scholarly journals Fluoroaluminate treatment of rat liver microsomes inhibits GTP-dependent vesicle fusion

1991 ◽  
Vol 280 (2) ◽  
pp. 335-340 ◽  
Author(s):  
J G Comerford ◽  
A P Dawson
Keyword(s):  
Polyethylene Glycol ◽  
Membrane Fusion ◽  
Rat Liver ◽  
Liver Microsomes ◽  
Vesicle Fusion ◽  
Rat Liver Microsomes ◽  
Gtpase Activity ◽  
Maximal Inhibition ◽  
High Affinity ◽  
Gtp Binding

1. Inhibition of GTP-dependent membrane fusion of rat liver microsomes requires preincubation of the membranes with GDP (17 microM) and relatively high Mg2+ concentration (0.5 mM) as well as AlCl3 (30 microM) and KF (5 mM). Preincubation is required for maximal inhibition (75%). 2. Vesicle fusion in rat liver microsomes has been demonstrated in the absence of polyethylene glycol (PEG). Further, inhibition by AlF4- of GTP-dependent vesicle fusion in the absence of PEG has been demonstrated. 3. Under similar preincubation conditions AlF4- can bring about inhibition (80%) of the high-affinity PEG-stimulated GTPase activity in rat liver microsomes, previously described by Nicchitta, Joseph & Williamson [(1986) FEBS Lett. 209, 243-248]. 4. Preincubation of small-Mr GTP-binding proteins (Gn proteins) on nitrocellulose strips with GDP (20 pM), AlCl3 (30 microM) and KF (5 mM) results in inhibition of binding of guanosine 5′-[gamma-[35S]thio]triphosphate to Gn proteins. The extent of inhibition of this binding differs for different Gn proteins.

scholarly journals The effect of limited proteolysis on GTP-dependent Ca2+ efflux and GTP-dependent fusion in rat liver microsomal vesicles

1989 ◽  
Vol 258 (3) ◽  
pp. 823-829 ◽  
Author(s):  
J G Comerford ◽  
A P Dawson
Keyword(s):  
Rat Liver ◽  
Molecular Mechanisms ◽  
Limited Proteolysis ◽  
Liver Microsomes ◽  
Vesicle Fusion ◽  
Proteinase K ◽  
Rat Liver Microsomes ◽  
Proteolytic Digestion ◽  
Gtp Binding ◽  
Pre Treatment

1. Limited proteolytic digestion of rat liver microsomes (microsomal fractions) with trypsin (5 micrograms/ml), proteinase K (1.0 microgram/ml) and Pronase (20 micrograms/ml final concns.) resulted in abolition of GTP-dependent vesicle fusion. 2. Vesicle fusion could be partially restored to microsomes which had undergone limited tryptic digestion, by the addition of untreated microsomal vesicles. 3. GTP-dependent Ca2+ efflux from rat liver microsomes was also observed to be inhibited by limited proteolysis with trypsin and proteinase K. 4. Limited proteolysis of rat liver microsomes had no effect on subsequent GTP-dependent phosphorylation of polypeptides of Mr 17,000 and 38,000, and thus it is unlikely that the phosphorylation of these proteins is involved in GTP-dependent Ca2+ efflux and GTP-dependent vesicle fusion. 5. GTP binding by Gn proteins [proteins which bind GTP after transfer to nitrocellulose, as defined by Bhullar & Haslam (1986) Biochem. J. 245, 617-620] was inhibited by pre-treatment of microsomes with trypsin, proteinase K and Pronase at concentrations similar to those which abolished GTP-dependent Ca2+ efflux and vesicle fusion. 6. We suggest that one or more of the Gn proteins may be involved in the molecular mechanisms of GTP-dependent vesicle fusion and Ca2+ efflux in rat liver microsomes and that limited proteolytic digestion may be a useful tool in further investigation of these processes.

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

FEBS Letters ◽  
1989 ◽  
Vol 245 (1-2) ◽  
pp. 274-278 ◽  
Author(s):  
Jochen Kleineke ◽  
Andrea Schröder ◽  
Hans-Dieter Söling
Keyword(s):  
Rat Liver ◽  
Liver Microsomes ◽  
Vesicle Fusion ◽  
Rat Liver Microsomes

Induction of a high affinity nitrosamine demethylase in rat liver microsomes by acetone and isopropanol

1983 ◽  
Vol 44 (3) ◽  
pp. 247-260 ◽  
Author(s):  
Yityoong Yong Tu ◽  
Renxiu Peng ◽  
Zee-Fen Chang ◽  
Chung S. Yang
Keyword(s):  
Rat Liver ◽  
Liver Microsomes ◽  
Rat Liver Microsomes ◽  
High Affinity

