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G Y Sun

Publications and source records attributed to G Y Sun.

At least 91 records · Page 5Linked to original sources
PubMed1990

Decapitation-induced changes in inositol phosphates in rat brain.

Decapitation resulted in a time-dependent production of inositol phosphates in rat brain. This production was analyzed by measuring both the radioactivity and the concentrations of inositol phosphates generated from [3H]inositol-labeled phospholipids. Both measurements produced the same time-dependent changes, including a rapid decrease in inositol 1,4,5-trisphosphate within 1.5 min, a 6-fold increase in inositol 1,4-bisphosphate to a maximum at 1.5 min, a 5-fold rise in inositol 4-monophosphate to a maximum at 2.5 min, and little change in inositol 1-monophosphate. The temporal changes in the mass and radioactivity of these compounds, together with the decrease in labeling of phosphatidylinositol 4,5-bisphosphates, support the idea that the inositol phosphates originate from the hydrolysis of phosphatidylinositol 4,5-bisphosphates and not from either the direct hydrolysis of phosphatidylinositol 4-phosphates or phosphatidylinositols.

Animals↗
PubMed1990

Metabolism of phosphatidylinositol in plasma membranes and synaptosomes of rat cerebral cortex: a comparison between endogenous vs exogenous substrate pools.

The metabolism of phosphatidylinositols (PI) labeled with [14C]arachidonic acid within plasma membranes or synaptosomes was compared to the metabolism of PI prelabeled with [14C]arachidonic acid and added exogenously to the same membranes. Incubation of membranes containing the endogenously-labeled PI pool in the presence of Ca2+ resulted in the release of labeled arachidonic acid, as well as a small amount of labeled diacylglycerol. Labeled arachidonic acid was effectively reutilized and returned to the membrane phospholipids in the presence of adenosine triphosphate (ATP), CoA, and lysoPI. Although Ca2+ promoted the release of labeled diacylglycerol from prelabeled plasma membranes, this amount was only 17% of the maximal release, i.e., release in the presence of deoxycholate and Ca2+. This latter condition is known to fully activate the PI-phospholipase C, and incubation of prelabeled plasma membranes resulted in a six-fold increase in labeled diacylglycerols. On the other hand, when exogenously labeled PI were incubated with plasma membranes in the presence of Ca2+, the labeled diacylglycerols released were 59% of that compared to the fully activated condition. The phospholipase C action was calcium-dependent, regardless of whether exogenous or endogenous substrates were used in the incubation. In contrast to plasma membranes, intact synaptosomes had limited ability to metabolize exogenous PI even in the presence of Ca2+, although the activity of phospholipase C was similar to that in the plasma membranes when assayed in the presence of deoxycholate and Ca2+. These results suggest that discrete pools of PI are present in plasma membranes, and that the pool associated with the acyltransferase is apparently not readily accessible to hydrolysis by phospholipase C.

Animals↗
PubMed1990

A comparative blinded study in miniature swine of whole blood-, hemoglobin-, platelet-, plasma-, and lymphocyte-associated acetaldehyde as markers for ethanol intake.

Blood samples were obtained from miniature swine maintained on 0, 2, or 6 g/kg/24 hr ethanol for 8 months (N = 6 in each group). Samples from drinking pigs were taken after 8 hr of ethanol abstinence and all were coded and sent for "blinded" analysis. A fluorigenic high performance liquid chromatographic assay was used to quantify whole blood-associated acetaldehyde, hemoglobin-associated acetaldehyde, plasma-associated acetaldehyde, platelet-associated acetaldehyde, and lymphocyte-associated acetaldehyde. Detectable levels of acetaldehyde were found in each sample in both drinking and nondrinking pigs. Analysis of whole blood-associated acetaldehyde was most discriminatory in distinguishing nondrinking from drinking pigs (mean 21.4 +/- 1.0 microM for nondrinkers vs. 24.6 +/- 1.5 SD for the group consuming 2 g/kg ethanol, p = 0.001). Measurements of hemoglobin-associated acetaldehyde normalized to protein concentration (250 +/- 47 nmoles/g vs. 203 +/- 33 SD, p less than 0.05 drinking vs. nondrinking pigs) and platelet-associated acetaldehyde (0.46 0.34 vs. 0.15 +/- 0.16 nmoles/3 x 10(8) platelets, p = 0.05 drinking vs. nondrinking pigs) were also useful in discriminating drinking from nondrinking animals. Analysis of plasma-associated acetaldehyde and lymphocyte-associated acetaldehyde were not useful as markers of ethanol consumption.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetaldehyde↗
PubMed1990

Dynamic turnover of mouse brain phospholipids during normal aging and response to ischemia.

