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High-fat (HF) diet–induced obesity precipitates multiple metabolic disorders including insulin resistance, glucose intolerance, oxidative stress, and inflammation, resulting in the initiation of cell death programs. Previously, we demonstrated murine germline knockout of calcium-independent phospholipase A2γ (iPLA2γ) prevented HF diet–induced weight gain, attenuated insulin resistance, and decreased mitochondrial permeability transition pore (mPTP) opening leading to alterations in bioenergetics. To gain insight into the specific roles of hepatic iPLA2γ in mitochondrial function and cell death under metabolic stress, we generated a hepatocyte-specific iPLA2γ-knockout (HEPiPLA2γKO). Using this model, we compared the effects of an HF diet on wild-type versus HEPiPLA2γKO mice in eicosanoid production and mitochondrial bioenergetics. HEPiPLA2γKO mice exhibited higher glucose clearance rates than WT controls. Importantly, HF-diet induced the accumulation of 12-hydroxyeicosatetraenoic acid (12-HETE) in WT liver which was decreased in HEPiPLA2γKO. Furthermore, HF-feeding markedly increased Ca2+ sensitivity and resistance to ADP-mediated inhibition of mPTP opening in WT mice. In contrast, ablation of iPLA2γ prevented the HF-induced hypersensitivity of mPTP opening to calcium and maintained ADP-mediated resistance to mPTP opening. Respirometry revealed that ADP-stimulated mitochondrial respiration was significantly reduced by exogenous 12-HETE. Finally, HEPiPLA2γKO hepatocytes were resistant to calcium ionophore-induced lipoxygenase-mediated lactate dehydrogenase release. Collectively, these results demonstrate that an HF diet increases iPLA2γ-mediated hepatic 12-HETE production leading to mitochondrial dysfunction and hepatic cell death.

Recently, eicosanoid-lysophospholipids were identified as novel metabolites generated from the direct cyclooxygenase- or lipoxygenase-catalyzed oxidation of 2-arachidonoyl-lysophospholipids produced from either phospholipase A(1)-mediated hydrolysis of diacyl arachidonoyl-phospholipids or through the cytochrome c-catalyzed oxidative hydrolysis of the vinyl ether linkage of arachidonoyl-plasmalogens. Although the metabolic pathways generating eicosanoid-lysophospholipids have been increasingly appreciated, the signaling functions of eicosanoid-lysophospholipids remain largely unknown. Herein, we demonstrate that 2-12(S)-HETE-lysophospholipids as well as nonesterified 12(S)-HETE are potent lipid mediators that activate THP-1 human monocytic cells to generate tumor necrosis factor α (TNFα) and interleukin 8 (IL8). Remarkably, low nanomolar concentrations of 12(S)-HETE-lysophospholipids, but not other oxidized signaling lipids examined activated THP-1 cells resulting in the production of large amounts of TNFα. Moreover, TNFα release induced by 12(S)-HETE-lysophospholipids was inhibited by the TNFα converting enzyme inhibitor TAPI-0 indicating normal processing of TNFα in THP-1 cells stimulated with these agonists. Western blotting analyses revealed that 12(S)-HETE-lysophospholipids activated the phosphorylation of NFκB p65, suggesting activation of the canonical NFκB signaling pathway. Importantly, activation of THP-1 cells to release TNFα was stereoselective with 12(S)-HETE favored over 12(R)-HETE. Furthermore, the EC(50) of 2-12(S)-HETE-lysophosphatidylcholine in activating THP-1 cells was 2.1 nm, whereas the EC(50) of free 12(S)-HETE was 23 nm. Additionally, lipid extracts of activated platelets were separated by RP-HPLC demonstrating the coelution of 12(S)-HETE with fractions initiating TNFα release. Collectively, these results demonstrate the potent signaling properties of 2-12(S)-HETE-lysophospholipids and 12(S)-HETE by their ability to release TNFα and activate NFκB signaling thereby revealing a previously unknown role of 2-12(S)-HETE-lysophospholipids in mediating inflammatory responses.

The canonical pathway of eicosanoid production in most mammalian cells is initiated by phospholipase A(2)-mediated release of arachidonic acid, followed by its enzymatic oxidation resulting in a vast array of eicosanoid products. However, recent work has demonstrated that the major phospholipase in mitochondria, iPLA(2)γ (patatin-like phospholipase domain containing 8 (PNPLA8)), possesses sn-1 specificity, with polyunsaturated fatty acids at the sn-2 position generating polyunsaturated sn-2-acyl lysophospholipids. Through strategic chemical derivatization, chiral chromatographic separation, and multistage tandem MS, here we first demonstrate that human platelet-type 12-lipoxygenase (12-LOX) can directly catalyze the regioselective and stereospecific oxidation of 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC) and 2-arachidonoyl-lysophosphatidylethanolamine (2-AA-LPE). Next, we identified these two eicosanoid-lysophospholipids in murine myocardium and in isolated platelets. Moreover, we observed robust increases in 2-AA-LPC, 2-AA-LPE, and their downstream 12-LOX oxidation products, 12(S)-HETE-LPC and 12(S)-HETE-LPE, in calcium ionophore (A23187)-stimulated murine platelets. Mechanistically, genetic ablation of iPLA(2)γ markedly decreased the calcium-stimulated production of 2-AA-LPC, 2-AA-LPE, and 12-HETE-lysophospholipids in mouse platelets. Importantly, a potent and selective 12-LOX inhibitor, ML355, significantly inhibited the production of 12-HETE-LPC and 12-HETE-LPE in activated platelets. Furthermore, we found that aging is accompanied by significant changes in 12-HETE-LPC in murine serum that were also markedly attenuated by iPLA(2)γ genetic ablation. Collectively, these results identify previously unknown iPLA(2)γ-initiated signaling pathways mediated by direct 12-LOX oxidation of 2-AA-LPC and 2-AA-LPE. This oxidation generates previously unrecognized eicosanoid-lysophospholipids that may serve as biomarkers for age-related diseases and could potentially be used as targets in therapeutic interventions.

