A new method for cell permeabilization reveals a cytosolic protein requirement for Ca2+ -activated secretion in GH3 pituitary cells.
Ca2+ is a major regulator of exocytosis in secretory cells, however, the biochemical mechanisms underlying regulation remain to be identified. To render the secretory apparatus accessible for biochemical studies, we have developed a cell permeabilization method (cell cracking) which utilizes mechanical shear. GH3 pituitary cells subjected to cracking were permeable to macromolecules but retained a normal cytoplasmic ultrastructure including secretory granules. Incubation of the permeable cells at 30-37 degrees C with 0.1-1.0 microM Ca2+ and millimolar MgATP resulted in the release of the secretory proteins, prolactin (PRL) and a proteoglycan, but not lysosomal enzymes. Extensively washed permeable cells were incapable of releasing PRL in response to Ca2+ and MgATP addition. However, addition of cytosol was found to restore Ca2+-activated, MgATP-dependent PRL release. The cytosolic factor responsible for activity was thermolabile and protease sensitive. The protein was partially purified, and its molecular mass was estimated to be equivalent to that of a globular protein of 200-350 kDa by molecular sieve chromatography. Inhibitors of calmodulin or protein kinase C (trifluroperazine, calmidazolium, H-7) failed to inhibit Ca2+-activated PRL release, and the required cytosolic protein could not be replaced by purified calmodulin, calmodulin-dependent protein kinase II, protein kinase C, or calpactin I. Further purification and characterization of the cytosolic protein should reveal the nature of biochemical events involved in regulated secretory exocytosis.
Martin
T F
TF
Department of Zoology, University of Wisconsin, Madison 53706.
Walent
J H
JH
D000818:Animals; D001921:Brain / Q000502:physiology; D002118:Calcium / Q000502:physiology*; D002460:Cell Line; D002463:Cell Membrane Permeability; D003600:Cytosol / Q000502:physiology; D007700:Kinetics; D008854:Microscopy, Electron; D009363:Neoplasm Proteins / Q000502:physiology; D010911:Pituitary Neoplasms / Q000378:metabolism / Q000473:pathology / Q000648:ultrastructure; D011388:Prolactin / Q000378:metabolism*; D011506:Proteins / Q000502:physiology*; D051381:Rats
D009363:Neoplasm Proteins; D011506:Proteins; D011388:Prolactin; D002118:Calcium