An in vivo labeling procedure was used to probe the dynamic turnover of mouse brain phospholipids and to evaluate the response of these phospholipids toward decapitation ischemic insult. Within 4 hr after intracerebral injection of [32P]-ATP, the label was effectively incorporated into all phospholipids and uniquely in all subcellular membrane fractions examined. With respect to age, synaptosomes isolated from the 27-month-old mice group showed a higher incorporation of label into polyphosphoinositides and phosphatidic acids and a lower incorporation into the neutral phospholipids than the 10-month-old group. The increase in labeling of these phospholipids with age seems to reflect the increase in basal level of substrates for phosphorylation of phosphoinositol 4-phosphate and diacylglycerols. The increase in substrate level is probably related to a decrease in metabolic turnover of the lipids underlying the phosphoinositide cycle. Decapitation ischemic insult is known to result in a rapid time-dependent breakdown of the polyphosphoinositides in brain. However, a comparison of the ischemia-induced breakdown of polyphosphoinositide between the 10 and 27 month-old groups did not reveal obvious age differences in the rate of their disappearance.

Aging↗
PubMed1989

Purification of lysophosphatidylcholine transacylase from bovine heart muscle microsomes and regulation of activity by lipids and coenzyme A.

Heart muscle microsomes catalyze the transacylation of lysophosphatidylcholine (lyso PC) to produce phosphatidylcholine (PC). The enzyme which catalyzes this reaction, lyso PC:lyso PC transacylase, has been isolated and characterized from bovine heart muscle microsomes. The purification of the enzyme was achieved by a procedure involving extraction with 3-[3-cholamidopropyl)dimethylammonio)-1-propanesulfonate (CHAPS) detergent and chromatography on DEAE-cellulose, Reactive blue agarose, and Matrex gel green A. The purified enzyme was nearly homogeneous and consisted of a single molecular species of 128 kDa as determined by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate. The catalytic activity of the enzyme was dependent on the presence of either CoA or acyl-CoA, both of which maximally stimulated at concentrations of approx. 10 microM. Analysis of the PC produced in the reaction showed that the enzyme catalyzed a transacylation in which both acyl groups arose from lyso PC. Furthermore, the enzyme did not possess acyl-CoA:lyso PC acyltransferase activity, lysophospholipase or acyl-CoA hydrolase activity, nor did it catalyze transacylation from lyso PC to lysophosphatidylethanolamine, lysophosphatidylinositol or lysophosphatidylserine. Although transacylation was highly specific for lyso PC as the substrate, various unsaturated fatty acyl-CoA derivatives served as activators. Palmitoyl-CoA and stearoyl-CoA did not significantly activate, although acetyl-CoA was an effective activator. Further modulation of activity was produced by palmitic acid and PC, both of which further activated the enzyme in the presence of oleoyl-CoA, whereas arachidonic acid, oleic acid, phosphatidylethanolamine and phosphatidylserine had no effect on activity. The high activity of this transacylase and its regulation by lipids suggests an important role for disaturated PC species in membranes and a mechanism for controlling the metabolism of lyso PC.

Acyl Coenzyme A↗
PubMed1989

Effects of free fatty acids and acyl-coenzyme A on diacylglycerol kinase in rat brain.

