• International Journal of Industrial Entomology and Biomaterials
• /
• v.15 no.2
• /
• pp.115-122
• /
• 2007

As the genome of B.mori is available in GenBank and the EST database of B.mori is expanding, identification of novel genes of B.mori is conceivable by data-mining techniques. We used the in silico cloning method to get the vacuolar-type $H^+-ATP$ synthetase (V-ATPase) c subunit (16 kDa proteolipid subunit) gene of B.mori and analysed with bioinformatics tools. The result was confirmed by RT-PCR and sequencing. The V-ATPase c subunit cDNA contains a 468 bp ORF. The ORF encoded a 155-residue protein that showed extensive homology with V-ATPase c subunits from other 15 species and contained four membrane-spanning helices. Tissue expression pattern analysis revealed that V-ATPase c expressed strongly in Malpighian tubules, not in fat body. This gene has been registered in GenBank under the accession number EU082222.

• The Korean Journal of Physiology
• /
• v.10 no.1
• /
• pp.15-24
• /
• 1976

The action of aconite on the sodium plus potassium activated ATPase activity in the rabbit red cell membrane has been investigated and the experiments were also designed to determine the mechanism of action of aconite on the ATPase activity. The following results were observed. 1. The activity of the NaK ATPase from red cell membrane is stimulated by aconite, and the concentration of aconite for maximal activity is about 80 mg%. The pH optimum for the aconite sensitive component is 8.0. 2. The activating effect of aconite on the ATPase, with a given concentration of sodium in the medium, is increased by raising the potassium concentration but activity ratio is decreased. 3. The activating effect of aconite on the ATPase, with a given concentration of potassium in the medium, is increased by raising the sodium concentration but activity ratio is decreased. 4. The action of aconite on the ATPase activity is inhibited by calcium ions and the effect of inhibition is increased by small amounts of calcium but decreased by larger amounts. 5. The activating effect of aconite on the ATPase was not related to the sulfhydryl group of cysteine, the amino group of lysine, the hydroxyl group of threonine or the imidazole group of histidine. 6. The action of aconite on the ATPase activity is due to carboxyl group of the enzyme of NaK ATPase.

• The Korean Journal of Physiology
• /
• v.11 no.1
• /
• pp.11-20
• /
• 1977

The action of pilocarpine on the sodium plus potassium activated ATPase activity in the rabbit red cell membrane has been investigated and the experiments were also designed to determine the mechanism of action of pilocarpine on the ATPase activity. The following results were observed. 1. The activity of the NaK ATPase from red cell membrane is stimulated by pilocarpine, and the concentration of pilocarpine for maximal activity is about 3 mM. The pH optimum for the pilocarpine sensitive component is 8.0. 2. The activating effect of pilocarpine on the ATPase, with a given concentration of sodium .in the medium, is increased by raising the potassium concentration but activity ratio is decreased 3. The activating effect of pilocarpine on the ATPase, with a given concentration of Potassium in the medium, is increased by raising the sodium concentration but activity ratio is decreased 4. The NaK ATPase activity is increased by small amounts of calcium but decreased by 'larger amounts. The activity ratio of the enzyme by pilocarpine is decreased by small amounts .of calcium but decreased by larger amounts. 5. The activating effect of pilocarpine on the ATPase was not related to the sulfhydryl group of cysteine, the hydroxyl group of threonine or the imidazole group of histidine. 6. The activating effect of pilocarpine on the ATPase is due to amino group and carboxyl group of the enzyme of NaK ATPase

• Journal of Life Science
• /
• v.6 no.1
• /
• pp.1-5
• /
• 1996

Using differential hybridization, a cDNA clone was isolated fortuitously from Amaranthus viridis and sequenced. This nucleotide sequence exhibited 55.1% identity with vma6 which encodes the 36-kD subunit of the vacuolar proton transporting ATPase in Saccharmoyces cerevisiae. The predicted open reading frame encodes a protein of 221 amino acid sequence with a calculated molecular weight of 25,452 and reveals high levels of similarity with subunit D polypeptide of vacuolar H -ATP(e.g., 48.5, 52.1 and 49.3% identity to the vacuolar 36-kD chain of yeast, vacuolar 32-kD polypeptide IV of human and vacuolar 28-kD protein of bovine chromaffin granules, respectively). The hydropathy index computation revealed that this predicted protein is a peripheral protein. These results indicated that the predicted protein may play a sturctural role in the vaculor H -ATPase as does gamma subunit in V-type ATPase.

