Akira Hachimori

Cloning and expression of a unique inorganic pyrophosphatase from Bacillus subtilis: evidence for a new family of enzymes

An open reading frame located in the COTF‐TETB intergenic region of Bacillus subtilis was cloned and expressed in Escherichia coli and shown to encode inorganic pyrophosphatase (PPase). The isolated enzyme is Mn2+‐activated, like the authentic PPase isolated from B. subtilis. Although 13 functionally important active site residues are conserved in all 31 soluble PPase sequences so far identified, only two of them are conserved in B. subtilis PPase. This suggests that B. subtilis PPase represents a new family of soluble PPases (a Bs family), putative members of which were found in Archaeoglobus fulgidus, Methanococcus jannaschii, Streptococcus mutans and Streptococcus gordonii.

Replacement of L7/L12.L10 Protein Complex in Escherichia coli Ribosomes with the Eukaryotic Counterpart Changes the Specificity of Elongation Factor Binding

The L8 protein complex consisting of L7/L12 and L10 in Escherichia coli ribosomes is assembled on the conserved region of 23 S rRNA termed the GTPase-associated domain. We replaced the L8 complex in E. coli 50 S subunits with the rat counterpart P protein complex consisting of P1, P2, and P0. The L8 complex was removed from the ribosome with 50% ethanol, 10 mm MgCl2, 0.5 m NH4Cl, at 30 °C, and the rat P complex bound to the core particle. Binding of the P complex to the core was prevented by addition of RNA fragment covering the GTPase-associated domain of E. coli 23 S rRNA to which rat P complex bound strongly, suggesting a direct role of the RNA domain in this incorporation. The resultant hybrid ribosomes showed eukaryotic translocase elongation factor (EF)-2-dependent, but not prokaryotic EF-G-dependent, GTPase activity comparable with rat 80 S ribosomes. The EF-2-dependent activity was dependent upon the P complex binding and was inhibited by the antibiotic thiostrepton, a ligand for a portion of the GTPase-associated domain of prokaryotic ribosomes. This hybrid system clearly shows significance of binding of the P complex to the GTPase-associated RNA domain for interaction of EF-2 with the ribosome. The results also suggest that E. coli 23 S rRNA participates in the eukaryotic translocase-dependent GTPase activity in the hybrid system.

In vitro reconstitution of the GTPase-associated centre of the archaebacterial ribosome: The functional features observed in a hybrid form with Escherichia coli 50S subunits

We cloned the genes encoding the ribosomal proteins Ph (Pyrococcus horikoshii)-P0, Ph-L12 and Ph-L11, which constitute the GTPase-associated centre of the archaebacterium Pyrococcus horikoshii. These proteins are homologues of the eukaryotic P0, P1/P2 and eL12 proteins, and correspond to Escherichia coli L10, L7/L12 and L11 proteins respectively. The proteins and the truncation mutants of Ph-P0 were overexpressed in E. coli cells and used for in vitro assembly on to the conserved domain around position 1070 of 23S rRNA (E. coli numbering). Ph-L12 tightly associated as a homodimer and bound to the C-terminal half of Ph-P0. The Ph-P0.Ph-L12 complex and Ph-L11 bound to the 1070 rRNA fragments from the three biological kingdoms in the same manner as the equivalent proteins of eukaryotic and eubacterial ribosomes. The Ph-P0.Ph-L12 complex and Ph-L11 could replace L10.L7/L12 and L11 respectively, on the E. coli 50S subunit in vitro. The resultant hybrid ribosome was accessible for eukaryotic, as well as archaebacterial elongation factors, but not for prokaryotic elongation factors. The GTPase and polyphenylalanine-synthetic activity that is dependent on eukaryotic elongation factors was comparable with that of the hybrid ribosomes carrying the eukaryotic ribosomal proteins. The results suggest that the archaebacterial proteins, including the Ph-L12 homodimer, are functionally accessible to eukaryotic translation factors.