Cardiolipin (CL) is a dimeric phospholipid with critical roles in mitochondrial bioenergetics and signaling. Recently, inhibition of the release of oxidized fatty acyl chains from CL by the calcium-independent phospholipase A2γ (iPLA2γ)-selective inhibitor (R)-BEL suggested that iPLA2γ is responsible for the hydrolysis of oxidized CL and subsequent signaling mediated by the released oxidized fatty acids. However, chemical inhibition by BEL is subject to off-target pharmacologic effects. Accordingly, to unambiguously determine the role of iPLA2γ in the hydrolysis of oxidized CL, we compared alterations in oxidized CLs and the release of oxidized aliphatic chains from CL in experiments with purified recombinant iPLA2γ, germ-line iPLA2γ-/- mice, cardiac myocyte-specific iPLA2γ transgenic mice, and wild-type mice. Using charge-switch high mass accuracy LC-MS/MS with selected reaction monitoring and product ion accurate masses, we demonstrated that iPLA2γ is the major enzyme responsible for the release of oxidized aliphatic chains from CL. Our results also indicated that iPLA2γ selectively hydrolyzes 9-hydroxy-octadecenoic acid in comparison to 13-hydroxy-octadecenoic acid from oxidized CLs. Moreover, oxidative stress (ADP, NADPH, and Fe3+) resulted in the robust production of oxidized CLs in intact mitochondria from iPLA2γ-/- mice. In sharp contrast, oxidized CLs were readily hydrolyzed in mitochondria from wild-type mice during oxidative stress. Finally, we demonstrated that CL activates the iPLA2γ-mediated hydrolysis of arachidonic acid from phosphatidylcholine, thereby integrating the production of lipid messengers from different lipid classes in mitochondria. Collectively, these results demonstrate the integrated roles of CL and iPLA2γ in lipid second-messenger production and mitochondrial bioenergetics during oxidative stress.

Calcium-independent phospholipase A2γ (iPLA2γ) is a mitochondrial enzyme that produces lipid second messengers that facilitate opening of the mitochondrial permeability transition pore (mPTP) and contribute to the production of oxidized fatty acids in myocardium. To specifically identify the roles of iPLA2γ in cardiac myocytes, we generated cardiac myocyte-specific iPLA2γ knock-out (CMiPLA2γKO) mice by removing the exon encoding the active site serine (Ser-477). Hearts of CMiPLA2γKO mice exhibited normal hemodynamic function, glycerophospholipid molecular species composition, and normal rates of mitochondrial respiration and ATP production. In contrast, CMiPLA2γKO mice demonstrated attenuated Ca(2+)-induced mPTP opening that could be rapidly restored by the addition of palmitate and substantially reduced production of oxidized polyunsaturated fatty acids (PUFAs). Furthermore, myocardial ischemia/reperfusion (I/R) in CMiPLA2γKO mice (30 min of ischemia followed by 30 min of reperfusion in vivo) dramatically decreased oxidized fatty acid production in the ischemic border zones. Moreover, CMiPLA2γKO mice subjected to 30 min of ischemia followed by 24 h of reperfusion in vivo developed substantially less cardiac necrosis in the area-at-risk in comparison with their WT littermates. Furthermore, we found that membrane depolarization in murine heart mitochondria was sensitized to Ca(2+) by the presence of oxidized PUFAs. Because mitochondrial membrane depolarization and calcium are known to activate iPLA2γ, these results are consistent with salvage of myocardium after I/R by iPLA2γ loss of function through decreasing mPTP opening, diminishing production of proinflammatory oxidized fatty acids, and attenuating the deleterious effects of abrupt increases in calcium ion on membrane potential during reperfusion.

A highly sensitive, specific, and robust method for the analysis of oxidized metabolites of linoleic acid (LA), arachidonic acid (AA), and docosahexaenoic acid (DHA) was developed using charge-switch derivatization, liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI MS/MS) with selected reaction monitoring (SRM) and quantitation by high mass accuracy analysis of product ions, thereby minimizing interferences from contaminating ions. Charge-switch derivatization of LA, AA, and DHA metabolites with N-(4-aminomethylphenyl)-pyridinium resulted in a 10- to 30-fold increase in ionization efficiency. Improved quantitation was accompanied by decreased false positive interferences through accurate mass measurements of diagnostic product ions during SRM transitions by ratiometric comparisons with stable isotope internal standards. The limits of quantitation were between 0.05 and 6.0pg, with a dynamic range of 3 to 4 orders of magnitude (correlation coefficient r(2)>0.99). This approach was used to quantitate the levels of representative fatty acid metabolites from wild-type (WT) and iPLA2γ(-/-) mouse liver identifying the role of iPLA2γ in hepatic lipid second messenger production. Collectively, these results demonstrate the utility of high mass accuracy product ion analysis in conjunction with charge-switch derivatization for the highly specific quantitation of diminutive amounts of LA, AA, and DHA metabolites in biologic systems.

Herein, we demonstrate that calcium-independent phospholipase A(2)γ (iPLA(2)γ) is a critical mechanistic participant in the calcium-induced opening of the mitochondrial permeability transition pore (mPTP). Liver mitochondria from iPLA(2)γ(-/-) mice were markedly resistant to calcium-induced swelling in the presence or absence of phosphate in comparison with wild-type littermates. Furthermore, the iPLA(2)γ enantioselective inhibitor (R)-(E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one ((R)-BEL) was markedly more potent than (S)-BEL in inhibiting mPTP opening in mitochondria from wild-type liver in comparison with hepatic mitochondria from iPLA(2)γ(-/-) mice. Intriguingly, low micromolar concentrations of long chain fatty acyl-CoAs and the non-hydrolyzable thioether analog of palmitoyl-CoA markedly accelerated Ca(2+)-induced mPTP opening in liver mitochondria from wild-type mice. The addition of l-carnitine enabled the metabolic channeling of acyl-CoA through carnitine palmitoyltransferases (CPT-1/2) and attenuated the palmitoyl-CoA-mediated amplification of calcium-induced mPTP opening. In contrast, mitochondria from iPLA(2)γ(-/-) mice were insensitive to fatty acyl-CoA-mediated augmentation of calcium-induced mPTP opening. Moreover, mitochondria from iPLA(2)γ(-/-) mouse liver were resistant to Ca(2+)/t-butyl hydroperoxide-induced mPTP opening in comparison with wild-type littermates. In support of these findings, cytochrome c release from iPLA(2)γ(-/-) mitochondria was dramatically decreased in response to calcium in the presence or absence of either t-butyl hydroperoxide or phenylarsine oxide in comparison with wild-type littermates. Collectively, these results identify iPLA(2)γ as an important mechanistic component of the mPTP, define its downstream products as potent regulators of mPTP opening, and demonstrate the integrated roles of mitochondrial bioenergetics and lipidomic flux in modulating mPTP opening promoting the activation of necrotic and necroapoptotic pathways of cell death.

Background: Maintenance of cardiolipin molecular speciation by remodeling directly regulates mitochondrial bioenergetic efficiency. Results: Transgenic expression of cardiolipin synthase accelerates cardiolipin remodeling, improves mitochondrial function, modulates mitochondrial signaling, and attenuates mitochondrial dysfunction during diabetes. Conclusion: Cardiolipin synthase integrates multiple aspects of mitochondrial bioenergetic and signaling functions. Significance: Cardiolipin synthase expression attenuates mitochondrial dysfunction in diabetic myocardium.

The enzyme phosphofructokinase-1 (PFK-1) catalyzes the first committed step of glycolysis and is regulated by a complex array of allosteric effectors that integrate glycolytic flux with cellular bioenergetics. Here, we demonstrate the direct, potent, and reversible inhibition of purified rabbit muscle PFK-1 by low micromolar concentrations of long chain fatty acyl-CoAs (apparent Ki ∼1 μm). In sharp contrast, short chain acyl-CoAs, palmitoylcarnitine, and palmitic acid in the presence of CoASH were without effect. Remarkably, MgAMP and MgADP but not MgATP protected PFK-1 against inhibition by palmitoyl-CoA indicating that acyl-CoAs regulate PFK-1 activity in concert with cellular high energy phosphate status. Furthermore, incubation of PFK-1 with [1-14C]palmitoyl-CoA resulted in robust acylation of the enzyme that was reversible by incubation with acyl-protein thioesterase-1 (APT1). Importantly, APT1 reversed palmitoyl-CoA-mediated inhibition of PFK-1 activity. Mass spectrometric analyses of palmitoylated PFK-1 revealed four sites of acylation, including Cys-114, Cys-170, Cys-351, and Cys-577. PFK-1 in both skeletal muscle extracts and in purified form was inhibited by S-hexadecyl-CoA, a nonhydrolyzable palmitoyl-CoA analog, demonstrating that covalent acylation of PFK-1 was not required for inhibition. Tryptic footprinting suggested that S-hexadecyl-CoA induced a conformational change in PFK-1. Both palmitoyl-CoA and S-hexadecyl-CoA increased the association of PFK-1 with Ca2+/calmodulin, which attenuated the binding of palmitoylated PFK-1 to membrane vesicles. Collectively, these results demonstrate that fatty acyl-CoA modulates phosphofructokinase activity through both covalent and noncovalent interactions to regulate glycolytic flux and enzyme membrane localization via the branch point metabolic node that mediates lipid flux through anabolic and catabolic pathways.

Phospholipases are critical enzyme mediators participating in many aspects of cellular function through modulating the generation of lipid 2nd messengers, membrane physical properties, and cellular bioenergetics. Here, we demonstrate that mice null for calcium-independent phospholipase A(2)γ (iPLA(2)γ(-/-)) are completely resistant to high fat diet-induced weight gain, adipocyte hypertrophy, hyperinsulinemia, and insulin resistance, which occur in iPLA(2)γ(+/+) mice after high fat feeding. Notably, iPLA(2)γ(-/-) mice were lean, demonstrated abdominal lipodystrophy, and remained insulin-sensitive despite having a marked impairment in glucose-stimulated insulin secretion after high fat feeding. Respirometry of adipocyte explants from iPLA(2)γ(-/-) mice identified increased rates of oxidation of multiple different substrates in comparison with adipocyte explants from wild-type littermates. Shotgun lipidomics of adipose tissue from wild-type mice demonstrated the anticipated 2-fold increase in triglyceride content after high fat feeding. In sharp contrast, the adipocyte triglyceride content was identical in iPLA(2)γ(-/-) mice fed either a standard diet or a high fat diet. Respirometry of skeletal muscle mitochondria from iPLA(2)γ(-/-) mice demonstrated marked decreases in state 3 respiration using multiple substrates whose metabolism was uncoupled from ATP production. Shotgun lipidomics of skeletal muscle revealed a decreased content of cardiolipin with an altered molecular species composition thereby identifying the mechanism underlying mitochondrial uncoupling in the iPLA(2)γ(-/-) mouse. Collectively, these results identify iPLA(2)γ as an obligatory upstream enzyme that is necessary for efficient electron transport chain coupling and energy production through its participation in the alterations of cellular bioenergetics that promote the development of the metabolic syndrome.

Herein, we report the heterologous expression of the human peroxisomal 63-kDa calcium-independent phospholipase A2γ (iPLA2γ) isoform in Sf9 cells, purification of the N-terminal His-tagged enzyme by affinity chromatography, and the identification of its remarkable substrate selectivity that results in the highly selective generation of 2-arachidonoyl lysophosphatidylcholine. Mass spectrometric analyses demonstrated that purified iPLA2γ hydrolyzed saturated or monounsaturated aliphatic groups readily from either the sn-1 or sn-2 positions of phospholipids. In addition, purified iPLA2γ effectively liberated arachidonic acid from the sn-2 position of plasmenylcholine substrates. In contrast, incubation of iPLA2γ with 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine resulted in the rapid release of palmitic acid and the selective accumulation of 2-arachidonoyl lysophosphatidylcholine (LPC), which was not metabolized further by iPLA2γ. The putative regiospecificity of the 2-arachidonoyl LPC product was authenticated by its diagnostic fragmentation pattern during tandem mass spectrometric analysis. To identify the physiological relevance of iPLA2γ-mediated 2-arachidonoyl LPC production utilizing naturally occurring membranes, we incubated purified rat hepatic peroxisomes with iPLA2γ and similarly identified the selective accumulation of 2-arachidonoyl LPC. Furthermore, tandem mass spectrometric analysis demonstrated that 2-arachidonoyl LPC is a natural product in human myocardium, a tissue in which iPLA2γ expression is robust. Because 2-arachidonoyl LPC represents a key branch point intermediate that can potentially lead to a variety of bioactive molecules in eicosanoid signaling (e.g. arachidonic acid, 2-arachidonoylglycerol), these results have uncovered a novel eicosanoid selective pathway through iPLA2γ-mediated 2-arachidonoyl LPC production to amplify and diversify the repertoire of biologic lipid second messengers in response to cellular stimulation.

