Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

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Enzymatic Assays 349 10. Wash each column with three consecutive 1-mL aliquots of water and collect the flowthrough in 15-mL scintiverse cocktails. 11. Count the cocktails in a scintillation counter (see Note 10). 12. Determine enzyme activity from the specific activity of the radioactive substrate. 3.7. MLCK Assay MLCK activity is absolutely dependent on Ca2+ and CaM. This assay measures the incorporation of 32P from [γ-32P]ATP into LC20 at a fixed time following addition of radiolabeled ATP to start the reaction. Figure 4 shows the concentration dependence of CaM activation of MLCK. To conduct this assay: 1. To 15 µL of buffer in a plastic 1.5-mL microfuge tube (without cap) on ice, add LC 20 (10 µM), CaM (0–1 µM), and MLCK (0.05 µg/mL). 2. Add distilled, deionized H 2O to give a total volume of 29 µL. 3. Mix the solution well by vortexing and let it equilibrate to 30°C in the water bath. 4. Start the reaction by adding 1 µL of stock [γ- 32 P]ATP. 5. Incubate the reaction mixture at 30°C for 10 min. 6. With a pipetman, transfer 20 µL of the reaction mixture to a square (1 × 1 cm) of P81 paper and immerse immediately in a glass 600-mL beaker containing 500 mL of 0.5% (v/v) H 3PO 4 and a stainless steel wire mesh basket. This stops the reaction. 7. Wash the paper squares three times for 5 min each with stirring in 500 mL of 0.5% (v/v) H 3PO 4 and once for 2 min with acetone. 8. Remove the wire basket from the beaker, place on a paper towel and dry the P81 papers with a hair dryer. 9. Transfer the dried paper squares to plastic scintillation vials and quantify 32 P by ˆ Cerenkov counting (no scintillant or other liquid) in a scintillation counter using 3 H window settings. 10. Determine enzyme activity from the specific activity of the radiolabeled ATP. 3.8. CaM Kinase II Assay This assay measures the incorporation of 32P from [γ-32P]ATP into caldesmon at a fixed time following addition of radiolabeled ATP to start the reaction. Figure 5 shows the characterization of the CaM kinase II/caldesmon preparation: caldesmon phosphorylation is observed only in the presence of both Ca2+ and CaM, and is completely inhibited by [Ala9]autocamtide 2, a CaM kinase II inhibitor. Figure 6 shows the concentration dependence of CaM activation of CaM kinase II. To conduct this assay: 1. To 15 µL of buffer in a plastic 1.5-mL microfuge tube (without cap) on ice, add CaM (0–10 µM) and caldesmon containing CaM kinase II (0.2 mg/mL). 2. Add distilled, deionized H 2O to give a total volume of 27 µL.
350 Walsh et al. Fig. 4. CaM-dependent activation of MLCK. MLCK activity was measured at the indicated concentrations of bovine brain CaM. Values represent the mean ± SEM (n = 3 – 7, each done in duplicate or triplicate). Maximal activity corresponds to 12.7 µmol P i incorporated/min.mg MLCK. 3. Mix the solution well by vortexing and let it equilibrate to 30°C in a water bath. 4. Start the reaction by adding 3 µL of stock [γ- 32 P]ATP. 5. Continue from #5 of MLCK assay (see Subheading 3.7.). 4. Notes 4.1. General Considerations 1. For any assay, it is important that the solution be mixed thoroughly after each addition. For the continuous assays, this is achieved by placing Parafilm over the cuvet and mixing the solution before placing it in the fluorimeter or spectrophotometer. Subsequent additions to the cuvet are generally mixed by placing a pipet tip in the cuvet and rapidly drawing 150 µL of solution in and out of the pipet tip five times. Alternatively, a stirring stick or a magnetic stir bar can be used. For the discontinuous assays vortexing is used to assure homogeneity after each addition. 2. For any enzyme assay, it is essential to determine the time over which the reaction is linear. For the continuous assays, this can be done by simply following the change in fluorescence or absorption as a function of time. It is imperative that any CaM dose- response curves or inhibitor studies be conducted while the change in signal is in the linear phase. Depending on the time of linearity, this may allow determination of the effect of one or many [CaM] per assay. For example, the continuous PDE assay is so rapid (over in 3–4 min) that we add one [CaM] per assay. The continuous CaN assay remains linear for 25 min, even with maximal [CaM], and it is, therefore, possible to determine the effect of several [CaM] in one assay. If the CaM dose-response curve allows you to use multiple
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Methods in Molecular BiologyTM Biol
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M E T H O D S I N M O L E C U L A R
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© 2002 Humana Press Inc. 999 River
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Preface Calcium plays an important
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Contents Dedication ...............
