Identification of endogenous 1-O-alk-1'-enyl-2-acyl-sn-glycerol in myocardium and its effective utilization by choline phosphotransferase

Recently we have identified a novel choline and ethanolamine specific phospholipase C in myocardium and have hypothesized that this enzyme is responsible for the introduction of the vinyl ether linkage into plasmenylcholine by shuttling 1-O-alk-1'-enyl-2-acyl-sn-glycerol fragments from plasmenylethanolamine to plasmenylcholine (Wolf, R. A., and Gross, R. W. (1985) J. Biol. Chem. 260, 7295-7303). The present study demonstrates that rabbit myocardium contains endogenous 1-O-hexadec-1'-enyl-2-acyl-sn-glycerol (0.46 micrograms/g) and that these moieties are selectively utilized by myocardial choline phosphotransferase to generate plasmenylcholine. The apparent Michaelis constant of CDP-choline for microsomal choline phosphotransferase was 9 microM with a corresponding Vmax of 18 pmol/mg.min utilizing endogenous 1-O-alk-1'-enyl-2-acyl-sn-glycerol as substrate. The flux of CDP-choline into plasmenylcholine or phosphatidylcholine was similar despite the fact that the mass of endogenous 1,2-diacyl-sn-glycerol was over 20 times the mass of endogenous 1-O-alk-1'-enyl-2-acyl-sn-glycerol. Augmentation of endogenous 1-O-alk-1'-enyl-2-acyl-sn-glycerol content by pretreatment of myocardial microsomes with exogenous phospholipase C resulted in an 8-fold increase in plasmenylcholine synthesis. The results suggest that myocardial plasmenylcholine biosynthesis occurs by polar head group remodeling utilizing endogenous 1-O-alk-1'-enyl-2-acyl-sn-glycerol as a synthetic intermediate. Flux through this pathway is likely regulated by physiologic increments in endogenous 1-O-alk-1'-enyl-2-acyl-sn-glycerol content and cytosolic CDP-choline concentration.     

The primary determinant of rabbit myocardial ethanolamine phosphotransferase substrate selectivity is the covalent nature of the sn-1 aliphatic group of diradyl glycerol acceptors.

Plasmenylethanolamines represent the major endogenous phospholipid storage depot of arachidonic acid in many mammalian cells. To elucidate the biochemical mechanisms contributing to the high plasmalogen content and arachidonic acid enrichment present in myocardial ethanolamine glycerophospholipids, the substrate specificity of rabbit myocardial ethanolamine phosphotransferase (EPT) was quantified utilizing multiple molecular species of each subclass of diradyl glycerol substrate. Myocardial EPT demonstrated over a 16-fold selectivity for 1-O-alk-1'-enyl-2-acyl-sn-glycerol (AAG) compared to 1,2-diacyl-sn-glycerol (DAG) substrate utilizing individual molecular species of each subclass dispersed in Tween 20. The selective utilization of AAG by EPT was substantiated utilizing two independent assay systems which employed either the presentation of substrate to enzyme as a substitutional impurity in Triton X-100 mixed micelles or the obligatory utilization of endogenously generated diradyl glycerol substrates. Although rabbit myocardial microsomes contained over a 20-fold molar excess of endogenous DAG to AAG mass, incubation of rabbit myocardial microsomes with CDP-ethanolamine resulted in the highly selective synthesis of plasmenylethanolamines which were predominantly comprised of molecular species containing arachidonic acid at the sn-2 position (greater than 75%). Endogenous AAG molecular species in rabbit myocardial microsomes were similarly enriched in arachidonic acid, and the distribution of AAG molecular species closely paralleled the distribution of plasmenylethanolamine (but not plasmenylcholine) molecular species. Thus, the subclass and molecular species distribution of the ethanolamine glycerophospholipids synthesized by rabbit myocardial EPT reflects independent contributions from the subclass selectivity of EPT for AAG substrate in conjunction with the enrichment of arachidonic acid in microsomal AAG molecular species.     

