A multinuclear, high-resolution NMR study of bovine casein micelles and submicelles

High-resolution, natural abundance 13C[1H] (100.5 MHz), 31P[1H] (161.8 MHz) and 1H (400.0 MHz) NMR spectroscopy was used to identify the calcium-binding sites of bovine casein and to ascertain the dynamic state of amino acid residues within the casein submicelles (in 125 mM KCl, pD = 7.4) and micelles (in 15 mM CaCl2/80 mM KCl, pD = 7.2). The presence of numerous, well-resolved peaks in the tentatively assigned 13C-NMR spectra of submicelles (90 A radius) and micelles (500 A radius) suggests considerable segmental motion of both side chain and backbone carbons. The partly resolved 31P-NMR spectra concur with this. Upon Ca2+ addition, the phosphoserine beta CH2 resonance (65.8 ppm vs DSS) shifts upfield by 0.2 ppm and is broadened almost beyond detection; a general upfield shift (up to 0.3 ppm) is also observed for the 31P-NMR peaks. The T1 values of the alpha CH envelope for submicelles and micelles are essentially identical corresponding to a correlation time of 8 ns for isotropic rotation of the caseins. Significant changes in the 31P T1 values accompany micelle formation. Data are consistent with a loose and mobile casein structure, with phosphoserines being the predominant calcium-binding sites.     

Thermodynamic and molecular determinants of sterol solubilities in bile salt micelles

We examined, by reverse-phase high performance liquid chromatography (HPLC), the hydrophilic-hydrophobic balance of cholesterol and 12 non-cholesterol sterols and related this property to their equilibrium micellar solubilities in sodium taurocholate and sodium glycodeoxycholate solutions. Sterols investigated exhibited structural variations in the polar function (3 alpha-OH, 3 beta-OH, 3 beta-SH), nuclear double bonds (none, delta 5, or delta 7), side chain length (C27, C28, C29) and side chain double bonds (none, delta 22, or delta 24). In general, a sterol's hydrophilic-hydrophobic balance became progressively more hydrophobic (as exemplified by increasing HPLC retention values, k') with additions of side chain methyl (C28) and ethyl (C29) groups and with 3 beta-SH substitution of the 3-OH polar function. Side chain delta 22 and especially delta 24 double bonds rendered the sterols appreciably more hydrophilic, whereas a single nuclear double bond had little influence. Sterol solubilities (24 degrees C, 0.15 M Na+) were uniformly greater in 50 mM solutions of sodium glycodeoxycholate (range 0.15 to 2.5 mM) than in equimolar solutions of the more hydrophilic bile salt, sodium taurocholate (range 0.07 to 0.67 mM). For each bile salt system, a strong inverse correlation existed between micellar solubilities of sterols and their HPLC k' values, indicating that more hydrophilic sterols had greater micellar solubilities than the more hydrophobic ones. Based upon the aqueous monomeric solubilities of cholesterol (C27) and beta-sitosterol (C29) at 24 degrees C, we derived free energy changes associated with micellar binding and found that solubilization of both sterols was more energetically favored in glycodeoxycholate solutions. Although cholesterol exhibited a higher binding affinity than beta-sitosterol in glycodeoxycholate micelles, solubilization of beta-sitosterol in taurocholate micelles was more energetically favored than cholesterol by -0.6 kcal/mol. Based upon these results we offer a thermodynamic explanation for the greater micellar solubilities of more hydrophilic sterols and suggest that the high affinity, but low capacity, of a typical phytosterol for binding to trihydroxy bile salt micelles may provide a physical-chemical basis for its inhibition of intestinal cholesterol absorption.     