Membrane-associated calcium-independent phospholipase A2gamma (iPLA2gamma) contains four potential in-frame methionine start sites (Mancuso, D.J. Jenkins, C.M. & Gross, R.W. (2000) J. Biol. Chem.275, 9937-9945), but the mechanisms regulating the types, amount and subcellular localization of iPLA2gamma in cells are incompletely understood. We now: (a) demonstrate the dramatic transcriptional repression of mRNA synthesis encoding iPLA2gamma by a nucleotide sequence nested in the coding sequence itself; (b) localize the site of transcriptional repression to the most 5' sequence encoding the iPLA2gamma holoprotein; (c) identify the presence of nuclear protein constituents which bind to the repressor region by gel shift analysis; (d) demonstrate the translational regulation of distinct iPLA2gamma isoforms; (e) identify multiple novel exons, promoters, and alternative splice variants of human iPLA2gamma; (f) document the presence of dual-competing subcellular localization signals in discrete isoforms of iPLA2gamma; and (g) demonstrate the functional integrity of an N-terminal mitochondrial localization signal by fluorescence imaging and the presence of iPLA2gamma in the mitochondrial compartment of rat myocardium. The intricacy of the regulatory mechanisms of iPLA2gamma biosynthesis in rat myocardium is underscored by the identification of seven distinct protein products that utilize multiple mechanisms (transcription, translation and proteolysis) to produce discrete iPLA2gamma polypeptides containing either single or dual subcellular localization signals. This unanticipated complex interplay between peroxisomes and mitochondria mediated by competition for uptake of the nascent iPLA2gamma polypeptide identifies a new level of phospholipase-mediated metabolic regulation. Because uncoupling protein function is regulated by free fatty acids in mitochondria, these results suggest that iPLA2gamma processing contributes to integrating respiration and thermogenesis in mitochondria.

Barth syndrome is a complex metabolic disorder caused by mutations in the mitochondrial transacylase tafazzin. Recently, an inducible tafazzin shRNA knockdown mouse model was generated to deconvolute the complex bioenergetic phenotype of this disease. To investigate the underlying cause of hemodynamic dysfunction in Barth syndrome, we interrogated the cardiac structural and signaling lipidome of this mouse model as well as its myocardial bioenergetic phenotype. A decrease in the distribution of cardiolipin molecular species and robust increases in monolysocardiolipin and dilysocardiolipin were demonstrated. Additionally, the contents of choline and ethanolamine glycerophospholipid molecular species containing precursors for lipid signaling at the sn-2 position were altered. Lipidomic analyses revealed specific dysregulation of HETEs and prostanoids, as well as oxidized linoleic and docosahexaenoic metabolites. Bioenergetic interrogation uncovered differential substrate utilization as well as decreases in Complex III and V activities. Transgenic expression of cardiolipin synthase or iPLA2γ ablation in tafazzin-deficient mice did not rescue the observed phenotype. These results underscore the complex nature of alterations in cardiolipin metabolism mediated by tafazzin loss of function. Collectively, we identified specific lipidomic, bioenergetic, and signaling alterations in a murine model that parallel those of Barth syndrome thereby providing novel insights into the pathophysiology of this debilitating disease.

We report the cDNA sequences of rat colipase, rat pancreatic lipase (rPL), and a rat pancreatic lipase-related protein (rPLRP). Comparison to the human PLRP cDNA suggests that the isolated clone encodes rPLRP-2. Both cDNA and a third cDNA encoding rPLRP-1 are secreted from Sf9 cells infected with recombinant baculovirus. rPL and rPLRP-2 hydrolyze triolein, 8.0 and 4.4 mumol.min-1.microgram-1, respectively. They are inhibited by bile salts, and activity is restored by (pro)colipase. PLRP-1 has barely detectable activity against triolein, even with (pro)colipase present. The pattern of mRNA expression during development in the rat reveals that all mRNA are low in the fetal rat pancreas. Both PLRP mRNA rise just before birth to a maximum 12 h after birth. They fall to low levels in the adult. In contrast, the PL mRNA is low at birth and rises rapidly during the suckling-weanling transition. In conclusion, the rat has at least three genes encoding different lipases, and these related genes have separate regulatory controls.

The endothelium comprises a cellular barrier between the circulation and tissues. We have previously shown that activation of protease-activated receptor 1 (PAR-1) and PAR-2 on the surface of human coronary artery endothelial cells by tryptase or thrombin increases group VIA phospholipase A2(iPLA2β) activity and results in production of multiple phospholipid-derived inflammatory metabolites. We isolated cardiac endothelial cells from hearts of iPLA2β-knockout (iPLA2β-KO) and wild-type (WT) mice and measured arachidonic acid (AA), prostaglandin I2(PGI2), and platelet-activating factor (PAF) production in response to PAR stimulation. Thrombin (0.1 IU/ml) or tryptase (20 ng/ml) stimulation of WT endothelial cells rapidly increased AA and PGI2release and increased PAF production. Selective inhibition of iPLA2β with ( S)-bromoenol lactone (5 μM, 10 min) completely inhibited thrombin- and tryptase-stimulated responses. Thrombin or tryptase stimulation of iPLA2β-KO endothelial cells did not result in significant PAF production and inhibited AA and PGI2release. Stimulation of cardiac endothelial cells from group VIB (iPLA2γ)-KO mice increased PAF production to levels similar to those of WT cells but significantly attenuated PGI2release. These results indicate that cardiac endothelial cell PAF production is dependent on iPLA2β activation and that both iPLA2β and iPLA2γ may be involved in PGI2release.

The digestion of dietary triglycerides occurs in the duodenum through the action of triglyceride lipase, a pancreatic exocrine protein. The activity of pancreatic lipase is inhibited by the bile salts normally found in the gut lumen. Another pancreatic exocrine protein, colipase, restores the lipolytic activity of triglyceride lipase. The synthesis and secretion of both triglyceride lipase and colipase is increased by dietary fats and secretin. An increase in mRNA accompanies the increased activity, suggesting that the genes for triglyceride lipase and colipase contain nucleotide elements responsive to dietary fats or secretin or both. To study the regulation of colipase expression, we have first isolated the gene for human colipase from a cosmid library with a cDNA probe. The gene was localized to chromosome 6 and is organized into three exons contained in a single 3.3-kb BamHI fragment. The 5'-flanking region of the gene contains a TATA box, a GC box, and a 28-bp region with homology to the rat pancreatic-specific enhancer. This region directs the tissue-specific expression of the chloramphenicol acetyltransferase gene in a transfected rat pancreatic acinar cell line, AR42-J. The same construct is inactive in HEPG2, C2C12, and COS-1 cells. These results demonstrate that the isolated gene for human colipase contains tissue-specific promoter activity in the 5'-flanking DNA. The 28-bp region specifically binds to a factor in nuclear extracts.

