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1.
sn-1,2-Diacylglycerol kinase of Escherichia coli. Purification, reconstitution, and partial amino- and carboxyl-terminal analysis 总被引:4,自引:0,他引:4
The sn-1,2-diacylglycerol kinase structural gene from Escherichia coli was demonstrated to be the dgkA locus previously sequenced (Lightner, V. A., Bell, R. M., and Modrich, P. (1983) J. Biol. Chem. 258, 10856-10861). The dgkA gene product was shown by maxicell analysis to be an Mr = 14,000 membrane-bound protein. When dgkA was placed on a hybrid plasmid under control of the lambda pL promoter, a 100-fold overproduction of diacylglycerol kinase activity was obtained. Diacylglycerol kinase was solubilized from membranes with 2-propanol/heptane/trifluoroacetic acid and purified to near homogeneity by high performance liquid chromatography. Activity was reconstituted in a mixed micellar assay containing beta-octylglucoside, cardiolipin, and sn-1,2-dioleoylglycerol. Amino acid analysis, partial NH2-terminal analysis and COOH-terminal analysis permitted alignment of the polypeptide on the sequenced gene. The data establish that dgkA is the structural gene for the diacylglycerol kinase and establish the primary structure of the enzyme of 122 residues, 13,245 daltons. Secondary structure analysis predicted a protein conformation consisting of three transmembrane alpha-helical segments, an amphipathic helix, and an alpha-helix. Taken together, the predicted helical segments comprise more than 75% of the polypeptide. 相似文献
2.
sn-1,2-diacylglycerol kinase of Escherichia coli. Diacylglycerol analogues define specificity and mechanism 总被引:2,自引:0,他引:2
A detailed structure/function analysis of the substrate specificity of Escherichia coli sn-1,2-diacylglycerol kinase was performed with three goals in mind: (a) to define the substrate specificity; (b) to discover inhibitors; and (c) to elucidate the specificity of diacylglycerol-dependent inactivation. Forty-seven structural analogues of sn-1,2-diacylglycerol were prepared and examined as substrates, inhibitors, and irreversible inactivators of the enzyme using mixed micellar assay methods. Modification of the acyl chains or the sn-2 ester affected the apparent Km but had only small effects on Vm; modifications of the sn-1 ester, sn-3 methylene, or sn-3 hydroxyl had large effects on the apparent Vm and smaller effects on Km. Consistent with these observations, diacylglycerol analogues modified only in the acyl chains or sn-2 ester were not diacylglycerol kinase inhibitors, whereas analogues with substitutions of the sn-1 ester or sn-3 hydroxyl frequently caused inhibition. A hydrogen bond-donating group was required for an analogue to be a diacylglycerol kinase inhibitor. Studies of diacylglycerol kinase inactivation by the various analogues were consistent with the previous conclusion that this process involves an interaction of diacylglycerols with an enzyme conformation different from that active in catalysis (Walsh, J. P., and Bell, R. M. (1986) J. Biol. Chem. 261, 15062-15069). Studies with a water-soluble diacylglycerol, sn-1,2-dibutyrylglycerol, allowed direct comparison of diacylglycerol kinase activity in mixed micelles with that in native membranes. The results are discussed in relation to the structural requirements of other diacylglycerol-dependent enzymes. 相似文献
3.
Efficient delivery of hydrophobic water-insoluble substrates and cofactors to membrane-bound enzymes is a recurring problem which has impeded kinetic analyses. Kinetic analysis of the Escherichia coli sn-1,2-diacylglycerol kinase, an extremely hydrophobic integral membrane protein of 122 residues, was facilitated by the development of a mixed micellar assay. beta-Octyl glucoside micelles quantitatively solubilized diacylglycerol kinase from membranes of strains which overproduced the enzyme up to 250-fold and provided an effective method to disperse and deliver the hydrophobic water-insoluble substrate, sn-1,2-dioleoyglycerol. Diacylglycerol kinase was active in mixed micelles containing octyl glucoside and dioleoyglycerol. Several phospholipids stimulated activity up to 6-fold, suggesting a cofactor function. Activation by phospholipids was not stereospecific and was mimicked partially by fatty acids. Half-maximal activation was observed at 1 mol % cardiolipin, suggesting that a small number of phospholipids are sufficient to activate the enzyme. Activity was dependent on the mole fractions of dioleoylglycerol and phospholipid in the mixed micelles, but independent of micelle number. Several lines of evidence indicated that the transfer of diacylglycerol between micelles was much more rapid than its utilization by the enzyme. Diacylglycerol kinase exhibited Michaelis-Menten kinetics with respect to diacylglycerol and MgATP. A second Mg2+ ion (in addition to MgATP) was required for activity. When Mg2+ was excluded from the assay, Mn2+, Zn2+, Cd2+, and Co2+ supported activity to lesser extents. These data establish a suitable system for in-depth kinetic analysis of the E. coli diacylglycerol kinase and its phospholipid cofactor requirements. 相似文献
4.
