Osmotic fragility of chromaffin granules prepared under isoosmotic or hyperosmotic conditions and localization of acetylcholinesterase

Burgun, Claude; Muñoz, D Martínez de; Aunis, Dominique

doi:10.1016/0304-4165(85)90001-7

Cited by 24 publications

(9 citation statements)

References 31 publications

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“…The resistance of these granules, which are markedly less dense that the major granule fraction, is probably due to an incomplete load of catecholamines; they may have lost a fraction of their catecholamines by leakage during subcellular fractionation, or may represent an original population of granule precursors which have not yet acquired their full catecholamine complement. Chromaffin granules are known to be heterogeneous in their sensitivity to osmotic shock [27]. The presence of a soluble form of AChE in chromaffin granules is also consistent with the inaccessibility of this enzyme to a non-permeant inhibitor [27].…”

Section: Subcellular Localization Qf the Various Acheformsmentioning

confidence: 86%

“…Chromaffin granules are known to be heterogeneous in their sensitivity to osmotic shock [27]. The presence of a soluble form of AChE in chromaffin granules is also consistent with the inaccessibility of this enzyme to a non-permeant inhibitor [27].…”

Section: Subcellular Localization Qf the Various Acheformsmentioning

confidence: 86%

“…Submitting isolated chromaffin granules to a series of hypoosmotic shocks, Burgun et al [27] found that the release of catecholamines and dopamine P-hydroxylase followed the resulting changes in turbidity, while the release of AChE appeared to be significantly delayed. The released enzyme was not inhibited by application of echothiopate, a non-permeant inhibitor, during homogenization of the adrenal gland, and was therefore originally contained in closed vesicles, rather than present on membrane fragments which might be secondarily associated with the granule preparation.…”

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confidence: 99%

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Subcellular distribution of acetylcholinesterase forms in chromaffin cells

Bon

Bader

Aunis

et al. 1990

European Journal of Biochemistry

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The presence of acetylcholinesterase (AChE) in chromaffin granules has been controversial for a long time. We therefore undertook a study of AChE molecular forms in chromaffin cells and of their distribution during subcellular fractionation. We characterized four main AChE forms, three amphiphilic forms (G1a· G2a and G4a), and one non‐amphiphilic form (G4na). Each form shows the same molecular characteristics (sedimentation, electrophoretic migration, lectin interactions) in the different subcellular fractions. All forms are glycosylated and seem to possess both N‐linked and O‐linked carbohydrate chains. There are differences in the structure of the glycans carried by the different forms, as indicated by their interaction with some lectins. Glycophosphatidylinositol‐specific phospholipases C converted the G2a form, but not the other amphiphilic forms, into non‐amphiphilic derivatives. The distinct patterns of AChE molecular forms observed in various subcellular compartments indicate the existence of an active sorting process. G4na was concentrated in fractions of high density, containing chromaffin granules. We obtained evidence for the existence of a lighter fraction also containing chromogranin A, tetrabenazine‐binding sites and G4na AChE, which may correspond to immature, incompletely loaded granules or to partially emptied granules. The distribution of G4na during subcellular fractionation suggested that this form is largely, but not exclusively, contained in chromaffin granules, the membranes of which may contain low levels of the three amphiphilic forms.

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Section: Subcellular Localization Qf the Various Acheformsmentioning

confidence: 86%

Section: Subcellular Localization Qf the Various Acheformsmentioning

confidence: 86%

mentioning

confidence: 99%

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Subcellular distribution of acetylcholinesterase forms in chromaffin cells

Bon

Bader

Aunis

et al. 1990

European Journal of Biochemistry

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“…As shown in Figure 1, absorbance at 540 nm of the granule suspension was proportional to amylase activity pelletable at 12,000 g. This relation between the absorbance at 540 nm and the amount of intact zymogen granules in suspension has been routinely used for zymogen granules [1,2,4,16] as well as chromaffin granules [19,20]. Absorbance at 540 nm was measured as an index of zymogen granule lysis.…”

