Tatsuya Samejima

Optical rotatory dispersion and circular dichroism of rice dwarf virus ribonucleic acid

Abstract The optical rotatory dispersion and circular dichroism of a double-stranded ribonucleic acid from rice dwarf virus have been measured. The RNA exhibited multiple Cotton effects with two peaks (282 and 228 mμ) and two troughs (250 and 220 mμ) between 210 and 350 mμ, which resembled the profiles of normal RNAs. The circular dichroic spectrum showed an intense positive band at 260 mμ, close to the cross-over point of the Cotton effect. The magnitude of the Cotton effect and the intensity of the circular dichroic band are the greatest ever observed in nucleic acids. Heat denaturation drastically reduces the magnitude of the 282 mμ peak and 250 mμ trough and causes shifts to the red of their positions. The denaturation curve measured by the change in height of the 282 mμ peak gives a melting temperature of 79 °C in 1/100 standard saline citrate solution, with a sharp transition region which provides evidence of a doublestranded helical structure. The melting temperature measured using the circular dichroic band at 260 mμ was 79 °C, in good agreement with the value 81 °C from the hyperchromicity at 258 mμ.

Purification and properties of glutamine synthetase from Bacillus stearothermophilus

Abstract Glutamine synthetase (EC 6.3.1.2) has been purified from Bacillus stearothermophilus. The molecular weight of the enzyme was found to be 630 000, and that of a component obtained on treating the enzyme with 4 M urea or 1% sodium dodecyl-sulfate plus 10 mM 2-mercaptoethanol 540 000 or 500 000, respectively, suggesting that the enzyme consists of 12 subunits. The enzyme requires divalent cations for activity, Mg2+ being the most effective activator. The pH and temperature optima in the presence of Mg2+ or Mn2+ are 7.3 or 6.5 and 70 or 75 °C, respectively. The enzyme is inactivated on exposure to 70 °C, but the inactivation is partially protected by Mg2+ (Mn2+ or glutamate) and completely by Mg2+ (Mn2+), NH4Cl and glutamate. Thermodynamic quantities for the enzyme reaction show a conformational transition at 58 °C. Glycine, alanine, serine, tryptophan, histidine, AMP and CTP inhibit the enzyme in the presence of Mg2+ or Mn2+. The inhibition of the Mg2+- or Mn2+-activated enzyme by these compounds seems to be cumulative, except for the combined effects of amino acids on the Mn2+-activated enzyme. Circular dichroism analyses of the enzyme show an α-helix, s-structure and unfolded conformation. Addition of Mg2+ or Mn2+ results in an increase of the α-helix content accompanied by a decrease of the unfolded conformation content.

Optical rotatory dispersion and circular dichroism of sulfur-containing nucleosides and nucleotide and of the ribonuclease-thionucleotide complex

Abstract Optical rotatory dispersion (ORD) and circular dichroism (CD) of sulfur-containing nucleosides (4-thiouridine, 6-thioguanosine and 6-thioinosine) and a nucleotide (4-thiouridine 2′(3′)-monophosphate) as well as a complex between ribonuclease (EC 2.7.7.16) and the nucleotide were measured in the near-ultraviolet and ultraviolet regions. All thionucleosides and the nucleotide displayed multiple Cotton effects which were correlated with their absorption maxima. Their CD spectra corresponded to the multiple Cotton effects except for 6-thioguanosine and 6-thioinosine which exhibited such low intensities that their bands could not be detected. The Cotton effects and CD bands were displaced toward shorter wavelengths upon alkaline treatment. The ribonuclease-thionucleotide complex showed ORD and CD near the absorption maximum at 331 mμ of the nucleotide quite different from the curves computed on the basis of the constituents. This suggests that the change in dissymmetry in the nucleotide molecule occurred on the formation of the complex. The ultraviolet ORD and CD were almost the same as those of the constituents, indicating the absence of any drastic conformational change in the protein molecule accompanying complex formation.

