BBA - Molecular and Cell Biology of Lipids (v.1771, #9)
Editorial Board (ii).
Ezetimibe in search of receptor(s)—Still a never-ending challenge in cholesterol absorption and transport by Friedrich Spener (1113-1116).
Statins and foam cell formation: Impact on LDL oxidation and uptake of oxidized lipoproteins via scavenger receptors by Oliver Hofnagel; Birgit Luechtenborg; Gabriele Weissen-Plenz; Horst Robenek (1117-1124).
The uptake of oxidized lipoproteins via scavenger receptors and the ensuing formation of foam cells are key events during atherogenesis. Foam cell formation can be reduced by treatment with 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins). The efficacy of statins is evidently due not only to their cholesterol-lowering properties, but also to lipid-independent pleiotropic effects. This review focuses on lipid-independent pleiotropic effects of statins that influence foam cell formation during atherogenesis, with special emphasis on oxidative pathways and scavenger receptor expression.
Keywords: Statin; Foam cell formation; Atherogenesis; oxLDL; Scavenger receptor;
Emerging roles of PPARδ in metabolism by Udo Seedorf; Jens Aberle (1125-1131).
PPARδ differs from the other two PPAR isotypes (α and γ) by its more wide-spread tissue-specific expression pattern, its involvement in developmental processes and its profound impact on muscle and heart fat metabolism. Activation of PPARδ modulates inflammatory responses of macrophages and is linked to altered lipoprotein metabolism, most importantly a significant raise of HDL cholesterol. PPARδ activation in the liver decreases hepatic glucose output, thereby contributing to improved glucose tolerance and insulin sensitivity. Several studies have shown that PPARδ polymorphisms are associated with plasma lipid levels, body mass index and the risk for diabetes and coronary heart disease. These findings support that high affinity PPARδ agonists may be promising drugs of the future to treat the metabolic syndrome which is an expanding overweight-related health threat characterized by insulin resistance, hyperglycemia, dyslipidemia, hypertension, and accelerated atherosclerosis.
Keywords: Peroxisome proliferator activated receptor; Metabolic syndrome; Diabetes type 2; Insulin resistance; HDL cholesterol; Dyslipidemia; Obesity;
Class B type I scavenger receptor is responsible for the high affinity cholesterol binding activity of intestinal brush border membrane vesicles by Eric D. Labonté; Philip N. Howles; Norman A. Granholm; Juan C. Rojas; Joanna P. Davies; Yiannis A. Ioannou; David Y. Hui (1132-1139).
Recent studies have documented the importance of Niemann–Pick C1-like 1 protein (NPC1L1), a putative physiological target of the drug ezetimibe, in mediating intestinal cholesterol absorption. However, whether NPC1L1 is the high affinity cholesterol binding protein on intestinal brush border membranes is still controversial. In this study, brush border membrane vesicles (BBMV) from wild type and NPC1L1 −/− mice were isolated and assayed for micellar cholesterol binding in the presence or absence of ezetimibe. Results confirmed the loss of the high affinity component of cholesterol binding when wild type BBMV preparations were incubated with antiserum against the class B type 1 scavenger receptor (SR-BI) in the reaction mixture similar to previous studies. Subsequently, second order binding of cholesterol was observed with BBMV from wild type and NPC1L1 −/− mice. The inclusion of ezetimibe in these in vitro reaction assays resulted in the loss of the high affinity component of cholesterol interaction. Surprisingly, BBMVs from NPC1L1 −/− mice maintained active binding of cholesterol. These results documented that SR-BI, not NPC1L1, is the major protein responsible for the initial high affinity cholesterol ligand binding process in the cholesterol absorption pathway. Additionally, ezetimibe may inhibit BBM cholesterol binding through targets such as SR-BI in addition to its inhibition of NPC1L1.
