caa a22 4500 477119883 CHVBK 20180405111633.0 cr unu---uuuuu 170330e19960201xx s 000 0 eng 10.1007/BF00229309 doi (NATIONALLICENCE)springer-10.1007/BF00229309 The uptake of calcium by isolated chromaffin granules of the adrenal medulla [Elektronische Daten] [Sandra Fitzpatrick, David Waisman] Bovine chromaffin secretory granules were purified by isopycnic Metrizamide gradient centrifugation and their Ca2+ sequestration pathways were characterized. The rate of Ca2+ sequestration at 37°C was first order, with a maximal uptake of 26.9 ±0.46 (mean ± S.D., n = 3) nmol Ca2+/mg protein and a first order rate constant (k) of 0.046 ± 0.002 min−1. At 4°C the rate of uptake was substantially attenuated, with only 2.47 ± 0.2 (mean ± S.D, n = 3) nmol Ca2+/mg protein sequestered in 60 min. Ca2+ sequestration was 93% inhibited by 180 mM NaCl [I50% of 78.7 ± 9.3 mM NaCl (mean ± S.D., n = 11)] but only slightly inhibited by KCl or MgCl2. Ca 2+ sequestration was not stimulated by incubation with MgATP but was inhibited by 57% after incubation with 30 μM monensin. Ca 2+ sequestration was dependent on extravesicular Ca 2+ with half-maximal sequestration at pCa2+ 6.81 ± 0.028 (mean ± S.D., n = 3). Sequestered Ca2+ could be exchanged with external 45Ca2+, the exchange rate was first order (k of 0.042 ± 0.004: mean ± S.D., n = 3) and saturated at 27.7 ± 1.1 nmol Ca2+/mg (mean ± S.D., n = 3). The Ca2+/Ca2+ exchange system was totally inhibited by NaCl or KCl but only slightly by MgCl2. About 75% of sequestered 45Ca2+ could be released by incubation with NaCl, but only 8% was released by incubation with KCI. Half-maximal release of sequestered 45Ca2+ required 69.3 ± 12.2 mM NaCl (mean ± S.D., n = 3). The Na+-induced release of sequestered 45Ca2+ was rapid, t0.5 of 2.80 ± 0.63 min (mean ± S.D., n = 3) and inhibited at 4°C. The concurrent incubation of chromaffin granules with 45Ca2+ and either annexin proteins V or VI resulted in attenuated uptake of 45Ca2+. These results suggest that Ca2+ uptake in adrenal chromaffin granules is regulated by Na+ and Ca2+ gradients and also possibly by annexins V and VI. Kluwer Academic Publishers, 1996 calcium uptake nationallicence chromaffin granules nationallicence annexins nationallicence EGTA : ethylene glycol bis (β-aminoethyl ether)-N,-N,N′,N′-tetraacetic acid nationallicence SDS : Sodium dodecyl sulphate nationallicence PAGE : Polyacrylamide gel electrophoresis nationallicence BSA : bovine serum albumin nationallicence AI : Annexin I nationallicence AIIt : Annexin II tetramer nationallicence AIII : Annexin III nationallicence AIV : Annexin IV nationallicence AV : Annexin V nationallicence AVI : Annexin VI nationallicence k : first order rate constant nationallicence AT : total extent of Ca2+ uptake (nmol) nationallicence BufferA : 300 mM sucrose, 10 mM potassium phosphate (pH 7.0), 5 mM EGTA nationallicence Buffer B : 300 mM sucrose, 10 mM potassium phosphate (pH 7.0) and 1 mM EGTA nationallicence Buffer C : 300 mM sucrose, 10 mM potassium phosphate (pH 7.0) nationallicence Buffer D : 300 mM sucrose, 10 mM potassium phosphate (pH 7.0), 0.5 mM EGTA and 0.65 MM CaCl2 nationallicence Buffer E : 300 mM sucrose, 10 mM potassium phosphate (pH 7.0), 0.25 mM EGTA and 0.325 mM CaCl2 nationallicence Fitzpatrick Sandra Cell Regulation Research Group, Department of Medical Biochemistry, University of Calgary, Health Sciences Centre, 3330 Hospital Drive N. W., T2N 4N1, Alberta, Canada aut Waisman David Cell Regulation Research Group, Department of Medical Biochemistry, University of Calgary, Health Sciences Centre, 3330 Hospital Drive N. W., T2N 4N1, Alberta, Canada aut Molecular and Cellular Biochemistry Kluwer Academic Publishers 155/2(1996-02-01), 121-130 0300-8177 155:2<121 1996 155 11010 https://doi.org/10.1007/BF00229309 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/BF00229309 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Fitzpatrick Sandra Cell Regulation Research Group, Department of Medical Biochemistry, University of Calgary, Health Sciences Centre, 3330 Hospital Drive N. W., T2N 4N1, Alberta, Canada aut NATIONALLICENCE 700 1- Waisman David Cell Regulation Research Group, Department of Medical Biochemistry, University of Calgary, Health Sciences Centre, 3330 Hospital Drive N. W., T2N 4N1, Alberta, Canada aut NATIONALLICENCE 773 0- Molecular and Cellular Biochemistry Kluwer Academic Publishers 155/2(1996-02-01), 121-130 0300-8177 155:2<121 1996 155 11010 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer