Kazutaka Murayama

Bach2 is a transcriptional repressor that plays an important role in the differentiation of T-cells and B-cells. Bach2 is functionally regulated by heme binding, and possesses five CP-motifs as the heme binding site. To reveal the molecular mechanism of heme binding by Bach2, the intrinsically disordered heme binding region (a.a. 331-520; Bach2^331-520) and its CP-motif mutant were prepared and characterized with and without heme, by UV-Vis spectroscopy and thermal profiles. In addition, the charge-state-distributions (CSDs) were assessed by electrospray ionization mass spectrometry. The UV-Vis spectroscopy revealed a lack of 5-coordinated heme binding in the CP-motif mutant of Bach2^331-520. The thermal profile and CSDs of Bach2^331-520 indicated that heme binding induces the destabilization of Bach2^331-520. The thermal profile revealed that the wild type Bach2^331-520 was destabilized more than the CP-motif mutant. The shift in the CSDs by heme binding suggested that heme binding causes Bach2^331-520 to adopt a more compact conformation. In addition, heme binding to the CP-motif could reduce the flexibility of Bach2^331-520. Consequently, the 5-coordinated heme binding destabilizes Bach2^331-520, by reducing the flexibility of the polypeptide chain.

The clostridial collagenases, H and G, play key roles in pancreatic islet isolation. Collagenases digest the peptide bond between Yaa and the subsequent Gly in Gly-Xaa-Yaa repeats. To fully understand the pancreatic islet isolation process, identification of the collagenase substrates in the tissue is very important. Although collagen types I and III were reported as possible substrates for collagenase H, the substrate for collagenase G remains unknown. In this study, collagen type V was focused upon as the target for collagenases. In vitro digestion experiments for collagen type V were performed and analyzed by SDS-PAGE and mass spectrometry. Porcine pancreatic tissues were digested in vitro under three conditions and observed during digestion. The results revealed that collagen type V was only digested by collagenase G and that the digestion was initiated from the N-terminal part. Tissue degradation during porcine islet isolation was only observed in the presence of both collagenases H and G. These findings suggest that collagen type V is one of the substrates for collagenase G. The enzymatic activity of collagenase G appears to be more important for pancreatic islet isolation in large mammals such as pigs and humans. The clostridial collagenases, H and G, play key roles in pancreatic islet isolation. Collagenases digest the peptide bond between Yaa and the subsequent Gly in Gly-Xaa-Yaa repeats. To fully understand the pancreatic islet isolation process, identification of the collagenase substrates in the tissue is very important. Although collagen types I and III were reported as possible substrates for collagenase H, the substrate for collagenase G remains unknown. In this study, collagen type V was focused upon as the target for collagenases. In vitro digestion experiments for collagen type V were performed and analyzed by SDS-PAGE and mass spectrometry. Porcine pancreatic tissues were digested in vitro under three conditions and observed during digestion. The results revealed that collagen type V was only digested by collagenase G and that the digestion was initiated from the N-terminal part. Tissue degradation during porcine islet isolation was only observed in the presence of both collagenases H and G. These findings suggest that collagen type V is one of the substrates for collagenase G. The enzymatic activity of collagenase G appears to be more important for pancreatic islet isolation in large mammals such as pigs and humans.

This chapter provides structural data, mainly metal binding sites/modes, observed in crystal structures of alkali metal ion complexes containing low-molecular-weight ligands of biological relevance, mostly obtained from the Cambridge Structural Database (the CSD version 5.35 updated to February 2014). These ligands include (i) amino acids and small peptides, (ii) nucleic acid constituents (excluding quadruplexes and other oligonucleotides), (iii) simple carbohydrates, and (iv) naturally occurring antibiotic ionophores. For some representative complexes of these ligands, some details on the environment of the metal coordination and structural characteristics are described.

Objectives To obtain an ethanolamine plasmalogen (PlsEtn)-hydrolyzing enzyme and to develop an assay that would help determine PlsEtn concentrations in human serum as an indicator of Alzheimer-type dementia and of arteriosclerosis. Results Phospholipase A₁s, SaPLA1 and SvPLA1 from, respectively, Streptomyces albidoflavus NA297 and S. avermitilis JCM5070—but not phospholipase B from Streptomyces sp. NA684, PLA2-Nagase from S. avermitilis, PLA₂IIL from S. violaceoruber nor LIPOMOD 699L (porcine phospholipase)—hydrolyzed choline plasmalogen (PlsCho) and PlsEtn (PlsCho preferred over PlsEtn). Using a combination of SaPLA1, lysoplasmalogen-specific phospholipase D (LyPls-PLD), with amine oxidase, an end-point assay was developed for measuring serum PlsEtn concentration. The standard curve, generated using various amounts of PlsEtn in this assay, was linear between 0 and 0.2 mM. PlsEtn concentrations in forty-seven serum samples, determined independently by this enzyme-based assay and ¹²⁵I-HPLC method, exhibited a linear relationship, indicating that the assay is suitable for fast and accurate measurement of serum PlsEtn concentration. Conclusions An assay, developed using SaPLA1, LyPls-PLD, and AOX, selectively measured PlsEtn levels in blood samples. This assay could be a useful diagnostic tool for early stage detection of diseases such as Alzheimer-type dementia and arteriosclerosis.

The clostridial collagenases G and H are multidomain proteins. For collagen digestion, the domain arrangement is likely to play an important role in collagen binding and hydrolysis. In this study, the full-length collagenase H protein from Clostridium histolyticum was expressed in Escherichia coli and purified. The N-terminal amino acid of the purified protein was Ala31. The expressed protein showed enzymatic activity against azocoll as a substrate. To investigate the role of Ca ²⁺ in providing structural stability to the full-length collagenase H, biophysical measurements were conducted using the recombinant protein. Size exclusion chromatography revealed that the Ca ²⁺ chelation by EGTA induced interdomain conformational changes. Dynamic light scattering measurements showed an increase in the percent polydispersity as the Ca ²⁺ was chelated, suggesting an increase in protein flexibility. In addition to these conformational changes, differential scanning fluorimetry measurements revealed that the thermostability was decreased by Ca ²⁺ chelation, in comparison with the thermal melting point ( T _m ). The melting point changed from 54 to 49°C by the Ca ²⁺ chelation, and it was restored to 54°C by the addition of excess Ca ²⁺ . These results indicated that the interdomain flexibility and the domain arrangement of full-length collagenase H are reversibly regulated by Ca ²⁺ .

Metal ions play a key role in nucleic acid structure and activity. Elucidation of the rules that govern the binding of metal ions is therefore an essential step for better understanding of the nucleic acid functions. This review is as an update to a preceding one (Metal Ions Biol. Syst., 1996, 32, 91-134), in which we offered a general view of metal ion interactions with mono-, di-, tri-, and oligonucleotides in the solid state, based on their crystal structures reported before 1994. In this chapter, we survey all the crystal structures of metal ion complexes with nucleotides involving oligonucleotides reported after 1994 and we have tried to uncover new characteristic metal bonding patterns for mononucleotides and oligonucleotides with A-RNA and A/B/Z-DNA fragments that form duplexes. We do not cover quadruplexes, duplexes with metal-mediated base-pairs, tRNAs, rRNAs in ribosome, ribozymes, and nucleic acid–drug and –protein complexes. Factors that affect metal binding to mononucleotides and oligonucleotide duplexes are also dealt with.