Limited hydrolysis of the polypeptide chain elongation factor Tu by trypsin. Isolation and characterization of the polypeptide fragments.

Limited hydrolysis of the polypeptide chain elongation factor Tu by trypsin. Isolation and characterization of the polypeptide fragments. Research Abstract Details 

Research Abstract Table of Contents

Jump to the:

Limited hydrolysis of the polypeptide chain elongation factor Tu by trypsin. Isolation and characterization of the polypeptide fragments. Abstract Text:

K Arai,S Nakamura,T Arai,M Kawakita,Y Kaziro,

The digestion of EF-Tu-GDP (or EF-Tu-GTP) by trypsin [EC 3.4.21.4] under native conditions has been shown to proceed through two different and characteristic stages. 1. In the first phase, the protein is transformed into a fragment (Fragment A) with a molecular weight of 39,000 by exposure to trypsin for a relatively short period of time. Fragment A is unable to catalyze the binding of aminoacyl-tRNA to ribosomes. The ability to promote two partial steps of the binding reaction, i.e., formation of the aminoacyl-tRNA-EF-Tu-GTP ternary complex as well as the methanol-stimulated, ribosome dependent GTPase reaction, was rapidly destroyed. On the other hand, the ability to interact with guanine nucleotides as well as EF-Ts survived well during prolonged digestion. 2. In the second phase of digestion, a nick is introduced in Fragment A to yield two subfragments (Fragments B and C). These two fragments exist as a hybrid molecule which migrates as a single peak on a Sephadex G-75 column, and which dissociates into Fragments B and C only in the presence of 6 M guanidine hydrochloride or 5% sodium dodecyl sulfate. The molecular weights of Fragments B and C, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, were 22,000 and 12,000 respectively. The hybrid molecule still retained one mole of bound guanine nucleotide and was resistant to further tryptic digestion. 3. Three sulfhydryl groups of EF-Tu were found to be present in Fragment B, both by amino acid analysis of the purified fragments and also by electrophoresis of tryptic digests labeled with N-ethyl[14C]maleimide. 4. The tryptic digestion of EF-Tu-GDP (or EF-Tu-GTP) labeled with N-(1-anilinonaphthyl-4)maleimide (ANM) at SH2 (the second SH), caused a 30% decrease in the fluorescence emission during the first rapid phase of digestion. This indicates that destruction of the hydrophobic environment near SH2 of EF-Tu occurred in the early phase of tryptic digestion. 5. The kinetic studies on the reaction of ANM with EF-Tu before and after tryptic digestion indicated that both Fragment A and the hybrid molecule reacted with ANM in the presence of GTP three to four times more rapidly than in the presence of GDP. Thus, it appears that the ability to induce conformational transition near SH2 by a change of nucleotide ligands is still retained in the hybrid molecule consisting of Fragments B and C.

Limited hydrolysis of the polypeptide chain elongation factor Tu by trypsin. Isolation and characterization of the polypeptide fragments. Publishing Authors By Initials

K Arai,S Nakamura,T Arai,M Kawakita,Y Kaziro,

For similar enzymes and coenzymes: enzymes: hydrolases: peptide hydrolases: endopeptidases: serine endopeptidases: trypsin research abstracts see: enzymes and coenzymes: enzymes: hydrolases: peptide hydrolases: endopeptidases: serine endopeptidases: trypsin research

PUBMED ID PMID:

MEDLINE DATE:

Limited hydrolysis of the polypeptide chain elongation factor Tu by trypsin. Isolation and characterization of the polypeptide fragments. Journal Published:

PUBLICATION TYPE: Journal Article

Journal: Journal of biochemistry

VOLUME: 79

Page Numbers: 69-83

Journal Abbreviation: J. Biochem.

ISSN: 0021-924X

DAY: 19

MONTH: Jan

YEAR: 1976

Limited hydrolysis of the polypeptide chain elongation factor Tu by trypsin. Isolation and characterization of the polypeptide fragments. Information

Number of References:

LANGUAGE: eng

NlmUniqueID: 376600

Limited hydrolysis of the polypeptide chain elongation factor Tu by trypsin. Isolation and characterization of the polypeptide fragments. Keywords Mesh Terms:

KEYWORDS: Trypsin

MESH TERMS: metabolism

Chemical & Substance for Abstract: Limited hydrolysis of the polypeptide chain elongation factor Tu by trypsin. Isolation and characterization of the polypeptide fragments. Information

Substance Name: Trypsin

Registry Number: EC 3.4.21.4

Grant and Affiliation Information for Limited hydrolysis of the polypeptide chain elongation factor Tu by trypsin. Isolation and characterization of the polypeptide fragments.