Amiteye S Kobayashi K Imamura D Hosoya S Ogasawara N Sato T 《Journal of bacteriology》2003,185(17):5306-5309
The sn-1,2-diacylglycerol kinase homologue gene, dgkA, is a sporulation gene indispensable for the maintenance of spore stability and viability in Bacillus subtilis. After 6 h of growth in resuspension medium, the endospore morphology of the dgkA mutant by standard phase-contrast microscopy was normal; however, after 9 h, the endospores appeared mostly dark by phase-contrast microscopy, suggesting a defect in the spores. Moreover, electron microscopic studies revealed an abnormal cortex structure in mutant endospores 6 h after the onset of sporulation, an indication of cortex degeneration. In addition, a significant decrease in the dipicolinic acid content of mutant spores was observed. We also found that dgkA is expressed mainly during the vegetative phase. It seems likely that either the DgkA produced during growth prepares the cell for an essential step in sporulation or the enzyme persists into sporulation and performs an essential function. 相似文献
5.
The Escherichia coli sn-1,2-diacylglycerol (DAG) kinase has been successfully expressed in COS cells. The E. coli dgkA locus which contains the coding sequences for DAG kinase was subcloned into an eukaryotic expression vector, pMT2. COS cells transfected with the vector pMT2dgk expressed the DAG kinase as shown by Western analysis. Immunofluorescence studies revealed that the E. coli DAG kinase was prominently but not exclusively located in the endoplasmic reticulum. In addition, mixed micellar assays in beta-octyl glucoside revealed that membranes prepared from pMT2dgk-transfected COS cells contained over a 1500-fold increase in DAG kinase activity: 107 nmol/min/mg compared with only 0.067 nmol/min/mg for controls. DAG kinase activity from the E. coli enzyme was distinguished from endogenous COS cell activity based on differences in thermolability and the ability of the E. coli enzyme to use ceramide as a substrate. No ceramide kinase activity was detected in control COS cells, so the activity detected in pMT2dgk transfectants must have resulted from the expressed E. coli DAG kinase. The Km values for DAG kinase derived from E. coli and COS cells were nearly identical. Finally, transfected COS cells were labeled with [32P]Pi to investigate possible perturbations in lipid composition induced by the action of the E. coli DAG kinase. Ceramide (generated by the action of sphingomyelinase) was also used to clearly implicate the E. coli enzyme. Levels of ceramide phosphate increased more than 150-fold in pMT2dgk-transfected cells relative to controls. The results of these studies show that the E. coli enzyme expressed in COS cells is active and perturbs lipid composition in the intact cell system; the absolute lipid cofactor requirement of E. coli DAG kinase can be satisfied in COS cells. 相似文献
6.
The stereospecificity of monoacylglycerol acyltransferase from rat intestinal mucosa and suckling rat liver microsomes was examined using sn-1,2-diacylglycerol kinase from Escherichia coli. With 2-monooleoyl glycerol and palmitoyl-CoA, 88 and 87.9% of the diacylglycerol synthesized by the intestinal mucosa and suckling liver, respectively, was demonstrated to be the sn-1,2-isomer. Analysis of similar preparations of these diacylglycerol products by gas-liquid chromatography-mass spectrometry indicated that most of the remaining diacylglycerol was the 1,3-isomer that probably arose via acyl-migration. These results indicate that monoacylglycerol acyltransferase is stereospecific. Measurement of acyltransferase activities in microsomes using 1- and 2-monoacyl- and monoalkylglycerols as substrates indicated that the monoacylglycerol acyltransferases from suckling liver and intestinal mucosa have different substrate specificities. 相似文献
7.