• 한국유가공학회:학술대회논문집
• /
• 1997.05a
• /
• pp.23-34
• /
• 1997

Calcium-stimulated ATPase ($Ca^{2+}$-ATPase) which has optimal pH value at 7.0 was found in the membrane fraction of human milk, and its enzymatic properties were studied. The purified $Ca^{2+}$-ATPase required 0.45 mM Ca ion for maximal activity. Among the nucleosides, $Ca^{2+}$-ATPase showed a higher substrate specificity to ATP and UTP than to CTP and GTP. $Ca^{2+}$-ATPase had apparent Km value of 0.065, and V max of 7.63 mol ATP hydrolyzed/mg pro-tein per min, respectively. $Ca^{2+}$-ATPase was potently inhibited by lanthanide, vanadate, and p-chloromercuribenzoate, and inactivated by EDTA, and CDTA and EGTA, but were unaffected by N-ethylmaleimide, $NaN_3$, ouabain, or oligomycin, and was completely inactivated by heating at $60^{\circ}C$ for 10 min. This enzyme activity was concentrated in the membrane fraction of the cream and skim milk membrane, but not founded in bovine milk.

• Applied Biological Chemistry
• /
• v.40 no.3
• /
• pp.202-208
• /
• 1997

In order to investigate the mechanisms of epithelial ion transports, microsomes of soybean roots were prepared and the activity of microsomal ATPases was measured by an enzyme-coupled assay. The effects of various ions were evaluated on the total activity of microsomal ATPases and the average activity was 190 nmol/min/mg protein in the control solution containing $10\;mM\;Na^+\;and\;120\;mM\;K^+$. The activities were increased to 150% and decreased to 63% of the control activity in the solution containing $130\;mM\;K^+$ without $Na^+$ and in the solution containing $130\;mM{\;}Na^+$ without $K^+$, respectively. In general, the activity of microsomal ATPase was increased by$K^+$ in a concentration-dependent manner The activity was also increased at lower pH and relatively higher activities were observed in the pH range of $6{\sim}7$. However, the activity was decreased at weak alkaline $pH\;and{\sim}80%$ of the activity was inhibited at pH 9. Since intracellular $Ca^{2+}$ has been known to control the activity of various enzymes, we have investigated the effects of intra-and extrarnicrosomal $Ca^{2+}$ on the activity of microsomal ATPases. The maximal activity was obtained at the extrarnicrosomal $Ca^{2+}$ concentrations below 1 nM. The activity was gradually decreased by increasing $‘Ca^{2+}’$ concentration and 50% inhibition was observed at ${\sim}500{\;}{\mu}M{\;}Ca^{2+}$. The increase in luminal $Ca^{2+}$ concentration also inhibited the activity of microsomal ATPase. When the influx of external $Ca^{2+}$ was induced by $Ca^{2+}$ ionophore A23187 treatment, the activity was decreased by 30%; however, it was recovered by EGTA-induced chelation of $Ca^{2+}$. These results suggest that the presence of $Ca^{2+}$ regulation sites on both cytoplasmi and luminal sides of microsomal ATPases.

• Journal of Microbiology and Biotechnology
• /
• v.9 no.3
• /
• pp.235-242
• /
• 1999

We investigated the growth-inhibitory mechanism of Helicobacter pylori by omeprazole (OMP) and its activated sulfenamide (OAS). Using dithiothreitol (DTT) and 5,5'-dithio-bis[2-nitrobenzoic acid] (DTNB; Ellman's reagent), we first determined the relationship between the binding capacity of these compounds to H. pylori membrane and its significance to membrane P-type ATPase activity. After incubation of the intact H. pylori cells with either OMP or OAS, the residual quantity of free SH-groups on the cell membrane was measured, and, the resulting values were plotted as a function of time. From this experiment, we found that there was a considerable difference in the membrane-binding rates between OMP and OAS. At neutral pH, the disulfide bond formation on H. pylori membrane was completed within 2 min of incubation of the intact cells with OAS. By OMP, however, it was gradually formed, exceeding 10 min of incubation for completion, whereby, the extent of P-type ATPase inhibition appeared to be proportional to the disulfide forming rate. From this data, it was suggested that the disulfide formation might directly affect enzyme activity. Since OMP per se cannot yield a disulfide bond with cysteine, it is predicted that the enzyme inactivation must be caused by the OAS form. Accordingly, we postulated that, under the neutral pH, OMP could be converted to OAS in the course of transport. By extrapolating the inhibitory slopes, we could evaluate K₁ values, relating to their minimal inhibitory concentrations (MICs) for H. pylori growth. In these MIC ranges, H. pylori uptake or vesicular export of nutrients such as peptides were totally prohibited, but their effect in Escherichia coli were negligible. From these observations, we strongly suggest that the P-type ATPase activity is essential for the survival of H. pylori cells in particular.