Section: Turbidimetry Measurement Of Granule Iysismentioning

confidence: 98%

In vitro stability of pancreatic zymogen granules: roles of pH and calcium

Lebel¹

1988

Biology of the Cell

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Purified preparations of pancreatic zymogen granules have the peculiar property of lysing instantaneously at neutral pH, a property clearly irreconcilable with the cytoplasmic pH of the acinar cell. Two important factors known for regulating the stability of secretory granules are calcium and pH. Fluorescence microscopy of acinar cells in the presence of weak bases showed that zymogen granules have an acidic pH. In vivo, abolition of the delta pH by NH4Cl did not induce any lysis of the granules. In vitro, with purified granules, an acidic intragranular pH was measured. This delta pH was produced by a Donnan potential. The importance for granule stability of keeping the intragranular pH acidic has been confirmed in vitro by addition of K+ and nigericin to the suspension medium. These conditions produced alkalinization of the granule matrix and caused instantaneous solubilization of the granules. Concentrations of 15 mM total, and 10 mM free calcium were measured in purified granules. The importance of intragranular Ca2+ was evaluated by means of the ionophore A23187 which induced calcium efflux and granule lysis. The lysis induced by the calcium ionophore was in direct relation with the calcium efflux, since addition of Ca2+ to the medium, at concentrations corresponding to that measured in the granule, relieved the effect. The role of calcium-binding sites on the cytoplasmic surface of the granules was investigated with Ca2+, EGTA, and La3+. Calcium did not have any damaging effects; EGTA induced a slight lysis, while lanthanum yielded a strong and spontaneous lysis at micromolar concentrations. In addition to calcium-binding sites, La3+ would bind to specific sites on the granule that would be directly coupled to maintenance of its stability. These findings suggest that the intragranular acidic pH and calcium are both important for the in vitro stability of the zymogen granule and that purified granules have lost, in the course of purification, some cytoplasmic factors that in vivo, control the permeability of the membrane to protons, and chloride more particularly. Calcium-binding sites and other specific sites probed with La3+, presumably on proteins at the surface of the granule, are also believed to have key roles in preserving the integrity of the membrane and the resulting stability of the granule.

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“…Recently, acetylcholinesterase and other enzyme markers for plasma membrane have also been found as constituents of chromaffin granule membranes [24]. Nevertheless, these enzymatic markers will provide us with a real approach to study the subcellular distribution of glucose transporters.…”

Section: G 24mentioning

confidence: 99%

Glucose transporters in chromaffin cells: Subcellular distribution and characterization

Delicado

Torres

1988

FEBS Letters

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The glucose transporter was identified and characterized by cytochalasin B binding in subcellular membrane fractions of chromaffin tissue. The binding was saturable with a Kd of about 0.3/zM for each subceilular fraction. The Bm~ capacity was 12-16 pmol/mg protein for enriched plasma membrane fractions, 6.3 pmol/mg protein for mierosomal membrane preparations and 5.4 pmol/mg protein for chromaitin granule membranes. Irreversible photoaffinity labelling of the glucose-protectable binding sites with [aH]cytochaiasin B followed by solubilization and polyacrylamide gel electrophoresis from enriched plasma membrane preparations demonstrated the presence of three molecular species: 97+ 10, 51.5 + 6 and 30 + 4 kDa. The chromaffin granule membranes showed only a molecular species of 80__+ 10 kDa.

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Osmotic fragility of chromaffin granules prepared under isoosmotic or hyperosmotic conditions and localization of acetylcholinesterase

Cited by 24 publications

References 31 publications

Subcellular distribution of acetylcholinesterase forms in chromaffin cells

Subcellular distribution of acetylcholinesterase forms in chromaffin cells

In vitro stability of pancreatic zymogen granules: roles of pH and calcium

Glucose transporters in chromaffin cells: Subcellular distribution and characterization

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