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Contents xi 26 Enzymatic Assays to
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xiv Contents of Companion Volume 15
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xvi Contributors LESLIE D. HICKS
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20 Dean, Kelsey, and Re
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2 Yazawa
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4 Yazawa Fig. 1. Schematic represen
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6 Yazawa Fig. 2. Shop drawing for t
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8 Yazawa DuPont-NEN and the molar c
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10 Yazawa Fig. 4. Examples of the C
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12 Yazawa 5. Plot the calculated Ca
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14 Yazawa 12. Starovasnik, M. A., D
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16 Fig. 1. Molecular structures and
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18 Linse 7. 0.1 M EDTA. Dissolve 37
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20 Linse iterate the other paramete
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22 Linse tive to small alterations
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24 Linse References 1. Tsien, R. Y.
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26 Haiech and Kilhoffer to use the
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28 Haiech and Kilhoffer where γ is
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30 Haiech and Kilhoffer reporter gr
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32 Haiech and Kilhoffer tein with i
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34 Haiech and Kilhoffer Calmodulin
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36 Haiech and Kilhoffer Fig. 3. Mec
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38 Haiech and Kilhoffer We may sugg
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40 Haiech and Kilhoffer 29. Stewart
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42 Haiech and Kilhoffer 62. Becking
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44 Martin and Bayley Fig. 1. Absorp
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46 Martin and Bayley evaporation. C
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48 Martin and Bayley 4. Set the tem
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50 Martin and Bayley Fig. 2. Near-U
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52 Martin and Bayley discussed by M
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54 Martin and Bayley References 1.
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20 Dean, Kelsey, and Reik
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58 Fabian and Vogel are equipped wi
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60 Fabian and Vogel The penetration
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62 Fabian and Vogel perature, ionic
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64 Fabian and Vogel Fig. 3. IR spec
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66 Fabian and Vogel Fig. 4. Lower t
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68 Fabian and Vogel These correlati
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70 Fabian and Vogel Fig. 5. Amide I
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72 Fabian and Vogel 4. ATR-FTIR spe
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74 Fabian and Vogel 25. Georg, H.,
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76 Weljie and Vogel Fig. 1. Simplif
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78 Weljie and Vogel Fig. 3. Steady-
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80 Weljie and Vogel VIS spectrophot
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82 Weljie and Vogel 4. A series of
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84 Table 1 Advanced Fluorescence Me
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86 Weljie and Vogel References 1. L
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90 Johnson and Tikunova is removed
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92 Johnson and Tikunova function of
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94 Johnson and Tikunova Fig. 2. Rat
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96 Johnson and Tikunova Fig. 3. Rat
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98 Johnson and Tikunova Fig. 4. Ca
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100 Johnson and Tikunova cence inte
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102 Johnson and Tikunova 6. Johnson
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104 Julenius Fig. 1. Under conditio
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106 Julenius 3. Mix 50 µL of NHS s
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108 Julenius Fig. 3. A typical sens
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110 Julenius (2), is used in the Ia
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114 Lopez and Makhatadze Fig. 1. Th
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116 Lopez and Makhatadze 6. Buffers
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118 Lopez and Makhatadze The excess
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122 Lopez and Makhatadze Fig. 1. Is
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124 Lopez and Makhatadze 2.5 mL are
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126 Lopez and Makhatadze pendence o
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128 Hicks et al. equipped with a pu
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130 Hicks et al. Fig. 2. Debye plot
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132 Hicks et al. and 1.0 mg/mL for
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134 Hicks et al. Fig. 3. Sedimentat
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136 Hicks et al. 5. Hayes, D. B. (M
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138 Trewhella and Krueger This chap
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140 Trewhella and Krueger of the sc
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142 Trewhella and Krueger Table 1 C
