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Y Kuriki

Publications and source records attributed to Y Kuriki.

At least 19 recordsLinked to original sources
PubMed1996

Synthesis of alpha-helix-forming peptides by gene engineering methods and their characterization by circular dichroism spectra measurements.

Two kinds of peptides which were considered to form alpha-helices were designed and characterized. One was "alpha(3)-peptide' with 21 residues comprising three repeats of the seven-residue sequence Leu-Glu-Thr-Leu-Ala-Lys-Ala. This peptide appeared to be amphipathic due to a hydrophobic surface of Leu residues and a hydrophilic surface of Lys and Glu residues, thus forming a bundle structure. The other was "alpha(3)-GPRRG-alpha(3) peptide' with 47 residues in which two alpha(3)-peptides were connected by the five-residue sequence Gly-Pro-Arg-Arg-Gly. The genes encoding these peptides were fused to the adenylate kinase gene via a methionine codon. The resulting fused protein was expressed as an inclusion body, and the peptides were purified after cleavage with BrCN. The stability of the peptides in various buffers was then examined by measuring their circular dichroism spectra. The alpha(3)-peptide showed concentration-dependent stabilization of the alpha-helix. Sedimentation equilibrium ultracentrifugation indicated that it formed a bundle structure composed of four polypeptide chains, and a dimer intermediate during oligomerization was also detected by analytical gel-filtration. The stability of the alpha(3)-peptide was decreased by shifting the pH to 2 or 12, due to electrostatic repulsion of charged residues. Thus, the alpha(3)-peptide was stabilized by increasing the ionic strength, particularly in acidic or alkaline buffer, through the masking of the repulsion by high salt concentration. In buffer of neutral pH and a high salt concentration, the alpha(3)-peptide at high concentration formed visible aggregates, due possibly to the exposed hydrophobic surfaces of the alpha-helical bundles. On the other hand, alpha(3)-GPRRG-alpha(3) peptide did not show concentration-dependent reversible dissociation and association. It was shown to exist as a trimer even at low concentration, indicating very tight association of the alpha(3)-GPRRG-alpha(3) peptide. In contrast to the alpha(3)-peptide, the alpha(3)-GPRRG-alpha(3) peptide was very stable at various pH values and salt concentrations. This seemed to be due to increased hydrophobic interactions resulting from the increase in the number of seven-residue repeats from three to six, even though each group of three repeats was separated by a five-residue sequence.

Amino Acid Sequence↗
PubMed1993

Temperature-sensitive amber suppression of ompF'-'lacZ fused gene expression in a supE mutant of Escherichia coli K12.

In an ompF'-'lacZ fusion system carried by the open reading frame vector pORF1 in a supE mutant of Escherichia coli K12, read-through of an amber codon was decreased at temperatures higher than 40 degrees C. This effect of temperature was dependent on the nucleotide sequence surrounding the amber codon, which was inserted into a site between the ompF and lacZ cistrons. Upon a temperature shift-up from 30 to 42 degrees C, beta-galactosidase synthesis directed by this fusion showed a transient arrest.

Bacterial Outer Membrane Proteins↗
PubMed1990

A nucleotide sequence in the translation start signal region is involved in heat shock-induced translation arrest in Escherichia coli.

In Escherichia coli synthesis of several proteins is transiently depressed upon heat shock treatment. A comparison of nucleotide sequences of the genes encoding these proteins revealed the occurrence of a consensus sequence, GAGGAA(N)3-6ATG, in their translation start signal region. To examine whether this sequence is involved in heat shock-induced depression of protein synthesis, DNA segments corresponding to this region of four of these genes, fusA, rpoB, glnS, and pheT, were synthesized, and each of them was fused in frame with the lacZ gene on the open reading frame vector pORF1. The effect of heat shock on the synthesis of beta-galactosidase encoded by these fused genes was then studied in E. coli. It was thus found that beta-galactosidase synthesis starting from the inserted translation start signal was arrested transiently upon temperature shift-up from 30 to 42 degrees C. I conclude that the heat shock-induced depression of gene expression is an event taking place at the initiation of translation.

