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

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.     

Guanosine 5'-triphosphate releases calcium from rat liver and guinea pig parotid gland endoplasmic reticulum independently of inositol 1,4,5-trisphosphate

GTP releases calcium from rat liver microsomes and guinea pig parotid gland microsomal subfractions independently of the presence of inositol 1,4,5-trisphosphate (IP3). Non-hydrolyzable guanine nucleotide analogues have no effect and inhibit the effect of GTP. The mechanism of GTP-mediated calcium release differs from IP3-mediated calcium release as indicated by the following findings: GTP-induced calcium release depends on the presence of compounds which increase the viscosity of the medium (polyethylene glycol, polyvinylpyrrolidone, or bovine serum albumin); GTP-mediated calcium release is much slower; GTP-mediated calcium release is strongly temperature-dependent, whereas IP3-mediated calcium release is not; GTP-mediated calcium release is much more sensitive to a decrease of intravesicular free calcium than IP3-mediated calcium release.     

Evidence for a GTP-dependent increase in membrane permeability for calcium in NG108-15 microsomes

The effect of GTP on Ca2+ uptake and release was studied in a microsomal fraction isolated from neuroblastoma x glioma hybrid NG108-15 cells. GTP did not alter the ATP-dependent initial uptake of Ca2+ but markedly enhanced the efflux of Ca2+ from microsomes. GTP-dependent Ca2+ release requires the presence of millimolar concentration of Mg2+. The effect of GTP was not mimicked by other nucleotides and was competitively blocked by the thiophosphate analogue of GTP, GTP gamma S but not by the non-hydrolyzable nucleotide GMP-PNP. Addition of an inhibiting concentration of GTP gamma S after completion of GTP-induced calcium release did not result in a re-uptake of Ca2+, showing the irreversibility of the releasing effect of GTP. Our data are consistent with the hypothesis of Ca2+-dependent GTP-induced opening of a channel responsible for vectorial transport of Ca2+ ions from one intracellular compartment to another. A model is proposed suggesting that the GTP-binding protein is a GTP-specific diacylglycerol kinase.     

Polyethylene glycol-stimulated microsomal GTP hydrolysis. Relationship to GTP-mediated Ca2+ release

It has recently been observed that GTP mediates Ca2+ release from internal Ca2+ stores. In contrast to effects on permeabilized cells, GTP-dependent Ca2+ release in isolated microsomes requires the presence of polyethylene glycol (PEG). We have investigated the effects of PEG on microsomal GTPase activity and report that PEG stimulates a high-affinity (Km = 0.9 microM) GTPase. The effects of PEG reflect an increase in the Vmax of this activity; no effects on Km were observed. The concentration dependence for PEG-dependent stimulation of the high-affinity GTPase exactly mimicked that for GTP-dependent Ca2+ release. The stimulation of GTP hydrolysis by PEG was specific for the microsome fraction; only small effects were obtained with plasma membrane or cytosol fractions. As observed for GTP-dependent Ca2+ release, the microsomal PEG-stimulated GTPase was competitively inhibited by the GTP analog GTP gamma S (Ki = 60 nM). It is proposed that the PEG-stimulated GTPase may represent an intrinsic activity of the guanine nucleotide binding protein involved in the regulation of reticular Ca2+ fluxes.     

GTP-dependent membrane fusion during hepatocarcinogenesis and liver regeneration

Rough microsomes were isolated from homogenates of livers of rats bearing hepatomas as well as from homogenates of livers of rats 24 and 48 h after partial hepatectomy. When incubated in the presence of GTP in a cell-free system to assay membrane fusion these membranes were observed to have a greater capacity (1.4 to 5 fold) for GTP-dependent fusion than homologous membranes from control non-proliferating liver tissue. The enhanced GTP-dependent membrane fusion may reflect changes in membrane properties related to cell proliferation.     