Arachidonic acid mobilization among phospholipids in murine mastocytoma P-815 cells: role of ether-linked phospholipids

The ethanolamine-containing glycerophospholipids, choline-containing glycerophospholipids, and phosphatidylinositol fractions are major sources of arachidonic acid in murine mastocytoma P-815 cloned cells. The choline-linked fraction contained high arachidonic acid contents in 1-O-alkyl-2-acyl- (18%) and 1,2-diacyl-sn-glycero-3-phosphocholine (11%), with smaller amounts in 1-O-alk-1'-enyl-2-acyl species, whereas the arachidonic acid content of the ethanolamine-linked fraction was high in 1-O-alk-1'-enyl-2-acyl (26%) and 1,2-diacyl species (15%) and low in 1-O-alkyl-2-acyl species. The uptake and transfer of [3H]arachidonic acid into the 1,2-diacyl and ether classes of choline-containing glycerophospholipids and ethanolamine-containing glycerophospholipids in mastocytoma cells were examined. There was very rapid incorporation of radioactive arachidonic acid into mastocytoma cells that leveled off after 30 min. By labeling cells with [3H]arachidonic acid for 7.5 min, the radioactivity was recovered in the choline-containing glycerophospholipids (43%), phosphatidylinositol (32%), and ethanolamine-containing glycerophospholipids (20%) with little in other phospholipids, neutral lipid, or free fatty acid fractions. Upon reincubation of the mastocytoma cells in the radiolabel-free medium, the [3H]arachidonate radioactivity was gradually lost from the choline-containing glycerophospholipids fraction and, concomitantly, increased in ethanolamine-containing glycerophospholipids. At the zero time of reincubation, most of the radioactivity was recovered in the 1,2-diacyl species of both choline-containing glycerophospholipids and ethanolamine-containing glycerophospholipids.(ABSTRACT TRUNCATED AT 250 WORDS)     

豚鼠心脏CDP-乙醇胺磷酸转移酶的动力学研究

为探讨不同的二价基甘油对三种乙醇胺甘油磷脂生物合成能力的影响,通过对豚鼠乙醇胺磷酸转移酶动力学研究,发现磷脂酰乙醇胺的合成可被1-烷基-2-脂酰甘油和1-烯醚基-2-脂酰甘油抑制,而缩醛磷脂酰乙醇胺的生成不受1,2-二脂酰甘油影响,并提示不同的二价基甘油对乙醇胺磷酸转移酶的抑制作用呈非竞争性抑制,此有利于对三种乙醇胺磷脂酰甘油生物合成的相互协调作用。     

Plasmenylethanolamine synthesis in Leishmania major

Ethanolamine glycerophospholipids are ubiquitous cell membrane components. Trypanosomatid parasites of the genus Leishmania synthesize the majority of their ethanolamine glycerophospholipids as 1‐O‐alk‐1′‐enyl‐2‐acyl‐sn‐glycero‐3‐phosphoethanolamine or plasmenylethanolamine (PME) through the Kennedy pathway. PME is a subtype of ether phospholipids also known as ethanolamine plasmalogen whose functions are not well characterized. In this study, we investigated the role of PME synthesis in Leishmania major through the characterization of an ethanolamine phosphotransferase (EPT) mutant. EPT‐null parasites are largely devoid of PME and fully viable in regular medium but fail to proliferate in the absence of fetal bovine serum. They exhibit significant abnormalities in the synthesis and localization of GPI‐anchored surface molecules. EPT‐null mutants also show attenuated virulence in BALB/c mice. Furthermore, in addition to PME synthesis, ethanolamine also contributes to the production of phosphatidylcholine, the most abundant class of lipids in Leishmania. Together, these findings suggest that ethanolamine production is likely required for Leishmania promastigotes to generate bulk phospholipids, to handle stress, and to control the expression of membrane bound virulence factors.     

Serine utilization as a precursor of phosphatidylserine and alkenyl-(plasmenyl)-, alkyl-, and acylethanolamine phosphoglycerides in cultured glioma cells

In several tissues and cell lines, serine utilized for phosphatidylserine (PS) synthesis is an eventual precursor of the base moiety of ethanolamine phosphoglycerides (PE). We investigated the biosynthesis and decarboxylation of PS in cultured C6 glioma cells, with particular attention to 1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine (plasmenylethanolamine) biosynthesis. Incorporation of [3H]serine into PS reached a maximum within 4-8 h, and label in nonplasmenylethanolamine phosphoglyceride (NP-PE) and plasmenylethanolamine was maximal by 12-24 h and 48 h, respectively. After 8 h, label in PS decreased even though 40-60% of initial label remained in the culture medium. Serial additions of fresh [3H]serine restored PS synthesis to higher levels of labeled PS accumulation followed by a subsequent decrease in 4-8 h. High performance liquid chromatographic analyses confirmed that medium serine was depleted by 8 h, and thereafter metabolites, including acetate and formate, accounted for radioactivity in the medium. The rapid but transient appearance of labeled glycine and ATP inside the cells indicated conversion of serine by hydroxymethyltransferase. 78-85% of label from serine was in headgroup of PS or of PE formed by decarboxylation. A precursor-product relationship was suggested for label from [3H]serine appearing in the headgroup of diacyl, alkylacyl, and alkenylacyl subclasses of PE. By 48 h, a constant specific activity, ratio of approximately 1:1 was reached between plasmenylethanolamine and NP-PE, similar to the molar distribution of these lipids. In contrast, equilibrium was not achieved in cells incubated with [1,2-14C]ethanolamine; plasmenylethanolamine had 2-fold greater specific activity than labeled NP-PE by 72-96 h. These observations indicate that in cultured glioma cells 1) serine serves as a precursor of the head group of PS and of both plasmenyl and non-plasmenyl species of PE; 2) exchange of headgroup between NP-PE and plasmenylethanolamine may involve different donor pools of PE depending on whether the headgroup originates with exogenous serine or ethanolamine; 3) serine is rapidly converted to other metabolites, which limits exogenous serine as a direct phospholipid precursor.     

Phospholipid (diacyl, alkylacyl, alkenylacyl) and fatty acyl chain composition in murine mastocytoma cells

The phospholipids from murine mastocytoma FMA3 and P-815 clone cells were quantitatively analyzed, and the major glycerophospholipids were examined for their fatty acyl chain distribution. In these cells, the content of histamine was less than 1/100 of normal mouse mast cells, and FMA3 cells had 1.5-fold as much histamine content as P-815 cells. The predominant phospholipid species of both mastocytoma FMA3 and P-815 were choline-containing glycerophospholipids (48%) and ethanolamine-containing glycerophospholipids (29%). The remaining minor constituents were sphingomyelin (6%, 7%), phosphatidylinositol (7%, 5%), phosphatidylserine (2%, 5%), cardiolipin (4%, 3%), and phosphatidic acid (2%, 1% for FMA3 and P-815, respectively). The choline-containing glycerophospholipids consisted of high amounts of 1-O-alkyl-2-acyl type (31%, 25%) and 1,2-diacyl type (63%, 66%) and a smaller amount of 1-O-alk-1'-enyl-2-acyl type (7%, 8%). In contrast, ethanolamine-containing glycerophospholipids were characterized by high contents of 1-O-alk-1'-enyl-2-acyl type (36%, 31%) and 1,2-diacyl type (55%, 58%), and a lower level of 1-O-alkyl-2-acyl type (12% and 11% for FMA3 and P-815, respectively). Unlike choline-containing glycerophospholipids and sphingomyelin that were rich in palmitic acid, ethanolamine-containing glycerophospholipids, phosphatidylserine and phosphatidylinositol showed a high proportion of stearic acid in the overall fatty acid composition. The content of arachidonic acid was highest in phosphatidylinositol. Sphingomyelin had a large amount of long chain and polyunsaturated fatty acids. In both choline- and ethanolamine-containing glycerophospholipids, the predominant fatty acids in the sn-1-position were palmitic, stearic, and oleic acid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential metabolism of diradyl glycerol molecular subclasses and molecular species by rabbit brain diglyceride kinase