The occurrence of two hepatic microsomal sites for amobarbital hydroxylation

1983 ◽  
Vol 61 (1) ◽  
pp. 67-71 ◽  
Author(s):  
P. A. Reilly ◽  
B. K. Tang ◽  
D. J. Stewart ◽  
W. Kalow
Keyword(s):  
Rat Liver ◽  
Human Liver ◽  
Liver Microsomes ◽  
Isolated Hepatocytes ◽  
Rat Liver Microsomes ◽  
Low Velocity ◽  
Whole Cells ◽  
High Affinity ◽  
High Affinity Site

Amobarbital metabolism in human liver and in rat liver, lung, kidney, and small intestine was measured in vitro using thin-layer chromatography (TLC) for separation of metabolites generated from incubation with [2-14C]amobarbital. Formation of 3′-hydroxyamobarbital (C-OH) occurred primarily in the liver. The kinetics of C-OH formation by rat liver microsomes or isolated hepatocytes could be described by a Michaelis–Menten model incorporating two metabolic sites, one characterized by high-affinity and low-velocity constants (Km = 0.054 ± 0.012 mM, Vmax = 16.89 ± 4.27 nmol C-OH∙g liver−1∙min−1), the other by low-affinity and high-velocity (Km = 0.679 ± 0.097 mM, Vmax = 66.0 ± 5.41 nmol C-OH∙g liver−1∙min−1). The kinetic parameters of the high-affinity site differed significantly between whole cells and homogenates. Pretreatment with phenobarbital for 3 days induced only the high-affinity site. Quantitation of C-OH formation in four human liver samples from several sources showed that metabolism may conform to the two-site model observed in rat liver.

scholarly journals Effects of CoA and acyl-CoAs on GTP-dependent Ca2+ release and vesicle fusion in rat liver microsomal vesicles

1993 ◽  
Vol 289 (2) ◽  
pp. 561-567 ◽  
Author(s):  
J G Comerford ◽  
A P Dawson
Keyword(s):  
Chain Length ◽  
Rat Liver ◽  
Liver Microsomes ◽  
Carbon Chain ◽  
Vesicle Fusion ◽  
Carbon Chain Length ◽  
Rat Liver Microsomes ◽  
Low Concentrations ◽  
Ic50 Values ◽  
Pumping Activity

(1) CoA (IC50 23 microM) and acyl-CoAs (IC50 values 15-18 microM) inhibit GTP-dependent vesicle fusion in rat liver microsomal vesicles. Acyl-CoAs of carbon chain length C8 and C20 are much less effective than acyl-CoAs of carbon chain length C14-C18. The effect of CoA is mimicked by dephospho-CoA, but not by desulpho-CoA. High acyl-CoA concentrations (50 microM) appear to favour formation of small vesicles (budding), while 50 microM CoA does not. (2) Low concentrations of CoA (EC50 2 microM) and palmitoyl-CoA (10 microM) cause re-accumulation of Ca2+ released in response to GTP. This re-accumulation is into an Ins(1,4,5)P3-sensitive compartment. By investigation of the effects of CoA and palmitoyl-CoA on the thapsigargin-induced passive leak rate of Ca2+, and on the latency of the mannose-6-phosphatase of the vesicles, we conclude that CoA and palmitoyl-CoA cause decreased vesicle permeability rather than stimulation of Ca2+ pumping activity. (3) It is suggested that GTP-induced membrane fusion in rat liver microsomes involves an as yet uncharacterized acylation-deacylation reaction which is required to produce complete vesicle sealing.

scholarly journals Evidence that a low-molecular-mass GTP-binding protein is required for store-activated Ca2+ inflow in hepatocytes

1997 ◽  
Vol 328 (2) ◽  
pp. 463-471 ◽  
Author(s):  
C. Kekulu FERNANDO ◽  
B. Roland GREGORY ◽  
Frosa KATSIS ◽  
E. Bruce KEMP ◽  
J. Greg BARRITT
Keyword(s):  
Endoplasmic Reticulum ◽  
Molecular Mass ◽  
G Protein ◽  
Rat Liver ◽  
Liver Microsomes ◽  
Brefeldin A ◽  
Rat Hepatocytes ◽  
Rat Liver Microsomes ◽  
Isolated Rat Hepatocytes ◽  
Gtp Binding