Accumulation of polyunsaturated free fatty acids coincident with the fusion of rough endoplasmic reticulum membranes.

The accumulation of polyunsaturated free fatty acids (PUFAs) was observed coincident with GTP-dependent fusion of liver rough microsomes. Whereas 0.5 mM NADPH led to a parallel reduction (greater than 50%) in membrane fusion and PUFA accumulation, indomethacin (50 microM) either had little effect or slightly augmented both processes. CTP was observed to stimulate accumulation of PUFAs and diacylglycerol (DAG). Therefore PUFAs may be relevant for GTP-dependent membrane fusion and together with DAG may play a role in fusion stimulated in the presence of CTP.     

The interaction of albumin and fatty-acid-binding protein with membranes: oleic acid dissociation

Bovine serum albumin or fatty-acid-binding protein rapidly lose oleic acid when incubated in the presence of dimyristoyl lecithin liposomes. The phenomenon is dependent on vesicle concentration and no measurable quantities of protein are found associated with liposomes. Upon gel filtration on Sepharose CL-2B of incubated mixtures of microsomes containing [1-14C] oleic acid and albumin or fatty-acid-binding protein, association of fatty acid with the soluble proteins could be demonstrated. Both albumin and fatty-acid-binding protein stimulated the transfer of oleic acid from rat liver microsomes to egg lecithin liposomes. These results indicate that albumin is more effective in the binding of oleic acid than fatty-acid-binding protein, which allows a selective oleic acid dissociation during its interaction with membranes.     

Energy-dependent calcium uptake activity of microsomes from the aorta of normal and hypertensive rats.

Energy-dependent calcium uptake activity of microsomes isolated from the rat aorta has been characterized. The microsomes consist of smooth membrane vesicles which in the presence of MG-ATP as an energy source continuously sequester calcium over a 60-min period. This calcium uptake is greatly stimulated by oxalate anion which serves as a calcium trapping agent. Unlike the calcium uptake of mitochondria this uptake is not inhibited by sodium azide. Sucrose density gradient analysis of the microsomal calcium uptake suggests that the system is associated with the sarcoplasmic reticulum. In presence of 5 mM Mg-ATP and 20 muM calcium approximately 38 nmol of calcium per mg of microsomal protein are taken up in 20 min. In the absence of ATP, less than 2 nmol of calcium per mg of protein are taken up in the first 2 min with no further uptake of calcium in subsequent time periods. When calcium uptake activity is plotted against calcium or ATP concentration of the medium, half maximal activity is calculated for 24.3 muM calcium and for 1.6 mM ATP. The calcium uptake characteristics of the rat aorta microsomes are compatible with a postulated role in the relaxation of the vascular smooth muscle and the provision of an intracellular calcium store for muscle contraction. Aorta microsomes from SHR rats (a genetic strain that is spontaneously hypertensive) have a significantly reduced uptake when compared with the corresponding nonhypertensive control strain. The level of calcium and ATP for half maximal activity of the rat aorta microsomal calcium uptake system is approximately the same in the SHR and the control strain. The rate of release of calcium from rat aorta microsomes is apparently identical in SHR strain and control. The calcium uptake activity of kidney and liver microsomes isolated from the SHR strain and control. The calcium uptake activity of kidney and liver microsomes isolated from the SHR rat appears to be identical to that found in the control strain.     

Synthesis of CDP-diacylglycerol by rat liver rough microsomes as visualized by electron microscopic autoradiography: relationship to GTP-stimulated membrane fusion