Mass measurement of inositol 1,4,5-trisphosphate and sn-1,2-diacylglycerol in bombesin-stimulated Swiss 3T3 mouse fibroblasts. 总被引:4,自引:0,他引:4
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Two specific and selective assays were used to measure changes in the mass of Ins(1,4,5)P3 and sn-1,2-diacylglycerol in bombesin-stimulated Swiss 3T3 cells. The results demonstrate that the increase in Ins(1,4,5)P3 was extremely rapid, but transient, returning to basal levels by 30 s. In contrast, the increase in sn-1,2-diacylglycerol was biphasic: the first phase mirrored the transient Ins(1,4,5)P3 response, whereas the second phase was sustained and occurred in the absence of elevated Ins(1,4,5)P3. The possible source of the second phase of diacylglycerol is discussed. 相似文献
8.
Diacylglycerol kinase is though to play a central role in the metabolism of diacylglycerol second messengers in agonist-stimulated cells. A series of diacylglycerol analogs were tested for their ability to act as substrates or inhibitors of diacylglycerol kinase with the goal of determining the substrate specificity of the enzyme, and of discovering inhibitors. Screening of these compounds was performed using a partially purified diacylglycerol kinase from pig brain. Modified assays for this enzyme using co-sonicated mixtures of diacylglycerol and anionic phospholipids were developed. This enzyme was found to be quite specific for sn-1,2-diacylglycerol (KM 24 microM for dioctanoyl-glycerol). Among the analogs investigated, only 1,2-dioctanoyl-2-amino-1,3-propanediol was utilized at a significant rate. Two analogs, dioctanoylethylene glycol (KI 58 microM) and 1-monooleoylglycerol (KI 91 microM), were potent inhibitors in vitro. These compounds were tested for effects on diacylglycerol formation and metabolism in thrombin-stimulated human platelets. Dioctanoylethylene glycol inhibited diacylglycerol phosphorylation in platelets (70-100% at 100 microM) leading to a longer-lived diacylglycerol signal. This compound may be a useful tool for studies of diacylglycerol kinase in other cell types. 1-Monooleoylglycerol treatment elevated diacylglycerol levels up to 4-fold in unstimulated platelets and up to 10-fold in thrombin-stimulated platelets. The implications with regard to the pathways of diacylglycerol metabolism in human platelets are discussed. 相似文献
9.
Distribution of distinct arachidonoyl-specific and non-specific isoenzymes of diacylglycerol kinase in baboon (Papio cynocephalus) tissues. 总被引:1,自引:0,他引:1
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We investigated the diacyglycerol kinase species present in several baboon tissues using the substrates sn-1-stearoyl-2-arachidonoyl diacylglycerol and sn-1,2-didecanoyl diacylglycerol. Chromatography of octyl glucoside extracts of the baboon (Papio cynocephalus papio) tissues on hydroxyapatite columns revealed the presence of three diacylglycerol kinase species with different substrate preferences. One species markedly 'preferred' the substrate sn-1-stearoyl-2-arachidonoylglycerol, the two other species preferred sn-1,2-didecanoylglycerol. Measurement of the activity of the baboon brain diacylglycerol kinases toward diacylglycerols with a range of different fatty acid chains revealed a strict preference of the arachidonoyl diacylglycerol kinase for sn-1-acyl-2-arachidonoyl diacylglycerol, whereas the other enzymes showed no preference toward several long-chain-fatty-acid-containing diacylglycerols. The arachidonoyl diacylglycerol kinase was particularly abundant in brain and testis, whereas liver was practically devoid of this enzyme. The arachidonoyl diacylglycerol kinase from baboon brain was found to be predominantly associated with the particulate fraction and exhibited an apparent molecular mass of 130 kDa. 相似文献
10.
