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 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

scholarly journals Evidence that β-hydroxyacyl-CoA dehydrase purified from rat liver microsomes is of peroxisomal origin

1992 ◽  
Vol 287 (1) ◽  
pp. 91-100 ◽  
Author(s):  
L Cook ◽  
M N Nagi ◽  
S K Suneja ◽  
A R Hand ◽  
D L Cinti
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Competitive Inhibition ◽  
Liver Microsomes ◽  
Carbon Chain ◽  
Enzymic Activity ◽  
Chain Elongation ◽  
Rat Liver Microsomes ◽  
Ph Optimum ◽  
Fatty Acid Chain

The present study provides strong evidence that the previously isolated hepatic microsomal beta-hydroxyacyl-CoA dehydrase (EC 4.2.1.17), believed to be a component of the fatty acid chain-elongation system, is derived, not from the endoplasmic reticulum, but rather from the peroxisomes. The isolated dehydrase was purified over 3000-fold and showed optimal enzymic activity toward beta-hydroxyacyl-CoAs or trans-2-enoyl-CoAs with carbon chain lengths of 8-10. The purified preparation (VDH) displayed a pH optimum at 7.5 with beta-hydroxydecanoyl-CoA, and at 6.0 with beta-hydroxystearoyl-CoA. Competitive-inhibition studies suggested that VDH contained dehydrase isoforms, and SDS/PAGE showed three major bands at 47, 71 and 78 kDa, all of which reacted to antibody raised to the purified preparation. Immunocytochemical studies with anti-rabbit IgG to VDH unequivocally demonstrated gold particles randomly distributed throughout the peroxisomal matrix of liver sections from both untreated and di-(2-ethylhexyl) phthalate-treated rats. No labelling was associated with endoplasmic reticulum or with the microsomal fraction. Substrate-specificity studies and the use of antibodies to VDH and to the peroxisomal trifunctional protein indicated that VDH and the latter are separate enzymes. On the other hand, the VDH possesses biochemical characteristics similar to those of the D-beta-hydroxyacyl-CoA dehydrase recently isolated from rat liver peroxisomes [Li, Smeland & Schulz (1990) J. Biol. Chem. 265, 13629-13634; Hiltunen, Palosaari & Kunau (1989) J. Biol. Chem. 264, 13536-13540]. Neither enzyme utilizes crotonoyl-CoA or cis-2-enoyl-CoA as substrates, but both enzymes convert trans-2-enoyl substrates into the D-isomer only. In addition, the VDH also contained beta-oxoacyl-CoA reductase (beta-hydroxyacyl-CoA dehydrogenase) activity, which co-purified with the dehydrase.

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.

Assessment of cytochrome P450 inhibition and induction potential of lupeol and betulin in rat liver microsomes

2016 ◽  
Vol 31 (2) ◽  
Author(s):  
Madhav Seervi ◽  
Shweta Lotankar ◽  
Shrikant Barbar ◽  
Sadhana Sathaye
Keyword(s):  
Cytochrome P450 ◽  
Rat Liver ◽  
Liver Microsomes ◽  
Rat Liver Microsomes ◽  
Significant Modification ◽  
Ic50 Values ◽  
Cyp 1A2 ◽  
Inhibition Studies

AbstractLupeol and betulin are triterpenoids that are majorly found in dietary substances. The aim of present study was to investigate the inhibition and induction potential of lupeol and betulin on cytochrome P450 (CYP)1A2, CYP2C11, CYP2D6 and CYP3A2 activities in rat liver microsomes.The inhibition and induction studies were conducted using ethoxy resorufin-The IC50 values in inhibition studies were found to be 59.42 μM (CYP1A2), >100 μM (CYP2C11, CYP2D6, CYP3A2) for lupeol, 52.24 μM (CYP1A2), and >100 μM (CYP2C9, CYP2D6, CYP3A2) for betulin. There was no significant modification observed in the CYP450 isoforms, indicating neither inhibition nor induction potential of lupeol and betulin.Lupeol and betulin have very low propensity to interact with CYP enzyme, suggesting no CYP inhibitory and inducing potential in rat liver microsomes.

scholarly journals Inhibition of UGT2B7 Enzyme Activity in Human and Rat Liver Microsomes by Herbal Constituents