Using detergent-free conditions of incubation for the analysis of liponucleotide synthesis, we compared GTP-dependent formation of CDP-diacylglycerol (CDP-DG) and membrane fusion in RNA-depleted rough microsomes from rat liver. After incubation of stripped rough microsomes (SRM) in the presence of GTP and [5-3H]-CTP, radioactivity was recovered in lipid extracts and identified by thin-layer chromatography as a single spot which co-migrated with CDP-DG. The nucleotide requirement for CDP-DG synthesis and that for membrane fusion were observed to be identical. We next carried out an electron microscopic autoradiographic analysis on incubated membranes to determine the site of incorporation of [5-3H]-CTP. Silver grains were observed directly over the unilamellar membranes of natural vesicles. In confirmation of the biochemical data, quantitation of silver grain density indicated more grains over membranes incubated in the presence of GTP than over those incubated in the absence of this nucleotide. For membranes incubated in the presence of GTP, the grain density was similar over fused and unfused membranes in the same preparation. When SRM were incubated with the enzyme co-factors required for synthesis of phosphatidylinositol, a GTP-independent membrane fusion was observed by both transmission and freeze-fracture electron microscopy. Together with the biochemical and autoradiographic data, this suggests that phospholipid metabolism may be activated by GTP and lead to the fusion of RER membrane.     

Cytosolic PLA2 in zymogen granule fusion and amylase release: inhibition of GTP-induced fusion by arachidonyl trifluoromethyl ketone points to cPLA2 in G-protein-mediated secretory vesicle fusion

Previously we reported that the G-protein Galphai3 localized in pancreatic zymogen granule (ZG) membrane participates in vesicular fusion at the cell plasma membrane (PM). In the present study, the presence of cytosolic phosholipase A2 (cPLA2) in rat ZGs was demonstrated and its potential role in G-protein-mediated ZG-PM fusion was investigated. In vitro fusion assays utilizing both enzymatic and fluorimetric techniques demonstrate that ZGs fuse with PM with a greater potency in the presence of GTP. Arachidonyl trifluoromethyl ketone (AACOCF3) at 40 microM reduces GTP-induced ZG-PM fusion by 25-50%. Anti-cPLA2 antibody reduces ZG-PM fusion in a dose-dependent manner and a 50% reduction of the fusion takes place in the range of 0.48-0.64 ratios of cPLA2 antibody to ZG proteins. PLAP, a cPLA2 activator synthetic peptide increases ZG-PM fusion in a limited dose-dependent manner and tends to inhibit at higher concentrations. Exogenous arachidonic acid inhibits GTP-induced ZG-PM fusion in a dose-dependent manner. Furthermore, a non-hydrolysable GTP analogue, Gpp(NH)p, reduces PLAP effect in ZG-PM fusion; and the net effect of Gpp(NH)p and PLAP differs significantly from the net effect of GTP and PLAP on ZG-PM fusion suggesting that cPLA2 is involved in G-protein-mediated secretory vesicle fusion.     

Nucleotide specificity of cardiac sarcoplasmic reticulum. GTP-induced calcium accumulation and GTPase activity

We previously demonstrated that the hydrolysis of GTP by canine cardiac sarcoplasmic reticulum is not sensitive to calcium and does not support the translocation of calcium and oxalate into the vesicular space. In response to GTP, however, calcium is accumulated into a compartment which is sensitive to pH and ionophore. In the present paper, we further explored the relationship between GTP hydrolysis and GTP-induced calcium accumulation. Both ATP- and GTP-induced calcium accumulation were prevented by the sulfhydryl reagent, N-ethylmaleimide (NEM; I50 = 0.2 mM). In contrast, the sensitivity of NTP hydrolysis to NEM differed markedly; GTPase activity was not affected by NEM, whereas ATPase activity was markedly inhibited. Conversely, although the GTPase was noncompetitively inhibited by the ATP analogue, adenylyl imidodiphosphate (Ki = 8 microM), and was competitively inhibited by the GTP analogue, guanylyl imidodiphosphate (Ki = 60 microM), GTP-induced calcium accumulation was not affected by the NTP analogues at any concentration. Therefore, the GTP-dependent accumulation of calcium into the pH- and ionophore-sensitive compartment of cardiac SR may not require GTP hydrolysis but may be dependent on GTP binding. The previously reported noncompetitive inhibition of the GTPase by ATP was also observed when the calcium-dependent hydrolysis of ATP was prevented by NEM (Ki = 1.2 microM). Along with the noncompetitive inhibition of the GTPase by adenylyl imidodiphosphate, the inhibition of the GTP by ATP in the presence of NEM suggests that ATP binding may be involved in the observed inhibition. The Ki for the noncompetitive inhibition of GTPase activity is compatible with ATP binding to the high affinity catalytic site of the ATPase. Thus, although GTP-induced calcium accumulation differs somewhat from ATP-dependent calcium translocation, the similarities between the two processes (i.e. similar time courses and sensitivity to pH, ionophore, and sulfhydryl modification) suggest that they may be related in some manner.     

The effect of inositol 1,4,5-trisphosphate and GTP on calcium release from rat liver microsomes

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and GTP mobilized 8% and 90% of the ionophore-releaseable Ca2+ pool from rat liver microsomes, respectively. In contrast to GTP, which acted after a lag-time, the Ins(1,4,5)P3-induced Ca2+ release was immediate. Poly(ethylene glycol) inhibited the effect of Ins(1,4,5)P3 and enhanced that of GTP. Ins(1,4,5)P3 accelerated and enhanced the GTP-induced Ca2+ release. Guanylyl imidodiphosphate inhibited competitively the GTP stimulated Ca2+ release, but not the GTP-dependent phosphorylation of the Mr 17,000 and 38,000 protein bands.     

Characteristics of GTP-mediated microsomal Ca2+ release

Guanosine triphosphate (GTP) can release Ca2+ and enhance responses to D-myo-inositol 1,4,5-trisphosphate (IP3) in crude liver microsomes in the presence of polyethylene glycol (PEG) (Dawson et al. (1986) Biochem. J. 234, 311-315). The mechanism of these responses has been further investigated. GTP gamma S which antagonizes the actions of GTP on microsomes, does not promote Ca2+ re-uptake when added after the completion of GTP-mediated Ca2+ release. However, the effects of GTP could be reversed by washing or dilution of the microsomes. Addition of PEG to the incubation medium promoted the aggregation of microsomes. Electron microscopy provided no evidence for the fusion of microsomal vesicles in the presence or absence of GTP. In the presence of PEG, GTP produced an alteration of the permeability properties of the microsomal membrane as indicated by increased leakage of an intraluminal esterase, a reduction in the mean buoyant density of the vesicles, and a decrease in the latency of mannose 6-phosphate hydrolysis. All three effects developed relatively slowly, whereas the effects of GTP on Ca2+ fluxes occurred more rapidly (complete within 15 min). A low permeability to mannose 6-phosphate was restored upon washing away the GTP. These results suggest that non-specific permeability changes may underly the effects of GTP on Ca2+ release and that, under certain conditions, GTP can reversibly modulate the permeability of a transmembrane 'pore' in microsomal membranes that can pass ions and macromolecules. The possibility that such a pore serves to link IP3-sensitive vesicles with other Ca2+-containing compartments is discussed.     

Energy-dependent calcium uptake activity of microsomes from the aorta of normal and hypertensive rats