Elevations in the mass of ether-linked diglycerides (i.e. 1-O-alk-1'-enyl-2-acyl-sn-glycerol (AAG) and 1-O-alkyl-2-acyl-sn-glycerol (Alkyl AG)) during cellular activation are prolonged in comparison to their 1,2-diacyl-sn-glycerol (DAG) counterparts. Since the metabolic removal of DAG is determined, in large part, by the rate of its phosphorylation by diglyceride kinase, we quantified differences in the activity of diglyceride kinase utilizing individual subclasses of diradyl glycerols as substrate. Rabbit brain microsomal diglyceride kinase activity was over 30-fold greater utilizing DAG as substrate (25.8 nmol.mg-1.min-1) in comparison to AAG (0.8 nmol.mg-1.min-1). No alterations in the affinity of microsomal diglyceride kinase for ATP were present (Km approximately 0.5 mM) utilizing each diradyl glycerol subclass. Similar subclass specificities for diglyceride kinase (i.e. DAG greater than Alkyl AG much greater than AAG) were present in brain and liver cytosol as well as in liver microsomes utilizing multiple assay conditions. In sharp contrast, Escherichia coli diglyceride kinase phosphorylated DAG, Alkyl AG, or AAG diradyl glycerol molecular subclasses at identical rates. Furthermore, although DAG was rapidly hydrolyzed by diglyceride lipase, catabolism of AAG or Alkyl AG by plasmalogenase, alkyl ether hydrolase, or diglyceride/monoglyceride lipase was undetectable. Collectively, these results demonstrate the importance of the differential catabolism of each diradyl glycerol molecular subclass as a primary determinant of their biologic half-lives. Since individual subclasses of diglycerides have distinct physical properties and physiologic functions, these results underscore the importance of lipid subclass specific metabolism in tailoring individual cellular responses during activation.     

Isolation of soluble proteins capable of stimulating aerobic plasmalogen biosynthesis

The terminal step during aerobic plasmalogen biosynthesis is catalyzed by a microsomal desaturase system which converts 1-O-alkyl-2-acyl-sn-glycerophosphoethanolamine to 1-O-alk-1'-enyl-2-acyl-sn-glycerophosphoethanolamine (ethanolamine plasmalogen). The reaction depends on oxygen and NAD(P)H and is stimulated 3-10-fold by soluble activating factors contained in the 100 000 X g supernatant. Two stimulating proteins have been isolated from pig kidney; the partially purified proteins have identical molecular weights (27 000) but differ in their respective isoelectric points (protein I, 5.1 and protein II, 4.9). Both proteins behave identically in the biochemical studies conducted. Exogenous substrate binds to the stimulating proteins; the transfer of ethanolamine, but not of choline phospholipids, from liposomes to microsomes is enhanced by the stimulating proteins. They stimulate plasmalogen synthesis from either exogenous or endogenous substrate (synthesized from alkylglycerophosphoethanolamine by microsomal transacylases). The stimulating proteins have no enzymatic activity themselves; it is suggested that they affect events within the membrane and function as specific mediators between the membrane-bound enzyme system and the lipophilic substrate.     

Turnover Rates of the Molecular Species of Ethanolamine Plasmalogen of Rat Brain

1,2-Diradyl-3-acetylglycerols prepared from 1-O-alk-1'-enyl-2-acylglycero-3-phosphoethanolamine (alkenylacyl-GPE, ethanolamine plasmalogen) and 1-alkyl-2-acylglycero-3-phosphoethanolamine (alkylacyl-GPE) of rat brain at 18 days of age were subfractionated into six species by AgNO3-impregnated TLC. The percent compositions of substractions were compared with that of 1,2-diacylglycero-3-phosphoethanolamine (diacyl-GPE). The incorporation rate of [1-3H]glycerol into each molecular species was also estimated to examine the turnover rate and selective synthesis of molecular species of ethanolamine phosphoglycerides (EPG). Among the molecular species of EPG, a major proportion contained polyunsaturated fatty chains, and the sum of tetraene-, pentaene-, and hexaene-containing species was greater than 65% in common with three classes of EPG. It was possible to calculate the turnover time, synthesis rate, and synthesis rate constant of ethanolamine plasmalogen in myelinating rat brain by the equation of Zilversmit et al. since the time-dependent change of specific activity and the distribution of molecular species indicated that each molecular species of alkenylacyl-GPE is synthesized from the corresponding species of alkylacyl-GPE. The observed turnover time of ethanolamine plasmalogen was about 5 h. The observed turnover times of the various molecular species were of the order: tetraene greater than or equal to hexaene greater than pentaene greater than or equal to monoene greater than or equal to diene. The synthesis rate constants of each molecular species, in the formation of alkenylacyl-GPE from alkylacyl-GPE, were of the order: hexaene greater than tetraene greater than pentaene greater than diene greater than or equal to monoene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functions and biosynthesis of plasmalogens in health and disease