The roles of a monomeric GTP-binding regulatory protein in the activation of store-activated plasma membrane Ca2+ channels and in the release of Ca2+ from the smooth endoplasmic reticulum (SER) in rat liver parenchymal cells were investigated with the use of freshly isolated rat hepatocytes and rat liver microsomes. A low concentration (approx. 130 μM intracellular) of guanosine 5ʹ-[γ-thio]triphosphate (GTP[S]) activated Ca2+ inflow in intact hepatocytes in the absence of an agonist, whereas a high concentration (approx. 530 μM intracellular) of GTP[S] or guanosine 5ʹ-[βγ-imido]triphosphate (p[NH]ppG) inhibited the Ca2+ inflow induced by inhibitors of the activity of the endoplasmic-reticulum Ca2+-ATPase (SERCA) and by vasopressin. GTP (530 μM) prevented the inhibition of Ca2+ inflow by GTP[S] and p[NH]ppG. Brefeldin A and the peptide human Arf-1-(2-17), which inhibit many functions of ADP ribosylation factor (Arf) proteins, inhibited the Ca2+ inflow induced by SERCA inhibitors and vasopressin, and altered the profile of Ca2+ release from the SER. These effects were observed at concentrations of Brefeldin A and Arf-1-(2-17) comparable with those that inhibit the functions of Arf proteins in other systems. Succinylated Arf-1-(2-17) had a negligible effect on Ca2+ inflow. GTP[S] and Arf-1-(2-17) completely inhibited the synergistic action of GTP and Ins(1,4,5)P3 in releasing 45Ca2+ from rat liver microsomes loaded with 45Ca2+. AlF4- (under conditions expected to activate trimeric G-proteins) and succinylated Arf-1-(2-17) had no effect on GTP/Ins(1,4,5)P3-induced 45Ca2+ release, and a mastoparan analogue caused partial inhibition. Arf-1-(2-17) did not inhibit 45Ca2+ release induced by either thapsigargin or ionomycin. It is concluded that a low-molecular-mass G-protein, most probably a member of the Arf protein family, is required for store-activated Ca2+ inflow in rat hepatocytes. The idea that the role of this G-protein is to maintain a region of the SER in the correct intracellular location is discussed briefly.

High affinity binding of [3H] cocaine to rat liver microsomes

Life Sciences ◽  
1988 ◽  
Vol 42 (17) ◽  
pp. 1675-1682 ◽  
Author(s):  
Essam A. El-Maghrabi ◽  
David O. Calligaro ◽  
Mohyee E. Eldefrawi
Keyword(s):  
Rat Liver ◽  
Liver Microsomes ◽  
Affinity Binding ◽  
Rat Liver Microsomes ◽  
High Affinity Binding ◽  
High Affinity

Solubilization of receptors for pancreatic polypeptide from rat liver membranes

1995 ◽  
Vol 268 (2) ◽  
pp. G215-G223
Author(s):  
T. D. Nguyen ◽  
M. S. Wolfe ◽  
G. G. Heintz
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Pancreatic Polypeptide ◽  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Liver Microsomes ◽  
Rat Liver Microsomes ◽  
Receptor Complexes ◽  
High Affinity

We have previously identified, on rat liver microsomes and plasma membranes, proteins that bind pancreatic polypeptide (PP) with high affinity and specificity and that may serve as receptors for a hepatic effect of PP (J. Biol. Chem. 267: 9416-9421, 1992). Further characterization of these proteins requires the solubilization of receptors with conserved ability to bind PP selectively and efficiently. In this report, using 6 mM of the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), we solubilized, from liver microsomes, receptors that bound PP with high affinity (dissociation constant 6.15 +/- 1.6 nM) and specificity (no interaction with the homologous peptides neuropeptide Y and peptide YY). Gel filtration chromatography showed different degrees of receptor aggregation related to different concentrations of CHAPS in the eluent. To characterize the structure of these solubilized receptors, the chemical cross-linker N-(5-azido-2-nitrobenzoyloxy)succinimide was used to covalently bind these receptors to radiolabeled PP, and the resulting PP-receptor complexes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A radioactive band with an apparent molecular weight (M(r)) of 46,000 was detected that was inhibited by unlabeled PP with a half-maximal inhibitory concentration of approximately 10(-8) M. It most likely reflected a PP receptor with an estimated M(r) of 42,000, excluding the molecular weight of PP. The migration of this complex was not affected by the reducing agent dithiothreitol, suggesting the absence of disulfide bonding. The solubilization and identification of a bioactive hepatic PP receptor will allow further characterization and purification of this receptor and will lead to the clarification of the interaction between PP and the digestive system.

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