Estela L. Arrese Bertha I. Rojas-Rivas Michael A. Wells 《Archives of insect biochemistry and physiology》1996,31(3):325-335
The pathway for the synthesis of sn-1,2-diacylglycerol stimulated by the action of adipokinetic hormone (AKH) in the insect fat body is unknown. Previous results from this laboratory suggested that the hydrolysis of stored triacylglycerol to sn-2-monoacylglycerol followed by the stereospecific acylation of sn-2-monoacylglycerol catalyzed by a monoacylglycerol-acyltransferase (MGAT) could be the major route of AKH-stimulated sn-1,2-diacylglycerol synthesis. Thus, MGAT might represent a key enzyme of this pathway. In this study we characterized the MGAT activity from the Manduca sexta fat body. The activity, which was assayed by acylation of 2-monoolein using radioactive labeled palmitoyl-CoA, was found to be primarily a microsomal enzyme. The products of the acylation of 2-monoolein were 1,2-diacylglycerol (40–50%), 1,3-diacylglycerol (20–30%), and triacylglycerol (30–40%). The presence of triacylglycerol as a product revealed the presence of diacylglycerol-acyltransferase activity in the fat body microsomes. The pH optimum of MGAT activity was 7.0, and the dependence of the activity on the concentration of 2-monoolein showed saturation kinetics. An endogenous MGAT activity, which represented 20% of the maximal activity observed with added substrate, was detected. Optimal concentrations of palmitoyl-CoA ranged between 0.10–0.20 mM. The specific activity of MGAT, measured under optimal conditions, was about 0.6 nmol DG formed/min-mg protein. MGAT activity was greatest with 2-monoolein, and lower activity was observed when a saturated 2-monoacylglycerol was employed. The activity observed with sn-1-monoacylglycerol was lower than that observed with sn-2-monoacylglycerol. AKH did not stimulate MGAT activity, suggesting that either the enzyme is not under hormonal regulation or the monoacylglycerol pathway is not involved in the AKH-stimulated production of sn-1,2-diacylglycerol in the M. sexta fat body. © 1996 Wiley-Liss, Inc. 相似文献
11.
Diacylglycerol mass measurements in stimulated HL-60 phagocytes 总被引:3,自引:0,他引:3
The mass of sn-1,2-diacylglycerol in crude lipid extracts from differentiated HL-60 phagocytes was measured by quantitative conversion of the diacylglycerol to [32P]-labeled phosphatidic acid catalyzed by E. coli diacylglycerol kinase. The chemotactic peptide N-formyl-Met-Leu-Phe caused a time- and concentration-dependent increase in diacylglycerol that was maximal at 4 min. Diacylglycerol returned toward basal levels by 15 min. The basal level of diacylglycerol was 290 +/- 25 pmol/10(7) cells (n = 36). Maximally effective concentrations of N-formyl-Met-Leu-Phe and N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys increased diacylglycerol to 176% +/- 16 of basal (n = 8) and 198% +/- 15 of basal (n = 4), respectively. t-Boc-Phe-Leu-Phe-Leu-Phe, a competitive antagonist of formyl peptide receptor function, competitively inhibited the N-formyl-Met-Leu-Phe-induced diacylglycerol increase. Pretreatment of the cells with pertussis toxin abolished the stimulated rise in diacylglycerol, whereas depletion of extracellular Ca2+ markedly inhibited the increase. The Ca2+ ionophore A23187 stimulated a large (450% of basal) and persistent (greater than 30 min) increase in diacylglycerol. These data suggest that agents which raise intracellular Ca2+ levels in differentiated HL-60 cells produce a prolonged increase in cellular diacylglycerol which may activate protein kinase C. 相似文献
12.
Correction of abnormal small intestinal cytosolic protein kinase C activity in streptozotocin-induced diabetes by insulin therapy. 总被引:2,自引:0,他引:2
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Diabetes was induced in rats by administration of a single intraperitoneal injection of streptozotocin (50 mg/kg body wt). After 7 days, one group of diabetic animals was treated with insulin for an additional 5 days. Control, diabetic and diabetic + insulin rats were then killed, their distal small intestines were removed and the epithelial cells were examined and compared with respect to polyphosphoinositide turnover, total protein kinase C activity and cellular distribution, and 1,2-diacylglycerol mass and production. The results of these experiments demonstrated that, compared with their control counterparts, the intestines from diabetic rats had a decreased turnover of polyphosphoinositides, but an increase in 1,2-diacylglycerol mass which was a result, at least in part, of an increase in the synthesis of this lipid de novo. Total protein kinase C activity was decreased in the diabetic rats due to a decrease in cytosolic activity, with no significant change in particulate activity. Moreover, insulin administration for 5 days to diabetic animals did not affect their lowered intestinal polyphosphoinositide turnover, but did further accentuate their increased 1,2-diacylglycerol mass and synthesis de novo; this treatment also corrected total protein kinase C activity by increasing the cytosolic activity of this enzyme. These results indicate that signalling mechanisms involving polyphosphoinositides, 1,2-diacylglycerol and protein kinase C are abnormal in the intestines of diabetic rats and that some of these biochemical parameters can be modulated by insulin administration in vivo. 相似文献
13.
