| Popis: |
Publisher Summary This chapter presents a protocol for the isolation and purification of inositol 1, 4, 5-triphosphate and kinase from brain. To demonstrate the purification process, 5-phosphatase or 3-kinase was associated with a given protein band on sodium dodecyl sulfate (SDS) gels, regeneration of activities was performed after SDS-polyacrylamide gel electrophoresis. It was proposed that the existence of three distinct 5-phosphatases: types I and II were present in rat or bovine brain, and type III has been purified in platelets (Mr = 75,000) and cloned. 3-Kinase appeared to exist in two forms: 3-kinase A was present in rat, bovine and human brain, and 3-kinase B had been isolated as a cDNA clone from a human hippocampus cDNA library. 3-Kinases A and B were associated with different genes on different chromosomes. The rat brain was homogenized and centrifuged for separation of supernatant. This supernatant was introduced into Blue-Sepharose column. The column was washed with buffer A. The elution of the enzyme is largely influenced by both salt and detergent concentrations. The pooled peak fractions were immediately concentrated to approximately 1 ml using an Amicon PM10 ultrafiltration membrane and verified to be pure by SDS-polyacrylamide gel electrophoresis. |
