Kinetics of growth and fatty acid production of Rhodotorula glutinis

Summary The effect of culture conditions, especially nitrogen/carbon (N/C) ratio and temperature, on acyl lipid profiles in the oleaginous yeast Rhodotorula glutinis NRRL Y 1091 have been investigated. Cultivation of the microorganism under N-limited conditions (N/C < 0.14 g/g) resulted in enhanced fatty acid (FA) cell content but a reduced relative amount of polyunsaturated fatty acids (PUFA). The maximal FA productivity was obtained for a 0.025 N/C ratio resulting from the arrangement between the specific rate of FA synthesis and the concentration of lipid-free biomass. Under nitrogen-non-limiting conditions, cells grown at lower temperatures had a higher PUFA content and the maximal productivity of -linolenic acid was obtained by shifting the temperature of the culture from 30° C to 25° C. Offprint requests to: A. Pareilleux     

Mathematical modeling of production of microbial lipids

As a part of the investigations on the microbial lipid production using the yeast Rhodotorula gracilis, CFR-1, kinetics of the biomass synthesis has been studied using shake flask experiments. Using a medium containing a carbon to nitrogen ratio of 701, the rates of biomass production were followed at different initial substrate concentrations in the range of 20–100 kg/m³. A logistic model was found to be reasonably adequate to describe the kinetics of the growth of biomass; the maximum specific growth rate of 0.105 h^–1 was applicable for substrate concentrations less than 60 kg/m³, which gave reasonable agreement between predicted and actual biomass concentration values.List of Symbols S ₀, X ₀ kg/m³ Initial concentrations of sugar, non lipid biomass respectively - X, X(t) kg/m³ Concentrations of non lipid biomass at any time t - dX/dt kg/(m³ · h) Rate of biomass growth - h^–1 Specific growth rate - _max h^–1 Maximum specific growth rate - K _s mol/dm³ Monods constant - X _max kg/m³ Maximum biomass reached in a run     

Lipid formation by Cryptococcus albidus in nitrogen-limited and in carbon-limited chemostat cultures

Summary Cryptococcus albidus var. Albidus CBS 4517 was grown in nitrogen-limited and in carbon-limited chemostat cultures. The effect of growth rate and limiting nutrient on lipid accumulation and fatty acid composition was investigated.The maximum lipid content in the biomass was, in both cultivation systems, observed at the lowest dilution rate (growth rate) tested. At this dilution rate, D=0.31 h^-1, cells from the nitrogen-limited culture contained 41% (w/w) lipid and cells from the carbon-limited culture 37%. These results indicate the ability of C. albidus, unlike other oleaginous yeasts, to accumulate lipid also in carbon-limited chemostats.The yield of lipid from carbon source was about the same at D=0.031 h^-1 in nitrogen-limited (Y _L/S=0.16 g/g) as in carbon-limited (Y _L/S=0.17 g/g) cultures and decreased with increasing growth rates. In the nitrogen-limited culture, the lipid productivity was about constant at low growth rates (0.031–0.056 h^-1) and a slight decrease was observed at D=0.08 h^-1, while the specific lipid productivity, q _L, increased to 27.5 mg/g per hour. In the carbon-limited culture, however, lipid productivity increased with increasing growth rates and reached its maximum value near _max, whereas q _L was about constant at 20 mg/g per hour.The fatty acid composition was influenced by the specific growth rate in nitrogen-limited as well as in carbon-limited cultures, although the changes were more pronounced during carbonlimitation. A decrease in the degree of unsaturation (/mole) was also observed with increasing lipid content in the cells.     