sn-1,2-Diacylglycerol kinase of Escherichia coli. Structural and kinetic analysis of the lipid cofactor dependence 总被引:4,自引:0,他引:4
The lipid cofactor requirement of Escherichia coli sn-1,2-diacylglycerol kinase was studied using a beta-octylglucoside mixed micellar assay (Walsh, J. P., and Bell, R. M. (1986) J. Biol. Chem. 261, 6239-6247). The enzyme was shown to have an absolute requirement for a lipid activator. sn-1,2-Dioleoylglycerol was both an activator and a substrate for the enzyme, 1,3-dioleoylglycerol was an activator but not a substrate, and sn-1,2-dioctanoylglycerol was a substrate but not an activator. Activation was observed with a large number of phospholipids, sulfolipids, neutral lipids, and detergents. Lipids with longer alkyl/acyl chains stimulated activity to a greater extent and at lower concentrations than their shorter chain homologs. Anionic lipids were the best activators, and neutral lipids were somewhat less effective. Cationic lipids were poor activators. Lipid activation was cooperative in all cases, with Hill coefficients ranging from 2.9 to 4.7. Lipid activators stabilized the enzyme against inactivation induced by diacylglycerols. The effectiveness of several lipids in stabilizing the enzyme correlated with their effectiveness as kinetic activators, suggesting a common mechanism. Kinetic analyses also suggested that a lipid cofactor-induced conformational change occurs as a part of the activation process. beta-Octylglucoside was shown not to function as a lipid cofactor for diacylglycerol kinase. The requirement for detergent in the assay was related, instead, to the need to disperse and deliver water-insoluble substrates and cofactors to the enzyme. beta-Octylglucoside also provided an inert matrix to which lipid substrates and cofactors could be added, enabling study of their concentration dependencies. 相似文献
14.
Dioctanoylglycerol and phorbol diesters enhance phosphorylation of phosphoprotein B-50 in native synaptic plasma membranes 总被引:5,自引:0,他引:5
J Eichberg P N de Graan L H Schrama W H Gispen 《Biochemical and biophysical research communications》1986,136(3):1007-1012
The short chain diacylglycerol, 1,2-dioctanoylglycerol, at concentrations of 100-300 microM stimulated phosphorylation of the nervous system-specific membrane protein B-50 (Mr 48 kDa, IEP 4.5) in isolated synaptic plasma membranes both in the presence and absence of exogenous protein kinase C. Comparable enhancement of histone phosphorylation by purified protein kinase C was achieved with 1 microM neutral lipid. Phorbol dibutyrate was 100 times more potent than the diacylglycerol in stimulating endogenous B-50 kinase in the membranes, whereas 4-alpha-phorbol was without effect. These results further confirm that B-50 is phosphorylated physiologically by a C kinase. Our data are consistent with a negative feedback mechanism in which generation of 1,2-diacylglycerol by enhanced phosphatidylinositol-4,5-bisphosphate hydrolysis could stimulate B-50 phosphorylation, thereby diminishing phosphatidylinositol-4-phosphate kinase activity and decreasing phosphatidylinositol-4,5-bisphosphate biosynthesis. 相似文献
15.