The conformational changes of catalase molecule caused by ligand molecules

Abstract Circular dichroism and optical rotatory dispersion of bovine liver catalase have been measured over wide wavelength range of 200–650 mμ. Native catalase showed a small negative CD band in the Soret absorption region, which corresponded to a small negative Cotton effect. The denaturation with acid (pH 3.0), alkali (pH 12.0) and 8 M urea caused almost complete loss of this Soret CD band and the Cotton effect. Addition of the ligand molecules such as KCN and NaN3 to native catalase caused an appearance of new small CD band near 450 mμ. In the near-ultraviolet region native molecule exhibited a CD band at 285 mμ, while the denaturations caused blue shift of the band with a large enhancement in intensity at pH 12.0 as well as diminutions at pH 3.0 and by 8 M urea. The liganded catalase molecules showed a new band near 340 mμ in addition to the CD band at 280–290 mμ. These CD bands may be related to aromatic amino acids and also their interaction with the heme groups in the molecules. In the ultraviolet region all proteins except for the denatured one with 8 M urea exhibited two intense negative CD bands and a deep trough of Cotton effect at 233 mμ, which indicated the presence of α-helical structure in their molecules. It was concluded from these data that the helix content of the native molecule was about 50%, whereas the acid- and alkali-denatured molecules showed the less helical content of 20–25% and 15–20%, respectively. Treatment with 8 M urea seemed to cause complete loss of helical conformation in the molecule. However, addition of ligands (KCN and NaN3) to the heme groups of catalase caused reduction of helical content in the molecule whose enzymic activity was completely lost. The estimated helical content was approx. 30%.

On the conformation of porcine ceruloplasmin

Abstract The ultracentrifugal behavior and optical properties of porcine ceruloplasmin have been measured to elucidate the conformation of the protein molecule. The molecular weights of apo- and reduced porcine ceruloplasmin are almost identical with that of native protein, i.e., about 150,000, whereas their sedimentation velocities are slightly lower. No dissociation of the protein molecule into subunits occurs upon removal or reduction of the copper atoms, while minor unfolding seems to occur in the apoprotein and further extended unfolding occurs in the reduced protein molecule. Measurements of CD and ORD 4 revealed that native ceruloplasmin shows a small positive CD band at 590 mμ and a negative one at 450 mμ, whereas these bands disappear completely upon removal or reduction of the copper atoms. The ultraviolet CD and ORD data indicate the presence of β-structure in the protein moiety of native, apo- and reduced ceruloplasmin; a distinct negative band at 219 mμ and a positive one at 199 mμ are observed in the CD spectra, while the Cotton effect shows a trough at 230 mμ and a peak at 208 mμ, with crossover at 220 mμ. From these optical assays it is estimated that ceruloplasmin is a composite of β-structures and random coil. This is also evidenced by infrared spectroscopic analysis.

Authentic and recombinant bilirubin oxidases are in different resting forms.

Myrothecium verrucaria bilirubin oxidase expressed in Aspergillus oryzae is in a resting form different from that of the authentic bilirubin oxidase, but reaches the resting form of the authentic enzyme after one cycle of reduction and reoxidation with dioxygen as shown by the absorption and electron paramagnetic resonance spectra.

Comparison of the γ-crystallins isolated from eye lenses of shark and carp Unique secondary and tertiary structure of shark γ-crystallin

γ‐Crystallin isolated from the shark of cartilaginous fishes was compared with the cognate γ‐crystallin from the carp of bony fishes. Distinct differences in amino acid compositions, primary, secondary and tertiary structures were found. The most salient features of shark γ‐crystallin lie in the fact that this crystallin possessed a significant α‐helical structure in the peptide backbone as revealed by circular dichroism study, in contrast to those orthologous γ‐crystallins from other vertebrate species including bony fishes which all show a predominant β‐sheet secondary structure. The tertiary structure as reflected in the intrinsic microenvironments of various aromatic amino acids in the native crystallins also shows unambiguous differences between these two classes of γ‐crystallins. N‐Terminal sequence analysis corroborates the structural differences between shark and carp γ‐crystallins. γ‐Crystallin from the more primitive shark seems to be more in line with the main evolutionary phylogeny leading to the modern mammalian γ‐crystallin.