Plasmalogens (1-O-alk-1'-enyl-2-acyl glycerophospholipids) constitute a special class of phospholipids characterized by the presence of a vinyl-ether bond at the sn-1 position. Although long considered as biological peculiarities, interest in this group of phospholipids has grown in recent years, thanks to the realization that plasmalogens are involved in different human diseases. In this review, we summarize the current state of knowledge with respect to the enzymatic synthesis of plasmalogens, the characteristic topology of the enzymes involved and the biological roles that have been assigned to plasmalogens.     

Calcium ion activation of rabbit liver alpha 1,2-mannosidase

Rabbit liver alpha 1,2-mannosidase is a calcium ion requiring enzyme involved in processing the asparagine-linked oligosaccharides of glycoproteins. Ca2+ activation occurs with an apparent Ka of 1.1 microM. The major effect of the metal ion activator is on Km rather than Vmax. The kinetic mechanism of the enzyme is that of an ordered equilibrium in which Ca2+ must bind before substrate and the metal ion cannot release once the substrate has added to the enzyme. Several other divalent cations including Co2+, Mn2+, and Zn2+ were competitive with Ca2+ and inhibited the enzyme. Significantly, Mg2+ had no effect on enzyme activity. 1-Deoxymannojirimycin and Tris, which inhibit glycoprotein processing in vivo, are inhibitors of the mannosidase competitive with substrate. The effect of Ca2+ on the affinity of the enzyme for substrate may be a determinant in regulation of enzyme activity in vivo.     

5'-Nucleotidase I from rabbit heart

5'-Nucleotidase I (N-I) from rabbit heart was purified to homogeneity. After ammonium sulfate precipitation, the purification involved chromatography on phosphocellulose, DEAE-Sepharose, AMP-agarose, and ADP-agarose. The pure enzyme has a specific activity of 318 mumol (mg of protein)-1 min-1. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate yields a subunit molecular weight of 40,000. N-I is activated by ADP but not by ATP, in contrast to the 5'-nucleotidase (N-II) purified by Itoh et al. (1986), which is activated by ATP and, less well, by ADP. N-I displays sigmoidal saturation kinetics in the absence of ADP and hyperbolic kinetics in the presence of ADP. Partially purified N-I was previously shown to prefer AMP over IMP as substrate (Truong et al., 1988); this has been confirmed for pure N-I. Comparison of AMP and ADP concentrations reported to occur in heart with the kinetic behavior of N-I implicates N-I as the enzyme responsible for producing adenosine under conditions leading to a rise in ADP and AMP, such as hypoxia or increased workload. N-I is not activated by the ADP analogue adenosine 5'-methylenediphosphonate (AOPCP) and is only weakly inhibited by relatively high concentrations of AOPCP, in contrast to 5'-nucleotidase from plasma membrane, which is powerfully inhibited by this analogue. N-I shows an absolute dependence on Mg2+ ions. Mn2+ and Co2+ ions can replace Mg2+ ions as activator; Ni2+ and Fe2+ are much less effective, while Ca2+, Ba2+, Zn2+, and Cu2+ fail to activate the enzyme.     

Stimulated neutrophils produce an ethanolamine plasmalogen analog of platelet-activating factor