Sucrose and starch metabolism in carrot (Daucus carota L.) cell suspensions analysed by 13-labelling: indications for a cytosol and a plastid-localized oxidative pentose phosphate pathway

Cells were grown in batch culture on a mixture of 50 mM glucose and fructose as the carbon source; either the glucose or the fructose was [1-¹³C]-labelled. In order to investigate the uptake and conversion of glucose and fructose during long-term labelling experiments in cell suspensions of Daucus carota L., samples were taken every 2 d during a 2 week culture period and sucrose and starch were assayed by means of HPLC and ¹³C-nuclear magnetic resonance. The fructose moieties of sucrose had a lower labelling percentage than the glucose moieties. Oxidative pentose phosphate pathway activity in the cytosol is suggested to be responsible for this loss of label of especially C-1 carbons. A combination of oxidative pentose phosphate pathway activity, a relatively high activity of pathway to sucrose synthesis and a slow equilibration between glucose-6-phosphate and fructose-6-phosphate could explain these results. Starch contained glucose units with a much lower labelling percentage than glucose moieties of sucrose: it was concluded that a second, plastid-localized, oxidative pentose phosphate pathway was responsible for removal of C-1 carbons of the glucosyl units used for synthesis of starch. Redistribution of label from [1-¹³C]-hexoses to [6-¹³C]-hexoses also occurred: 18-45% of the label was found at the C-6 carbons. This is a consequence of cycling between hexose phosphates and those phosphates in the cytosol catalysed by PFP. The results indicate that independent (oxidative pentose phosphate pathway mediated) sugar converting cycles exist in the cytosol and plastid.Key words: Daucus carotaL., cell suspensions, carbon-13 nuclear magnetic resonance, ¹³C-NMR, carbohydrate cycling, oxidative pentose phosphate pathway, plastid.      

Temperature dependence of the vesicle-micelle transition of egg phosphatidylcholine and octyl glucoside

The temperature dependence of octyl glucoside micellization was determined and compared to the phase behavior of the octyl glucoside--egg phosphatidylcholine (PC) mixed system in excess water to help elucidate the process of vesicle formation from mixed surfactant-phospholipid micelles. The critical micelle concentration of octyl glucoside (OG) was determined from the sharp increase of ANS fluorescence at micellization in an NaCl buffer at temperatures ranging from 5 to 40 degrees C. The cmc decreased with increasing temperature from 31 mM at 5 degrees C to 16 mM at 40 degrees C. A similar but less steep temperature dependence is observed for the solubilization of egg PC vesicles by OG as monitored by the surfactant-dependent changes in (1) solution turbidity and (2) the resonance energy transfer between NBD-PE and Rho-PE incorporated in the vesicles. These assays identify two breakpoints, most likely the boundaries of the cylindrical micelle and spheroidal micelle coexistence region. The [OG]aq values at these two breakpoints have similar temperature dependencies. However, the cylindrical mixed micelles at the boundary have nearly identical OG:PC ratios over the temperature range studied, whereas the spheroidal mixed micelles have relatively more OG at the higher temperatures (OG:PC ratio increases from 2.92 to 3.72 between 5 and 35 degrees C). Estimation of the acyl volume to surface area ratio for the compositions observed suggests that this parameter remains constant over temperature. The spheroidal mixed micelles, but not the cylindrical PC-OG micelles, exhibit ideal mixing between the two components at all temperatures (5-35 degrees C). This temperature sensitivity may be utilized to improve the efficacy of membrane protein reconstitution.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of free cholesterol on the solubilization of cholesteryl oleate in phosphatidylcholine bilayers: A 13C-NMR study

The solubilization of cholesteryl oleate in sonicated phosphatidylcholine vesicles containing between 0 and 50 mol% cholesterol was studied by 13C-NMR using isotopically enriched [carbonyl-13C]cholesteryl oleate. The carbonyl-13C chemical shift from cholesteryl oleate in the phospholipid/cholesterol bilayer was significantly downfield from that for cholesteryl oleate in an oil phase and the peak area, relative to that of the phospholipid carbonyl, was used to determine bilayer solubility of the ester. The solubility (with respect to phospholipid) in the phospholipid bilayer without cholesterol (2.9 mol%) was only moderately reduced (to 2.3 mol%) at cholesterol levels up to 33 mol% but showed a more marked reduction to 1.4 mol% at 40 mol% cholesterol or 1.2 mol% at 50 mol% cholesterol. Since the vesicles containing 50 mol% cholesterol were larger (520 +/- 152 A diameter) than those with no cholesterol (291 +/- 97 A diameter), we measured the solubility of cholesteryl oleate in large vesicles with no cholesterol, prepared by extrusion through polycarbonate membrane filters, and found it similar to that in small, sonicated vesicles with no cholesterol. Therefore, the larger size of vesicles was not the factor responsible for the decreased cholesteryl oleate solubility at high cholesterol contents. A more direct effect of cholesterol is envisioned where the ester becomes displaced to deeper regions of the bilayer.     

