The use of high pressure liquid chromatography (HPLC) for the separation of radiolabeled arachidonic acid and its metabolites produced by thrombin-treated human platelets. II. Establishment of optimal assay conditions.

We have utilized HPLC to develop optimal conditions for assaying the transformation of arachidonic acid in thrombin-treated human platelets. In the presence of increasing amounts of albumin, the total amount of radioactivity released from thrombin-treated platelets pre-labeled with 3H-arachidonic acid is first enhanced and then inhibited. Maximal release, reflecting primarily enhanced amounts of free labeled arachidonic acid, occurs at a final albumin concentration of 0.5 mg/ml. Calcium promoted the release of all radiolabeled metabolites, but it specifically enhanced HETE formation and release. Magnesium was without effect. Cyclo-oxygenase derived products constituted the bulk of released label at short time intervals, but after ten minutes exposure to thrombin in the presence of albumin (0.5 mg/ml) and 3 mM calcium, radioactivity in the released products was equally distributed among cyclo-oxygenase derived products (TXB2 + PGD2 + HHT), HETE and free arachidonic acid.     

Polyphosphate-deficient mutants of Anacystis nidulans.

Polyphosphate-deficient mutants of Anacystis nidulans have been isolated by either ethyl methanesulfonate (EMS) or N-methyl nitrosoguanidine (NTG) mutagenesis and penicillin-enrichment techniques. Mutagenised stock was preincubated in a medium lacking sulfate, then transferred to a phosphate-lacking medium before penicillin treatment. Many single-colony isolates, in contrast to wild-type, show little growth in absence of phosphate, and have altered polyphosphate, and have altered polyphosphate kinase levels indicating that the lesions affect either the activity or the expression of this enzyme. In these same mutants radioactive phosphate incorporation is severely retarded. Electron micrographs confirm the absence of polyphosphate granules in some mutants.