Ribosomal Proteins at the Stalk Region Modulate Functional rRNA Structures in the GTPase Center

Replacement of the L10·L7/L12 protein complex and L11 in Escherichia coli ribosomes with the respective rat counterparts P0·P1/P2 and eukaryotic L12 causes conversion of ribosomal specificity for elongation factors from prokaryotic elongation factor (EF)-Tu/EF-G to eukaryotic EF (eEF)-1α/eEF-2. Here we have investigated the effects of protein replacement on the structure and function of two rRNA domains around positions 1070 and 2660 (sarcin/ricin loop) of 23 S rRNA. Protein replacement at the 1070 region in E. coli 50 S subunits was demonstrated by chemical probing analysis. Binding of rat proteins to the 1070 region caused increased accessibility of the 2660 and 1070 regions to ligands for eukaryotic ribosomes: the ribotoxin pepocin for the 2660 region (E. coli numbering), anti-28 S autoantibody for the 1070 region, and eEF-2 for both regions. Moreover, binding of the E. coli L10·L7/L12 complex and L11 to the 1070 region was shown to be responsible for E. coli ribosomal accessibility to another ribotoxin, gypsophilin. Ribosomal proteins at the 1070 region appear to modulate the structures and functions of the 2660 and 1070 RNA regions in slightly different modes in prokaryotes and eukaryotes.

Binding study of metal ions to S100 protein: 43Ca, 25Mg, 67Zn and 39K n.m.r.*

The interactions of the S100 protein (S100) with metal cations such as Ca2+, Mg2+, Zn2+ and K+ were studied by the metal n.m.r. spectroscopy. The line widths of 43Ca, 25Mg, 67Zn and 39K n.m.r. markedly increased by adding all S100s. A broad 43Ca n.m.r. band of Ca(2+)-S100a solution was not affected by Zn2+ and K+, while it was greatly decreased by adding Mg2+. The 43Ca n.m.r. spectra of Ca(2+)-S100a0 and -S100b solutions consisted of two slow-exchangeable signals which corresponded to Ca2+ bound to two environmentally different sites of the S100a0. These two 43Ca n.m.r. signals were not affected by Zn2+ and K+. The line width of broad 25Mg n.m.r. band of the Mg(2+)-S100 solution greatly decreased by adding Ca2+, while it did not change by adding Zn2+ and K+. Further, the addition of Ca2+, Mg2+ and K+ did not affect the line width of the 67Zn n.m.r. of the Zn(2+)-S100 solutions. These findings suggest that: (1) Mg2+ binds to all S100s, and at least one of the Mg2+ binding sites of S100 molecule is the same as the Ca2+ binding site; (2) Zn2+ binds to S100s, although the binding site(s) is/are different from Ca(2+)- or Mg(2+)-binding site(s), and the environment of Zn2+ nuclei will not change even though Ca2+ binds to S100s.

Interaction of albumin with polysaccharides containing ionic groups

It is very important to clarify the mechanisms of the interaction between components of organisms. In this report, the interaction between bovine serum albumin (BSA) and ionic polysaccharides were discussed. The fluorescence spectrum of tryptophan (Trp) in BSA changed as its conformation changed. On adding polysaccharide containing sulfonic acid residues (sulfonic-type) at pH 7.4, a remarkable blue shift of the emission maximum (λem) of Trp was observed. This blue shift was decreased by adding NaCl. In contrast, polysaccharide containing carboxylic acid residues (carboxy-type) scarcely changed Trp fluorescence at the same pH. At a pH lower than the isoelectric point (PI = 4.7–4.9) of BSA, a remarkable blue shift was observed not only by adding sulfonic-type polysaccharide but also by adding carboxy-type polysaccharide. Moreover, using the energy transfer method, in the pH region higher than the PI of BSA, carboxylic-type polysaccharides may interact relatively weakly with the binding site II of BSA, but sulfonic-type ones can selectively interact with binding site I weakly and with binding site II strongly. And in the pH region lower than the PI of BSA, carboxylic-type polysaccharides interact with binding site II strongly. On the other hand, sulfonic-type polysaccharides are bound to both binding site I and binding site II very strongly. Copyright