Keywords: Niemann–Pick C1-like 1 protein (NPC1L1); Micelle; Brush border membrane vesicle (BBMV); Cholesterol transport proteins; Ezetimibe;
Multiple plasma membrane receptors but not NPC1L1 mediate high-affinity, ezetimibe-sensitive cholesterol uptake into the intestinal brush border membrane by Martin Knöpfel; Joanna P. Davies; Phu T. Duong; Lisbet Kværnø; Erick M. Carreira; Michael C. Phillips; Yiannis A. Ioannou; Helmut Hauser (1140-1147).
We compared cholesterol uptake into brush border membrane vesicles (BBMV) made from the small intestines of either wild-type or Niemann–Pick C1-like 1 (NPC1L1) knockout mice to elucidate the contribution of NPC1L1 to facilitated uptake; this uptake involves cholesterol transport from lipid donor particles into the BBM of enterocytes. The lack of NPC1L1 in the BBM of the knockout mice had no effect on the rate of cholesterol uptake. It follows that NPC1L1 cannot be the putative high-affinity, ezetimibe-sensitive cholesterol transporter in the brush border membrane (BBM) as has been proposed by others. The following findings substantiate this conclusion: (I) NPC1L1 is not a brush border membrane protein but very likely localized to intracellular membranes; (II) the cholesterol absorption inhibitor ezetimibe and its analogues reduce cholesterol uptake to the same extent in wild-type and NPC1L1 knockout mouse BBMV. These findings indicate that the prevailing belief that NPC1L1 facilitates intestinal cholesterol uptake into the BBM and its interaction with ezetimibe is responsible for the inhibition of this process can no longer be sustained.
Keywords: Intestinal cholesterol absorption; Niemann–Pick C1 like 1; wt and NPC1L1−/− mouse brush border membrane vesicle; Inhibitors of cholesterol absorption; Ezetimibe; Apolipoprotein A-I;
Induction of choline kinase alpha by carbon tetrachloride (CCl4) occurs via increased binding of c-jun to an AP-1 element by Chieko Aoyama; Kozo Ishidate; Hiroyuki Sugimoto; Dennis E. Vance (1148-1155).
The mechanism by which treatment of mice with CCl4 induces an increase in choline kinase α has been investigated. Nuclear run on assays demonstrated a major increase in the transcript for choline kinase α in livers from mice 3 h and 6 h after administration of CCl4 compared to vehicle (olive oil). 5′deletion analyses of choline kinase α promoter-luciferase constructs expressed in Hepa-1 cells identified a promoter element between − 875 and − 866 that was nearly identical to an AP-1 consensus site. Mutation of this AP-1 site caused a striking decrease in the expression of choline kinase α promoter-luciferase constructs. Electromobility shift assays with nuclear extracts from mouse liver demonstrated that c-Jun, but not c-fos, bound oligonucleotides with the AP-1 site. The amount of c-jun bound was greatly increased when hepatic nuclear extracts from mice treated with CCl4 were used. Chromatin immunoprecipitation assays confirmed that c-jun binds to the choline kinase α promoter. The results from these studies provide strong evidence that the choline kinase α promoter has a distal element (− 875/− 867) that binds c-jun and the binding of c-jun is enhanced by treatment with CCl4.
Keywords: Choline kinase; Alpha carbon tetrachloride; AP-1 element;
Expression of 15-lipoxygenase type-1 in human mast cells by Magdalena Gulliksson; Åsa Brunnström; Malin Johannesson; Linda Backman; Gunnar Nilsson; Ilkka Harvima; Barbro Dahlén; Maria Kumlin; Hans-Erik Claesson (1156-1165).
