Methods in Molecular Biology • 16 Enzymes of Molecular Biology

254 Sharp and Slater Enzyme Data 2.1. Purification Purification of the nuclease activity from mung-bean sprouts has been report ...

Mung-Bean Nuclease I 255 Laskowski (3) found that a temperature of 37°C improved the hydro- lysis of native biosynthetic d(A-T)n ...

256 Sharp and Slater 2.7. Specificity Mung-bean nuclease 1 has more than one activity. Its predominant mode of action, and that ...

Mung-Bean Nuclease I 257 The relative base preference of mung-bean nuclease is A > T(U) > C > G as reported by Mikulski ...

258 Sharp and Slater stored in 50% (w/v) glycerol, has been shown to be stable for at least 6 mo at 20°C. Johnson and Laskowski ...

Mung-Bean Nuclease I 259 5' end~ 3' end IIIIIIIIIIIIIII 3' end 5' end Fig. 1. Conversion of protruding termini to blunt ends. E ...

260 Sharp and Slater Precipitate the DNA with 2 vol of ethanol at 0°C. Recover the DNA by centrifugation at 12,000g for 10 min ...

Mung-Bean Nuclease I 261 Wingert, L. and Von Hippel, P. H. (1968) The conformation dependent hydroxylysis of DNA by micrococcal ...

CHAPTER 13 RNase A (EC 3.1.27.5) Michael M. Burrell Introduction The term ribonuclease (RNase) is an imprecise term and is used ...

264 Burrell nus destroys enzyme activity. There is a considerable literature on the folding of the RNase A polypeptide and the i ...

RNase A 265 0~I--OH CH 2 0 OH O~l '--OH <~=ffi=~ C 2 Base H f I ? 0 OH \/ //P\ O OH CH20H '1 'r I Fig. 1. Cleavage of RNA. ~= ...

266 Burrell concentrations of enzyme, RNA with secondary structures will not be cleaved. If it is important to achieve total RNA ...

RNase A 267 Other mechanisms of inhibiting RNase activity during RNA extrac- tion include the use of sodium dodecyl sulfate, die ...

268 Burrell From the preceding discussion in this chapter, it is clearly important that, to obtain reproducible results, the pur ...

RNase A 269 Incubate at 37°C for 15-30 min. l 1. Extract protein with an equal volume phenol:chloroform solution. Retain aqueou ...

270 Burrell Bain, J. A. and Rusch, H. P. (1944) A method for the assay of in biological material. J. Biol. Chem. 153, 659-667. ...

CHAPTER 14 Pronase (EC 3.4.24.4) Patricia J. Sweeney and John M. Walker Introduction Pronase is the name given to a group of pr ...

272 Sweeney and Walker sion of substrates (5). Pronase has also been used to study membrane topology and protein membrane transl ...

Pronase 273 incubated at 40°C. After 10 min, 2 mL of protein precipitation agent are added (1.63 g trichloroacetic acid, 0.82 g ...

274 Sweeney and Walker Inhibitors Among the alkaline proteases, there are at least three that are inhib- ited by diisopropyl ...