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H Fukutome

Publications and source records attributed to H Fukutome.

16 recordsLinked to original sources
PubMed1984

A highly sensitive photometric method for proton release or uptake: difference protometry.

A highly sensitive quantitative method was developed to detect protons released or taken up upon ligand binding. A small change in pH due to proton release or uptake was detected by measuring the difference in the absorbance of a pH indicator upon ligand addition. Owing to the difference detection of protons, the uncertainty of pH due to CO2 dissolution and unknown buffering capacities of sample solutes could be compensated with easy manipulations. Precise calibration of the absolute amount of protons could also be made very easily. The amount of protons measurable by the method is as small as 0.5 nmol that is 10 to 30 times more sensitive than the pH-stat method. We measured the Mg2+ ion-induced proton releases of ADP to confirm the accuracy and reliability of the method and of Escherichia coli ribosomes to show the improvement in sensitivity. The method is useful for protometric studies of biomolecules that are difficult to obtain in large amount.

Acid-Base Equilibrium↗
PubMed1984

Mg2+-induced proton release from Escherichia coli ribosome and ribosomal RNA.

Escherichia coli ribosome released protons upon addition of Mg2+. The Mg2+-induced proton release was studied by means of the pH-stat technique. The number of protons released from a 70 S ribosome in the Mg2+ concentration range 1-20 mM was about 30 at pH 7 and 7.6, and increased to about 40 at pH 6.5. The rRNA mixture extracted from 70 S ribosome showed proton release of amount and of pH dependence similar to those of the 70 S ribosome but the ribosomal protein mixture released few. This indicates that rRNA is the main source of the protons released from ribosome. The pH titration of rRNA showed that the pKa values of nucleotide bases were downward shifted upon Mg2+ binding. This pKa shift can account for the proton release. The Scatchard plots of proton release from rRNA and ribosome were concave upward, showing that the Mg2+-binding sites leading to proton release were either heterogeneous or had a negative cooperativity. A model assuming heterogeneous Mg2+-binding sites is shown to be unable to explain the proton release. Electrostatic field effect models are proposed in which Mg2+ modulates the electrostatic field of phosphate groups and the potential change induces a shift of the pKa values of bases that leads to the proton release. These models can explain the main features of the proton release.

Binding Sites↗
PubMed1983

Magnesium ion induced proton release as a probe for the polyelectrolytic structure of ribosomal RNAs and subunits.

E coli ribosomes and rRNA's released 20 to 50 protons upon jump of magnesium ion concentration from 1 mM to 20 mM. The Mg2+-induced proton release was measured separately for 16S rRNA, 23S rRNA, 30S subunit, and 50S subunit by a new spectrophotometric method that had a much better sensitivity than the pH-stat method. The proton release from the subunits and rRNA's were similar in the number of protons, the pH dependence that had a minimum at neutral pH, and the upward concaveness of the Scatchard plot. From these results, the main source of protons in ribosomal subunits was assigned to nucleotide bases of rRNA's that showed a downward pKa shift upon Mg2+-ion binding. The subunits and rRNA's, however, differed in the proton release. 16S rRNA released protons somewhat more effectively than 23S rRNA, while 30S subunit released protons 2 to 5 times more effectively than 50S subunit. The marked difference between the two subunits suggest that ionizable bases in 16S and 23S rRNA's are covered and their pKa values are shifted by ribosomal proteins to different extents. The association of 30S and 50S subunits induced little proton release, showing that few ionizable groups with pKa near neutral pH are involved in the association. E. coli tRNA and poly U also showed Mg2+-induced proton release. The amounts of protons released from rRNA's, tRNA, and poly U were roughly proportional to the amount of bases not hydrogen bonded. The Mg2+-induced proton release from the natural and synthetic RNA's can be explained by the electrostatic field effect of polyphosphate backbones on bases not hydrogen bonded, as proposed in a previous paper. It also reflects the conformational structure of each RNA molecule.

Escherichia coli↗
PubMed1981

Conformational studies of Escherichia coli ribosomes with the use of acridine orange as a probe.

The interaction of E. coli vacant ribosomes with acridine orange (AO) was studied, to obtain conformational information about rRNAs in ribosomes. Acridine orange binds to an RNA in two different modes: cooperative outside binding with stacking of bound AO's and intercalation between nucleotide bases. Free 16S and 23S rRNAs have almost identical affinities to AO. At 1 mM Mg2+, AO can achieve stacking binding on about 40% of rRNA phosphate groups. The number of stacking binding sites falls to about 1/3 in the 30S subunit in comparison with free 16S rRNA. In the 50S subunit, the number of stacking binding sites is only 1/5 in comparison with free 23S rRNA. Mg2+ ions are more inhibitory for the binding of AO to ribosomes than to free rRNAs. The strength of stacking binding appears to be more markedly reduced by Mg2+ in active ribosomes than in rRNAs. "Tight couple" 70S particles are less accessible for stacking binding than free subunits. The 30S subunits that have irreversibly lost the capability for 70S formation under low Mg2+ conditions have an affinity to AO that is very different from that of active 30S but similar to that of free rRNA, though the number of stacking binding sites is little changed by the inactivation. 70S and 30S ribosomes with stacking bound AO's have normal sedimentation constants, but the 50S subunits reversibly form aggregates.

Acridine Orange↗
PubMed1975

Substituent effects on substrate activation and Michaelis-Menten Kinetic parameters in the alpha-chymotrypsin-catalyzed hydrolysis of phenyl acetates.

The effects of substituents on the steady state and pre-steady state kinetics in alpha-chymotrypsin [EC 3.4.21.1]-catalyzed hydrolysis were studied using substituted phenyl acetates. In the steady state hydrolysis, substrate activation, which had been observed and studied previously for p-nitrophenyl acetate, was also observed for p-bromo, p-chloro-, and m-methylphenyl acetates. Little activation was observed for p-acetyl-, m-nitro-, p-methyl-, and p-methoxyphenyl acetates. Addition of p-dichlorobenzene increased kcat for all substrates examined and greatly diminished the substrate activation for the activatable substrate(s) to activator binding site(s). The value of kcat decreased in accordance with increase of the sigma-value of substituents. On the other hand, kcat/Km (app) showed an opposite sigma- dependence, as was previously observed. In pre-steady state measurements, little burst was observed for more electron-donating substituents than m-nitro. The sigma dependence of kcat is apparently not consistent with the prediction derived from that of kcat/Km (app) on the basis of the usual two-step mechanism with a common acetyl-enzyme intermediate.

Binding Sites↗