RELEASE OF CA2- EVIDENCE AGAINST INVOLVEMENT OF METALLOENDOPROTEASES IN CA2+ SEQUESTRATION BY THE ENDOPLASMIC-RETICULUM( FROM INTRACELLULARORGANELLES BY PEPTIDE ANALOGS )

N-Benzyloxycarbonyl-Gly-Phe-amide (Z-Gly-Phe-NH2), a competitive subst rate for metalloendoproteases, mobilizes intracellular Ca2+ and suppre sses protein synthesis and processing in a Ca2+-dependent, reversible manner. To ascertain whether Z-Gly-Phe-NH2 acts at Ca2+-storing organe lles, effects of the dipeptide on Ca2+ sequestration by saponin-porate d GH(3) pituitary cells were examined. Porated preparations sequestere d Ca2+ into two compartments with different Ca2+ affinities. Ca2+ accu mulation at nM concentrations of free Ca2+ was inhibited by thapsigarg in and inositol 1,4,5-triphosphate [Ins(1,4,5)P-3], enhanced by oxalat e and unaffected by oligomycin. Cation accumulation at mu M concentrat ions of free Ca2+ was sensitive to oligomycin but not to thapsigargin. Z-Gly-Phe-NH2 reduced Ca2+ sequestration by both compartments. The di peptide mobilized Ca2+ from the high-affinity compartment within 1-2 m in without affecting Ca2+ uptake. Ca2+ was mobilized more rapidly by Z -Gly-Phe-NH2 and thapsigargin together than by either agent alone. The presence of a thiol-reducing agent was required for Ca2+ mobilization by Z-Gly-Phe-NH2 but not by thapsigargin or Ins(1,4,5)P-3. Ca2+ mobil ization by Z-Gly-Phe-NH2 could not be attributed to effects on anion-p ermeability or to actions at Ins(1,4,5)P-3 or ryanodine receptors. Res ults with assorted peptide analogues did not favour suppression of met alloendoprotease activity in the Ca2+-mobilizing action of Z-Gly-Phe-N H2. The more hydrophobic analogue Z-L-Tyr-p-nitrophenyl ester was 60-8 0-fold more potent in mobilizing Ca2+ from intact and porated cells an d perturbed the high-affinity Ca2+-sequestering compartment selectivel y. Z-Gly-Phe-NH2 and Z-L-Tyr-p-nitrophenyl ester are proposed to relea se Ca2+ from the endoplasmic reticulum through an ion pore with affini ty for hydrophobic molecules containing internal peptide bonds.