D R Jones T R Pettitt M A Sanjuán I Mérida M J Wakelam 《The Journal of biological chemistry》1999,274(24):16846-16852
Phosphatidic acid generation through activation of diacylglycerol kinase alpha has been implicated in interleukin-2-dependent T-lymphocyte proliferation. To investigate this lipid signaling in more detail, we characterized the molecular structures of the diradylglycerols and phosphatidic acids in the murine CTLL-2 T-cell line under both basal and stimulated conditions. In resting cells, 1,2-diacylglycerol and 1-O-alkyl-2-acylglycerol subtypes represented 44 and 55% of total diradylglycerol, respectively, and both showed a highly saturated profile containing primarily 16:0 and 18:1 fatty acids. 1-O-Alk-1'-enyl-2-acylglycerol represented 1-2% of total diradylglycerol. Interleukin-2 stimulation did not alter the molecular species profiles, however, it did selectively reduce total 1-O-alkyl-2-acylglycerol by over 50% at 15 min while only causing a 10% drop in 1,2-diacylglycerol. When radiolabeled CTLL-2 cells were challenged with interleukin-2, no change in the cellular content of phosphatidylcholine nor phosphatidylethanolamine was observed thereby ruling out phospholipase C activity as the source of diradylglycerol. In addition, interleukin-2 failed to stimulate de novo synthesis of diradylglycerol. Structural analysis revealed approximately equal amounts of 1,2-diacyl phosphatidic acid and 1-O-alkyl-2-acyl phosphatidic acid under resting conditions, both containing only saturated and monounsaturated fatty acids. After acute (2 and 15 min) interleukin-2 stimulation the total phosphatidic acid mass increased, almost entirely through the formation of 1-O-alkyl-2-acyl species. In vitro assays revealed that both 1,2-diacylglycerol and 1-O-alkyl-2-acylglycerol were substrates for 1,2-diacylglycerol kinase alpha, the major isoform in CTLL-2 cells, and that the lipid kinase activity was almost totally inhibited by R59949. In conclusion, this investigation shows that, in CTLL-2 cells, 1,2-diacylglycerol kinase alpha specifically phosphorylates a pre-existing pool of 1-O-alkyl-2-acylglycerol to form the intracellular messenger 1-O-alkyl-2-acyl phosphatidic acid. 相似文献
16.
The mouse genome contains a retrovirus-like sequence, designated VL30, which is expressed at high levels in transformed cells and which can be induced by exogenously supplied epidermal growth factor (EGF). Binding of EGF to the EGF receptor produces changes in intracellular calcium levels and phospholipase activity which indirectly lead to activation of protein kinase C. We treated AKR-2B cells, Swiss 3T3 cells, and the 3T3 variants NR6 (EGF receptorless) and TNR9 (phorbol ester nonresponsive) with various phorbol ester tumor promoters and with the synthetic diacylglycerol sn-1,2-dioctanoylglycerol. Tumor-promoting phorbol esters (e.g. 12-O-tetradecanoyl phorbol acetate (TPA] increased the level of VL30 expression. Stimulation with either TPA or EGF produced a similar time course of VL30 expression. TPA induced VL30 expression in the EGF-receptorless NR6 cell line, indicating that neither EGF ligand-receptor binding nor phosphorylation of the EGF receptor was required for induction of VL30 expression. Protein synthesis was not required for the TPA-mediated increase in VL30 expression, as pretreatment with cycloheximide did not block or reduce the TPA effect. VL30 expression was also stimulated by treatment with sn-1,2-dioctanoylglycerol, an analog of a probable endogenous activator of protein kinase C. These results suggest that activation of protein kinase C plays a direct role in regulating VL30 expression. 相似文献
17.
Diacylglycerol metabolism and arachidonic acid release in human fetal membranes and decidua vera 总被引:2,自引:0,他引:2
T Okazaki N Sagawa J R Okita J E Bleasdale P C MacDonald J M Johnston 《The Journal of biological chemistry》1981,256(14):7316-7321
Diacylglycerol lipase activity has been demonstrated in human fetal membranes and decidua vera tissues. The specific activity of the enzyme is highest in the microsomal fraction of decidua vera tissue. The acylester bond at the sn-1 position of 1,2-diacyl-sn-glycerol is hydrolyzed followed by release of the fatty acid at the sn-2 position. The diacylglycerol lipase activity present in the microsomal fraction of decidua vera tissue hydrolyzes preferentially a diacylglycerol containing an arachidonoyl group in the sn-2 position. Monoacylglycerol lipase activity was also demonstrated in these tissues. The specific activity of monoacylglycerol lipase was significantly greater than that of diacylglycerol lipase and catalyzed preferentially the hydrolysis of monoacylglycerols containing an arachidonyl group in the sn-2 position. Based on the subcellular distribution and the differential effects of various inhibitors, we suggest that the monoacylglycerol lipase and diacylglycerol lipase in decidua vera tissue are 2 distinct enzymes. Diacylglycerol kinase specific activity was examined also and was found to be 4-5 times greater in amnion than in either chorion laeve or decidua vera. The importance of diacylglycerol metabolism in the mechanism of arachidonic acid release and prostaglandin biosynthesis is discussed. 相似文献
18.