• Bulletin of the Korean Chemical Society
• /
• v.30 no.8
• /
• pp.1724-1728
• /
• 2009

Severe acute respiratory syndrome coronavirus (SARS-CoV) helicase separates the double-stranded nucleic acids using the energy from ATP hydrolysis. We have measured ATPase activity of SARS-CoV helicase in the presence of various types of nucleic acids. Steady state ATPase analysis showed that poly(U) has two-times higher turnover number than poly(C) with lower Michaelis constant. When M13 single-stranded DNA is used as substrate, the Michaelis constant was about twenty-times lower than poly(U), whereas turnover numbers were similar. However, stimulation of ATPase activity was not observed in the presence of double-stranded DNA. pH dependent profiles of ATP hydrolysis with the helicase showed that the optimal ATPase activities were in a range of pH 6.2 ~ 6.6. In addition, ATP hydrolysis activity assays performed in the presence of various divalent cations exhibited that $Mg^{2+}$ stimulated the ATPase activity with the highest rate and $Mn^{2+}$ with about 40% rate as compared to the $Mg^{2+}$.

• Journal of Plant Biology
• /
• v.24 no.4
• /
• pp.171-179
• /
• 1981

The membrane-bound ATPases were separated on sucrose gradient from corn roots and characterized by pH optima, sensitivity to monovalent salt, Km and Vmax. The pH optima for the activity of all the ATPases associated with 13, 000g pellet and 13, 000~80, 000g pellet were 5 and 9, respectively. The ATPases in Fractions B and C of the 13, 000 g pellet were more active at pH 5 than pH 9. While, in the case of Fractions D, E and F, they were reverse. The activities of the ATPase in Fractions A and C of the 13, 000~80, 000 g pellet were greater at pH 5 than pH 9. On the other hand, the ATPases in Fractions B, D, E, and F were more active at pH 9 than pH 5. The optimum concentraction of ATP for the assay was about 3 to 5 mM. The Km's for the membrane-bound ATPases in 13, 000g pellet and in 13, 000~80, 000 g pellet were 0.25 mM. While Vmax values for 13, 000g pellet were from 8.0 to 12.5 $\mu$M Pi/mg protein/hr. according to pH values, those for 13, 000~80, 000 g pellet were from 35.7 to 55.6 $\mu$M Pi/mg protein/hr. Activities of the membrane-bound ATPases in both 13, 000 g pellet and 13, 000~80, 000 g pellet were stimulated with increasing the concentration of $K^+$.

• The Korean Journal of Physiology
• /
• v.20 no.2
• /
• pp.257-270
• /
• 1986

It has been reported that the luteal function may be regulated by the intracellular calcium in luteal cells (Higuchi et al, 1976; Dorflinger et at, 1984; Gore and Behrman, 1984) which is adjusted partially by $Ca^{++}-ATPase$ activities in luteal cell membranes (Verma and Pennistion, 1981). However, the physicochemical and kinetic properties of $Ca^{++}-ATPase$ in luteal membranes were not fully characterized. This study was, therefore, undertaken to partially characterize the physicochemical and kinetic properties of $Ca^{++}-ATPase$ system in luteal membranes and microsomal fractions, known as an one of the major $Ca^{++}$ storge sites (Moore and Pastan, 1978), from the highly luteinized ovary Highly luteinized ovaries were obtained from PMSG-hCG injected immautre female rats. Light membrane and heavy membrane fractions and microsomal fractions were prepared by the differential and discontinuous sucrose density gradient centrifugation method desribed by Bramley and Ryan (1980). Light membrane and heavy membrane fractions and microsomal fractions from highly luteinized ovaries are composed of the two different kinds of $Ca^{++}-ATPase$ system. One is the high affinity $Ca^{++}-ATPase$ which is activated in low $Ca^{++}$ concentration (Km, 10-30 nM), the other is low affinity $Ca^{++}-ATPase$ activated in higher $Ca^{++}$ concentration $(K_{1/2},\;40\;{\mu}M)$. At certain $Ca^{++}$ concentrations, activities of high and low affinity $Ca^{++}-ATPase$ are the highest in light membrane fractions and are the lowest in microsomal fractions. It appeares that high affinity $Ca^{++}-ATPase$ system have 2 binding sites for ATP (Hill's coefficient; around 2 in all membrane fractions measured) and the positive cooperativity of ATP bindings obviously existed in each membrane fractions. The optimum pH for high affinity $Ca^{++}-ATPase$ activation is around S in all membrane fractions measured. The lipid phase transition temperature measured by Arrhenius plots of high affinity $Ca^{++}-ATPase$ activity is around $25^{\circ}C$. The activation energies of high affinity $Ca^{++}-ATPase$ below the transition temperature are similar in each membrane fractions, but at the above transition temperature, it is the hightest in heavy membrane fractions and the lowest in microsomal fractions. According to the above results, it is suggested that intracellular $Ca^{++}$ level, which may regulate the luteal function, may be adjusted primarily by the high affinity $Ca^{++}-ATPase$ system activated in intracellular $Ca^{++}$ concentration range $(below\;0.1\;{\mu}M)$.