Our earlier studies have indicated the presence of diacylglycerol kinase activity in rat brain cytosol as well as subcellular membrane fractions (Strosznajder et al.: Neurochemistry International 8(2):213-221, 1986). There is much evidence indicating the release of diacylglycerols due to stimulation of polyphosphoinositide hydrolysis by hormones and receptor agonists. In turn, diacylglycerols have been linked to a second messenger role for activation of protein kinase C. The present study tests the ability of free fatty acids and acyl-coenzyme A (acyl-CoA) to regulate diacylglycerol kinase activity. In a system containing brain cytosol and microsomes, addition of oleic acid (0.5 mM) resulted in large stimulation of diacylglycerol kinase activity as well as some translocation of the enzyme from cytosol to microsomes. On the other hand, oleoyl-CoA (0.1 mM), but neither palmitoyl-CoA nor arachidonoyl-CoA, was effective in translocation of the diacylglycerol kinase. Unlike oleic acid, which preferred to associate with membranes, most of the oleoyl-CoA remained in the cytosolic fraction. Since free fatty acids in brain are stringently controlled and are released during ischemic insult, a condition which also elicits the breakdown of polyphosphoinositide to diacylglycerols, results here suggest a plausible mechanism for regulation of diacylglycerol metabolism by free fatty acids and acyl-CoA.

Acyl Coenzyme A↗
PubMed1989

Ethanol alters the transfer of arachidonic acid to ethanolamine plasmalogens in C-6 glioma cells.

In this study, the effects of ethanol exposure on uptake and metabolism of arachidonic acid by C-6 glioma cells in culture was examined. Labeled arachidonic acid was effectively taken up by the phospholipids of these cells and radioactivity was initially incorporated into phosphatidylinositols and phosphatidylcholines, reaching a peak between 4 and 6 hours. However, the labeling of ethanolamine plasmalogens continued to show an increase with time after labeled arachidonic acid has been exhausted in the medium. Since over 90% of labeled arachidonic acid was already taken up by the cells after 4 hours of exposure, the continued increase in labeling of ethanolamine plasmalogens is attributed to a transacylation mechanism. Cells grown in 150 mM ethanol for 2 days did not show a change in the overall incorporation of labeled arachidonic acid into phospholipids but showed a significant increase in labeling of ethanolamine plasmalogens, which was marked by a concomitant decrease in labeling of phosphatidylcholines. Ethanol exposure also resulted in a significant increase in the transfer of labeled arachidonic acid to triacylglycerols. Changes in phospholipid and triacylglycerol labeling pattern positively correlated with increasing ethanol concentration from 75 to 300 mM. Besides, most ethanol effects were readily noticeable after 24 hours of exposure. These data suggest a specific effect of ethanol on promoting the transacylase process for biosynthesis of ethanolamine plasmalogens as well as the acyltransferase for biosynthesis of triacylglycerols.

Arachidonic Acid↗
PubMed1989

Purification and kinetic properties of lysophosphatidylinositol acyltransferase from bovine heart muscle microsomes and comparison with lysophosphatidylcholine acyltransferase.

The enzyme acyl-CoA:1-acyl-sn-glycero-3-phosphoinositol acyltransferase (LPI acyltransferase, EC 2.3.1.23) was purified approximately 11,000-fold to near homogeneity from bovine heart muscle microsomes. The purification was effected by extraction with the detergent 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate, followed by chromatography on Cibacron blue agarose, DEAE-cellulose, and Matrex gel green A. The isolated enzyme was a single protein of 58,000 Da as measured by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate. This purification procedure also allows isolation of the related enzyme lysophosphatidylcholine (LPC) acyltransferase, which was separated from LPI acyltransferase at the final chromatographic step. The purified LPI acyltransferase exhibits an absolute specificity for LPI as the acyl acceptor. Broader specificity was found for acyl-CoA derivatives as substrates, although the preferred substrates are long-chain, unsaturated derivatives: measured reactivities were in the order arachidonoyl-CoA greater than oleoyl-CoA greater than eicosadienoyl-CoA greater than linoleoyl-CoA. Little activity was found with palmitoyl-CoA or stearoyl-CoA as potential substrates. These properties are consistent with a role of the enzyme in controlling the acyl group composition of phosphoinositides. Comparison of LPC acyltransferase and LPI acyltransferase shows that these two enzymes have distinct kinetic and physical properties and are affected differently by local anesthetics, which are potent inhibitors.

1-Acylglycerophosphocholine O-Acyltransferase↗
PubMed1989

Membrane properties of dolichol in different age groups of mice.