Genetic ablation of calcium-independent phospholipase A(2)gamma (iPLA(2)gamma) results in profound alterations in hippocampal phospholipid metabolism and mitochondrial phospholipid homeostasis resulting in enlarged and degenerating mitochondria leading to autophagy and cognitive dysfunction. Shotgun lipidomics demonstrated multiple alterations in hippocampal lipid metabolism in iPLA(2)gamma(-/-) mice including: 1) a markedly elevated hippocampal cardiolipin content with an altered molecular species composition characterized by a shift to shorter chain length molecular species; 2) alterations in both choline and ethanolamine glycerophospholipids, including a decreased plasmenylethanolamine content; 3) increased oxidized phosphatidylethanolamine molecular species; and 4) an increased content of ceramides. Electron microscopic examination demonstrated the presence of enlarged heteromorphic lamellar structures undergoing degeneration accompanied by the presence of ubiquitin positive spheroid inclusion bodies. Purification of these enlarged heteromorphic lamellar structures by buoyant density centrifugation and subsequent SDS-PAGE and proteomics identified them as degenerating mitochondria. Collectively, these results identify the obligatory role of iPLA(2)gamma in neuronal mitochondrial lipid metabolism and membrane structure demonstrating that iPLA(2)gamma loss of function results in a mitochondrial neurodegenerative disorder characterized by degenerating mitochondria, autophagy, and cognitive dysfunction.

Previously, we identified a novel calcium-independent phospholipase, designated calcium-independent phospholipase A(2) gamma (iPLA(2)gamma), which possesses dual mitochondrial and peroxisomal subcellular localization signals. To identify the roles of iPLA(2)gamma in cellular bioenergetics, we generated mice null for the iPLA(2)gamma gene by eliminating the active site of the enzyme through homologous recombination. Mice null for iPLA(2)gamma display multiple bioenergetic dysfunctional phenotypes, including 1) growth retardation, 2) cold intolerance, 3) reduced exercise endurance, 4) greatly increased mortality from cardiac stress after transverse aortic constriction, 5) abnormal mitochondrial function with a 65% decrease in ascorbate-induced Complex IV-mediated oxygen consumption, and 6) a reduction in myocardial cardiolipin content accompanied by an altered cardiolipin molecular species composition. We conclude that iPLA(2)gamma is essential for maintaining efficient bioenergetic mitochondrial function through tailoring mitochondrial membrane lipid metabolism and composition.

Previously, we identified calcium-independent phospholipase A2gamma (iPLA2gamma) with multiple translation initiation sites and dual mitochondrial and peroxisomal localization motifs. To determine the role of iPLA2gamma in integrating lipid and energy metabolism, we generated transgenic mice containing the alpha-myosin heavy chain promoter (alphaMHC) placed proximally to the human iPLA2gamma coding sequence that resulted in cardiac myocyte-restricted expression of iPLA2gamma (TGiPLA2gamma). TGiPLA2gamma mice possessed multiple phenotypes including: 1) a dramatic approximately 35% reduction in myocardial phospholipid mass in both the fed and mildly fasted states; 2) a marked accumulation of triglycerides during brief caloric restriction that represented 50% of total myocardial lipid mass; and 3) acute fasting-induced hemodynamic dysfunction. Biochemical characterization of the TGiPLA2gamma protein expressed in cardiac myocytes demonstrated over 25 distinct isoforms by two-dimensional SDS-PAGE Western analysis. Immunohistochemistry identified iPLA2gamma in the peroxisomal and mitochondrial compartments in both wild type and transgenic myocardium. Electron microscopy revealed the presence of loosely packed and disorganized mitochondrial cristae in TGiPLA2gamma mice that were accompanied by defects in mitochondrial function. Moreover, markedly elevated levels of 1-hydroxyl-2-arachidonoyl-sn-glycero-3-phosphocholine and 1-hydroxyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine were prominent in the TGiPLA2gamma myocardium identifying the production of signaling metabolites by this enzyme in vivo. Collectively, these results identified the participation of iPLA2gamma in the remarkable lipid plasticity of myocardium, its role in generating signaling metabolites, and its prominent effects in modulating energy storage and utilization in myocardium in different metabolic contexts.

Genetic knockout of hormone-sensitive lipase in mice has implicated the presence of other intracellular triacylglycerol (TAG) lipases mediating TAG hydrolysis in adipocytes. Despite intense interest in these TAG lipases, their molecular identities thus far are largely unknown. Sequence data base searches for proteins containing calcium-independent phospholipase A2 (iPLA2) dual signature nucleotide ((G/A)XGXXG) and lipase (GXSXG) consensus sequence motifs identified a novel subfamily of three putative iPLA2/lipase family members designated iPLA2epsilon, iPLA2zeta, and iPLA2eta (previously named adiponutrin, TTS-2.2, and GS2, respectively) of previously unknown catalytic function. Herein we describe the cloning, heterologous expression, and affinity purification of the three human isoforms of this iPLA2 subfamily in Sf9 cells, and we demonstrate that each possesses abundant TAG lipase activity. Moreover, iPLA2epsilon, iPLA2zeta, and iPLA2eta also possess acylglycerol transacylase activity utilizing mono-olein as an acyl donor which, in the presence of mono-olein or diolein acceptors, results in the synthesis of diolein and triolein, respectively. (E)-6-(Bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one, a mechanism-based suicide substrate inhibitor of all known iPLA2s, inhibits the triglyceride lipase activity of each of the three isoforms similarly (IC50=0.1-0.5 microm). Quantitative PCR revealed dramatically increased expression of iPLA2epsilon and iPLA2zeta transcripts during the hormone-induced differentiation of 3T3-L1 cells into adipocytes and identified the presence of all three iPLA2 isoforms in human SW872 liposarcoma cells. Collectively, these results identify three novel TAG lipases/acylglycerol transacylases that likely participate in TAG hydrolysis and the acyl-CoA independent transacylation of acylglycerols, thereby facilitating energy mobilization and storage in adipocytes.

Cytosolic phospholipase A(2)gamma (cPLA(2)gamma) is a calcium-independent, membrane-associated phospholipase A(2) that possesses a C-terminal prenylation motif (-CCLA) whose covalent structure cannot be deduced from the primary sequence alone. Accordingly, we overexpressed human cPLA(2)gamma containing an N-terminal His tag ((His)(6)cPLA(2)gamma) in Sf9 cells and quantitatively solubilized and purified the enzyme by sequential immobilized metal affinity and Mono Q column chromatographies. The final preparation appeared as a single 61 kDa band after SDS-PAGE/silver-staining, possessed high lysophospholipase activity (50 micromol min(-1) mg(-1)), and was inhibited by, but did not hydrolyze, palmitoyl-CoA. Radiolabeling of recombinant human cPLA(2)gamma with [(3)H]-mevalonolactone in the absence of statins and subsequent cleavage of prenyl groups with Raney nickel revealed that the enzyme is only farnesylated and is not geranylgeranylated. Analysis of CNBr-digested cPLA(2)gamma by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI/TOF-TOF) mass spectrometry demonstrated the presence of a farnesyl moiety at Cys-538, cleavage of the Cys(538)-Cys(539) bond, and carboxymethylation of the resultant C-terminal prenylated cysteine. Collectively, these results describe the solubilization and purification of recombinant cPLA(2)gamma to homogeneity and identify cPLA(2)gamma as a farnesylated protein that undergoes at least three sequential posttranslational modifications that likely facilitate its targeting and interactions with its membrane substrates.