Human neutrophils stimulated by ionophore A23187 incorporate [3H]acetate into platelet-activating factor and an additional product which is chromatographically similar to phosphatidylethanolamine and accounts for approximately 25% of the [3H]acetate-containing lipids. Three general approaches indicated the sn-1 moiety of the unknown phospholipid is primarily alk-1'-enyl-linked: 1) approximately 80% of the intact phospholipid as well as its derivatives was highly sensitive to hydrolysis by HCl, 2) 80% of the product which resulted from treating the unknown with phospholipase C and acetylating the free hydroxyl group at the sn-3 position, chromatographed with authentic 1-O-alk-1'-enyl-2,3-diacetylglycerol, and 3) catalytic hydrogenation of the diacetylglycerol product described in 2) resulted in a product which chromatographed with alkyldiacetylglycerol and was not sensitive to strong acid. Treatment of the intact phospholipid with phospholipase A2 resulted in the release of 88% of the radiolabel into the acidified aqueous phase of the extraction mixture, indicating the moiety in the sn-2 position remained as acetate and had not been elongated to fatty acid. The head group was determined to be phosphoethanolamine based upon its complete conversion to the dinitro- and trinitrophenyl derivatives by the amine-derivatizing reagents fluorodinitrobenzene and trinitrobenzenesulfonic acid, respectively. From these data is was concluded that the unknown product is 1-O-alk-1'-enyl-2-acetyl-sn-glycero-3-phosphoethanolamine (80%), and 1-O-alkyl-2-acetyl-sn-glycero-3-phosphoethanolamine (10%). Sonicates prepared from neutrophils stimulated with ionophore A23187 contained an acetyltransferase activity capable of utilizing 1-O-alk-1'-enyl-2-lyso-sn-glycero-3-phosphoethanolamine and [14C]acetyl-CoA to produce the product identified as 1-O-alk-1'-enyl-2-acetyl-sn-glycero-3-phosphoethanolamine.     

ENZYMATIC BIOSYNTHESIS OF ETHANOLAMINE- AND CHOLINE-PHOSPHOGLYCERIDES IN RETINA DURING DEVELOPMENT. EFFECT OF LIGHT STIMULATION

Choline- and ethanolamine-phosphoglycerides (CPG and EPG) are the most abundant phospholipids of retinal membranes. We have investigated some regulatory mechanisms involved in the final steps of their biosynthesis, namely those catalysed by CDP-choline 1,2 diradyl-sn-glycerol choline phosphotransferase (CPT) and CDP-ethanolamine 1,2 diradyl-sn-glycerol ethanolamine phosphotransferase (EPT). We have studied both enzymes in the retina which offers an excellent model for the investigation of the molecular basis of the effect of its physiological stimulus, the light. In chick retina. the specific activity (SA) of EPT reached a maximum at the 18th day of embryonic life and decreased thereafter. In the case of CPT, a similar peak of SA was observed at hatching. The time of maximum SA of EPT and CPT corresponded to the period during which retinal rod outer segments are formed. The apparent K_m values of EPT and CPT determined with whole retinal homogenates for CDP-bases showed different profiles. The apparent K_m of EPT decreased during embryonic life and increased thereafter whereas the apparent K_m of CPT did not change during ontogenesis. Light stimulation of calf retinal homogenates had different effects on phosphotransferase activities. In the presence of only endogenous diacylglycerol (DAG) the SA of CPT was 2-fold higher for dark-adapted retinas, whereas no differences in EPT activities were observed. After addition of exogenous DAG (4mM) to the incubation medium, light stimulation of the retina led to a 50% increase of EPT activity whereas no effect was observed for CPT. These different effects could be related to the cyclic nucleotides present in retina before and after light stimulation. In addition all the data presented in this study indicate that, as in brain, CPT and EPT in retina are two different enzymes.     

Effects of Ca2+ on ethanolaminephosphotransferase and cholinephosphotransferase in rabbit platelets

The effects of Ca2+ on ethanolaminephosphotransferase [EC 2.7.8.1] and cholinephosphotransferase [EC 2.7.8.2] activities in rabbit platelet membranes were studied using endogenous diglyceride and CDP-[3H]ethanolamine or CDP-[14C]choline as substrates. Both transferases required Mn2+, Co2+, or Mg2+ as a metal cofactor and the optimal concentrations of the metals for both activities were about 5, 10, and 5 mM, respectively. When 5 mM Mg2+ was used as a cofactor, both transferase activities were inhibited by a low concentration of Ca2+ (half maximal inhibition at approx. 15 microM). In the presence of 5 mM Mn2+, however, approx. 5 mM Ca2+ was required to produce half maximal inhibition. The Ca2+-induced inhibition was reversible and the rate of the inhibition was not affected either by the concentrations of the CDP-compound or by exogenously added diacylglycerol. The relationship between Ca2+ and both Mg2+ and Mn2+ on the transferase activities was competitive. 45Ca2+ binding (and/or uptake) to the platelet membranes was inhibited by Mn2+, Mg2+, and Co2+, in a concentration-dependent manner. However, the inhibitory effects of the three metal ions on the total Ca2+ binding (and/or uptake) did not correlate with the activation of both transferase activities by the three metal ions in the presence of Ca2+. These results suggest that both transferase activities are regulated by low concentrations of Ca2+ in the presence of optimal concentrations of Mg2+, and that the inhibition is mediated directly by Ca2+, which interacts with a specific metal cofactor binding site(s) of the transferases.     

