Analysis of a Calcium Ion Binding System Composed of Two Different Sites

Ohnishi, Tsuyoshi; Masoro, Edward J.; Bertrand, Helen A.; Yu, Byung Pal

doi:10.1016/s0006-3495(72)86160-5

Cited by 22 publications

(5 citation statements)

References 16 publications

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“…Figure 6 shows the Scatchard plot of the binding of acridine orange to the phospholipid vesicles, obtained at room temperature by dialysis and filtration experiments. The dashed lines are obtained by resolution of the Scatchard plot according to Ohnishi et al (1972). The amount of low and high affinity sites is 108 and 50 nmol/µ P¡, respectively; the instability constants are 35 and 3.5 µ , respectively.…”

Section: Resultsmentioning

confidence: 99%

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Phosphatidic acid distribution on the external surface of mixed vesicles

Massari¹,

Pascolini²,

Veronese³

et al. 1977

Biochemistry

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A new method has been developed to detect the distribution of phosphatidic acid on the external surface of mixed phospholipid vesicles. Some positive dyes undergo large absorbance changes when the spatial separation between two or more dye molecules is smaller than a critical distance. When these dyes interact with mixed phospholipid vesicles, the absorbance changes may be utilized to calculate the amount of phosphatidic acid molecules which, on the external surface, occupy nearby positions not exceeding the critical dye distance, i.e., the amount of paired phosphatidic acid molecules. This amount was found to be higher than that calculated by statistical methods, indicating that phosphatidic acid molecules tend to be associated, in spite of the electrostatic repulsion between negative groups. The dependence of the amount of paired phosphatidic acid molecules on the pH, phosphatidylcholine:phosphatidic acid ratio, and temperature has been also analyzed.

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Section: Resultsmentioning

confidence: 99%

“…Resolution of the Scatchard plot into linear partial plots has been accomplished according to Ohnishi et al (1972) using an Olivetti P602 computer.…”

mentioning

confidence: 99%

Phosphatidic acid distribution on the external surface of mixed vesicles

Massari¹,

Pascolini²,

Veronese³

et al. 1977

Biochemistry

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“…Ca2+-occupancy titration curves for caldolysin were obtained spectrophotometrically by the titration of a murexide-containing Hepes buffer solution of caldolysin with Ca2+ (Harnach & Coolidge, 1963;Ohnishi et al, 1972;Kwak & Joshi, 1981;Dorogi & Neumann, 1981), and comparison with a similar titration curve in the absence of caldolysin. In both cases plots of AA (the absorbance difference between 507 nm and 535 nm) versus added Ca2+ were obtained.…”

Section: Determination Of Binding Constantsmentioning

confidence: 99%

“…A Scatchard plot was generated from the titration curves, and the two partial plots were derived as described by Ohnishi et al (1972). The best partial plots were derived by a process of mathematical iteration.…”

Section: Determination Of Binding Constantsmentioning

confidence: 99%

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Interactions of calcium and other metal ions with caldolysin, the thermostable proteinase from Thermus aquaticus strain T351

Khoo¹,

Cowan²,

Daniel³

et al. 1984

Biochemical Journal

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Caldolysin, the extracellular proteinase from the extreme thermophile Thermus aquaticus strain T351, is stabilized by Ca2+. A variety of metal ions were able to substitute for Ca2+. Most were unable to confer as much stability as Ca2+, with the exception of the lanthanide ions, which increased the half-life at 95 degrees C from 1 h to more than 4 h. Results from a variety of separation methods indicated that caldolysin binds 6 Ca2+ ions/molecule of enzyme. The presence of non-linear Ca2+ titration plots, and the removal of 4 Ca2+ ions/molecule by treatment with a cationic ion-exchange gel suggested that caldolysin possesses at least two different types of Ca2+-binding sites, with different affinities. Average binding constants of the two types of binding sites were 2.8 X 10(4)M-1 (for the low-affinity sites) and 7.5 X 10(5) M-1 (for the high-affinity sites). The total Ca2+-binding free energy for caldolysin was shown to be greater than for either thermolysin or Bacillus subtilis neutral proteinase. It appears that the higher thermostability of caldolysin is due to the presence of 6 Ca2+ ions rather than 4 Ca2+ ions/molecule.

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Calcium binding and fluorescence measurements of dansylaziridine-labelled troponin C in reconstituted thin filaments

Zot

Potter

1987

J Muscle Res Cell Motil

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The direct binding of Ca2+ to reconstituted thin filaments containing troponin C and the 5-dimethylaminonaphthalene-1-sulphonylaziridine (DANZ) fluorescent analogue of troponin C (TnCDANZ) was measured (25 degrees C) at three Mg2+ concentrations. Biphasic Scatchard plots were found for all binding curves reflecting the binding of Ca2+ to high- and low-affinity sites of troponin. The binding of Ca2+ to the high-affinity sites had a greater sensitivity to Mg2+ (KMg = 1 x 10(4)M-1) than the low-affinity sites (KMg = 1.2 x 10(3)M-1). The fluorescence change of thin filaments reconstituted with TnCDANZ was titrated with Ca2+ in the same solutions used for binding assays. The Ca2+-dependent fluorescence change had nearly the same sensitivity to Mg2+ (KMg = 9.4 x 10(2)M-1) as did Ca2+ binding to the low-affinity sites. The Ca2+ concentration at the midpoint of the fluorescence change was about 0.3 log units less than at the midpoint for Ca2+ binding to the low-affinity sites. A similar relationship between the fluorescence change and Ca2+ binding to the low-affinity sites of isolated TnCDANZ was measured (4 degrees C). From these results the binding of Ca2+ to either low-affinity site is concluded to produce the fluorescence change. In comparison with the low-affinity sites of isolated troponin and troponin-tropomyosin complex, the low-affinity sites of reconstituted thin filaments were consistently lower in Ca2+ affinity.

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Analysis of a Calcium Ion Binding System Composed of Two Different Sites

Cited by 22 publications

References 16 publications

Phosphatidic acid distribution on the external surface of mixed vesicles

Phosphatidic acid distribution on the external surface of mixed vesicles

Interactions of calcium and other metal ions with caldolysin, the thermostable proteinase from Thermus aquaticus strain T351

Calcium binding and fluorescence measurements of dansylaziridine-labelled troponin C in reconstituted thin filaments

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