Mast cells play a key role in the pathophysiology of asthma. These cells exert their effector functions by releasing a variety of proinflammatory and immunoregulatory compounds. Mast cells infiltrate the bronchial epithelium and smooth muscle to a higher degree in patients with asthma compared to control subjects. 15-Lipoxygenase type-1 (15-LO-1) is a prooxidant enzyme which is expressed in asthmatic lungs leading to formation of pro- and anti-inflammatory mediators. Here we report that interleukin-4 (IL-4) induced the expression of 15-LO-1 in human cord blood derived mast cells (CBMC) as demonstrated by RT-PCR, western blot and immunocytochemistry. The major metabolite of arachidonic acid formed via the 15-LO pathway in IL-4 treated CBMC was identified as 15-ketoeicosatetraenoic acid (15-KETE, also named 15-oxo-ETE) with smaller amounts of 15-hydroxyeicosatetraenoic acid (15-HETE) as identified by HPLC and mass spectrometry (MS/MS). Furthermore, immunohistochemical stainings demonstrated the expression of 15-LO-1 in mast cells in lung and skin in vivo. Osmotic activation of CBMC with mannitol resulted in activation of the 15-LO-1 pathway. In conclusion, the expression of 15-LO-1 and release of 15-LO-1 derived products by mast cells may contribute to the role of these cells in asthma and other inflammatory diseases.
Keywords: Mast cell; IL-4; 15-lipoxygenase; 15-hydroxyeicosatetraenoic acid; 15-ketoeicosatetraenoic acid;
Lovastatin enhances phenylbutyrate-induced MR-visible glycerophosphocholine but not apoptosis in DU145 prostate cells by Matthew Milkevitch; Thomas M. Jeitner; Nancy J. Beardsley; E. James Delikatny (1166-1176).
In this study the effects of lovastatin on DU145 prostate cancer cells treated with phenylbutyrate (PB) was investigated in order to determine the NMR-detectable metabolic changes resulting from the cooperative activity of these two agents. DU145 cells were perfused with PB in the presence or absence of 10 μM of the HMG-CoA reductase inhibitor lovastatin, and the results monitored by 31P and diffusion-weighted 1H NMR spectroscopy. Lovastatin had additive effects on the PB-induced NMR-visible total choline in 1H spectra, and glycerophosphocholine in 31P spectra but no significant effect on NMR-visible lipid. Moreover, lovastatin had no effect on the ability of PB to either promote the formation of oil red O-detectable lipid droplets or arrest the cell cycle. The most remarkable observations from these studies were that lovastatin enhanced the increase in glycerophosphocholine while reversing late markers of apoptosis and the loss of NTP caused by PB. These results identify a branch point separating the neutral lipid production and the apoptotic cell death caused by the actions of differentiating agents.
Keywords: Lovastatin; Phenylbutyrate; NMR; Differentiation; HMG-CoA reductase inhibitor; Apoptosis; NMR-visible lipids; Glycerophosphocholine;
Expression of the phospholipid scramblase (PLSCR) gene family during the acute phase response by Biao Lu; Peter J. Sims; Therese Wiedmer; Arthur H. Moser; Judy K. Shigenaga; Carl Grunfeld; Kenneth R. Feingold (1177-1185).
Phospholipid scramblase 1 (PLSCR1) is a member of PLSCR gene family that has been implicated in multiple cellular processes including movement of phospholipids, gene regulation, immuno-activation, and cell proliferation/apoptosis. In the present study, we identified PLSCR1 as a positive intracellular acute phase protein that is upregulated by LPS in liver, heart, and adipose tissue, but not skeletal muscle. LPS administration resulted in a marked increase in PLSCR1 mRNA and protein levels in the liver. This stimulation occurred rapidly (within 2 h), and was very sensitive to LPS (half-maximal response at 0.1 μg/mouse). Moreover, two other APR-inducers, zymosan and turpentine, also produced significant increases in PLSCR1 mRNA and protein levels, indicating that PLSCR1 was stimulated in a number of models of the APR. To determine signaling pathways by which LPS stimulated PLSCR1, we examined the effect of proinflammatory cytokines in vitro and in vivo. TNFα, IL-1β, and IL-6 all stimulated PLSCR1 in cultured Hep B3 hepatocytes, whereas only TNFα stimulated PLSCR1 in cultured 3T3-L1 adipocytes, suggesting cell type-specific effects of cytokines. Furthermore, the LPS-stimulated increase in liver PLSCR1 mRNA was greatly attenuated by 80% in TNFα and IL-1β receptor null mice as compared to wild-type controls. In contrast, PLSCR1 levels in adipose tissue were induced to a similar extent in TNFα and IL-1β receptor null mice and controls. These results indicate that maximal stimulation of PLSCR1 by LPS in liver required TNFα and/or IL-1β, whereas the stimulation of PLSCR1 in adipose tissue is not dependent on TNFα and/or IL-1β. These data provide evidence that PLSCR1 is a positive intracellular acute phase protein with a tissue-specific mechanism for up-regulation.