Energy-dependent calcium uptake activity of microsomes isolated from the rat aorta has been characterized. The microsomes consist of smooth membrane vesicles which in the presence of Mg · ATP as an energy source continuously sequester calcium over a 60-min period. This calcium uptake is greatly stimulated by oxalate anion which serves as a calcium trapping agent. Unlike the calcium uptake of miltochondria this uptake is not inhibited by sodium azide. Sucrose density gradient analysis of the microsomal calcium uptake suggests that the system is associated with the sarcoplasmic reticulum. In presence of 5 mM Mg · ATP and 20μM calcium approximately 38 nmol of calcium per mg of microsormal protein are taken up in 20 min. In the absence of ATP, less than 2 nmol of calcium per mg of protein are taken up in the first 2 min. with no further uptake of calcium in subsequent time periods. When calcium uptake activity is plotted against calcium or ATP concentration of the medium, half maximal activity is calculated for 24.3 μM calcium and for 1.6 mM ATP. The calcium uptake characteristics of the rat aorta microsomes are compatible with a postulated role in the relaxation of the vascular smooth muscle and the provision of an intracellular calcium store for muscle contraction.Aorta microsomes from SHR rats (a genetic strain that is spontaneously hypertensive) have a significantly reduced calcium uptake when compared with the corresponding nonhypertensive control strain. The level of calcium and ATP for half maximal activity of the rat aorta microsomal calcium uptake system is approximately the same in the SHR and the control strain. The rate of release of calcium from rat aorta microsomes is apparently identical in SHR strain and control. The calcium uptake activity of kidney and liver microsomes isolated from the SHR rat appears to be identical to that found in the control strain.Rats were treated with the steroid deoxycorticosterone acetate for ten and thirty days to induce hypertension. After ten days of deoxycorticosterone acetate although hypertension is present, there is no change in calcium uptake activity of aorta microsomes, renal microsomes or renal plasma membranes. After 30 days of deoxycorticosterone acetate treatment calcium uptake activity of renal microsomes is reduced. A variable decrease in calcium uptake activity is observed with aorta microsomes. Renal plasma membrane calcium uptake remains unchanged.     

Stimulation of monoacylglycerophosphate formation by Z protein

Z protein has been purified from 110,000 × g rat liver supernatant using Sephadex G-100 and DEAE Sephadex. Z protein obtained in this manner was superior to albumin in stimulating the esterification of sn-glycerol-3-phosphate in the presence of palmityl-CoA and rat liver microsomes. These observations constitute direct evidence for the possible role of Z protein in fatty acid metabolism.     

Chemical evidence for the difference between albumins from microsomes and serum and a possible precursor-product relationship

Immunologically isolated albumin from rat liver microsomes separates on DEAE-cellulose into almost equal proportions of an albumin-like protein and authentic albumin. Besides this similarity in immunological properties, both albumin species have almost the same molecular weight and amino acid composition. Furthermore, the amino acid sequences appear to be identical apart from an additional pentapeptide at the N-terminus of the albumin-like protein. It is suggested that the albumin-like protein represents a precursor which is converted to albumin by release of a pentapeptide from the N-terminus.     

Uptake and release of calcium by microsomes of nonvascular smooth muscle.

Microsomes prepared from guinea-pig ileum longitudinal smooth muscle and rat uterus continuously sequester calcium for a one hour period in the presence of Mg-ATP as an energy source and oxalate anion as a trapping agent. Dithiothreitol is essential for maximal calcium uptake activity of the rat uterus microsomes. On sucrose density gradients, calcium uptake of the smooth muscle microsomes appears to be associated with intracellular membrane (sarcoplasmic reticulum). Release of sequestered calcium from the longitudinal muscle microsomes is very slow (20% in 50 minutes). A small labile fraction (20%) is released by EGTA (1 mM) in 10 minutes. Rapid release of sequestered calcium (90% in 10 minutes) occurs in presence of the calcium ionophore A23187 (2 μM) or in the presence of chlorpromazine (1 mM).     

GTP hydrolysis is required for vesicle fusion during nuclear envelope assembly in vitro