Formal kinetics of poly-β-hydroxybutyric acid (PHB) production in Alcaligenes eutrophus H 16 and Mycoplana rubra R 14 with respect to the dissolved oxygen tension in ammonium-limited batch cultures

Summary Under chemolithoautotrophic growth conditions with the organism Alcaligenes eutrophus H16 the exponential growth phase is characterized by two different growth rates, each associated with different specific rates of ammonium consumption. On the basis of the analytical determination of Poly--hydroxybutyric acid (PHB), it can be conclusively shown that PHB is synthesized even during the exponential growth phase at a specific rate proportional to the specific growth rates of total biomass. After complete consumption of ammonium, the increase of biomass is exclusively due to PHB synthesis, whereas protein and rest biomass (cell dry weight minus PHB) remain constant. After an extended period of fermentation, the PHB content reaches a saturation value. The transient phase between the growth and the storage phase is very short in comparison to the duration of the whole fermentation. In the case of Alcaligenes eutrophus, strain H 16, high concentrations of dissolved oxygen strongly influence growth as well as PHB synthesis.Abbrevations ^cO₂,L concentration of oxygen in the liquid phase (dissolved oxygen tension: d.o.t) - ^cH₂,L concentration of hydrogen in the liquid phase - ^cCO₂,L concentration of carbon dioxide in the liquid phase - S limiting substrate, concentration of - X total biomass, concentration of; total cell dry weight - P product; PHB, concentration of - R rest biomass: X-P, concentration of - ^rX dX/dt growth rate - ^rP dP/dt rate of PHB synthesis - ^rR dR/dt rate of rest biomass production - ^r0 ^dcO₂,L/dt rate of oxygen consumption - X dX/dt·1/X=r_X·1/X specific growth rate - P dP/dt·1/P=r_P·1/P specific rate of product formation - R dR/dt·1/R=r_R·1/R specific rate of rest biomass formation - r₀/R specific respiration rate     

Lipid production by an unsaturated fatty acid auxotroph of the oleaginous yeast Apiotrichum curvatum grown in single-stage continuous culture

An unsaturated fatty acid auxotroph of the oleaginous yeast Apiotrichum curvatum, named UfaM3, blocked in the conversion of stearic to oleic acid was cultivated in single-stage continuous culture. The influence of consumed carbon to nitrogen ratios (C/N ratios, g g^–1) obtained at various dilution rates (D) on fatty acid (FA) accumulation and its profiles were studied. In continuous culture in N-limited medium a maximum FA accumulation of 45.6% (g g^–1 of dry biomass) was obtained at an optimal D of 0.049 h^–1, recording an efficiency of substrate conversion of 0.48 g g^–1 and 0.22 g g^–1 for biomass and lipids, respectively. The quality of lipid approached cocoa butter at an optimal C/N ratio of between 20 and 30. The C/N ratio in the incoming medium was 38.5 g g^–1 with 30 g l^–1 of glucose and both C and N sources were completely consumed at a critical D of 0.07 h^–1. The stability of the mutant was demonstrated in the steady-state conditions of the chemostat with regard to the FA composition of its lipids. Correspondence to: P. J. Blanc     

Specific inhibition of tetrapyrrole biosynthesis by 3-amino 2,3-dihydrobenzoic acid (gabaculin) in the cyanobacteriumSynechococcus 6301

Gabaculin (3-amino 2,3-dihydrobenzoic acid) inhibited the growth of cyanobacteria but not of other prokaryotes. Exposure of growing cultures ofSynechococcus 6301 to 50 M gabaculin resulted in an immediate and complete inhibition of the synthesis of chlorophylla and phycocyanin. With 8 M gabaculin, tetrapyrrole synthesis was suppressed for approximately 10 h and then resumed at a lower rate than in untreated organisms. The effect of 50 M gabaculin was reversed by transferring organisms to inhibitor-free medium; chlorophylla synthesis began within 5 h and exponential growth was re-established after about 25 h. Compared with 4,6-dioxoheptanoic acid (DA) and laevulinic acid (LA), gabaculin was a much more potent inhibitor of tetrapyrrole synthesis inSynechococcus 6301. The catalytic activity of -aminolaevulinic acid (ALA) dehydratase in vitro was inhibited by DA and LA but not by 1 mM gabaculin. However, the specific activity of the dehydratase was much lower in organisms exposed to the inhibitor for 36 h. Growing cultures and cell suspensions ofSynechococcus 6301 exposed to DA excreted appreciable quantities of ALA. In contrast, relatively small amounts of ALA accumulated in the presence of gabaculin alone and this inhibitor blocked the excretion of ALA caused by DA. This suggests that the primary effect of gabaculin is the specific inhibition of the C₅ pathway for the biosynthesis of ALA.Abbreviations ALA -aminolaevulinic acid - DA 4,6-dioxoheptanoic acid - LA laevulinic acid - GABA -aminobutyric acid     