Keywords: PLSCR; LPS; Acute phase response; Tumor necrosis factor; Interleukin;
Inhibition of sphingomyelin synthase (SMS) affects intracellular sphingomyelin accumulation and plasma membrane lipid organization by Zhiqiang Li; Tiruneh K. Hailemariam; Hongwen Zhou; Yan Li; Dale C. Duckworth; David A. Peake; Youyan Zhang; Ming-Shang Kuo; Guoqing Cao; Xian-Cheng Jiang (1186-1194).
Sphingomyelin plays a very important role both in cell membrane formation that may well have an impact on the development of diseases like atherosclerosis and diabetes. However, the molecular mechanism that governs intracellular and plasma membrane SM levels is largely unknown. Recently, two isoforms of sphingomyelin synthase (SMS1 and SMS2), the last enzyme for SM de novo synthesis, have been cloned. We have hypothesized that SMS1 and SMS2 are the two most likely candidates responsible for the SM levels in the cells and on the plasma membrane. To test this hypothesis, cultured cells were treated with tricyclodecan-9-yl-xanthogenate (D609), an inhibitor of SMS, or with SMS1 and SMS2 siRNAs. Cells were then pulsed with [14C]-l-serine (a precursor of all sphingolipids). SMS activity and [14C]-SM in the cells were monitored. We found that SMS activity was significantly decreased in cells after D609 or SMS siRNA treatment, compared with controls. SMS inhibition by D609 or SMS siRNAs significantly decreased intracellular [14C]-SM levels. We measured cellular lipid levels, including SM, ceramide, phosphatidylcholine, and diacylglycerol and found that SMS1 and SMS2 siRNA treatment caused a significant decrease of SM levels (20% and 11%, respectively), compared to control siRNA treatment; SMS1 but not SMS2 siRNA treatment caused a significant increase of ceramide levels (10%). There was a decreasing tendency for diacylglycerol levels after both SMS1 and SMS2 siRNA treatment, however, it was not statistical significant. As shown by lipid rafts isolation and lipid determination, SMS1 and SMS2 siRNA treatment led to a decrease of SM content in detergent-resistant lipid rafts on the cell membrane. Furthermore, SMS1 and SMS2 siRNA-treated cells had a stronger resistance than did control siRNA-treated cells to lysenin (a protein that causes cell lysis due to its affinity for plasma membrane SM). These results indicate that both SMS1 and SMS2 contribute to SM de novo synthesis and control SM levels in the cells and on the cell membrane including plasma membrane, implying an important relationship between SMS activity and cell functions.
Keywords: Sphingomyelin; Sphingomyelin synthase 1 and 2; Lipid drafts; SMS1 and SMS2 siRNA;
Protein kinase C-dependent antilipolysis by insulin in rat adipocytes by Jiro Nakamura (1195-1201).
Recently, we have shown that protein kinase C (PKC) activated by phorbol 12-myristate 13-acetate (PMA) attenuates the β1-adrenergic receptor (β1-AR)-mediated lipolysis in rat adipocytes. Stimulation of cells by insulin, angiotensin II, and α1-AR agonist is known to cause activation of PKC. In this study, we found that lipolysis induced by the β1-AR agonist dobutamine is decreased and is no longer inhibited by PMA in adipocytes that have been treated with 20 nM insulin for 30 min followed by washing out insulin. Such effects on lipolysis were not found after pretreatment with angiotensin II and α1-AR agonists. The rate of lipolysis in the insulin-treated cells was normalized by the PKCα- and β-specific inhibitor Gö 6976 and PKCβ-specific inhibitor LY 333531. In the insulin-treated cells, wortmannin increased lipolysis and recovered the lipolysis-attenuating effect of PMA. Western blot analysis revealed that insulin slightly increases membrane-bound PKCα, βI, and δ, and wortmannin decreases PKCβI, βII, and δ in the membrane fraction. These results indicate that stimulation of insulin receptor induces a sustained activation of PKC-dependent antilipolysis in rat adipocytes.