Base Sequence↗
PubMed1989

Translational repression of TEM beta-lactamase synthesis as a response of Escherichia coli to heat shock.

As a result of a temperature shift-up from 30 degrees C to 42 degrees C, beta-lactamase synthesis in Escherichia coli carrying pBR322 ceased transiently, even though the level of beta-lactamase mRNA was not altered. pBR328-directed pre-beta-lactamase synthesis in an in vitro transcription-translation coupled system was also repressed by incubation at the higher temperature. Translation of the lacZ sequence from the amp translation start signal, inserted into the open reading frame vector pORF1, was also repressed transiently upon the temperature shift-up. Pre-heating of the in vitro coupled system at 45 degrees C specifically reduced its capacity for pre-beta-lactamase synthesis. This capacity was restored by the addition of a 160,000 x g supernatant prepared from E. coli grown at 30 degrees C, but not by the supernatant from the cells incubated at 42 degrees C. These and other results indicate that (i) the 160,000 x g supernatant contains a heat-labile protein(s) that is required for efficient initiation of the translation of pre-beta-lactamase mRNA, and (ii) the heat shock-induced repression of beta-lactamase synthesis is due to inactivation of the protein(s) in the 160,000 x g supernatant.

Ampicillin↗
PubMed1989

The translation start signal region of TEM beta-lactamase mRNA is responsible for heat shock-induced repression of amp gene expression in Escherichia coli.

pBR322 contains the amp gene encoding beta-lactamase. When Escherichia coli carrying this plasmid is exposed to heat shock, beta-lactamase synthesis is repressed transiently at the translational level. To identify the DNA element responsible for this translational repression, DNA segments containing the translation start region of the amp gene were excised from pAT153 and fused in frame with the lacZ reading frame in the open reading frame vector pORF1. These constructs were introduced into E. coli, and the effect of heat shock of the cells on the synthesis of beta-galactosidase starting from the amp start codon was examined. As is the case for pBR322-encoded synthesis of beta-lactamase, the synthesis of beta-galactosidase encoded by the fused genes also ceased transiently upon heat shock. It is concluded that the heat shock-induced repression of the amp gene occurs at the initiation step of translation. As far as the present study is concerned, the minimum DNA segment responsible for the repression is AT TGA AAA AGG AAG AGT ATG AG, which includes the Shine-Dalgarno sequence (AAGGA) and the initiation codon (ATG).

Base Sequence↗
PubMed1987

Heat shock inactivates a supernatant factor(s) specifically required for efficient expression of the amp gene in Escherichia coli.

A 160,000 x g supernatant of E. coli extract prepared from cells grown at 30 degrees C stimulated specifically the expression of the amp gene on pBR322 and pBR328 in an in vitro gene expression system from E. coli. This activity of the supernatant was markedly reduced when the cells were exposed to 42 degrees C for 30 min prior to preparing the supernatant. These results are consistent with the view that heat shock-induced repression of the amp gene expression is due to inactivation of a supernatant factor(s) required for effective expression of the amp gene.

Ampicillin Resistance↗
PubMed1987

Requirement of a heat-labile factor(s) for in vitro expression of the amp gene of pBR322.

The amp gene of pBR328 and pBR322 was expressed less efficiently at 45 than at 30 degrees C in an in vitro coupled transcription-translation system of Escherichia coli. Preincubation of S30 extract at 45 degrees C reduced specifically its ability to express the amp gene at 30 degrees C, indicating inactivation of a factor(s) required for efficient expression of the amp gene.

Acetyltransferases↗
PubMed1986

Stimulation in vitro of expression of the amp gene of pBR322 by soluble protein fractions isolated from E. coli.

