Abstract
This chapter describes the assay method, purification procedure, and properties of glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle. The enzyme is assayed spectrophotometrically at 340 nm by the method of Krebs except that Tris buffer is substituted for the pyrophosphate buffer. All steps in the purification scheme are carried out at 0–4°. In order to minimize the inactivating effects of heavy metal ions, the saturated ammonium sulfate solutions should be prepared with enzyme grade reagent. In addition, EDTA, KCN, and dithioerythritol (DTE) or dithiothreitol (DTT) are used throughout purification to protect the sulfhydryl groups of the enzyme. The percent saturation of the ammonium sulfate solutions is calculated by the formula of Kunitz. The pH of the ammonium sulfate solutions is determined with a pH meter after diluting 1:20. After specific inhibition of the rabbit muscle enzyme with [1- 14 C] iodoacetic acid and tryptic digestion, a single radioactive peptide is obtained, which contains [1-C 14 ]carboxymethylcysteine. The amino acid sequence of the “active-site” peptide is similar to those of the enzyme isolated from other sources.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