Interaction of guanosine cyclic 3',5'-phosphate dependent protein kinase with lin-benzoadenine nucleotides

Using the activated cGMP-dependent protein kinase in the presence of the phosphorylatable peptide [[Ala34]histone H2B-(29-35)], we found that lin-benzoadenosine 5'-diphosphate (lin-benzo-ADP) was a competitive inhibitor of the enzyme with respect to ATP with a Ki (22 microM) similar to the Kd (20 microM) determined by fluorescence polarization titrations. The Kd for lin-benzo-ADP determined in the absence of the phosphorylatable peptide, however, was only 12 microM. ADP bound with lower affinity (Ki = 169 microM; Kd = 114 microM). With [Ala34]histone H2B-(29-35) as phosphoryl acceptor, the Km for lin-benzo-ATP was 29 microM, and that for ATP was 32 microM. The Vmax with lin-benzo-ATP, however, was only 0.06% of that with ATP as substrate [0.00623 +/- 0.00035 vs. 11.1 +/- 0.17 mumol (min.mg)-1]. Binding of lin-benzo-ADP to the kinase was dependent upon a divalent cation. Fluorescence polarization revealed that Mg2+, Mn2+, Co2+, Ni2+, Ca2+, Sr2+, and Ba2+ supported nucleotide binding to the enzyme; Ca2+, Sr2+, and Ba2+, however, did not support any measurable phosphotransferase activity. The rank order of metal ion effectiveness in mediating phosphotransferase activity was Mg2+ greater than Ni2+ greater than Co2+ greater than Mn2+. Although these results were similar to those observed with the cAMP-dependent protein kinase [Hartl, F. T., Roskoski, R., Jr., Rosendahl, M. S., & Leonard, N. J. (1983) Biochemistry 22, 2347], major differences in the Vmax with lin-benzo-ATP as substrate and the effect of peptide substrates on nucleotide (both lin-benzo-ADP and ADP) binding were observed.     

Purification and characterization of an alpha-1,2-mannosidase involved in processing asparagine-linked oligosaccharides

A calcium-dependent alpha-1,2-mannosidase involved in the processing of asparagine-linked oligosaccharides was purified to homogeneity from rabbit liver microsomes. N-terminal amino acid analysis was consistent with the presence of a homogeneous protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under both reducing and nonreducing conditions, revealed a single protein band with an apparent molecular weight of 52,000. Gel filtration and sedimentation analysis under nondenaturing conditions suggested that the purified enzyme is a monomeric protein. The mannosidase is a glycoprotein based on the presence of protein-linked sugar and specific binding of the enzyme to concanavalin A-Sepharose. Purified mannosidase was optimally active between pH 5.0 and 6.0. The enzyme was inactive with p-nitrophenyl-alpha-D-mannopyranoside and was inhibited by deoxymannojirimycin but not by swainsonine. The enzyme was specifically activated by Ca2+, with half-maximal activation occurring at concentrations of 10 microM or less and was inhibited by Mn2+, Co2+, Ba2+, and Zn2+. Calcium ions protected the enzyme against inactivation by p-chloromercuribenzoate. Rabbit liver mannosidase hydrolyzed alpha-1,2-mannosyl-mannose linkages in a variety of substrates including methyl-2-O-alpha-D-mannopyranosyl-alpha-D-mannopyranoside (Schutzbach, J. S. (1987) Anal. Biochem. 167, 279-283), ovalbumin glycopeptide IV, and the high mannose chains of thyroglobulin and phytohemagglutinin-P. Approximately 70% of the alpha-1,2-linked mannosyl units in the oligosaccharides of thyroglobulin were accessible to rabbit liver alpha-mannosidase, whereas most of the alpha-1,2-mannosyl units in phytohemagglutinin were resistant to digestion prior to heat denaturation of the plant lectin.     