Keywords: Adipocyte; Lipolysis; β-adrenergic receptor; PKC; PMA; Insulin; Wortmannin;
Topology of acyltransferase motifs and substrate specificity and accessibility in 1-acyl-sn-glycero-3-phosphate acyltransferase 1 by Atsushi Yamashita; Hiroki Nakanishi; Hiroshi Suzuki; Ryo Kamata; Ken Tanaka; Keizo Waku; Takayuki Sugiura (1202-1215).
1-Acyl-sn-glycero-3-phosphate (AGP) acyltransferases (AGPAT) are involved in de novo biosynthesis of glycerolipids, such as phospholipids and triacylglycerol. Alignment of amino acid sequences from AGPAT, sn-glycerol-3-phosphate acyltransferase, and dihydroxyacetonephosphate acyltransferase reveals four regions with strong homology (acyltransferase motifs I–IV). The invariant amino acids within these regions may be part of a catalytically important site in this group of acyl-CoA acyltransferases. However, in human AGPAT1 a transmembrane domain is predicted to separate motif I on the cytosolic side from motifs II–III on the lumenal side, with motif IV near surface of the membrane. The topology of motifs I and III was confirmed by experiments with recombinant AGPAT1 containing potential glycosylation site near the motifs. This topology conflicts with the expectation that catalytically important sites are near one another, raising questions of whether the acyltransferase motifs really are important for AGPAT catalysis, and how substrates access motifs II–III on the lumenal side of the endoplasmic reticulum membrane. Using human AGPAT1 as a model, we have examined the catalytic roles of highly conserved residues in the four acyltransferase motifs by site-directed mutagenesis. Modifications of the sidechain structures of His104, Asp109, Phe146, Arg149, Glu178, Gly179, Thr180, Arg181 and Ile208 all affected AGPAT1 activity, indicating that the acyltransferase motifs indeed are important for AGPAT catalysis. In addition, we examined substrate accessibility to the catalytic domain of human AGPAT1 using a competition assay. Lysophosphatidic acid (LPA) with fatty acid chains shorter than 10 carbons did not access the catalytic domain, suggesting that LPA hydrophobicity is important. In contrast, short chain acyl-CoAs did access the catalytic domain but did not serve as the second substrate. These results suggest that motifs II and III are involved in LPA binding and motifs I and IV are involved in acyl-CoA binding.
Keywords: Lysophosphatidic acid; Acyl-CoA:1-acyl-sn-glycero-3-phosphate acyltransferase; Membrane topology; Substrate specificity;
Activation of the hepatic LDL receptor promoter by thyroid hormone by Dayami Lopez; Jose F. Abisambra Socarrás; Mohini Bedi; Gene C. Ness (1216-1225).
The question of whether mature sterol regulatory element binding protein-2 (SREBP-2) mediates transcriptional activation of the hepatic low density lipoprotein (LDL) receptor by thyroid hormone was investigated. Western blotting analysis and electrophoretic mobility shift assays demonstrated that mature nuclear SREBP-2 protein could be detected in liver nuclear extracts prepared from normal animals but not in extracts prepared from rats rendered hypothyroid either by hypophysectomy (Hx) or thyroidectomy (Tx). Treatment of Hx rats with T3 restored LDL receptor mRNA levels in about 1 h and caused a 6-fold increase 2.5 h after T3 administration. However, no detectable mature SREBP-2 was seen in this time period despite a substantial reduction in serum cholesterol levels caused by the T3 treatment. Deletion of the SRE region from the LDL receptor promoter did not decrease the T3 response. Thus, the possibility that T3 may be mediating LDL receptor induction directly via a thyroid response element (TRE) was investigated. Reporter gene analysis and electrophoretic mobility shift assays demonstrated that the rat LDL receptor promoter contains two functional TREs (US-TRE and 2H-TRE). Either one of these elements could support T3 induction. However, the stronger of these elements is US-TRE at-612 which binds TRβ1 more tightly and when mutated results in a diminished T3 response. These results indicate that the rapid induction of the hepatic LDL receptor by thyroid hormone is likely due to direct interaction with TREs rather than indirectly by a mechanism involving SREBP-2.