Very-long-chain acyl-CoA dehydrogenase (VLCAD) is one of a family of nuclear-encoded enzymes that catalyze the initial step in mitochondrial fatty acid β-oxidation (FAO). Previous studies have indicated that two other members of the AD gene family (medium-chain AD and long-chain AD) are controlled at the transcriptional level by nuclear hormone receptors. In this study, we have cloned and characterized the human VLCAD gene promoter region to identify cis -acting elements involved in its transcriptional control. VLCAD gene promoter-luciferase reporter (VLCAD-Luc) constructs were found to be transcriptionally active in a variety of mammalian cell lines and in primary rat cardiomyocytes when driven by varying lengths of the VLCAD promoter region. Removal of a 20-bp DNA segment of the proximal VLCAD gene promoter markedly reduced the transcriptional activity of VLCAD-Luc constructs. Gel mobility shift assays identified a DNA-binding activity in nuclear extracts prepared from human hepatoma G2 cells that interacted with the 20-bp regulatory region. Competition studies revealed that this DNA-binding activity could be abolished by a molar excess of unlabeled specific oligonucleotide as well as a DNA fragment containing an activator protein 2 (AP-2)-binding site but not by an unrelated nonspecific DNA fragment. These results provide an initial characterization of the human VLCAD gene promoter, identify AP-2 as a candidate activator of VLCAD gene transcription, and suggest that VLCAD gene transcription may be regulated by pathways distinct from that of other AD genes.

Mitochondrial long chain fatty acid beta-oxidation provides the major source of energy in the heart. Deficiencies of human beta-oxidation enzymes produce sudden, unexplained death in childhood, acute hepatic encephalopathy, skeletal myopathy, or cardiomyopathy. Long chain 3-hydroxyacyl-CoA dehydrogenase [LCHAD; long-chain-(S)-3-hydroxyacyl-CoA:NAD+ oxidoreductase, EC 1.1.1.211] catalyzes the third step in beta-oxidation, and this activity is present on the C-terminal portion of the alpha subunit of mitochondrial trifunctional protein. We used single-stranded conformation variance analysis of the exons of the human LCHAD (alpha subunit) gene to determine the molecular basis of LCHAD deficiency in three families with children presenting with sudden unexplained death or hypoglycemia and abnormal liver enzymes (Reye-like syndrome). In all families, the mothers had acute fatty liver and associated sever complications during pregnancies with the affected infants. The analysis in two affected children revealed a G to C mutation at position 1528 (G1528C) of the alpha subunit of the trifunctional protein on both alleles. This is in the LCHAD domain and substitutes glutamine for glutamic acid at position 474 of mature alpha subunit. The third child had this G1528C mutation on one allele and a different mutation (C1132T) creating a premature termination codon (residue 342) on the second allele. Our results demonstrate that mutations in the LCHAD domain of the trifunctional protein alpha subunit in affected offspring are associated with maternal acute fatty liver of pregnancy. This is the initial delineation of the molecular basis of isolated LCHAD deficiency.

We have isolated and sequenced a cDNA encoding rat cardiac troponin I. The predicted amino acid sequence was highly identical with previously reported chemically derived amino acid sequences for rabbit and bovine cardiac troponin I. Clones for slow skeletal muscle troponin I were also obtained from neonatal rat cardiac ventricle by the polymerase chain reaction. The nucleotide sequences of these clones were determined to be more than 99% identical with a previously reported rat slow skeletal troponin I cDNA [Koppe et al. (1989) J. Biol. Chem. 264, 14327-14333]. The troponin I clones hybridized to RNA from the appropriate muscle from adult animals. However, RNA from fetal and neonatal rat heart also hybridized with the slow skeletal troponin I cDNA, demonstrating its expression in fetal and neonatal rat heart. Slow skeletal troponin I steady-state mRNA levels decreased with increasing age, but cardiac troponin I mRNA levels increased through fetal and early neonatal cardiac development. Thus, during fetal and neonatal development, slow skeletal and cardiac troponin I isoforms are coexpressed in the rat heart and regulated in opposite directions. The degree of primary sequence differences in these isoforms, especially at phosphorylation sites, may result in important functional differences in the neonatal myocardium.

THE MOLECULAR BASIS OF INHERITED CARDIOMYOPATHY DUE TO VERY LONG CHAIN ACYL-CoA DEHYDROGENASE DEFICIENCY. • 125

The primary translation product of human intestinal apolipoprotein A-I mRNA was isolated from wheat germ and ascites cell-free translation systems. Comparison of its NH2-terminal sequence with that of plasma high density lipoprotein-associated A-I showed that it is initially synthesized as a preproprotein. Like rat preproapolipoprotein A-I, it contains an 18-amino acid prepeptide and a 6-amino acid propeptide. The highly unusual COOH-terminal Gln-Gln dipeptide present in the rat pro-segment is also represented at the same position in the human sequence. The functional division of the 24-amino acid NH2-terminal extention into pro- and presegments was verified by finding that the stable intracellular form of A-I in a human hepatoma cell line was the proprotein. Edman degradation of radiolabeled intracellular and extracellular A-I indicated that this apolipoprotein was secreted without proteolytic cleavage of its hexapeptide prosegment. Therefore, it appears that apolipoprotein A-I undergoes an additional proteolytic processing step before it is fully integrated into plasma high density lipoprotein. Two-dimensional gel electrophoresis of purified proapolipoprotein A-I isolated from the hepatocyte cell culture media indicated that it corresponds to isoforms 2 and 3, the basic A-I isoproteins which are the precursors of plasma A-I and the predominant plasma A-I isoforms found in patients with Tangier's disease (Zannis, V. I., Lees, A. M., Lees, R. S., and Breslow, J. L. (1982) J. Biol. Chem., 257, 4978-4986). Therefore this pathologic state probably arises from a defect in the conversion of proapolipoprotein A-I to apolipoprotein A-I.