Fatty acid composition of vitellogenin from four teleost species

The fatty acid compositions of vitellogenin and liver from cod (Gadus morhua), rainbow trout (Oncorhynchus mykiss), turbot (Scophthalmus maximus) and wolffish (Anarhichas lupus) were determined. Vitellogenin was isolated from plasma of estradiol-17-treated fish by precipitation with EDTA-Mg²⁺ and distilled water or by high-performance ion-exchange chromatography. In all investigated species, vitellogenin contained 16–18% (w/w) lipid, in which polyunsaturated fatty acids, predominantly 20:5 (n-3) and 22:6 (n-3), comprised about 50% of the total fatty acids. The proportions of saturated, monounsaturated, polyunsaturated and (n-3) fatty acids in vitellogenin of the different species were generally similar, although the relative content of specific fatty acids was distinctive for each species. The distribution of fatty acids in total lipids of vitellogenin was highly consistent among individual females of each species. In contrast, liver fatty acid composition varied considerably, both within and between species. Altogether, the differences in the fatty acid composition of vitellogenin and liver from each species indicate that a specific selection of fatty acids occurs during the lipidation of vitellogenin.Abbreviations BHT butylated hydroxytoluene - E-17 estradiol-17 - EDTA ethylenedinitrilo tetra-acetic acid disodium salt dihydrate - FA fatty acids - FAME fatty acid methyl esters - HDL high density lipoproteins - PUFA polyunsaturated fatty acids - SD standard deviation - TLC thin-layer chromatography - VHDL very high density lipoproteins - VLDL very low density lipoproteins - v/v volume per volume - w/v weight per volume - w/w weight per weight     

Effect of Inducers on the Production of 5‐Aminolevulinic Acid by Recombinant Escherichia coli

Abstract

Aminolevulinic acid (ALA) was produced by recombinant Escherichia coli BL21(DE3) (pET28‐A.R‐hemA) harboring the ALA synthase gene (hemA) from Agrobacterium radiobacter zju‐0121. The effects of inducers on the ALA synthase activity and ALA productivity were evaluated. The results indicated that a low isopropyl‐β‐D‐thiogalactoside (IPTG) concentration (0.05 mmol/L) was favorable for high expression of ALA synthase, which resulted in higher ALA productivity. For metabolic engineering applications, lactose was a better substitute of IPTG for active enzyme expression. When lactose concentration was 5 mmol/L, the specific ALA synthase activity and ALA productivity reached 16.7 nmol/(min · mg of protein) and 1.15 g/L, respectively, which were about 15% and 43% higher than those induced by IPTG.     

RISING WATER TEMPERATURES ALTER LIPID DYNAMICS AND REDUCE N‐3 ESSENTIAL FATTY ACID CONCENTRATIONS IN SCENEDESMUS OBLIQUUS (CHLOROPHYTA)1

