Isolation of the cytoplasmic granules of guinea pig basophilic leukocytes: identification of esterase and protease activities.
Procedures were developed for isolating highly purified cytoplasmic granules of basophilic leukocytes from guinea pig peripheral blood. The methods involved disruption of cells in 0.34 M sucrose followed by a series of membrane filtrations and fractionation on sucrose density gradients. These preparations, up to 95% pure basophil granules by electron microscopy, contained a mixture of neutral esterases-proteases including caseinolytic activity; both trypsin- and chymotrypsin-like serine hydrolases were identified by means of appropriate inhibitors. Localization of at least one such activity to the basophil granule was confirmed by a cytochemical method; this activity was absent in contaminating lymphocytes and eosinophils. By contrast, several lysosomal enzymes, lactic dehydrogenase, and plasminogen activator activity, present in cell homogenates, were absent from purified granules. The granule matrix of guinea pig basophils, unlike the cytoplasmic granules of other granulocytes or mast cells, was little altered by high or low salt concentration but was disrupted into insoluble fragments by 0.01 N HCl and by Triton X-100. Granules were solubilized by papain and by urea-SDS but enzyme activity was destroyed. Triton X-100 incubation with freeze-thawing proved to be the optimal method for extracting esterase activities. Esterase activities were not released from basophils under conditions of anaphylactic degranulation that liberated the great majority of basophil granule histamine.
D000818:Animals; D001491:Basophils / Q000032:analysis; D002457:Cell Extracts / Q000302:isolation & purification; D002458:Cell Fractionation; D003594:Cytoplasmic Granules / Q000032:analysis / Q000201:enzymology / Q000648:ultrastructure; D004950:Esterases / Q000378:metabolism*; D006168:Guinea Pigs; D006651:Histocytochemistry; D010447:Peptide Hydrolases / Q000378:metabolism*; D012995:Solubility
D002457:Cell Extracts; D004950:Esterases; D010447:Peptide Hydrolases