The primary translation product of human intestinal apolipoprotein A-IV mRNA was purified from ascites and wheat germ cell-free systems. Comparison of its NH2-terminal sequence with mature, chylomicron-associated apo-A-IV revealed that apo-A-IV was initially synthesized with a 20-amino acid long NH2-terminal extension: Met-X-Leu-X-Ala-Val-Val-Leu-X-Leu-Ala-Leu-Val-Ala-Val-Ala-Leu-X-X-Ala. Co-translational cleavage of the cell-free product as well as Edman degradation of the stable intracellular form of the protein recovered from Hep G2 cells indicated that this entire 20-amino acid sequence behaved as a signal peptide. There is at least 55% sequence homology between the rat and human apo-A-IV signal peptides and 33% homology between the human A-I and A-IV presegments. Agarose gel chromatography of Hep G2 culture media indicated that neither apo-A-IV nor -A-I is associated with particles that have physical properties resembling any of the plasma lipoprotein density classes. Incubation of plasma with Hep G2 media resulted in transfer of A-I but not A-IV to lipoproteins. Since the NH2 termini of co-translationally cleaved and chylomicron-associated apo-A-IV are identical, it is apparent that 1) this polypeptide does not undergo NH2-terminal post-translational proteolysis like proapo-A-II or proapo-A-I, and 2) regulation of A-IV-lipoprotein interaction is not dependent on any NH2-terminal proteolytic processing event.

Apolipoprotein A-II is the second most abundant polypeptide found in human plasma high density lipoprotein particles. The primary translation product of human apo-A-II mRNA is a prepropolypeptide. We have previously reported (Gordon, J. I., Sims, H. F., Edelstein, C., Scanu, A. M., and Strauss, A. W. (1984) J. Biol. Chem. 259, 15556-15563) that the prosegment of apo-A-II was removed following export from a human hepatoma cell line (Hep G2). This represented a novel processing compartment for prosegments terminating with paired basic residues and differed from the processing of proalbumin which occurred with high efficiency prior to export from these cells. We have now characterized the enzyme responsible for this extracellular cleavage. The proapo-A-II converting activity is blocked by the thiol protease inhibitors antipain, E-64, leupeptin, and Ala-Lys-Arg chloromethyl ketone. Incubation of 125I-iodotyrosylated Ala-Lys-Arg chloromethyl ketone with serum-free media harvested from cell cultures over a 12-h period revealed a time-dependent accumulation of a 54-kDa protease. Although small quantities of the 54-kDa protease were detected in cell lysates, the major intracellular sequences labeled by the affinity probe had masses of 31.5 and 6 kDa. The 54-kDa extracellular, as well as 31.5- and 6-kDa intracellular, species were all immunoprecipitated by monospecific anti-human liver cathepsin B IgG. Addition of this antibody to media inhibited extracellular conversion of proapo-A-II to the mature protein. Based on these observations, we conclude that a "pro" cathepsin B-like protease exported by Hep G2 cells is responsible for proapo-A-II prosegment removal. It appears that cathepsin B-like proteases exhibit a complex pattern of segregation within the secretory pathway and that larger molecular weight forms of cathepsin B-like proteases are capable of accurately processing propolypeptides.

The primary translation product of human apolipoprotein A-II was purified from wheat germ and ascites cell-free lysates programmed with RNA isolated from either a hepatocellular carcinoma cell line (HepG2) or intestinal epithelium. A-II mRNA represents 0.2% of the translatable RNA in these hepatocytes and in jejunal epithelium. Plasma high density lipoprotein-associated A-II is a 77-amino acid polypeptide. The primary translation product is 100 amino acids long and contains a 23-amino acid NH2-terminal extension. Cotranslational cleavage of the cell-free product indicated that this NH2-terminal sequence consists of an 18-amino acid long signal peptide, Met-Lys-Leu-Leu-Ala-Ala-X-Val-Leu-Leu-Leu-X-X-Cys-X-Leu-X-X-, and a 5-amino acid long propeptide, Ala-Leu-Val-Arg-Arg. This functional division was confirmed by sequencing the stable intracellular form of apolipoprotein A-II isolated from HepG2 cells. Approximately 45% of the proapo-A-II is cleaved to the mature form during export from HepG2 cells. The COOH-terminal dipeptide conforms to the rule that prosegments are cleaved after paired basic residues. We have previously shown (Gordon, J. I., Sims, H. F., Lentz, S. R., Edelstein, C., Scanu, A. M., and Strauss, A. W. (1983) J. Biol. Chem. 258, 4037-4044) that proapolipoprotein A-I is not cleaved during export from these cells and contains a prosegment with a COOH-terminal Gln-Gln dipeptide. Therefore, proteolytic processing of the two principal high density lipoprotein-associated apolipoproteins proceeds along different pathways.

17β-Estradiol dehydrogenase (EC 1.1.1.62) catalyzes the interconversion of estradiol and estrone in human term placenta. We have raised a specific polyclonal antibody to this abundant placental enzyme to study its role in late pregnancy events and its molecular biologic characteristics. In this work the 17β-estradiol dehydrogenase antibody was used to isolate and sequence a complementary deoxyribonucleic acid clone encoding about 98% of the amino acid sequence of the 17β-estradiol dehydrogenase molecule. This sequence verifies previous sequence data on the molecule's steroid binding site and also localizes a putative nicotinamide adenine dinucleotide binding region similar to that of many other pyridine nucleotide-dependent dehydrogenases. Isolation of the complementary deoxyribonucleic acid for 17β-estradiol dehydrogenase expands our knowledge of the structure-function relationships of the enzyme and is a major step in our understanding of its biologic function in pregnancy.

The two principal high-density lipoprotein apolipoproteins A-I and A-II are both initially synthesized as preproproteins. The prosegment of apo-A-I is unusual: it ends with paired glutamine residues and is removed extracellularly. The apo-A-II prosegment resembles the propeptides of prohormones and proalbumin: it ends with paired basic amino acids. We have studied the processing of proapo-A-II in a human hepatoma cell line (Hep G2) which is known to accurately and efficiently remove the prosegment from proalbumin prior to secretion. Pulse-chase experiments were performed in order to determine if the apo-A-II prosegment is removed prior to or after secretion. Apo-A-II was purified from cell lysates and media at various times during the chase and subjected to automated sequential Edman degradation. The results indicate that proteolytic processing of proapo-A-II is largely an extracellular event. These cells secrete the protease responsible for prosegment removal. The converting activity present in media is not blocked by serine protease inhibitors (phenylmethanesulfonyl fluoride, aprotinin, and furoyl saccharin) or by a metalloprotease inhibitor (o-phenanthroline). It is inhibited by the thiol protease reagents p-chloromercuribenezene-sulfonic acid and leupeptin. Prosegment removal changes the pI of the dominant apo-A-II isoform from 6.61 to 4.95. The presence of the propeptide does not prevent specific in vitro recombination of apo-A-II with high-density lipoprotein3 particles present in normolipemic serum. Extracellular processing after a single basic amino acid has been described for a variety of precursor proteins. Extracellular cleavage of the apo-A-II propeptide after paired COOH-terminal basic residues represents a novel processing pathway.