The biosynthesis of nutritionally important polyunsaturated fatty acids (PUFAs) in phytoplankton is influenced by environmental temperature. We investigated the potential of climate warming to alter lipid dynamics of Scenedesmus obliquus (Turpin) Kütz. by comparing lipid and fatty acid (FA) profiles as well as FA metabolism (using [1‐¹⁴C] acetate) at 20°C and 28°C. We documented an overall decline (53%–37%) in the proportion of n‐3 PUFA (in particular, of α‐linolenic acid [ALA; 18:3n‐3]), and a concomitant increase in saturated fatty acids (SAFAs) in total lipids (TLs) at 28°C, consistent with enhanced incorporation of radioactivity from [1‐¹⁴C] acetate into total 16:0, 18:1, and decreased incorporation into 18:2 and 18:3 FA (from 36% to 22% of the total) at 28°C. Glycerophospholipids were also affected by warming; ALA and stearidonic acids (SDAs; 18:4n‐3) both decreased (by 13% and 15%, respectively) in phosphatidylcholine (PC) and (by 24% and 20%, respectively) in phosphatidylethanolamine (PE). The characteristic FA in phosphatidylglycerol (PG; 16:1n‐13t) increased (by 22%) at 28°C. The activities of desaturases, which add double bonds to FA moieties, comprised the major suite of reactions affected by the temperature increase in TL and polar lipid (PL) classes. Climate modelers predict an increase in the number of extreme heat days in summer at temperate latitudes, with parallel projected increases in water temperatures of shallow water bodies. Our results suggest that the overall decrease in the essential n‐3 FA ALA in S. obliquus at higher water temperatures may lower food quality for higher tropic levels, adding another climate‐warming stress.     

Kinetic analysis of oil biosynthesis by an arachidonic acid-producing fungus, Mortierella alpina 1S-4

 Kinetic analysis of arachidonic acid (AA)-oil biosynthesis by Mortierella alpina 1S-4 growing under lipid-accumulating (LN medium) and non-lipid-accumulating (HN medium) conditions was investigated and compared with industrial AA fermentation. Various kinetic parameters of these cultivation processes demonstrate a characteristic pattern of the lipogenesis in this fungus, where growth phase, phase of oil accumulation and phase of AA synthesis are distinct from each other. The fungus utilizing LN medium synthesized 32.3 g fatty acid 100 g⁻¹ glucose on the 4th day of cultivation and reached the maximum daily fatty acid accumulation (expressed as differential specific rate q _D(FA/B)) of 9.5%. Our results also indicate that a q _D(FA/B) value of about 2.5% might be critical for lipid overproduction in M. alpina. AA was rapidly incorporated into triacylglycerols (90% of total AA) at the later cultivation phase and overall AA yield was directly related to the total yield of fatty acid. Received: 10 December 1999 / Accepted: 13 February 2000     

The effect of 2,4-dichlorophenoxyacetic acid and (2-chloroethyl)-trimethylammonium chloride on chlorophyll synthesis in barley leaves

Summary 2.4-dichlorophenoxyacetic acid (2.4-D) and (2-chloroethyl)-trimethylammonium chloride (CCC) inhibit chlorophyll synthesis and protochlorophyllide ₆₅₂ regeneration in 6–8 day old barley leaves whilst having little effect on the rates of protochlorophyll ₆₃₂ synthesis from exogenous -aminelevulinic acid (ALA) and ALA-dehydratase activity. Longer pretreatments with 2.4-D and CCC show it is only after 50 to 60 hr that the rates of P₆₃₂ production from exogenous ALA and ALA-dehydratase activity are affected. Similar response times were obtained for chloramphenicol (CAP). The results indicate that 2.4-D and CCC may act by directly inhibiting specific plastid-protein synthesis similar to CAP. Hence it seems that it is only those proteins (enzymes) having a rapid turnover that are affected first i.e. the enzymes necessary for ALA synthesis in the plastid.Abbreviations used ALA -aminolevulinic acid - CAP chloramphenicol - CCC (2-chloroethyl)-trimethylammonium chloride - 2.4-D 2-4-dichlorophenoxyacetic acid - P₆₅₂ prodochlorophyllide with maximum in-vivo absorption at 652 nm - P₆₈₄ chlorophyllide absorbing at 684 nm - P₆₇₀ chlorophyllide absorbing at 670 nm - P₆₃₂ pigment absorbing at 632 nm synthesised from exogenous ALA - PBG Porphobilinogen P. R. Shewry is in receipt of a Science Research Council Studentship award.     

