ATP-dependent calcium sequestration and calcium/ATP stoichiometry in isolated microsomes from guinea pig parotid glands

AbstractATP-dependent calcium uptake was studied in isolated guinea pig parotid gland microsomes. The apparent Km for free Ca2+ was 0.41 μM, the apparent Km for ATP·Mg2− 0.23 mM. The pH optimum was 6.8–7.0. Subfractionation of the microsomes revealed that the highest specific uptake activity resided in a rather dense fraction of the endoplasmic reticulum. The calcium uptake/ATPase stoichiometry was determined in the absence of exogenous magnesium in the submicrosomal fractions. It ranged from 1–2. It is concluded that in vivo the stoichiometry is the same as in sarcoplasmic reticulum, namely 2

GTP-dependent Ca2+ release from rat liver microsomes Vesicle fusion is not required

AbstractThe GTP-dependent calcium release from rat liver microsomes is known to be promoted in the presence of colloids like polyethyleneglycol (PEG), polyvinylpyrrolidine, or albumin. Dawson et al. [(1987) Biochem. J. 244, 87–92] using the ‘fusogen’ PEG have concluded that both GTP-induced calcium efflux and the enhancement of InsP3-promoted calcium release in the presence of GTP could be attributed to a GTP-dependent vesicle fusion. Here, using the more physiological colloid albumin we report that GTP-induced calcium release from rat liver microsomes may not be linked to vesicle fusion

KDEL Receptor (Erd2p)-mediated Retrograde Transport of the Cholera Toxin A Subunit from the Golgi Involves COPI, p23, and the COOH Terminus of Erd2p

A cholera toxin mutant (CTX–K63) unable to raise cAMP levels was used to study in Vero cells the retrograde transport of the toxin A subunit (CTX-A–K63), which possesses a COOH-terminal KDEL retrieval signal. Microinjected GTP-γ-S inhibits the internalization as well as Golgi–ER transport of CTX-A–K63. The appearance of CTX-A–K63 in the Golgi induces a marked dispersion of Erd2p and p53 but not of the Golgi marker giantin. Erd2p is translocated under these conditions most likely to the intermediate compartment as indicated by an increased colocalization of Erd2p with mSEC13, a member of the mammalian coat protein II complex. IgGs as well as Fab fragments directed against Erd2p, β-COP, or p23, a new member of the p24 protein family, inhibit or block retrograde transport of CTX-A–K63 from the Golgi without affecting its internalization or its transport to the Golgi. Anti-Erd2p antibodies do not affect the binding of CTX-A to Erd2p, but inhibit the CTX-K63–induced translocation of Erd2p and p53

Inositol 1,4,5-trisphosphate and 5′-GTP induce calcium release from different intracellular pools

AbstractIt has been shown recently by several groups that 5′-GTP can release calcium from intracellular compartments independently from inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) by a mechanism which seems to be different from that used by Ins(1,4,5)P3. We report here for the first time that the 5′-GTP-sensitive and the Ins(1,4,5)P3-sensitive calcium pools reside in different intracellular compartments