H Brockerhoff 《FEBS letters》1986,201(1):1-4
Membrane-bound proteins owe their retention and conformation in the lipid bilayer to hydrophobic peptide domains. Additional fixation, by protein-lipid hydrogen bonding, has been suggested, and recent reports on protein kinase C activation by diacylglycerol (DG) provide an unambiguous model for such bonding. The sn-1,2-diacylglycerol appears to donate a hydrogen bond from the sn-3 hydroxyl to the enzyme and to receive two hydrogen bonds, in the sn-1 and sn-2 ester CO groups, from the enzyme. This arrangement is confirmed in phorbol ester, a competitive inhibitor of DG for the kinase. This tumor promotor has a nearly identical spatial arrangement of hydrogen bond donor (9 alpha-OH) and acceptors (12 and 13 ester CO); so have two other tumor promotors, teleocidin and aplysiatoxin. There are reasons to believe that protein kinase C is not the only protein that is bound to membrane lipids by hydrogen bonding, and such bonding will have to be considered in membrane-associated events such as fusion, cross-membrane transport, or anesthesia. 相似文献
19.
Sphingosine inhibition of protein kinase C activity and of phorbol dibutyrate binding in vitro and in human platelets 总被引:41,自引:0,他引:41
Y A Hannun C R Loomis A H Merrill R M Bell 《The Journal of biological chemistry》1986,261(27):12604-12609
Sphingosine inhibited protein kinase C activity and phorbol dibutyrate binding. When the mechanism of inhibition of activity and phorbol dibutyrate binding was investigated in vitro using Triton X-100 mixed micellar methods, sphingosine inhibition was subject to surface dilution; 50% inhibition occurred when sphingosine was equimolar with sn-1,2-dioleoylglycerol (diC18:1) or 40% of the phosphatidylserine (PS) present. Sphingosine inhibition was modulated by Ca2+ and by the mole percent of diC18:1 and PS present. Sphingosine was a competitive inhibitor with respect to diC18:1, phorbol dibutyrate, and Ca2+. Increasing levels of PS markedly reduced inhibition by sphingosine. Since protein kinase C activity shows a cooperative dependence on PS, the kinetic analysis of competitive inhibition was only suggestive. Sphingosine inhibited phorbol dibutyrate binding to protein kinase C but did not cause protein kinase C to dissociate from the mixed micelle surface. Sphingosine addition to human platelets blocked thrombin and sn-1,2-dioctanoylglycerol-dependent phosphorylation of the 40-kDa (47 kDa) dalton protein. Moreover, sphingosine was subject to surface dilution in platelets. The mechanism of sphingosine inhibition is discussed in relation to a previously proposed model of protein kinase C activation. The possible physiological role of sphingosine as a negative effector of protein kinase C is suggested and a plausible cycle for its generation is presented. The potential physiological significance of sphingosine inhibition of protein kinase C is further established in accompanying papers on HL-60 cells (Merrill, A. H., Jr., Sereni, A. M., Stevens, V. L., Hannun, Y. A., Bell, R. M., Kinkade, J. M., Jr. (1986) J. Biol. Chem. 261, 12010-12615) and human neutrophils (Wilson, E., Olcott, M. C., Bell, R. M., Merrill, A. H., Jr., and Lambeth, J. D. (1986) J. Biol. Chem. 261, 12616-12623). These results also suggest that sphingosine will be a useful inhibitor for investigating the function of protein kinase C in vitro and in living cells. 相似文献
20.
Diacylglycerol acyltransferase (DGAT) catalyses the acylation of the sn-3 hydroxy group of sn-1,2-diacylglycerol using acyl-CoA. The gene encoding DGAT from Arabidopsis thaliana has been cloned and the function of the enzyme proved by expression of the coding sequence using a bacculovirus expression system in insect cell cultures. The expressed protein catalysed the synthesis of [(14)C]triacylglycerol from [(14)C]diacylglycerol and oleoyl-CoA. The heterologously expressed DGAT activity was found mostly associated with the 100000 g pellet. The optimum activity was achieved at a neutral pH, in the presence of Mg2+, and at an optimum oleoyl-CoA concentration of 20 microM. The DGAT used the substrates palmitoyl-CoA and oleoyl-CoA equally effectively. In these experiments, the inclusion of recombinant acyl-CoA binding protein had a relatively small effect upon DGAT activity. 相似文献