A Highly Sensitive Method for Measurement of Myosin ATPase Activity by Reversed-Phase High-Performance Liquid Chromatography

Samizo, Koichi; Ishikawa, Ryoki; Nakamura, Akio; Kohama, Kazuhiro

doi:10.1006/abio.2001.5146

Cited by 29 publications

(22 citation statements)

References 19 publications

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“…ADP Generation upon Actin Polymerization-The total amount of ADP was measured by the reverse-phase HPLC method, as described previously (24). Actin was polymerized at 25 M, as described under "Polymerization Assay," for 45 min.…”

Section: Methodsmentioning

confidence: 99%

Dual Roles of Gln137 of Actin Revealed by Recombinant Human Cardiac Muscle α-Actin Mutants

Iwasa

Maéda

Narita

et al. 2008

Journal of Biological Chemistry

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The actin filament is quite dynamic in the cell. To determine the relationship between the structure and the dynamic properties of the actin filament, experiments using actin mutants are indispensable. We focused on Gln 137 to understand the relationships between two activities: the conformational changes relevant to the G-to F-actin transition and the activation of actin ATPase upon actin polymerization. To elucidate the function of Gln 137 in these activities, we characterized Gln 137 mutants of human cardiac muscle ␣-actin. Although all of the single mutants, Q137E, Q137K, Q137P, and Q137A, as well as the wild type were expressed by a baculovirus-based system, only Q137A and the wild type were purified to high homogeneity. The CD spectrum of Q137A was similar to that of the wild type, and Q137A showed the typical morphology of negatively stained Q137A F-actin images. However, Q137A had an extremely low critical concentration for polymerization. Furthermore, we found that Q137A polymerized 4-fold faster, cleaved the ␥-phosphate group of bound ATP 4-fold slower, and depolymerized 5-fold slower, as compared with the wild-type rates. These results suggest that Gln 137 plays dual roles in actin polymerization, in both the conformational transition of the actin molecule and the mechanism of ATP hydrolysis.

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Section: Methodsmentioning

confidence: 99%

Dual Roles of Gln137 of Actin Revealed by Recombinant Human Cardiac Muscle α-Actin Mutants

Iwasa

Maéda

Narita

et al. 2008

Journal of Biological Chemistry

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“…modifications (Mukai and Okuno, 2004;Samizo et al, 2001). Sperm suspended with each test solution in microtubes were incubated in a CO 2 incubator (5% CO 2 , 37°C) for 30 min.…”

Section: Research Articlementioning

confidence: 99%

Glycolysis plays an important role in energy transfer from the base to the distal end of the flagellum in mouse sperm

Takei

Miyashiro

Mukai

et al. 2014

Journal of Experimental Biology

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Many studies have been conducted to elucidate the relationship between energy metabolic pathways (glycolysis and respiration) and flagellar motility in mammalian sperm, but the contribution of glycolysis to sperm motility has not yet been fully elucidated. In the present study, we performed detailed analysis of mouse sperm flagellar motility for further understanding of the contribution of glycolysis to mammalian sperm motility. Mouse sperm maintained vigorous motility in the presence of substrates either for glycolysis or for respiration. By contrast, inhibition of glycolysis by alphachlorohydrine caused a significant decrease in the bend angle of the flagellar bending wave, sliding velocity of outer doublet microtubules and ATP content even in the presence of respiratory substrates (pyruvate or β-hydroxybutyrate). The decrease of flagellar bend angle and sliding velocity are prominent in the distal part of the flagellum, indicating that glycolysis inhibition caused the decrease in ATP concentration threrein. These results suggest that glycolysis potentially acts as a spatial ATP buffering system, transferring energy (ATP) synthesized by respiration at the mitochondria located in the basal part of the flagellum to the distal part. In order to validate that glycolytic enzymes can transfer high energy phosphoryls, we calculated intraflagellar concentration profiles of adenine nucleotides along the flagellum by computer simulation analysis. The result demonstrated the involvement of glycolysis for maintaining the ATP concentration at the tip of the flagellum. It is likely that glycolysis plays a key role in energy homeostasis in mouse sperm not only through ATP production but also through energy transfer.

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“…2mg/ml of protein equivalent to mitochondria was incubated with 5mM citrate and 0.1mM NAD + for 1h at 37°C. The reaction was stopped at 4°C with dilute acid to precipitate the protein and membranes [24]. The supernatant was analyzed using a Waters Alliance HPLC equipped a C 18 -reverse phase column.…”

Section: -Nuclear Magnetic Resonance and Hplc Studiesmentioning

confidence: 99%

Aluminum-Induced Mitochondrial Dysfunction Leads to Lipid Accumulation in Human Hepatocytes: A Link to Obesity

Mailloux¹,

Lemire²,

Appanna³

2007

Cell Physiol Biochem

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Mitochondrial dysfunction is the cause of a variety of pathologies associated with high energy-requiring tissues like the brain and muscles. Here we show that aluminum (Al) perturbs oxidative-ATP production in human hepatocytes (HepG2 cells). This Al-induced mitochondrial dysfunction promotes enhanced lipogenesis and the accumulation of the very low density lipoprotein (VLDL). Al-stressed HepG2 cells secreted more cholesterol, lipids and proteins than control cells. The level of apolipoprotein B-100 (ApoB-100) was markedly increased in the culture medium of the cells exposed to Al. ¹³C-NMR and HPLC studies revealed a metabolic profile favouring lipid production and lowered ATP synthesis in Al-treated cells. Electrophoretic and immunoblot analyses pointed to increased activities and expression of lipogenic enzymes such as glycerol 3-phosphate dehydrogenase (G3PDH), acetyl CoA carboxylase (ACC) and ATP-citrate lyase (CL) in the hepatocytes exposed to Al, and a sharp diminution of enzymes mediating oxidative phosphorylation. D-Fructose elicited the maximal secretion of VLDL in the Al-challenged cells. These results suggest that the Al-evoked metabolic shift favours the accumulation of lipids at the expense of oxidative energy production in hepatocytes.

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A Highly Sensitive Method for Measurement of Myosin ATPase Activity by Reversed-Phase High-Performance Liquid Chromatography

Cited by 29 publications

References 19 publications

Dual Roles of Gln137 of Actin Revealed by Recombinant Human Cardiac Muscle α-Actin Mutants

Dual Roles of Gln137 of Actin Revealed by Recombinant Human Cardiac Muscle α-Actin Mutants

Glycolysis plays an important role in energy transfer from the base to the distal end of the flagellum in mouse sperm

Aluminum-Induced Mitochondrial Dysfunction Leads to Lipid Accumulation in Human Hepatocytes: A Link to Obesity

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