Keywords: LDL receptor promoter activity; Cholesterol domeostasis; Thyroid hormone response element; Rat liver; Thyroid receptor β1; SREBP-2; Sterol response element;
Alteration of ganglioside synthesis by GM3 synthase knockout in murine embryonic fibroblasts by Nikolai A. Shevchuk; Yetrib Hathout; Olga Epifano; Yan Su; Yihui Liu; Margaret Sutherland; Stephan Ladisch (1226-1234).
To probe the functions of membrane gangliosides, the availability of ganglioside-depleted cells would be a valuable resource. To attempt to identify a useful genetic model of ganglioside depletion, we assessed ganglioside metabolism in murine GM3 synthase (GM3S)−/− knockout primary embryonic fibroblasts (MEF), because normal fibroblast gangliosides (GM3, GM2, GM1, and GD1a), all downstream products of GM3S, should be absent. We found that heterozygote MEF (GM3S+/−) did have a 36% reduced content of qualitatively normal gangliosides (7.0 ± 0.8 nmol LBSA/mg cell protein; control: 11 ± 1.6 nmol). However, two unexpected findings characterized the homozygous (GM3−/−) MEF. Despite complete knockout of GM3S, (i) GM3−/− MEF retained substantial ganglioside content (21% of normal or 2.3 ± 1.1 nmol) and (ii) these gangliosides were entirely different from those of wild type MEF by HPTLC. Mass spectrometry identified them as GM1b, GalNAc–GM1b, and GD1α, containing both N-acetyl and N-glycolylneuraminic acid and diverse ceramide structures. All are products of the 0 pathway of ganglioside synthesis, not normally expressed in fibroblasts. The results suggest that complete, but not partial, inhibition of GM3 synthesis results in robust activation of an alternate pathway that may compensate for the complete absence of the products of GM3S.
Keywords: Gangliosides; GM3 synthase; Knockout; Mouse embryonic fibroblasts; 0-pathway;
Characterization of prostaglandin E2 generation through the cyclooxygenase (COX)-2 pathway in human neutrophils by Mireille St-Onge; Nicolas Flamand; Jordane Biarc; Serge Picard; Line Bouchard; Andrée-Anne Dussault; Cynthia Laflamme; Michael J. James; Gillian E. Caughey; Leslie G. Cleland; Pierre Borgeat; Marc Pouliot (1235-1245).
In the present study, we characterized the generation of prostaglandin (PG)E2 in human neutrophils. We found that the Ca2+-dependent type IV cytosolic phospholipase A2 (cPLA2) was pivotally involved in the COX-2-mediated generation of PGE2 in response to a calcium ionophore, as determined by the use of selected PLA2 inhibitors. PGE2 biosynthesis elicited by bacterial-derived peptides or by phagocytic stimuli acting on cell surface receptors also showed to be dependent on cPLA2 activity. We then assessed metabolism of unesterified arachidonic acid (AA), and observed that PGE2 production becomes favored over that of LTB4 with higher AA concentrations. Withdrawal of calcium prevented the generation of PGE2 in response to a calcium ionophore but did not affect the up-regulation of COX-2 or its capacity to convert AA, thus limiting its implication at the level of cPLA2 activation. Of the main eicosanoids produced by neutrophils, only LTB4 was able to up-regulate COX-2 expression. Finally, the only PGE synthase isoform found in neutrophils is microsomal PGE synthase-1; it co-localized with COX-2 and its expression appeared mainly constitutive. These results highlight key differences in regulatory processes of the 5-LO and COX pathways, and enhance our knowledge at several levels in the PGE2 biosynthesis in neutrophils.