Nuclear envelope assembly was studied in vitro using extracts from Xenopus eggs. Nuclear-specific vesicles bound to demembranated sperm chromatin but did not fuse in the absence of cytosol. Addition of cytosol stimulated vesicle fusion, pore complex assembly, and eventual nuclear envelope growth. Vesicle binding and fusion were assayed by light and electron microscopy. Addition of ATP and GTP to bound vesicles caused limited vesicle fusion, but enclosure of the chromatin was not observed. This result suggested that nondialyzable soluble components were required for nuclear vesicle fusion. GTP gamma S and guanylyl imidodiphosphate significantly inhibited vesicle fusion but had no effect on vesicle binding to chromatin. Preincubation of membranes with 1 mM GTP gamma S or GTP did not impair vesicle binding or fusion when assayed with fresh cytosol. However, preincubation of membranes with GTP gamma S plus cytosol caused irreversible inhibition of fusion. The soluble factor mediating the inhibition by GTP gamma S, which we named GTP-dependent soluble factor (GSF), was titratable and was depleted from cytosol by incubation with excess membranes plus GTP gamma S, suggesting a stoichiometric interaction between GSF and a membrane component in the presence of GTP gamma S. In preliminary experiments, cytosol depleted of GSF remained active for fusion of chromatin-bound vesicles, suggesting that GSF may not be required for the fusion reaction itself. We propose that GTP hydrolysis is required at a step before the fusion of nuclear vesicles.     

Comparison of the epinephrine-mediated activation of adenylate cyclase in plasma membranes from liver and ascites hepatomas of rats.

(1) The apparent [3H]epinephrine binding parameters of plasma membranes from rat liver and ascites hepatomas such as AH-7974, AH-371A and AH-130, as measured by equilibrium dialysis and/or Millipore filtration, were almost similar to each other. The epinephrine binding sites in the plasma membranes were heterogenous (alpha, beta-receptors and non specific sites), but the pattern of these binding sites in the liver membranes appeared almost similar to that in the hepatoma membranes. 2. The beta-receptor seemed to be specifically involved in the epinephrine-mediated activation of adenylate cyclase of the liver membranes. In spite of the presence of almost similar beta-receptors and adenylate cyclase, the adenylate cyclase of hepatoma membranes was found to be less sensitive to the epinephrine-mediated activation. 3. GTP alone was found to activate adenylate cyclase of liver and hepatoma membranes to some extents when the concentration of ATP was lower (0.3 mM). When GTP was added with epinephrine, a marked, synergistic activation of adenylate cyclase was observed in liver plasma membranes, but not in hepatoma ones. 4. The synergistic activation of adenylate cyclase by epinephrine plus GTP showed a characteristic kinetic feature, reaching a maximal peak within 1 min or so after mixing. 5. Binding of [3H]epinephrine to liver membranes proceeded monophasically in the absence of GTP, while it proceeded biphasically in the presence of GTP, showing the retardation of binding at some earlier stages. GTP added at the time of binding equilibrium induced the temporary release of bound epinephrine from the beta-receptors. The GTP-induced temporary release of bound epinephrine, occurring within 4-5 min after the addition of GTP, was less marked in the hepatoma membranes as compared with the liver membranes. 6. Possible impairment of the GTP-dependent coupling mechanism in the receptor-adenylate cyclase system of hepatoma plasma membranes was suggested.     

Liver cytosolic non-dialysable factor(s) can counteract GTP-dependent Ca2+ release in rat liver microsomal fractions

Readdition to rat liver microsomes of dialysed liver post-microsomal supernatant resulted in an almost complete inhibition of the Ca2+-releasing effect of GTP. Such inhibition was heat-labile, and was associated with non-ultrafiltrable supernatant components with a molecular weight higher than 30,000 D. A preliminary fractionation of liver supernatant showed that the inhibitory effect is recovered in the 40-50% ammonium sulfate-precipitated proteins, with an approx. 10-fold enrichment. The active ammonium sulfate fraction did not modify the GTP-induced Ca2+ increase of passive Ca2+ efflux from microsomes, nor did it affect microsomal GTP hydrolysis, which is likely required for its Ca2+ releasing effect. The active ammonium sulfate fraction appears to markedly favour the translocation of GTP-released Ca2+ into a microsomal GTP-insensitive pool. Separation of liver microsomes in smooth and rough fractions revealed that such GTP-insensitive Ca2+ pool is almost completely associated with smooth microsomes.