Previous studies have established that human hepatocellular carcinoma cells (Hep G2) secrete into serum-free medium the pro form of apolipoprotein A-I (proapo-A-I) suggesting that its conversion to mature apo-A-I occurs after secretion. In order to assess the mode and site of proapo-A-I to apo-A-I conversion, we incubated the medium from [3H]proline-labeled Hep G2 cells with either human plasma, serum, lymph, or fractions thereof obtained by density gradient ultracentrifugation. The conversion was monitored by two-dimensional gel electrophoresis and by Edman degradation. Human plasma, serum, or mesenteric lymph all induced proapo-A-I to apo-A-I conversion; this was time dependent, unaffected by the serine protease inhibitor phenylmethylsulfonyl fluoride and inhibited by EDTA. Purified radiolabeled proapo-A-I bound to lymph chylomicrons and plasma high density lipoproteins. The converting enzyme was associated with both of these particles. Activity was also found in the d greater than 1.21-g/ml fraction and may have been derived from high density lipoprotein after displacement by high salts and/or ultracentrifugal force. We conclude that the conversion of proapo-A-I to apo-A-I occurs extracellularly and is probably effected by a metallo-enzyme which may act at the amphiphilic surface of either chylomicrons or high density lipoproteins.

A method is presented for determining the extent of methylation of tRNAs synthesized in mammalian and bacterial cell systems and is based upon determining the distribution of radioactivity associated with the guanine constituents of total cellular tRNA preparations previously labeled with [2-14C]guanosine and with [methyl]-3H or -14C]methionine. Whereas labeling with guanosine provides a means of assessing the extent of methylation of the [2-14C]guanine residues incorporated into tRNA, methionine labeling provides a measure of the percentage of [methyl-3H or -14C]methylated constituents that are methylated guanines. Analyses such as the above reveal that the tRNA of KB cells acquires approximately three times as many methyl groups as that of E. coli B tRNA. Coupled with the knowledge that both mammalian and bacterial tRNA preparations contain an average of 24 guanine residues per molecule, the above analyses further reveal that 7.2 and 2.4 methyl groups are incorporated into each tRNA molecule synthesized in exponentially growing KB- and E. coli B-cells, respectively. Additional information regarding the extent of formation of individual methylated constituents per tRNA molecule synthesized is presented.

The steps involved in the initial assembly of apolipoproteins and lipids into supramolecular arrays (nascent lipoprotein particles) are largely unknown. Examination of the proteolytic processing and compartmentalization of the primary translation products of apolipoprotein mRNAs represents one approach to deciphering the molecular details of lipoprotein assembly. The structures of the primary translation products of seven mammalian apolipoprotein mRNAs has been determined in the past several years. The organization of apolipoprotein signal peptides is typical of eukaryotic prepeptides, although an unusual degree of sequence conservation is present among the signal segments of apo AI, AIV, and E. For those apolipoprotein sequences studied in detail, SRP-dependent cotranslational translocation and proteolytic processing appears to be highly efficient and results in sequestration of the processed protein within the lumen of the endoplasmic reticulum (ER). However the mechanism by which these lipid-binding proteins avoid arrest during their translocation through the lipid bilayer of the ER membrane remains obscure. The two principal human HDL apolipoproteins undergo novel extracellular post-translational proteolytic processing, which results in removal of nonhomologous propeptides. The proteases responsible for proapo AI and AII processing appear to be different. The processing of these proapolipoproteins provides a potential series of steps for regulating the ordered assembly of HDL constituents.

Antibodies specific for N6-methyladenosine (m6A) were immobilized on Sepharose and the resulting immunoadsorbent was tested for its ability to retain those Escherichia coli tRNAs containing the antigenic hapten, i.e., m6A. Results obtained with [32P]PO4- and [methyl-3H]-methionine-labeled tRNAs indicated that approximately 3 to 5% of the radioactive RNA was retained by the immunoadsorbent. Under identical conditions, but in the presence of m6A (1 mg/mL), less than 0.2% of the radioactivity was retained. Subsequent characterization of the retained tRNA via (a) analysis of methyl-3H-labeled, methylated nucleosides, (b) two-dimensional gel electrophoresis, and (c) analysis of the retention of [3H]aminoacyl-tRNA species led to the conclusion that the anti-m6A/Sepharose adsorbent quantitatively and exclusively retained a single tRNA species containing m6A, namely, tRNAVal.

Immature rats treated with estradiol for selected periods of time demonstrated both increased methylation of uterine transfer ribonucleic acid (tRNA) and methylase activities. Whereas the former parameter was assessed by incubating whole uteri with [methyl-14C]methionine and measuring the incorporation of isotope into the tRNA, methylase activity was obtained by measuring the rate of incorporation of methyl groups from S-adenosyl[methyl-14C]methionine into heterologous tRNA (Escherichia coli B) in the presence of uterine cytosol preparations (100,000g supernatants). Although increased methylation of tRNA during the estrogen response was demonstrated, additional studies indicated that these results were largely attributable to an increased rate of synthesis of tRNA rather than gross changes in either the type or amount of methylated constituents present. Evidence in this regard included the inability of estrogen treatment of alter significantly the (a) resulting patterns of methyl-14C-methylated constituents of uterine tRNA, (b) the extent ot which [2-14C]guanine residues, incorporated into tRNA, become methylated, (c) the extent of methylation of precursor tRNA in the absence of tRNA synthesis, and (d) the types of methylase activities expressed in vitro.

In an attempt to separate a number of methylated nucleoside constituents reportedly present in various mammalian RNA preparations, a comparison of the migration of selected nucleoside standards was assessed via thin-layer chromatographic techniques with respect to three solvent systems and an adsorbent of varying proportions of silica gel and microcrystalline cellulose. The results of these studies revealed that a mixture of seventeen methylated constituents could be separated following two-dimensional development on an adsorbent containing 60% cellulose and 40% silica gel. Constituents separated included the 2′-O-methylated deerivatives of adenosine, guanosine, cytidine and uridine, 1-, N6- and N6,N6-methyladenosines, 1- methylinosine, 1-, 7-, N2- and N2,N2-methylguanosines, 3-, 4- and 5-methylcytidines and 3- and 5-methyluridines. In addition, all of the above constituents were separated from their respective non-methylated nucleosides and bases.