Keywords: Inflammation; Polymorphonuclear leukocyte; Cyclooxygenase; Fatty acid; Eicosanoids; Prostaglandins;
Mechanistic studies of the long chain acyl-CoA synthetase Faa1p from Saccharomyces cerevisiae by Hong Li; Elaina M. Melton; Steven Quackenbush; Concetta C. DiRusso; Paul N. Black (1246-1253).
Long chain acyl-CoA synthetase (ACSL; fatty acid CoA ligase: AMP forming; EC 18.104.22.168) catalyzes the formation of acyl-CoA through a process, which requires fatty acid, ATP and coenzymeA as substrates. In the yeast Saccharomyces cerevisiae the principal ACSL is Faa1p (encoded by the FAA1 gene). The preferred substrates for this enzyme are cis-monounsaturated long chain fatty acids. Our previous work has shown Faa1p is a principal component of a fatty acid transport/activation complex that also includes the fatty acid transport protein Fat1p. In the present work hexameric histidine tagged Faa1p was purified to homogeneity through a two-step process in the presence of 0.1% η-dodecyl-β-maltoside following expression at 15 °C in Escherichia coli. In order to further define the role of this enzyme in fatty acid transport-coupled activation (vectorial acylation), initial velocity kinetic studies were completed to define the kinetic parameters of Faa1p in response to the different substrates and to define mechanism. These studies showed Faa1p had a V max of 158.2 nmol/min/mg protein and a K m of 71.1 μM oleate. When the concentration of oleate was held constant at 50 μM, the K m for CoA and ATP were 18.3 μM and 51.6 μM respectively. These initial velocity studies demonstrated the enzyme mechanism for Faa1p was Bi Uni Uni Bi Ping Pong.
Keywords: Long chain acyl CoA synthetase; Mechanistic enzymology; Fatty acid transport;
Distinct metabolic handling of 3β-hydroxy-17a-oxa-D-homo-5α-androstan-17-one by the filamentous fungus Aspergillus tamarii KITA: Evidence in support of steroid/hydroxylase binding hypothesis by A. Christy Hunter; Hild Bergin-Simpson (1254-1261).
Aspergillus tamarii KITA transforms progesterone in to testololactone in high yield through a sequential four-step enzymatic pathway which also has the flexibility to transform a range of steroidal substrates. This study has investigated the further metabolism of testololactone and a range of fully saturated steroidal lactone analogues. In contrast to testololactone, which even after 120 h incubation did not undergo further metabolism, the lactone analogues entered the minor hydroxylation pathway. Uniquely, after forming 3β-hydroxy-17a-oxa-D-homo-5α-androstan-17-one (48 h) 4 distinct positions on the steroid skeleton were monohydroxylated (11β, 6β, 7β, 11α) which geometrically relate to the four binding positions (normal, reverse, inverted normal and inverted reverse) possible within the steroidal hydroxylase(s). This is the first evidence demonstrating the four possible steroid/hydroxylase(s) binding interactions with a single molecule that has previously been hypothesized with a single organism. In addition a rare 1β-monohydroxylation was observed, this may be indicative of dehydration generating 1-ene functionality in A. tamarii rather than dehydrogenation as reported in man and microorganisms. The importance of these findings in relation to steroid/hydroxylase binding interactions is discussed.
Keywords: Hydroxylation; Steroid/hydroxylase; Binding orientation; Aspergillus tamarii; Steroidal lactone; Microbiological transformation; Enzymatic dehydration;
Corrigendum to “Suppression of mast cell degranulation by a novel ceramide kinase inhibitor, the F-12509A olefin isomer K1” [Biochim. Biophys. Acta 1738 (2005) 82–90] by Jin-Wook Kim; Yuichi Inagaki; Susumu Mitsutake; Nobuhiro Maezawa; Shigeo Katsumura; Yeon-Woo Ryu; Chang-Seo Park; Masaru Taniguchi; Yasuyuki Igarashi (1262).