Carbon-13 nuclear magnetic resonance study of microtubule protein: evidence for a second colchicine site involved in the inhibition of microtubule assembly

A 13C nuclear magnetic resonance study of bovine microtubule protein was carried out at 43 kG in the presence and absence of colchicine 13C labeled at the tropolone methoxy. Analysis indicated that tubulin has at least two colchicine binding sites: a quasi-irreversibly bound, high-affinity site (i.e., the KD less than 5 microM site generally accepted as the site of colchicine action) as well as a low-affinity site(s) (KD approximately 650 microM) with which free colchicine rapidly exchanges (greater than 100 s-1). The methoxy resonance is broadened to different apparent extents as a result of binding at these two sites (50- vs. 150-Hz broadening for the high- and low-affinity sites, respectively) but undergoes no change in chemical shift upon binding. The low-affinity sites are interpreted to be analogous to the sites deduced by Schmitt and Atlas [Schmitt, H., & Atlas, D. (1976) J. Mol. Biol. 102, 743-758] from labeling studies using bromocolchicine. These sites are likely to be the sites responsible for the abrupt halt in microtubule assembly ("capping") observed at high colchicine concentrations (greater than 20 microM)--a qualitatively different behavior from that observed at low colchicine concentrations [Sternlicht, H., Ringel, I., & Szasz, J. (1983) Biophys. J. 42, 255-267]. Carbon-13 spectra from the aliphatic carbons of microtubule protein consists of narrow resonances--many with line widths less than 30 Hz--superimposed on a broad background. The narrow resonances were assigned to flexible regions in nontubulin proteins [microtubule-associated proteins (MAPs)], in accord with an earlier 1H nuclear magnetic resonance study of microtubule protein [Woody, R. W., Clark, D. C., Roberts, G. C. K., Martin, S. R., & Bayley, P. M. (1983) Biochemistry 22, 2186-2192]. This assignment was supported by 13C NMR analysis of phosphocellulose-purified (MAP-depleted) tubulin as well as heat-stable MAPs. Aliphatic carbons in the MAP preparations were characterized by narrow resonances indicative of carbons with considerable motional freedom whereas the aliphatic regions of phosphocellulose-purified tubulin were, for the most part, characterized by broad resonances indicative of carbons with restricted mobility. However, a moderately narrow resonance (approximately less than 50-Hz line width) coincident with the C gamma resonance of glutamate was detected in 13C NMR spectra of tubulin which indicated that a fraction of the glutamic acid residues is relatively mobile.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of phosphatidylcholine on fatty acid and cholesterol absorption from mixed micellar solutions.

Using the experimental model of the everted sac prepared from rat jejuna, kinetic studies on [14C]oleic acid uptake from bile salt micelles were conducted in the presence and absence of phosphatidylcholine. The concentration of oleic acid was varied between 0.625 and 5 mM. At every level of fatty acid concentration studied the addition of 2 mM phosphatidylcholine produced a significant inhibition of fatty acid uptake. It was further noted that the intact phospholipid molecule was required for this effect as lysophosphatidylcholine produced little, if any, inhibition of [14C]oleic acid uptake. The effect of varying the concentration of phosphatidylcholine on fatty acid uptake was also studied. The degree of inhibition was noted to be correlated grossly with media concentrations of this phospholipid although the decrease of fatty acid uptake was not strictly proportional to concentration of this material in the medium. Studies were also performed analyzing in vitro absorption of [14C]oleic acid and [3H]cholesterol simultaneously from mixed micelles composed of sodium taurocholate, oleic acid, monoolein and cholesterol. Control medium contained no phospholipid while experimental medium contained either diester or diether phosphatidylcholine, 2 mM. Both types of phosphatidylcholine caused significant inhibition of fatty acid and cholesterol uptake. In vivo absorption studies were also performed using the isolated jejunal segment technique. A mixed micellar solution containing [3H]cholesterol and [14C]oleic acid was used as the test dose. Phospholipid in the test dose for controls was supplied as lysophosphatidylcholine and for experimentals it was in the form of diether phosphatidylcholine. Significantly less radioactively labeled cholesterol and fatty acid was absorbed by experimentals as compared to controls over a 10-min period. It is concluded that the intact molecule of phosphatidylcholine inhibits intestinal uptake of cholesterol and fatty acid from mixed micellar solutions under both in vitro and in vivo conditions.     