Metabolic engineering of lipid pathways in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> and staged bioprocess for enhanced lipid production and cellular physiology

Microbially produced lipids have attracted attention for their environmental benefits and commercial value. We have combined lipid pathway engineering in Saccharomyces cerevisiae yeast with bioprocess design to improve productivity and explore barriers to enhanced lipid production. Initially, individual gene expression was tested for impact on yeast growth and lipid production. Then, two base strains were prepared for enhanced lipid accumulation and stabilization steps by combining DGAT1, ΔTgl3 with or without Atclo1, which increased lipid content ~?1.8-fold but reduced cell viability. Next, fatty acid (FA) biosynthesis genes Ald6-SEACS^L641P alone or with ACC1** were co-expressed in base strains, which significantly improved lipid content (8.0% DCW, 2.6-fold than control), but severely reduced yeast growth and cell viability. Finally, a designed two-stage process convincingly ameliorated the negative effects, resulting in normal cell growth, very high lipid productivity (307 mg/L, 4.6-fold above control) and improved cell viability.     

Intramyelinic conversion of cerebrosides into acylgalactosylceramides

Acylgalactosylceramide (AGC) synthesis was measured in vivo, and in a cell free system. 24 hours post-injection of [³H]palmitic acid into rat brain, more than 60% of the AGC radioactivity was associated with an ester linkage. Isolated rat myelin was incubated in the presence of [¹⁴C]palmitic acid, 2mM ATP, 50 M CoA and 10 mM MgCl₂ and acylation of myelin cerebrosides occurred at a linear rate for at least 60 min. Incubation of isolated myelin under standard conditions with [³H] cerebrosides and [¹⁴C]palmitic acid produced double labeled AGC. Labeling of AGC was maximum at pH 7.5 and 37°C and appeared to be enzyme mediated inasmuch as it was reduced by myelin incubation with trypsin and drastically reduced by preheating the myelin for 5 min at 80°C. Omission of ATP, CoA, MgCl₂ or all three did not reduce fatty acid incorporation into AGC when compared to the values in the complete system. Addition of Triton X100 or Sodium Dodecyl Sulfate had little or no effect on the acylation of cerebrosides. Pulse chase experiments indicated that the reaction involved the net addition of fatty acid to the cerebrosides, rather than a rapid fatty acid exchange.     

Engineering <Emphasis Type="Italic">Escherichia coli</Emphasis> for efficient coproduction of polyhydroxyalkanoates and 5-aminolevulinic acid

Single-cell biorefineries are an interesting strategy for using different components of feedstock to produce multiple high-value biochemicals. In this study, a strategy was applied to refine glucose and fatty acid to produce 5-aminolevulinic acid (ALA) and polyhydroxyalkanoates (PHAs). To express the ALA and PHAs dual-production system efficiently and stably, multiple copies of the poly-β-3-hydroxybutyrate (PHB) synthesis operon were integrated into the chromosome of Escherichia coli DH5αΔpoxB. The above strain harboring the ALA C5 synthesis pathway genes hemA and hemL resulted in coproduction of 38.2% PHB (cell dry weight, CDW) and 3.2 g/L extracellular ALA. To explore coproduction of ALA and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), the PHBV synthetic pathway was also integrated into engineered E. coli and coexpressed with hemA and hemL; cells produced 38.9% PHBV (CDW) with 10.3 mol% 3HV fractions and 3.0 g/L ALA. The coproduction of ALA with PHB and PHBV can improve the utilization of carbon sources and maximize the value derived from the feedstock.     

Fatty acid composition of lipids from mushrooms belonging to the family Boletaceae

The non-polar lipid content and fatty acid (FA) composition of 11 mushroom species of the family Boletaceae were determined. The non-polar lipid content ranged from 2.0 (Leccinum aurantiacum and Boletus erythropus) to 5.4 % (w/w) d.w. (Suillus grevillei) with an average value of 2.9 %. More than 25 different FAs were found in the mushroom lipids. Unsaturated FAs, mainly linoleic and oleic acids, accounted for about 83 % of the total FAs, while palmitic acid was the main saturated FA. Some FAs are identified for the first time in Boletaceae and in higher Basidiomycetes (cis-11,12-methyleneoctadecanoic acid, 7-cis,10-cis hexadecadienoic) or in fungi (cis-11,12-methyleneoctadecanoic acid). There were significant differences (P < 0.05) in the contents of specific FAs between mushroom species.     

