Methods in Molecular Biology • 16 Enzymes of Molecular Biology

Carboxypeptidase Y 317 nmol of carboxypeptidase Y in 5-10 lxL of 0.1M pyridine acetate buffer, pH 5.6, thoroughly mix, incubate ...

318 Winder and Walker Hayashi, R. (1976) Carboxypeptidase Y, in Methods in Enzymology, vol. 45 (Lorand, L., ed.), Academic, New ...

CHAPTER 18 Aminopeptidases Aminopeptidase M (EC 3.4.11.2), Pyroglutamate Aminopeptidase (EC 3.4.19.3), and Prolidase (EC 3.4.13. ...

320 Sweeney and Walker CONH 2 CH2 ~H2 I H2NCHCO L H2.C CH 2 I O~.NJCHCO ~ + NH3 Fig. 1. The cyclization of N-terminal glutamine ...

Aminopeptidases 321 Enzyme Data 2.1. Pyroglutamate Aminopeptidase (Calf Liver) 2.1.1. Alternative Names These are L-pyroglutamy ...

322 Sweeney and Walker incubated at 37°C for 1 h. The solution is then cooled, and the absorbance recorded at 570 nm. The amount ...

Aminopeptidases 323 2.2.2. Specificity The enzyme cleaves N-terminal residues from all peptides having a free a-amino or o~-imin ...

324 Sweeney and Walker eral months at -20°C. The enzyme is reported to be stable at pH 7.0 at temperatures up to 65°C, and is st ...

Aminopeptidases 325 2.3.5. Assay An assay based on the hydrolysis of glycyl-L-proline at 37°C has been described. An incubation ...

326 Sweeney and Walker occasional mixing. Another 25 ~g of enzyme are then added, and incubation continued under nitrogen at roo ...

Aminopeptidases 327 References Himmelhoch, S. R. (1970) Leucine aminopeptidase from swine kidney. Meth- ods Enzymol. 19, 508-51 ...

328 Sweeney and Walker Light, A. (1972) Leucine aminopeptidase in sequence determination of pep- tides. Methods Enzymol. 25B, 2 ...

Aminopeptidases 329 Richter, A. M., Lancaster, G. L., Choy, F. Y. M., and Hechtman, P. (1989) Puri- fication and characterizati ...

CHAPTER 19 Alkaline Phosphatase (EC 3.1.3.1) Martin J. Maunders Introduction Alkaline phosphatases (or alkaline phosphomonoeste ...

332 Maunders Detection systems involve the use of an alkaline phosphatase mol- ecule conjugated to a second protein that specifi ...

Alkaline Phosphatase 333 coefficient of BAP, S°20,w, is 6.0 at pH 8.0 (11) and its isoelectric point pH 4.5 (12). Isozymes have ...

334 Maunders vented by the inclusion of Mg 2+ in the reaction buffer, which stimu- lates the monoesterase activity and almost co ...

Alkaline Phosphatase 335 Incubation with Zn 2+ can reverse some of the inactivation by these agents, but Mg 2÷ and Co 2÷ are muc ...

336 Maunders free of such activities as DNA endo- and exonucleases, RNase, Pro- tease, and adenosine deaminase. BAP is commonly ...

Alkaline Phosphatase 337 pH 8.0-9.0 (or 100 mM Glycine/NaOH, pH 10.5) and 0.1-1.0 mM ZnC12. In many cases, however, the presence ...