Metabolic pathways leading from amino acids to vitamin B12 in Propionibacterium shermanii, and the sources of the seven methyl carbons

The metabolic pathways leading from l-[2-13C]aspartic acid, [2-13C]glycine and l-[methyl-13C]methionine to vitamin B12 were investigated, focusing on the biosynthetic pathways leading to the aminopropanol moiety of vitamin B12 and on the role of the Shemin pathway leading to delta-aminolevulinic acid (a biosynthetic intermediate of tetrapyrrole), by means of feeding experiments with Propionibacterium shermanii in combination with 13C-NMR spectroscopy. The 13C-methylene carbons of l-[2-(13)C]aspartic acid, which is transformed to [2-13C]glycine via l-[2-13C]threonine, and [2-13C]glycine added to the culture medium served mainly to enrich the seven methyl carbons of the corrin ring through C-methylation by S-adenosyl-l-[methyl-13C]methionine derived from catabolically generated l-[methyl-13C]methionine in the presence of tetrahydrofolic acid. The results indicate that the catabolism of these amino acids predominates over pathways leading to (2R)-1-amino-2-propanol or delta-aminolevulinic acid in P. shermanii. Feeding of l-[methyl-13C]methionine efficiently enriched all seven methyl carbons. In the cases of [2-13C]glycine and l-[methyl-13C]methionine, the 13C-enrichment ratio of the methyl carbon at C-25 (the site of the first C-methylation) was less than those of the other six methyl carbons, probably due to the influence of endogenous d-glucose in P. shermanii. The almost identical 13C-enrichment ratios of the other six methyl carbons indicated that these C-methylations during vitamin B12 biosynthesis were completed before the amino acids were completely consumed. However, in the case of l-[2-13C]aspartic acid, the 13C-enrichment ratios of five methyl carbons were low and similar, whereas the last two sites of C-methylation (C-53 and C-35) were not labeled, presumably because of complete consumption of the smaller amount of added label. The ratios of 13C-incorporation into the seven methyl carbons are influenced by the conditions of amino acid feeding experiments in a manner that is dependent upon the order of C-methylation in the corrin ring of vitamin B12.     

Solubilization of Multilamellar Liposomes in the Presence of Non-ionized Drug

The solubilization of multilamellar liposomes by metoprolol tartrate (MPL) has been studied as a function of pH, [MPL], [dimyristoylphosphatidylcholine (DMPC)], temperature and lipid composition. The solubilization of liposomes at 37° C by 7.3 mM MPL occurred at different rates at different pH values. MPL completely solubilized by 7.2 mM DMPC liposomes after about 17 hat pH 12, but only a partial solubilization occurred at pH 10 and 11. Between pH 7 and 9 no change in turbidity was observed after 1 week. Addition of cholesterol (CHOL) to DMPC (2:1 mol) had very little effect on solubilization after 24 h, however with DMPC:CHOL (5:1 mol) the decrease in turbidity was observed after 24 h, even though solubilization was much less compared with that of DMPC alone. The rate of solubilizaiton was decreased when dipalmitoylphosphatidylcholine liposomes were employed. Addition of dicetylphosphate (DCP) to DMPC liposomes reduced the rate of solubilization significantly. The solubilization of liposomes by 7.3 mM MPL as a function of [DMPC], indicated that the lower the liposome concentration the greater the effect on solubilization. It is concluded that MPL in the non-ionized form has a solubilizing effect on liposomes, and addition of CHOL or DCP to DMPC has a stabilizing effect against solubilization.     

