Yumi Kawazoe

A novel ATP regeneration system using polyphosphate-AMP phosphotransferase and polyphosphate kinase

Polyphosphate-AMP phosphotransferase (PAP) and polyphosphate kinase (PPK) were used for designing a novel ATP regeneration system, named the PAP-PPK ATP regeneration system. PAP is an enzyme that catalyzes the phospho-conversion of AMP to ADP, and PPK catalyzes ATP formation from ADP. Both enzymes use inorganic polyphosphate [poly(P)] as a phosphate donor. In the PAP-PPK ATP regeneration system, ATP was continuously synthesized from AMP by the coupling reaction of PAP and PPK using poly(P). Poly(P) is a cheap material compared to acetyl phosphate, phosphoenol pyruvate and creatine phosphate, which are phosphate donors used for conventional ATP regeneration systems. To achieve efficient synthesis of ATP from AMP, an excessive amount of poly(P) should be added to the reaction solution because both PAP and PPK consume poly(P) as a phosphate donor. Using this ATP generation reaction, we constructed the PAP-PPK ATP regeneration system with acetyl-CoA synthase and succeeded in synthesizing acetyl-CoA from CoA, acetate and AMP. Since too much poly(P) may chelate MG2+ and inhibit enzyme activity, the Mg2+ concentration was optimized to 24 mM in the presence of 30 mM poly(P) in the reaction. In this reaction, ATP was regenerated 39.8 times from AMP, and 99.5% of CoA was converted to acetyl-CoA. In addition, since the PAP-PPK ATP regeneration system can regenerate GTP from GMP, it could also be used as a GTP regeneration system.

A separation method for DNA computing based on concentration control

A separation method for DNA computing based on concentration control is presented. The concentration control method was earlier developed and has enabled us to use DNA concentrations as input data and as filters to extract target DNA. We have also applied the method to the shortest path problems, and have shown the potential of concentration control to solve large-scale combinatorial optimization problems. However, it is still quite difficult to separate different DNA with the same length and to quantify individual DNA concentrations. To overcome these difficulties, we use DGGE and CDGE in this paper. We demonstrate that the proposed method enables us to separate different DNA with the same length efficiently, and we actually solve an instance of the shortest path problems.

Morphogenetic Study on the Maturation of Osteoblastic Cell as Induced by Inorganic Polyphosphate

Since inorganic polyphosphates [poly(P)] have an activity to induce bone differenciation in vitro and in vivo, we examined an effect of poly(P) on organelle by light microscopy and electron microscopy in Murine MC3T3-E1 osteoblastic cells. The MC3T3-E1 cells were ultrastructurally observed to possess morphological characteristics of osteoblasts. Cells cultured with poly(P) were strongly stained with an anti-collagen type I antibody but not in those cultured without poly(P). Ultrastructural analysis of cells cultured with poly(P) revealed a well-developed Golgi apparatus, swollen and elongated rough endoplasmic reticulum, large mitochondria and many coated pits. Since MC3T3-E1 cells can be transformed from a resting phase to an active blastic cell phase after supplementation with poly(P), it implies that poly(P) can be an effective material for bone regeneration.

Polyphosphate:AMP phosphotransferase as a polyphosphate-dependent nucleoside monophosphate kinase in Acinetobacter johnsonii 210A.

We have cloned the gene for polyphosphate:AMP phosphotransferase (PAP), the enzyme that catalyzes phosphorylation of AMP to ADP at the expense of polyphosphate [poly(P)] in Acinetobacter johnsonii 210A. A genomic DNA library was constructed in Escherichia coli, and crude lysates of about 6,000 clones were screened for PAP activity. PAP activity was evaluated by measuring ATP produced by the coupled reactions of PAP and purified E. coli poly(P) kinases (PPKs). In this coupled reaction, PAP produces ADP from poly(P) and AMP, and the resulting ADP is converted to ATP by PPK. The isolated pap gene (1,428 bp) encodes a protein of 475 amino acids with a molecular mass of 55.8 kDa. The C-terminal region of PAP is highly homologous with PPK2 homologs isolated from Pseudomonas aeruginosa PAO1. Two putative phosphate-binding motifs (P-loops) were also identified. The purified PAP enzyme had not only strong PAP activity but also poly(P)-dependent nucleoside monophosphate kinase activity, by which it converted ribonucleoside monophosphates and deoxyribonucleoside monophosphates to ribonucleoside diphosphates and deoxyribonucleoside diphosphates, respectively. The activity for AMP was about 10 times greater than that for GMP and 770 and about 1,100 times greater than that for UMP and CMP.

LOCAL SEARCH BY CONCENTRATION-CONTROLLED DNA COMPUTING

Concentration-controlled DNA computing is presented for accomplishing a local search for the solution of a shortest path problem. In this method, the concentrations of DNA representing edges are determined according to the costs on edges, and then the hybridization process is performed. Since the concentrations of hopeless candidate solutions tend to be small after the hybridization process, a local search by concentration-controlled DNA computing is a promising approach. In order to discuss about the relationship between given costs on edges in the graph and concentrations of generated DNA paths, a simulation model of the hybridization process is used and the results of a laboratory experiment are shown.

Polyphosphate-Mediated Inhibition of Tartrate-Resistant Acid Phosphatase and Suppression of Bone Resorption of Osteoclasts

Inorganic polyphosphate (poly(P)) has recently been found to play an important role in bone formation. In this study, we found that tartrate-resistant acid phosphatase (TRAP), which is abundantly expressed in osteoclasts, has polyphosphatase activity that degrades poly(P) and yields Pi as well as shorter poly(P) chains. Since the TRAP protein that coprecipitated with anti-TRAP monoclonal antibodies exhibited both polyphosphatase and the original phosphatase activity, poly(P) degradation activity is dependent on TRAP and not on other contaminating enzymes. The ferrous chelator α, α’-bipyridyl, which inhibits the TRAP-mediated production of reactive oxygen species (ROS), had no effect on such poly(P) degradation, suggesting that the degradation is not dependent on ROS. In addition, shorter chain length poly(P) molecules were better substrates than longer chains for TRAP, and poly(P) inhibited the phosphatase activity of TRAP depending on its chain length. The IC50 of poly(P) against the original phosphatase activity of TRAP was 9.8 µM with an average chain length more than 300 phosphate residues, whereas the IC50 of poly(P) with a shorter average chain length of 15 phosphate residues was 8.3 mM. Finally, the pit formation activity of cultured rat osteoclasts differentiated by RANKL and M-CSF were markedly inhibited by poly(P), while no obvious decrease in cell number or differentiation efficiency was observed for poly(P). In particular, the inhibition of pit formation by long chain poly(P) with 300 phosphate residues was stronger than that of shorter chain poly(P). Thus, poly(P) may play an important regulatory role in osteoclastic bone resorption by inhibiting TRAP activity, which is dependent on its chain length.

Solutions of Shortest Path Problems by Concentration Control

In this paper, we present a concentration control method that may become a new framework of DNA computing. In this method, the concentration of each DNA is used as input and output data. By encoding the numeric data into concentrations of DNAs, a shortest path problem, which is a combinatorial optimization problem, can be solved. The method also enables local search among all candidate solutions instead of a exhaustive search. Furthermore, we can reduce the costs of some experimental operations in detecting process of DNA computing, because we have only to extract and analyze relatively intensive bands. Solutions of a shortest path problem by using a simulator and by laboratory experiments are presented to show the effectiveness of the concentration control method.