun Sahakian ◽  
Karen Nazarian ◽  
Arcady Mushegian ◽  
Irina Sorokina
Keyword(s):  
Molecular Dynamics ◽  
Protein Folding ◽  
Amino Acid ◽  
Dynamics Simulation ◽  
Terminal Amino Acid ◽  
Peptide Backbone ◽  
Terminal Amino ◽  
Energy Dependent

Background: Proteins fold robustly and reproducibly in vivo, but many cannot fold in vitro in isolation from cellular components. Despite the remarkable progress that has been achieved by the artificial intelligence approaches in predicting the protein native conformations, the pathways that lead to such conformations, either in vitro or in vivo, remain largely unknown. The slow progress in recapitulating protein folding pathways in silico may be an indication of the fundamental deficiencies in our understanding of folding as it occurs in nature. Here we consider the possibility that protein folding in living cells may not be driven solely by the decrease in Gibbs free energy and propose that protein folding in vivo should be modeled as an active energy-dependent process. The mechanism of action of such a protein folding machine might include direct manipulation of the peptide backbone. Methods: To show the feasibility of a protein folding machine, we conducted molecular dynamics simulations that were augmented by the application of mechanical force to rotate the C-terminal amino acid while simultaneously limiting the N-terminal amino acid movements. Results: Remarkably, the addition of this simple manipulation of peptide backbones to the standard molecular dynamics simulation indeed facilitated the formation of native structures in five diverse alpha-helical peptides. Steric clashes that arise in the peptides due to the forced directional rotation resulted in the behavior of the peptide backbone no longer resembling a freely jointed chain. Conclusions: These simulations show the feasibility of a protein folding machine operating under the conditions when the movements of the polypeptide backbone are restricted by applying external forces and constraints. Further investigation is needed to see whether such an effect may play a role during co-translational protein folding in vivo and how it can be utilized to facilitate folding of proteins in artificial environments.

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