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144 Trewhella and Krueger beam, or
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146 Trewhella and Krueger At very l
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148 Trewhella and Krueger analytica
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150 Trewhella and Krueger collapsed
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152 Trewhella and Krueger P(r) func
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154 Trewhella and Krueger Fig. 3. S
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156 Trewhella and Krueger concentra
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158 Trewhella and Krueger by a Nati
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162 Doherty-Kirby and Lajoie metal
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164 Doherty-Kirby and Lajoie two Ca
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166 Doherty-Kirby and Lajoie 7. Pep
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168 Doherty-Kirby and Lajoie Fig. 1
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170 Doherty-Kirby and Lajoie Fig. 2
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172 Doherty-Kirby and Lajoie 5. Alt
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174 Doherty-Kirby and Lajoie phosph
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176 Shaw Fig. 1. Ribbon drawing of
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178 Shaw 2.3. Other 1. Chemicals fo
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180 Shaw 3.3.3. Purification 1. Con
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182 Shaw 10. Hodges, R. S., Semchuk
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184 Brokx and Vogel Table 1 An Over
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186 Brokx and Vogel Fig. 1. UV abso
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188 Brokx and Vogel Fig. 2. 20% SDS
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190 Brokx and Vogel it through an a
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192 Brokx and Vogel 8. Weljie, A. M
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196 Berliner Fig. 1. Aqueous X-band
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198 Berliner in the Bruker instrume
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200 Berliner Fig. 3. ESR spectra of
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202 Berliner Fig. 5. Low-temperatur
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204 Berliner 14. Musci, G., Reed, G
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206 Clarke and Vogel cal calcium-bi
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208 Clarke and Vogel Fig. 1. 113 Cd
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214 Clarke and Vogel The linewidth
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218 Drakenberg sands, of Hertz broa
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220 Drakenberg 1. Bloch equations m
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222 Drakenberg Fig. 2. Measurement
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224 Drakenberg Fig. 3. (A) 43 Ca NM
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226 Drakenberg Fig. 5. 43 Ca NMR sp
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228 Drakenberg NMR at sub-mM concen
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230 Drakenberg 30. Shimizu, T., Hat
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232 Weljie and Heringa Table 1 Amin
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234 Weljie and Heringa Table 2 Webs
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236 Weljie and Heringa diction meth
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238 Weljie and Heringa these databa
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240 Weljie and Heringa lineages. Th
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242 Weljie and Heringa compared, an
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244 Weljie and Heringa sequences as
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246 Weljie and Heringa much larger
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248 Weljie and Heringa ment require
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250 Weljie and Heringa 10. Kawasaki
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252 Weljie and Heringa 44. Thompson
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254 Weljie and Heringa
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256 Li et al. In order for these ex
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258 Li et al. 7. Mineral Mixture (s
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260 Li et al. 3. When the 45% D 2O/
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262 Li et al. sion system works eff
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264 Li et al. reliably produce high
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268 Mal et al. NMR data, X-PLOR (11
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270 Mal et al. Table 1 Simulated An
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272 Mal et al. 3.1.2.1. AMBIGUOUS D
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274 Mal et al. molecular dynamics a
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276 Mal et al. Assuming a rigid bod
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278 Mal et al. assign (segid A and
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280 Mal et al. References 1. Drenth
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282 Mal et al. 33. Jeener, J., Meie
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286 Werner et al. Our research has
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288 Fig. 1. (A) T 1, T 2 and the he
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290 Werner et al. tion, using gradi
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292 Werner et al. Fig. 3. (A) Rotat
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294 Werner et al. Fig. 4. (A) Line-
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296 Werner et al. Table 2 Models of
Page 317 and 318: 298 Werner et al. 9. Reinhardt, D.