Dolichol content and the effects of dolichol in vitro on synaptic plasma membrane (SPM) structure of different age groups of C57BL/6NNIA mice were examined. SPM of aged mice had higher amounts of dolichol as compared to younger animals. Age differences also were observed in dolichol isoprenologue distribution. In older animals, dolichol isoprenologue chain length was shifted towards shorter chain length values. The structure of SPM was determined by differential polarized phase fluorometry of 1,6-diphenyl-1,3,5-hexatriene (DPH). The SPM of old mice were more rigid, as indicated by higher limiting anisotropy, than those of young mice. Dolichol administered in vitro to SPM fluidized the membranes. However, dolichol administered in vitro fluidized SPM of younger mice significantly more than SPM of aged mice. The functional role of increased dolichol content in aged individuals is not known. Dolichol may act to regulate membrane fluidity of aged animals.

Aging↗
PubMed1989

Time course for labeling of brain membrane phosphoinositides and other phospholipids after intracerebral injection of [32P]-ATP. Evaluation by an improved HPTLC procedure.

An improved two-dimensional HPTLC procedure was developed for separating phospholipids including individual phosphoinositides, phosphatidic acids and plasmalogens. This procedure was used to examine the time course for uptake of label by phospholipids in brain subcellular membranes after intracerebral injection of [gamma-32P]-ATP. There were considerable differences in the phospholipid labeling pattern among different subcellular fractions. In particular, a high proportion of labeled phosphatidylinositol 4,5-bisphosphates and phosphatidic acids was found in the myelin fraction during the initial 4 hr after injection. In other subcellular fractions, labeling of phosphoinositides was maximum at 2 hr, but with prolonged time, poly-phosphoinositides started to show a decline in radioactivity whereas labeling of other phospholipids continued to show a steady increase instead. Results indicate at least two different modes for the uptake of label by brain membrane phospholipids after intracerebral injection of [32P]-ATP.

Adenosine Triphosphate↗
PubMed1989

Bradykinin effects on phospholipid metabolism and its relation to arachidonic acid turnover in neuroblastoma x glioma hybrid cells (NG 108-15).

In neuroblastoma x glioma hybrid cells (NG 108-15) labelled with [32P]-trisodium phosphate, [3H]-inositol and [14C]-arachidonic acid, bradykinin stimulated the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) while it had no effect on the release of [14C]-arachidonic acid (AA). The effect on PIP2 was time- and dose-dependent with a maximal effect on [3H]-inositol- and [32P]-labelled cells after 10-30 s of stimulation with 10(-6) M bradykinin. However, the hydrolysis of [14C]-AA labelled PIP2 was delayed compared to the effect on [3H]- and [14C]-PIP2 and was not detectable until after 60 s of stimulation. Bradykinin stimulation resulted in an increased formation of [3H]-inositol phosphates (IP) and [32P]- and [14C]-phosphatidic acid (PA) but the time course for PA formation did not follow the time-course for PIP2 hydrolysis. A reduced labelling of [32P]- and [14C]-phosphatidylcholine was also found in stimulated cells suggesting that PA may derive from other sources than PIP2. In conclusion, our results indicate that bradykinin activates phospholipase C, but not phospholipase A2, in NG 108-15 cells.

Animals↗
PubMed1988

Ion chromatographic determination of sugar phosphates in physiological samples.

Ion chromatography is shown to be capable of simultaneous determination of biologically important anions. Application of this technique is illustrated for the separation and quantification of the major anions present in rat brain and liver tissues. Sugar phosphates and carboxylic acids are separated on high-performance anion-exchange columns and are detected using chemically suppressed conductivity. Detection limits range from 20 to 100 pmol for the anions tested, including inositol phosphates, lactate, pyruvate, glucuronic acid-1-phosphate, fructose-6-phosphate and glucose-6-phosphate. The coefficient of variation for the determination of most anions was in the range 5-10%. Many of these anions are either difficult to separate with other methods, or require expensive radiochemical techniques for detection. This method should be applicable to other biological studies, from the flow of carbons in photosynthesis to the study of synaptic transmission.

Animals↗
PubMed1988

Phosphatidylinositol kinase, phosphatidylinositol-4-phosphate kinase and diacylglycerol kinase activities in rat brain subcellular fractions.