Molecules ◽  
2018 ◽  
Vol 23 (10) ◽  
pp. 2696 ◽  
Author(s):  
Nurul Abdullah ◽  
Sabariah Ismail
Keyword(s):  
Rat Liver ◽  
Liver Microsomes ◽  
Herbal Medicines ◽  
Inhibitory Effect ◽  
Intrinsic Clearance ◽  
Rat Liver Microsomes ◽  
Metabolizing Enzymes ◽  
Conventional Drug ◽  
Ic50 Values

The co-use of conventional drug and herbal medicines may lead to herb-drug interaction via modulation of drug-metabolizing enzymes (DMEs) by herbal constituents. UDP-glucuronosyltransferases (UGTs) catalyzing glucuronidation are the major metabolic enzymes of Phase II DMEs. The in vitro inhibitory effect of several herbal constituents on one of the most important UGT isoforms, UGT2B7, in human liver microsomes (HLM) and rat liver microsomes (RLM) was investigated. Zidovudine (ZDV) was used as the probe substrate to determine UGT2B7 activity. The intrinsic clearance (Vmax/Km) of ZDV in HLM is 1.65 µL/mg/min which is ten times greater than in RLM, which is 0.16 µL/mg/min. Andrographolide, kaempferol-3-rutinoside, mitragynine and zerumbone inhibited ZDV glucuronidation in HLM with IC50 values of 6.18 ± 1.27, 18.56 ± 8.62, 8.11 ± 4.48 and 4.57 ± 0.23 µM, respectively, hence, herb-drug interactions are possible if andrographolide, kaempferol-3-rutinoside, mitragynine and zerumbone are taken together with drugs that are highly metabolized by UGT2B7. Meanwhile, only mitragynine and zerumbone inhibited ZDV glucuronidation in RLM with IC50 values of 51.20 ± 5.95 μM and 8.14 ± 2.12 µM, respectively, indicating a difference between the human and rat microsomal model so caution must be exercised when extrapolating inhibitory metabolic data from rats to humans.

scholarly journals Ruthenium red-insensitive calcium transport in ascites-sarcoma 180/TG cells

1981 ◽  
Vol 200 (2) ◽  
pp. 343-348 ◽  
Author(s):  
F L Bygrave ◽  
T A Anderson
Keyword(s):  
Rat Liver ◽  
Microsomal Fraction ◽  
Initial Rate ◽  
Liver Microsomes ◽  
Ruthenium Red ◽  
Rat Liver Microsomes ◽  
Sarcoma 180 ◽  
Low Concentrations ◽  
Rat Liver Cells ◽  
Mouse Ascites

1. Ruthenium Red-insensitive Ca2+ transport in the mouse ascites sarcoma 180/TG is enriched in a ‘heavy’ microsomal fraction (microsomes) sedimented at 35 000 g for 20 min. The subcellular distribution of this Ca2+ transport differed from that of Ruthenium Red-sensitive Ca2+ transport and (Na+ + K+)-dependent ATPase activity, but was similar to that of glucose 6-phosphatase. 2. The affinity of this transport system for ‘free’ Ca2+ is high (Km approx. 6 microM) and that for MgATP somewhat lower (Km approx. 100 microM). Ca2+ transport by the tumour microsomes, by contrast with that by liver microsomes, was greatly stimulated by low concentrations of P1. 3. Although incubation of intact ascites cells with glucagon led to an increase in intracellular cyclic AMP, no stable increase in the initial rate of Ca2+ transport in the subsequently isolated ‘heavy’ microsomes could be detected as in similar experiments carried out previously with rat liver cells. Reconstitution experiments suggest that a deficiency exists in the tumour microsomal membrane such that an action of glucagon that is normally present in rat liver microsomes is not evoked.

scholarly journals The effect of heparin on the inositol 1,4,5-trisphosphate receptor in rat liver microsomes Dependence on sulphate content and chain length

FEBS Letters ◽  
1989 ◽  
Vol 252 (1-2) ◽  
pp. 105-108 ◽  
Author(s):  
Michael A. Tones ◽  
Martin D. Bootman ◽  
Bernard F. Higgins ◽  
David A. Lane ◽  
Graham F. Pay ◽  
...  
Keyword(s):  
Chain Length ◽  
Rat Liver ◽  
Liver Microsomes ◽  
Rat Liver Microsomes ◽  
Sulphate Content ◽  
Inositol 1,4,5 Trisphosphate ◽  
Trisphosphate Receptor
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