Dietary fats,selected body temperature and tissue fatty acid composition of agamid lizards (Amphibolurus nuchalis)

The composition of tissue and membrane fatty acids in ectothermic vertebrates is influenced by both temperature acclimation and diets. If such change in body lipid composition and thermal physiology were linked, a diet-induced change in body lipid composition should result in a change in thermal physiology. We therefore investigated whether the selected body temperature of the agamid lizardAmphibolurus nuchalis (body mass 20 g) is influenced by the lipid composition of dietary fatty acids and whether diet-induced changes in thermal physiology are correlated with changes in body lipid composition. The selected body temperature in two groups of lizards was indistinguishable before dietary treatments. The selected body temperature in lizards after 3 weeks on a diet rich in saturated fatty acids rose by 2.1 °C (photophase) and 3.3 °C (scotophase), whereas the body temperature of lizards on a diet rich in unsaturated fatty acids fell by 1.5 °C (photophase) and 2.0 °C (scotophase). Significant diet-induced differences were observed in the fatty acid composition of depot fat, liver and muscle. These observations suggest that dietary lipids may influence selection of body temperature in ectotherms via alterations of body lipid composition.Abbreviations bm body mass - FA fatty acid(s) - MUFA monounsaturated fatty acids - PUFA polyunsaturated fatty acids - SFA saturated fatty acids - T _a air temperature - T _b body temperature - UFA unsaturated fatty acids     

Optimization of cell density and dilution rate in <Emphasis Type="Italic">Pichia pastoris</Emphasis> continuous fermentations for production of recombinant proteins

This paper provides an approach for optimizing the cell density (X_c) and dilution rate (D) in a chemostat for a Pichia pastoris continuous fermentation for the extracellular production of a recombinant protein, interferon (INF-). The objective was to maximize the volumetric productivity (Q, mg INF- l^–1 h^–1), which was accomplished using response surface methodology (RSM) to model the response of Q as a function of X_c and D within the ranges 150 X_c 450 g cells (wet weight) l^–1 and 0.1 _mD0.9 _m (_m=0.0678 h^–1, the maximum specific growth rate obtained from a fed-batch phase controlled with a methanol sensor). The methanol and medium feed rates that resulted in the desired X_c and D were determined based on the mass balance. From the RSM model, the optimal X_c and D were 328.9 g l^–1 and 0.0333 h^–1 for a maximum Q of 2.73 mg l^–1 h^–1. The model of specific production rate (, mg INF- g^–1 cells h^–1) was also established and showed the optimal X_c=287.7 g l^–1 and D=0.0361 h^–1 for the maximum (predicted to be 8.92×10^–3 mg^–1 g^–1 h^–1). The methanol specific consumption rate (, g methanol g^–1 cells h^–1) was calculated and shown to be independent of the cell density. The relationship between and (specific growth rate) was the same as that discovered from fed-batch fermentations of the same strain. The approach developed in this study is expected to be applicable to the optimization of continuous fermentations by other microorganisms.     

Accumulation of lipid byRhodotorula glutinis in continuous culture

Summary Rhodotorula glutinis accumulated 35% (w/w) lipid when grown nitrogen-limited in a chemostat at a dilution rate (D) of 0.02^–1 . At D = 0.10 h^–1, the lipid content was only 15% (w/w). Dual limitation of nitrogen and phosphate increased neither the amount of lipid produced nor the lipid yield (14g lipid per 100g glucose consumed). The fatty acid composition was unchanged by the growth rate.     