SOME PROPERTIES OF INORGANIC PYROPHOSPHATASE FROM BACILLUS SUBTILIS

Inorganic pyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1; PPase) from Bacillus subtilis was purified to a homogeneous state electrophoretically when analysed by SDS-PAGE. The enzyme consists of six identical subunits; the molecular weight of the native enzyme estimated by gel filtration was approx. 120,000, and denaturing polyacrylamide gel electrophoresis gave a single band corresponding to 24,000. The enzyme absolutely required a divalent cation for its activity. Mg2+ was most effective, showing two steps of concentration-dependent activation. Mg2+ could be partially replaced by Mn2+ and Co2+. The enzyme was thermostable in the presence of Mg2+, and no loss of activity was observed on the incubation at 55 degrees C for an hour.

Quenching in the excited singlet state of amphiphilic copolymers with pendant carbazolylalkyl groups in aqueous solution

Amphiphilic polyanions containing 2-(9-carbazolyl)ethyl methacrylate and 3-(9-carbazolyl)propyl methacrylamide (r-CzEMA(x) and r-CzPMAm(x), respectively) were prepared and their fluorescence properties were compared with those of various N-vinylcarbazole-containing copolymers (r-VCz(x)). The fluorescence spectra for those former polymers with a higher carbazolyl (Cz) content showed monomeric but diminished emission in aqueous solution, indicating that some excited-state quenching occurred in these polymers. The energy transfer to hydrophobically bound perylene and the fluorescence quenching by fumaric acid (FA), a water-soluble electron acceptor, were facilitated for r-CzPMAm(x) with a higher Cz content when compared to that observed for r-VCz(x). These facts suggest a large effect of the excited-state quenching on the photochemical reactions, as well as providing a contribution of energy migration to such reactions.

Effects of Ca2+ and Zn2+ on trifluoperazine-S100 proteins interactions: Induced circular dichroism and fluorescence spectra

Interactions of trifluoperazine (TFP) with S100 proteins, EF-hand type Ca2+-binding proteins, in the presence of Ca2+ and Zn2+ were studied with induced circular dichroism (CD) and fluorescence spectra. The positive CD bands of TFP were induced at around 265 nm by adding either S100a or S100a0 protein in the presence of Ca2+. No CD band of TFP was, however, induced by adding S100b protein in the presence of Ca2+. Addition of Zn2+ to the TFP/S100 protein solutions did not induce any CD band at all. The fluorescence intensity of 2-p-toluidinylnaphthalene 6-sulfonate (TNS) bound to S100a or S100a0 protein decreased by adding TFP in the presence of Ca2+, while that bound to S100b protein decreased by adding TFP in the presence of Zn2+, indicating that TFP binds to S100a protein and S100a0 protein in a Ca2+-dependent manner and to S100b protein in a Zn2+-dependent manner. From these results together with other experimental findings it was suggested that (1) TFP binds to S100a protein and S100a0 protein in the presence of Ca2+, with half-saturation points of 18 and 3 microM, respectively, (2) TFP binds to S100b protein only in the presence of Zn2+, (3) alpha-subunit of S100 protein binds to TFP specifically in a Ca2+-dependent manner and beta-subunit in a Zn2+-dependent manner.

Steric effect on photochemistry of benzyl ester derivatives: 1. Photolysis of 1-(naphthyl)ethyl alkanoates in methanol

Abstract The photochemistry of naphthylmethyl (NpM) and 1-(naphthyl)ethyl (NpE) alkanoates, 1a–d and 2a–d, has been examined in methanol (MeOH). Measurements of quantum yields for fluorescence and intersystem crossing and lifetimes for excited singlet and triplet states established similar photophysical behavior for all the esters studied here. The irradiation produced methyl NpM (or NpE) ether 3 and in-cage coupled product 4 as the major ionic and radical products, respectively. The conversion was found to be substituent-dependent, varying from 95% for 1c (2-NpMA) to 52% for 2b (1-NpEP). In contrast, a poorer correlation between the product distribution and ester structure was observed. The results indicate an important contribution of steric hindrance around the ester bond to the photocleavage. Steric effect on the stabilization of ionic and radical intermediates I–III in Scheme. 1 also perturbed the product distribution.