Differential patterns of lipid-protein association in fast and slow cholesterol nucleating human gallbladder biles: implications for cholesterol nucleation from biliary lipid carriers

We compared the protein/lipid structure and Ch-nucleating capacity of individual lipid carriers in two groups of human gallbladder biles: 11 with Fast cholesterol nucleation (2.2 +/- 1.3 days) and 10 with Slow cholesterol nucleation (19.2 +/- 4.4 days). The groups had comparable cholesterol-saturation (1.31 vs. 1.28), total lipids (9.9 vs. 8.5 g/dl) and proteins (8.5 vs. 7.6 mg/ml). Bile was ultracentrifuged (2 h at 150,000 x g) and the resulting isotropic phase was incubated with [3H]Ch and [14C]lecithin and gel-chromatographed on a Superose 6 column with a buffer containing 7.0 mM sodium-taurocholate. Seven protein peaks were identified (280 nm and biochemistry), with the following molecular mass ranges (kDa): 1 (Void volume), 2 (155-205), 3 (50-79), 4 (20-29), 5 (6-15), 6 (3.5-6), 7 (2-3.5). Peaks 2 and 3 were identified as vesicles and micelles, respectively. Fast vs. Slow Ch nucleating biles had: (a) more (P less than 0.02) cholesterol coeluting with vesicles, (b) more (P less than 0.01) lecithin coeluting with low m.w. peaks (Nos. 5-6), (c) less (P less than 0.01) cholesterol and lecithin coeluting with micelles. An inverse correlation (P less than 0.001) was observed between the amount of proteins coeluting with the micellar peak and the cholesterol nucleation of both whole bile and isolated micellar fractions. A marked shift of cholesterol and lecithin from micelles to vesicles was apparent, in the whole bile, after cholesterol nucleation had occurred. Incubation and sequential analysis of isolated and radiolabeled micelles showed a progressive transfer of lecithin and cholesterol molecules to low molecular weight fractions and to vesicles before cholesterol nucleation. We conclude that pro-nucleating biliary vesicles develop from micelles, due to the phasing out and redistribution of micellar cholesterol and lecithin, which are probably induced by biliary proteins.     

Plasmodium berghei: gluconeogenesis in the infected mouse liver studied by 13C nuclear magnetic resonance

Using 13C nuclear magnetic resonance, we have compared the gluconeogenic activity of perfused livers isolated from normal starved mice and mice highly parasitized with Plasmodium berghei, using [2-13C]pyruvate as substrate. In both types of livers, 13C labeling of glucose carbons occurred in positions 1, 2, 5, and 6. The equal proportions of [1,6-13C]- and [2,5-13C]glucose in livers from malarial and normal mice suggests that pyruvate enters the gluconeogenic pathway directly and, to an equal extent, via the tricarboxylic acid cycle. The normalized signal heights indicated that at a given time after the addition of [2-13C]pyruvate the degree of 13C labeling in glucose carbons was reduced in livers from malarial animals, when compared to livers from normal animals. During the course of the perfusion experiment, the [2-13C]lactate resonance signal was always more intense from livers of malarial animals than from normal animals. A reduced activity of hepatic gluconeogenesis in malarial animals was further confirmed by a separate set of perfusion experiments which showed a 56% reduction of the measured rate of glucose production in livers from malarial animals, with respect to that of normal animals. A lowered NAD/NADH ratio in livers from malarial animals would explain the increased proportion of lactate observed in the spectra and be related to a decreased gluconeogenic rate. A more reduced oxidoreduction level in the hepatocytes of a malarial animal would result from a defect in the oxidative phosphorylation activity of mitochondria.     

