By Ian Bird
This wide-ranging selection of state of the art strategies for the assay of phospholipid and phospholipid-derived moment messengers permits the identity and quantification of sign pathway activation. The assays defined conceal the entire significant phospholipases (C, D, A2), in addition to sphingomyelinase and linked metabolites. extra protocols are supplied for the assay of phosphoinositide 3-, 4-, and 5-kinase job and for the separation and identity of phospholipids, diacylglycerol and sphingolipids, in addition to their phosphoinositol, choline, and fatty acid metabolites. targeted, step by step directions make this e-book appropriate for either the newcomer and the skilled investigator.
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See NIOSH Pocket Guzde to Chemzcul Hazards (DHHSNIOSH 90- 117, 1990) p. 144. As a practical matter, the mvestigator IS cautioned that not all scintillation fluors are created equal For example, we have compared the htgh-salt compatible INFLOW BD scintillant from INUS System to Ecolume@ (ICN Research Products, Costa Mesa, CA) and found that the counting efficiency at high salt is markedly different between the two. Indeed, whereas there was an improvement in the detection of all radioacttve inosttol phosphates with INFLOW BD, the greatest improvement was seen in the recovery of InsP species elutmg at htgh salt A comparison of the recovery of InsP, counts using Ecolume to that injected on the HPLC column and 18%, while m an identical sample detected using INFLOW BD, the recovery of counts was 41%.
1 A4 formic acid (which has a pH of approx. 7). This solvent B was subsequently found to be able to elute InsP, InsPz, InsP3, and InsP4 standards with good baseline separation (Fig. 5). 4. Optimization of pH of So/vent 8 Having established an Initial salt concentration and pH, the next stage is to fully optimize the pH of solvent B to be used in the final method. To do this, a standard mixture of isomeric forms of phosphoinosltols in the classesof mterest (or if these are not available, tissue extracts known to contam mixtures of isomers in each class of the phosphoinositols) should be separated on a linear gradient of O-100% B (30 min for initial investigations but 60 min for fine tuning of methods), where solvent B always has the same salt concentration, but the pH is adjusted to different values m each run.
Hawkins, P. , and Downes, C. P. (1989) Metabolic and structural evrdence for the existence of a thrrd species of polyphosphomositidem cells: D-phosphatidyl-myo-inositol3-phosphate. Biochem. J 259,267-276. 5. , Williams, B. C , and Walker, W. W. (1992) Identification and metabolism of phosphomositol species formed on angiotensin II stimulation of zona fasciculata-reticularts cells from the bovine adrenal cortex. MOE Cell Endocnnol. 83,29-38. , Willlams, B. , and Walker, S W (1990) Vasopressm stimulates cortisol secretion and phosphoinosttide catabohsm m cultured bovine adrenal fasciculata/reticularis cells.