Growth hormone stimulates hepatic lipid mobilization in rainbow trout,Oncorhynchus mykiss

Rainbow trout were used to characterize the direct influence of growth hormone on hepatic lipid mobilization in vitro. Liver was removed from fish fasted 72 h, sliced into 1-mm³ pieces and incubated in Hanks' medium containing ovine or salmon growth hormone (0.28 ng·ml^-1–28 g·ml^-1). Lipid mobilization, resulting from triacylglycerol hydrolysis, was evaluated by fatty acid and glycerol release into culture medium. Control rates of fatty acid release and glycerol release were 0.95±0.16 (mean ± SE) and 0.88±0.28 mol·l^-1·mg fresh weight, respectively. Both ovine growth hormone (28 ng·ml^-1) and salmon growth hormone (28 ng·ml^-1) stimulated fatty acid release into culture medium, increasing rates by 112% and 97%, respectively, during the course of a 24-h incubation. Glycerol release was similarly augmented by growth hormone treatment. Growth hormone-stimulated metabolite release became evident within 12 h and persisted for up to 72 h. The presence of amino acids in the culture medium potentiated the lipolytic action of growth hormone. Ovine growth hormone (28 ng·ml^-1) in the presence of amino acids stimulated a 53% increase in fatty acid, and a 108% increase in glycerol, release over rates observed in the absence of amino acids. Salmon growth hormone (28 ng·ml^-1) in the presence of amino acids stimulated a 53% increase in fatty acid, and a 44% increase in glycerol, release over rates observed in the absence of amino acids. Ovine growth hormone (28 ng·ml^-1) also stimulated gluconeogenesis, as indicated by a 75% increase in phosphoenolpyruvate carboxykinase activity in liver pieces incubated in the presence of amino acids. These results indicate that growth hormone directly stimulates lipid breakdown in the liver of trout and that amino acids potentiate growth hormone-stimulated lipolysis.Abbreviations AA amino acid(s) - dGDP deoxy-guanosine diphosphate - ED ₅₀ 50% effective dose - FA fatty acid(s) - fw fesh weight - GH growth hormone - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid - MS-222 tricaine methanesulfonate - MEM minimum essential medium - oGH ovine growth hormone - PEPCK phosphoenolpyruvate carboxykinase - PK_c protein kinase C - rpm revolutions per minute - sGH salmon growth hormone - TG triacylglycerol - w/v weight per volume This paper was presented in abstract form at the Annual Meeting of the American Society of Zoologists, Dec. 26–30, 1991, Atlanta, GA     

Protein acylation in the cardiac muscle like cell line,H9c2

Besides serving as oxidisable substrates, fatty acids (FA) are involved in co- and post-translational modification of proteins (protein acylation). Despite the high rate of fatty acid utilisation in the heart, information on protein acylation in cardiac muscle is scarce. To explore this subject in more detail, we used the H9c2 cell line as an experimental model. After incubation with ³H-palmitate or ³H-myristate, cells were lysed and proteins precipitated, followed by extensive delipidation. The delipidated proteins were subjected to SDS-PAGE and transferred to nitro-cellulose prior to autoradiography. In addition, TLC was used to separate the various lipid classes. The first aspect we addressed was the extent of protein acylation as a function of time, relative to fatty acid incorporation into various lipid classes. Cells were incubated for 30 min, 1 h and 2 h with 100 Ci palmitate (PA, 2.3 nmol) or 125 Ci myristate (MA, 2.5 nmol). Palmitoylation increased from 0.48 ± 0.25 to 1.25 ± 0.56 Ci/mg protein between 30 min to 2 h, while myristoylation increased from 0.25 ± 0.12 to 0.77 ± 0.36 Ci/mg protein. Furthermore, delipidated proteins subjected to autoradiography showed that a set of distinct proteins was labelled with ³H-palmitate. Incorporation into phospholipids (PL) increased from 40–60% of the total amount of radio-labelled PA or MA supplied between 30 min and 2 h. Only the FA pool differed between MA and PA, with a higher FA content present after incubations with MA. Second, we investigated palmitoylation and incorporation into cellular lipids as a function of the amount of PA applied. Palmitoylation showed saturation at high PA concentrations. The percentage incorporation of ³H-PA in the various lipids depended on the amount of PA added: a decline in the PL pool with a concomitant increase in the size of the diacylglycerol pool at high PA concentrations. Third, inhibition of palmitoylation by cerulenin and tunicamycin was investigated. While both were able to inhibit palmitoylation, cerulenin also inhibited the incorporation of PA into various lipid classes, indicating differences in inhibitory action.