Solid-state NMR structure determination of melittin in a lipid environment

Solid-state (13)C NMR spectroscopy was used to investigate the three-dimensional structure of melittin as lyophilized powder and in ditetradecylphosphatidylcholine (DTPC) membranes. The distance between specifically labeled carbons in analogs [1-(13)C]Gly3-[2-(13)C]Ala4, [1-(13)C]Gly3-[2-(13)C]Leu6, [1-(13)C]Leu13-[2-(13)C]Ala15, [2-(13)C]Leu13-[1-(13)C]Ala15, and [1-(13)C]Leu13-[2-(13)C]Leu16 was measured by rotational resonance. As expected, the internuclear distances measured in [1-(13)C]Gly3-[2-(13)C]Ala4 and [1-(13)C]Gly3-[2-(13)C]Leu6 were consistent with alpha-helical structure in the N-terminus irrespective of environment. The internuclear distances measured in [1-(13)C]Leu13-[2-(13)C]Ala15, [2-(13)C]Leu13-[1-(13)C]Ala15, and [1-(13)C]Leu13-[2-(13)C]Leu16 revealed, via molecular modeling, some dependence upon environment for conformation in the region of the bend in helical structure induced by Pro14. A slightly larger interhelical angle between the N- and C-terminal helices was indicated for peptide in dry or hydrated gel state DTPC (139 degrees -145 degrees ) than in lyophilized powder (121 degrees -139 degrees ) or crystals (129 degrees ). The angle, however, is not as great as deduced for melittin in aligned bilayers of DTPC in the liquid-crystalline state (approximately 160 degrees ). The study illustrates the utility of rotational resonance in determining local structure within peptide-lipid complexes.     

Effects of thiopental on transport and metabolism of glutamate in cultured cerebellar granule neurons

This study was performed to analyze the effects of the barbiturate thiopental on neuronal glutamate uptake, release and metabolism. Since barbiturates are known to bind to the GABA(A) receptor, some experiments were carried out in the presence of GABA. Cerebellar granule neurons were incubated for 2 h in medium containing 0.25 mM [U-(13)C]glutamate, 3 mM glucose, 50 microM GABA and 0.1 or 1 mM thiopental when indicated. When analyzing cell extracts, it was surprisingly found that in addition to glutamate, aspartate and glutathione, GABA was also labeled. In the medium, label was observed in glutamate, aspartate and lactate. Glutamate exhibited different labeling patterns, indicating metabolism in the tricarboxylic acid cycle, and subsequent release. A net uptake of [U-(13)C]glutamate and unlabeled glucose was seen under all conditions. The amounts of most metabolites synthesized from [U-(13)C]glutamate were unchanged in the presence of GABA with or without 0.1 mM thiopental. In the presence of 1 mM thiopental, regardless of the presence of GABA, decreased amounts of [1,2, 3-(13)C]glutamate and [U-(13)C]aspartate were found in the medium. In the cell extracts increased [U-(13)C]glutamate, [1,2, 3-(13)C]glutamate, labeled glutathione and [U-(13)C]aspartate were observed in the 1 mM thiopental groups. Glutamate efflux and uptake were studied using [(3)H]D-aspartate. While efflux was substantially reduced in the presence of 1 mM thiopental, this barbiturate only marginally inhibited uptake even at 3 mM. These results may suggest that the previously demonstrated neuroprotective action of thiopental could be related to its ability to reduce excessive glutamate outflow. Additionally, thiopental decreased the oxidative metabolism of [U-(13)C]glutamate but at the same time increased the detectable metabolites derived from the TCA cycle. These latter effects were also exerted by GABA.     

13C nuclear magnetic resonance and gas chromatography-mass spectrometry studies of carbon metabolism in the actinomycin D producer Streptomyces parvulus by use of 13C-labeled precursors.