Two soluble protein fractions isolated from E. coli were found to be required for efficient expression of the amp gene of pBR322 in an in vitro coupled transcription-translation system consisting of unwashed ribosomes and a polyethylene glycol-treated S30 extract from E. coli. Both fractions stimulated the pBR322-directed synthesis of the amp gene product, pre-beta-lactamase, in the in vitro system. However, pBR322-directed RNA synthesis was stimulated only by one fraction. It is therefore likely that one fraction enhances the transcription of the amp gene and the other is involved mainly in stimulation of the translation of pre-beta-lactamase mRNA.

Bacterial Proteins↗
PubMed1980

Calorimetric studies of ligand-induced modulation of calcium adenosine 5'-triphosphatase from sarcoplasmic reticulum.

The enthalpy change (delta H degrees ') associated with the binding of Mg2+ to the sarcoplasmic reticulum calcium adenosine 5'-triphosphatase [(Ca2+)ATPase] is -76 kcal/mol. The affinity constant for Mg2+ obtained from calorimetric measurements agrees with the Km value for Mg2+ in the phosphorylation of the enzyme by inorganic phosphate (Pi). The delta H degrees ' of binding of Pi to the enzyme is -23.5 kcal/mol, and the affinity constant for Pi obtained from the calorimetry also agrees with the Km value for Pi in the phosphorylation reaction. delta H degrees ' of Mg2+ binding is reduced to -35 kcal/mol in the presence of either 20 mM Pi or 1.2 mM Ca2+ without a significant change in the affinity of the enzyme for Mg2+. delta H degrees ' of Pi binding to the enzyme drops to -8.5 kcal/mol in the presence of 10 mM Mg2+ without a significant change in the affinity of the enzyme for Pi. On the other hand, the presence of Ca2+ does not affect the delta H degrees ' for the binding of the substrate analogue 5'-adenylyl beta,gamma-imidodiphosphate [App(NH)p], and the presence of this analogue does not affect the delta H degrees ' for Ca2+ binding. The results suggest a model in which a conformational change, largely controlled by Mg2+ binding to the enzyme, leads to the formation of the covalent phosphoprotein intermediate.

Animals↗
PubMed1976

Inhibition of (Na+, K+)adenosine triphosphatase and its partial reactions by quercetin.

The bioflavonoid, quercetin, inhibited the (Na+, K+)adenosine triphosphatase purified from the electric organ of electric eel (Electrophorus electricus) or from lamb kidney. An analysis of its mode of action revealed that the formation of phosphoenzyme from Pi but not from ATP was inhibited. Quercetin increased the amount of ADP-sensitive phosphoenzyme (E1--P), indicating an inhibition of the conversion of E1--P to the ADP-insensitive form (E2--P). The rate of dephosphorylation of the phosphoenzyme formed from ATP was slowed by quercetin. These results suggest that quercetin inhibits the formation of E2--P from either Pi or E1-P as well as the hydrolysis of the phosphoenzyme. Its mode of action is therefore different from that of ouabain and other inhibitors of the Na+, K+)adenosine triphosphatase.

Adenosine Triphosphatases↗
PubMed1976

Calorimetric studies of the interaction of magnesium and phosphate with Na+, K+) ATPase: evidence for a ligand-induced conformational change in the enzyme.

The phosphorylation of (Na+, K+)ATPase from the electric organ of the electric eel is dependent on Mg2+. The amount of phosphoenzyme formed was increased by K+ and decreased by Na+. Kinetic analyses indicate that a ternary complex of ATPase, Pi and Mg2+ is formed prior to phosphorylation of the protein. Calorimetric studies revealed extraordinarily large enthalpy changes associated with the binding of Mg2+ (-49 kcal/mol) and of Pi (-42 kcal/mol), indicating a thermodynamically significant conformational change in the enzyme. The dissociation constant for the binding of Mg2+ and Pi derived from calorimetric measurements is in good agreement with the value obtained from the kinetic studies. These results indicate that ion binding induces a conformational change in the enzyme which is a prerequisite for phosphorylation by Pi.

Adenosine Triphosphatases↗