On the specific association of porcine erythrocyte catalase caused by formation of disulfide cross-links

Porcine erythrocyte catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase, EC 1.11.1.6) has been purified from porcine blood by DEAE-cellulose column chromatography, ammonium sulfate fractionation and CM-cellulose column chromatography. The purified enzyme was found to associate into larger molecules than the native one when it was stored at 4degrees C for more than one week. The associated molecules can be detected by gel filtration on a Bio-gel A-1.5 m column and disc gel electrophoresis as well as ultracentrifugal analysis. Molecular weights of the associated catalase molecules were about 500 000, 750 000, 1 000 000 and so forth estimated by gel filtration and disc gel electrophoresis, corresponding to dimer, trimer and tetramer respectively, of a native molecule (monomer) with a molecular weight of about 250 000. The association of catalase molecules is found to be time-dependent and to proceed seemingly from monomer through dimer as an intermediate. From the effects of several thiol reagents or reducing reagents on the association process and spectrophotometric titration of SH groups, it is inferred that this specific association of procine erythrocyte catalase is caused by formation of intermolecular disulfide cross-links due to air oxidation of SH groups in the protein moiety.

Interaction of 6-thioguanylic acid homologues with ribonuclease T1 Spectrophotometric and gel filtration studies

The interaction between ribonuclease T1 (RNAase T1) (EC 2.7.7.26) and 6-thioguanylic acid homologues, characterized by an absorption band centered at 342 nm, was compared with that between RNAase T1 and guanylic acids. The difference absorption spectra of the complexes of RNAase T1 with thioguanosine 2′(3′)-phosphate, thioguanosine 2′(3′),5′-diphosphate and thioguanosine 2′,3′-cyclic phosphate 5′-phosphate were similar in shape to those of the complexes with guanosine 2′(3′)-phosphate and guanosine 2′(3′),5′-diphosphate, but were at about 60–80 nm longer wavelengths, with maxima at 326 and 351 nm at pH 5.6. The gel filtration studies demonstrated that stoichiometric complexes were formed between the enzyme and these thioguanylic acids. These results seem to indicate that the modes of interaction of RNAase T1 with these thionucleotides are similar to those with guanylic acids. The circular dichroism band (negative) of thioguanosine 2′(3′),5′-diphosphate around 342 nm increased its intensity when it was complexed with RNAase T1, suggesting conformational changes in the nucleotide and/or interaction between the base moiety of the nucleotide and chromophores of the enzyme. The order of the affinity of guanosine and thioguanosine derivatives for RNAase T1, estimated from spectrophotometric and gel filtration studies, was as follows: guanosine 2′(3′)-phosphate, guanosine 2′(3′),5′-diphosphate > thioguanosine 2′(3′)-phosphate, thioguanosine 2′(3′),5′-diphosphate, thioguanosine 2′,3′-cyclic phosphate 5′-phosphate > thioguanosine 5′-phosphate > thioguanosine 2′,3′-cyclic phosphate, thioguanosine. The present data suggest that the enzyme possesses a subsite which is responsible for specific interaction with the 5′-phosphoryl groups of substrates or substrate analogues.

The change in optical activity of amino acid-specific Escherichia coli transfer RNA containing 4-thiouridylate by chemical modifications

Abstract Circular dichroism (CD) spectra of five species of 4-thiouridine containing amino acid-specific tRNA from Escherichia coli were measured in the near ultraviolet region. In the 300–400 nm region, near 335 nm, which correlated with a minor absorption maximum, tRNA II Tyr showed a particular profile of CD having three bands, whereas all other tRNA (tRNA F Met , tRNA I Val , tRNA Phe , and tRNA Arg showed essentially similar spectra having two bands. This suggests that two adjacent 4-thiouridylate residues in tRNA II Tyr have quite different optical activity from the others due to the excitation splitting of the CD band by the nearest neighbor interaction. Thus, the CD profiles in the near ultraviolet region show dissymmetry near the thionucleotide in tRNA molecule. Oxidation of tRNA II Tyr with iodine showed a gross change of CD in the near ultraviolet region, the sign of which converted from positive to negative; tRNA F Met and tRNA I Val showed their characteristic bands in the near ultraviolet region after iodine oxidation. Treatment of tRNA with cyanogen bromide gave essentially similar profiles of CD as for the intact molecules. Treatment with S -benzylthioisothiourea resulted in an unique CD spectrum for tRNA II Tyr , just a reversed profile of its iodine oxidation. No significant change in CD in the ultraviolet region was detected even after subjecting these tRNA to the three previously mentioned chemical modifications.