Subcellular fractions isolated and purified from rat brain cerebral cortices were assayed for phosphatidylinositol (PI-), phosphatidylinositol-4-phosphate (PIP-), and diacylglycerol (DG-) kinase activities in the presence of endogenous or exogenously added lipid substrates and [gamma-32P]ATP. Measurable amounts of all three kinase activities were observed in each subcellular fraction, including the cytosol. However, their subcellular profiles were uniquely distinct. In the absence of exogenous lipid substrates, PI-kinase specific activity was greatest in the microsomal and non-synaptic plasma membrane fractions (150-200 pmol/min per mg protein), whereas PIP-kinase was predominantly active in the synaptosomal fraction (136 pmol/min per mg protein). Based on percentage of total protein, total recovered PI-kinase activity was most abundant in the cytosolic, synaptosomal, microsomal and mitochondrial fractions (4-11 nmol/min). With the exception of the microsomal fraction, a similar profile was observed for PIP-kinase activity when assayed in the presence of exogenous PIP (4 nmol/20 mg protein in a final assay volume of 0.1 ml). Exogenous PIP (4 nmol/20 mg protein) inhibited PI-kinase activity in most fractions by 40-70%, while enhancing PIP-kinase activity. PI- and PIP-kinase activities were observed in the cytosolic fraction when assayed in the presence of exogenously added PI or PIP, respectively, but not in heat-inactivated membranes containing these substrates. When subcellular fractions were assayed for DG-kinase activity using heat-inactivated DG-enriched membranes as substrate, DG-kinase specific activity was predominantly present in in the cytosol. However, incubation of subcellular fractions in the presence of deoxycholate resulted in a striking enhancement of DG-kinase activities in all membrane fractions. These findings demonstrate a bimodal distribution between particulate and soluble fractions of all three lipid kinases, with each exhibiting its own unique subcellular topography. The preferential expression of PIP-kinase specific activity in the synaptic membranes is suggestive of the involvement of PIP2 in synaptic function, while the expression of PI-kinase specific activity in the microsomal fraction suggests additional, yet unknown, functions for PIP in these membranes.

1-Phosphatidylinositol 4-Kinase↗
PubMed1988

Arachidonic acid uptake by phospholipids and triacylglycerols of rat brain subcellular membranes.

In the presence of ATP, MgCl2 and CoASH, different subcellular membrane fractions isolated from rat cerebral cortex exhibited characteristic profiles for the incorporation of [1-14C]arachidonic acid into phospholipids and triacylglycerols. In general, uptake of label by phosphatidylcholines was higher in the synaptic membranes, and that by phosphatidylinositols was higher in the microsomes and somal plasma membranes. A substantial amount of the labeled arachidonate was also incorporated into triacylglycerols, especially in the somal plasma membranes and microsomes. Enzymes mediating the transfer of arachidonic acid to phospholipids were unstable with respect to sample storage and exposure to elevated temperatures. In contrast, the acyltransferase for triacylglycerols was more stable to these factors. Washing the membranes with bovine serum albumin resulted in an enhancement of the incorporation of label into phosphatidylinositols without affecting that of phosphatidylcholines, but the incorporation into triacylglycerols was inhibited. Treatment of synaptosomes and plasma membranes with saponin resulted in an enhancement in the labeling of phospholipids, but the labeling of triacylglycerols was inhibited. Thus, although labeled arachidonic acid was incorporated into phospholipids and triacylglycerols in brain subcellular membranes, these two types of acyltransferases exhibited different properties and responded differently to membrane perturbing agents.

Adenosine Triphosphate↗
PubMed1988

Acylation of lysophosphatidylcholine in bovine heart muscle microsomes: purification and kinetic properties of acyl-CoA:1-acyl-sn-glycero-3-phosphocholine O-acyltransferase.