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Page 321 and 322: 302 Boyd et al. utilize residual di
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Page 325 and 326: 306 Boyd et al. or significant peak
Page 327 and 328: 308 Boyd et al. Fig. 3. Histogram o
Page 329 and 330: 310 Boyd et al. total and NOE energ
Page 331 and 332: 312 Boyd et al. 2. When studying mu
Page 333 and 334: 314 Boyd et al. 4. Downing, A. K.,
Page 335 and 336: 316 Boyd et al. 35. Schulte-Herbrug
Page 337 and 338: 318 Yap et al. image representation
Page 339 and 340: 320 Yap et al. modified EF-hand is
Page 341 and 342: 322 Table 1 Angle and Distance Outp
Page 343 and 344: 324 Yap et al. 2. Ikura, M. (1995)
Page 345 and 346: 326 Kobayashi that S-100A1 and S-10
Page 347 and 348: 328 Kobayashi 3.2. Coupling of Crom
Page 349 and 350: 330 Kobayashi Fig. 2. Tricine/SDS/P
Page 351 and 352: 332 Kobayashi 0.1% TFA at a flow ra
Page 353 and 354: 334 Kobayashi Both recombinant S-10
Page 355 and 356: 336 Kobayashi Fig. 6. Affinity chro
Page 357 and 358: 20 Dean, Kelsey, and Reik
Page 359 and 360: 340 Walsh et al. verts CaM from an
Page 361 and 362: 342 Walsh et al. 11. Bovine serum a
Page 363 and 364: 344 Walsh et al. Fig. 1. CaM-depend
Page 367: 348 Walsh et al. 3.5. NOS Reduction
Page 373 and 374: 354 Walsh et al. 3. Cho, M. J., Vag
Page 375 and 376: 356 Hughes et al. The electroporati
Page 377 and 378: 358 Hughes et al. 4. 1 M Na 2CO 3.
Page 379 and 380: 360 Hughes et al. range, repeat ste
Page 381 and 382: 362 Hughes et al. Table 1 Examples
Page 383 and 384: 20 Dean, Kelsey, and Reik
Page 385 and 386: 366 Persechini of the different CaM
Page 387 and 388: 368 Persechini Fig. 1. Schematic re
Page 389 and 390: 370 Persechini 2. 1 L of Terrific b
Page 391 and 392: 372 Persechini Fig. 4. Titration wi
Page 393 and 394: 374 Persechini Table 1 Parameters f
Page 395 and 396: 376 Persechini Fig. 6. The [Ca 2+ -
Page 397 and 398: 378 Persechini determined if the di
Page 399 and 400: 380 Persechini relatively insensiti
Page 401 and 402: 382 Persechini 18. Chafouleas, J. G
Page 403 and 404: 384 Török et al. actions in the c
Page 405 and 406: 386 Török et al. 3. The reaction
Page 407 and 408: 388 Török et al. Fig. 2. Electros
Page 409 and 410: 390 Török et al. Fig. 3. Peptides
Page 411 and 412: 392 Török et al. Table 1 Peptide
Page 413 and 414: 394 Török et al. Fig. 7. Nanospra
Page 415 and 416: 396 Török et al. Fig. 9. Nanospra
Page 417 and 418: 398 Török et al. Fig. 11. Electro
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400 Török et al. 3 µM FL-calmodu
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402 Török et al. Fig. 14. Localiz
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404 Török et al. Fig. 16. Indicat
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406 Török et al. the significantl
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408 Török et al.
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410 Index substitutes, see Fluoresc
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412 Index Stern-Volmer plot, 78, 81
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414 Index Phenothiazines, see Calmo
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METHODS IN MOLECULAR BIOLOGY TM •
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