AFFILIATION:

Country: JAPAN

AGENCY:

GRANT:

ACRONYM:

MEDLINETA: J Biochem

REFSOURCE:

DATABASENAME:

ACCESSION NUMBER:

Number Hits: 0

Limited hydrolysis of the polypeptide chain elongation factor Tu by trypsin Isolation and characterization of the polypeptide fragments Related Publications

• A new extragenic suppressor of cya mutation. Mutant cyclic AMP receptor protein with an increased affinity for cyclic AMP.
• A new protein factor required for polypeptide elongation in mammalian tissues.
• Binding of aminoacyl-tRNA to ribosomes promoted by elongation factor Tu. Studies on the role of GTP hydrolysis.
• Conformational transition of polypeptide chain elongation factor G as determined by electron spin resonance.
• Conformational transitions of polypeptide chain elongation factor Tu. I. Studies with hydrophobic probes.
• Conformational transitioons of polypeptide chain elongation factor Tu. II. Further studies by electron spin resonance.
• Coordination of levels of elongation factors Tu, Ts, and G, and ribosomal protein SI in Escherichia coli.
• Distribution of the low molecular weight form of eukaryotic elongation factor 1 in various tissues.
• Formation of a binary complex between elongation factor G and guanine nucleotides.
• Further studies on the interaction of the polypeptide chain elongation factor G with guanine nucleotides.
• Further studies on the properties of the polypeptide chain elongation factors Tu and Ts: hydrogen-tritium exchange, optical rotatory dispersion, and intrinsic fluorescence.
• Interactions between elongation factor tu-guanosine triphosphate and ribosomes and the role of ribosome-bound transfer RNA in guanosine triphosphatase reaction.
• Involvement of 30S ribosomal protein S1 in poly(U)-directed polyphenylalanine synthesis.
• Limited hydrolysis of the polypeptide chain elongation factor Tu by trypsin. Isolation and characterization of the polypeptide fragments.
• Mechanism of action of chromomycin A3. 1. Inhibition of nucleic acid metabolism in Bacillus subtilis cells.
• Mechanism of the ribosome-dependent uncoupled GTPase reaction catalyzed by polypeptide chain elongation factor G.
• Proceedings: Polypeptide chain elongation in bacterial and mammalian systems.
• Properties and function of the sulfhydryl group in the polypeptide chain elongation factor G from E. coli.
• Purification and properties of polypeptide chain elongation factor-1 beta gamma from pig liver.
• Purification and properties of polypeptide chain elongation factor-1alpha from pig liver.
• Purification and properties of the polypeptide chain release factor (RF-4) from an extreme thermophile, Thermus thermophilus HB8.
• Purification of tubulin from bovine brain and its interaction with guanine nucleotides.
• Role of GTP in the assembly of microtubules.
• Studies on amino-acyl-sRNA synthetases from Pseudomonas and methionyl-sRNA. I. Partial purification of leucyl-, phenylalanyl-, tyrosyl-, and synthetases.
• Studies on polypeptide elongation factor 2 from pig liver. I. Purification and properties.
• Studies on polypeptide elongation factor 2 from pig liver. III. Interaction with guanine nucleotides in the presence and absence of ribosomes.
• Studies on the polypeptide elongation factors form E. coli. VI. Characterization of sulfhydryl groups in EF-Tu and EF-Ts.
• Studies on the polypeptide elongation factors from E. coli. IV. Crystalline Tu-GTP, Tu-Gpp(CH2)p, and phenylalanyl-tRNA-Tu-GTP complex.
• Studies on the polypeptide elongation factors from E. coli. V. Properties of various complexes containing EF-Tu and EF-Ts.
• Translocation reaction promoted by polypeptide chain elongation factor-2 from pig liver.