Bovine heart muscle microsomes rapidly convert lysophosphatidylcholine (LPC) into phosphatidylcholine (PC) in the presence of oleoyl-CoA. Both substrates are incorporated into the product, although the rate of incorporation of radiolabel into PC from 1-[14C]palmitoyl-LPC was approximately threefold higher than the rate of incorporation from [14C]oleoyl-CoA. Furthermore, the rate of incorporation of radiolabel from [14C]LPC was stimulated fivefold by the presence of oleoyl-CoA. These results demonstrate the presence of both acyl-CoA:1-acyl-sn-glycero-3-phosphocholine O-acyltransferase (EC 2.3.1.23) and an LPC:LPC transacylase (EC 3.1.1.5) in microsomes. Separation of the two enzymatic activities and purification of the acyltransferase was achieved by a procedure involving extraction with 3-[3-cholamidopropyl)dimethylammonio)-1-propanesulfonate detergent and chromatography on DEAE-cellulose, Reactive blue agarose, and Matrex gel green A. The isolated acyltransferase was a single species of 64,000 Da as judged by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate. The substrate specificity of the enzyme was studied by using a series of lysophospholipids as acyl acceptors and acyl-CoA derivatives as acyl donors. The enzyme was catalytically active with LPC as acyl acceptor but displayed little or no activity with lysophosphatidylethanolamine, lysophosphatidylinositol, or lysophosphatidylserine. Of the LPC derivatives tested, the highest activity was obtained with 1-palmitoyl-LPC. Wider specificity was exhibited for the nature of the acyl donor, for which arachidonoyl-CoA, linoleoyl-CoA, and oleoyl-CoA were highly active substrates. These properties of the acyltransferase are in accord with a role of the enzyme in determining the composition of PC in myocardium.

1-Acylglycerophosphocholine O-Acyltransferase↗
PubMed1988

Effects of ATP on phosphatidylinositol-phospholipase C and inositol 1-phosphate accumulation in rat brain synaptosomes.

Incubation of rat brain synaptosomes prelabeled with [2-3H]inositol resulted in a time-dependent release of labeled inositol 1-phosphate. This process was Ca2+ dependent, and ATP (1 mM) enhanced the inositol 1-phosphate formation three- to fivefold. Using [1-14C]arachidonoyl-phosphatidylinositol which was introduced into saponin-permeabilized synaptosomes, ATP (1 mM) and free Ca2+ (approximately 20 microM) enhanced the phospholipase C hydrolysis of this substrate to form labeled diacylglycerol. When the same permeabilized synaptosomal preparation was incubated with [2-3H]inositol-phosphatidylinositol, ATP not only enhanced the formation of labeled inositol 1-phosphate, but also inhibited the conversion of inositol 1-phosphate to inositol. Furthermore, ATP appeared to reduce the Ca2+ requirement of the phosphatidylinositol-phospholipase C. Inhibition of the conversion of inositol 1-phosphate to inositol could not be overcome by increasing the Mg2+ concentration in the incubation medium. Although the ATP effect is not viewed as a receptor-mediated event, it is possible that such an event may occur in synaptosomes under conditions in which intrasynaptic Ca2+ concentration becomes elevated.

Adenosine Triphosphate↗
PubMed1988

Effects of ethanol on arachidonic acid incorporation into lipids of a plasma membrane fraction isolated from brain cerebral cortex.

In the presence of ATP, MgCl2, and CoASH, somal plasma membranes isolated from rat cerebral cortex were active in transferring arachidonic acid to phosphatidylinositols, phosphatidylcholines, and triacylglycerols. Ethanol (350-525 mM) added to the incubation mixture inhibited arachidonic acid incorporation into phospholipids, while it enhanced the incorporation into triacylglycerols. Under these conditions, ethanol was found to react with arachidonic acid to form arachidonoyl ethyl ester. The incorporation of labeled arachidonic acid into glycerolipids as well as the synthesis of ethyl esters required the presence of ATP and CoASH for maximal activity. Nevertheless, each uptake process exhibited a unique pH profile. The esterification of arachidonic acid was not specific for ethanol as other aliphatic alcohols (e.g., propanol and butanol) were also able to react with labeled arachidonic acid to form the respective esters. Somal plasma membranes isolated from mice after chronic ethanol administration showed an increase in arachidonoyl transfer to both phospholipids and triacylglycerols. When these membranes were challenged with ethanol (325 mM), those isolated from the chronic ethanol group showed a greater increase in the labeling of triacylglycerols and ethyl esters than those from controls. Thus, different acyltransferases exhibite different responses to the effects of ethanol in vitro and in vivo.

Acyltransferases↗