Effect of a fish oil diet on the composition of rat neutrophil lipids and the molecular species of choline and ethanolamine glycerophospholipids

When rats were fed a corn oil versus a corn oil-fish oil diet the overall phospholipid content and composition as well as the subclass distribution of the choline- and ethanolamine-containing glycerophospholipids from neutrophils were not altered. The serine-containing glycerophospholipids were characterized by high levels of stearic and oleic acids. When fish oil was added to the diet it replaced some of the arachidonate in both the inositol- and the serine-containing glycerophospholipids. In the corn oil-fed animals, 25.2 and 33.6 mole %, respectively, of the molecular species of 1,2-diacyl- and 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine contained arachidonate. The values for 1,2-diacyl and 1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine were, respectively, 41 and 55.8 mole %. When half of the 5% corn oil in the diet was replaced by fish oil, there was a 53, 38, 27, and 25% reduction, respectively, in the level of arachidonate in these four lipid subclasses. The amount of 5,8,11,14,17-eicosapentaenoic acid incorporated into these four subclasses was always less than the decline in arachidonic acid. This was due, in part, to the acylation of small amounts of 22-carbon (n-3) acids into these lipids. Molecular species analysis demonstrated that 5,8,11,14,17-eicosapentaenoic acid paired with the same components at the sn-1 position, and in the same ratio, as did arachidonic acid. The amounts of 16- and 18-carbon saturated and unsaturated fatty acid at the sn-2 position were not altered by dietary change. Collectively, these findings suggest that 5,8,11,14,17-eicosapentaenoic and arachidonic acids are metabolized in a similar way by neutrophils. These studies also support the concept that neutrophils contain two metabolic pools of phospholipids. One pool is altered by dietary fat change while the pool containing 16- and 18-carbon acids is resistant to change when fish oil is included in the diet.     

Formation of complex ether lipids from 1-O-alkylglycerols in cell suspension cultures of rape

Photomixotrophic cell suspension cultures of rape, Brassica napus, were incubated with rac-1-O-[1′-¹⁴C]hexadecylglycerol. Radioactivity was incorporated predominantly into choline glycerophospholipids. Prolonged incubation led also to considerable proportions of labeled ethanolamine glycerophospholipids. In addition to these ionic lipids,isomeric hexadecylacylglycerols as well as hexadecyldiacylglycerols were formed. About a third of the hexadecylglycerol supplied as substrate was cleaved within 48 h incubation. The palmitic acid formed by oxidative cleavage of the substrate was incorporated predominantly into choline glycerophospholipids, ethanolamine glycerophospholipids, and triacylglycerols. Incubation of an equimolar mixture of homologous saturated rac-1-O-[1′¹⁴C]alkylglycerols (C₁₄, C₁₆, C₁₈, C₂₀) with rape cells showed that alkylglycerols with alkyl moieties having 16 and 18 carbon atoms were incorporated preferentially. Incubation of labeled hexadecyglycerol with a homogenate of rape cells led also predominantly to choline glycerophospholipids; highest yields were obtained at pH 7. Neither the 1-O-alkyl moieties in choline glycerophospholipis nor those in ethanolamine glycerophospholipids were desaturated to 1-O-alk-1′-enylmoieties. The results of these experiments led to the following conclusions: (1) The acylation of 1-O-alkylglycerols to isomeric alkylacylglycerols is catalyzed by two acyltransferases differing in their specificity with regard to the chain length of the alkyl moiety in the substrate. (2) CDP-Choline: diacylglycerol cholinephosphotransferase and CDP-ethanolamine: diacylglycerol ethanolaminephosphotransferase are two enzymes differing in various respects. Cholinephosphotransferase exhibits a much higher affinity for 1-O-alkyl-2-O-acylglycerols than ethanolaminephosphotransferase. The two enzymes show marked differences with regard to their specificity for 1-O-alkyl-2-O-acylglycerols differing in the chain lengths of their alkyl moieties.