Fructose and glutamate metabolism was monitored in cell suspensions of streptomyces parvulus by 13C nuclear magnetic resonance. The experiments were performed for cells grown with various 13C sources in a growth medium containing D-[U-13C]fructose, L-[13C]glutamate, or L-[U-13C]aspartate and with nonlabeled precursors to compare intracellular pools in S. parvulus cells at different periods of the cell life cycle. The transport of fructose into the cells was biphasic in nature; during rapid transport, mannitol, fructose, and glucose 6-phosphate were accumulated intracellularly, whereas during the passive diffusion of fructose, the intracellular carbohydrate pool comprised mainly trehalose (1,1'-alpha-alpha-D-glucose). The regulation of fructokinase activity by the intracellular intermediates may play an important role in fructose catabolism in S. parvulus. Transaldolase activity in S. parvulus was determined from the 13C nuclear magnetic resonance labeling pattern of trehalose carbons obtained from cells grown in medium containing either L-[U-13C]aspartate or L-[U-13C]glutamate. Only carbons 4, 5, and 6 of the disaccharide were labeled. Isotopomer analysis of the trehalose carbons led us to conclude that the flux through the reverse glycolytic pathway, condensation of glyceraldehyde 3-phosphate with dihydroxyacetone phosphate, makes at best a minor contribution to the 13C-labeled glucose units observed in trehalose. The pentose pathway and transaldolase activity can explain the labeling pattern of 4,5,6-13C3 of trehalose. Moreover, the transfer of the 13C label of L-[U-13C]aspartate into the different isotopomers of trehalose C4, C5, and C6 by the transaldolase activity allowed us to calculate the relative fluxes from oxaloacetate via gluconeogenesis and through the tricarboxylic acid cycle. The ratio of the two fluxes is approximately 1. However, the main carbon source for trehalose synthesis in S. parvulus is fructose and not glutamate or aspartate. The 13C enrichment and isotopomer population, measured by nuclear magnetic resonance and gas chromatography-mass spectrometry, of the actinomycin D peptide ring enabled us to specify the origins of the five amino acids of actinomycin D. Threonine and proline exhibited isotopomer populations similar to that of the extracellular L-[13C]glutamate, indicating that protein catabolism is the origin of their 13C label, whereas the isotopomer populations of sarcosine and N-methylvaline were similar to those of the new intracellular pool of S. parvulus that originated from D-[U-13C]fructose during the production of actinomycin D.     

13C NMR study of effects of fasting and diabetes on the metabolism of pyruvate in the tricarboxylic acid cycle and the utilization of pyruvate and ethanol in lipogenesis in perfused rat liver

13C NMR has been used to study the competition of pyruvate dehydrogenase with pyruvate carboxylase for entry of pyruvate into the tricarboxylic acid (TCA) cycle in perfused liver from streptozotocin-diabetic and normal donor rats. The relative proportion of pyruvate entering the TCA cycle by these two routes was estimated from the 13C enrichments at the individual carbons of glutamate when [3-13C]alanine was the only exogenous substrate present. In this way, the proportion of pyruvate entering by the pyruvate dehydrogenase route relative to the pyruvate carboxylase route was determined to be 1:1.2 +/- 0.1 in liver from fed controls, 1:7.7 +/- 2 in liver from 24-fasted controls, and 1:2.6 +/- 0.3 in diabetic liver. Pursuant to this observation that conversion of pyruvate to acetyl coenzyme A (acetyl-CoA) was greatest in perfused liver from fed controls, the incorporation of 13C label into fatty acids was monitored in this liver preparation. Livers were perfused under steady-state conditions with labeled substrates that are converted to either [2-13C]acetyl-CoA or [1-13C]acetyl-CoA, which in the de novo synthesis pathway label alternate carbons in fatty acids. With the exception of the repeating methylene carbons, fatty acyl carbons labeled by [1-13C]acetyl-CoA (from [2-13C]pyruvate) gave rise to resonances distinguishable on the basis of chemical shift from those observed when label was introduced by [3-13C]alanine plus [2-13C]ethanol, which are converted to [2-13C]acetyl-CoA. Thus, measurement of 13C enrichment at several specific sites in the fatty acyl chains in time-resolved spectra of perfused liver offers a novel way of monitoring the kinetics of the biosynthesis of fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhanced solubilization and intestinal absorption of cholesterol by oxidized linoleic acid

Solubilization of cholesterol in the intestinal lumen by bile acids and the subsequent formation of mixed micelles is an important step in the absorption of cholesterol. We propose that oxidized fatty acids (ox-FA) may mimic bile acids and form mixed micelles with cholesterol much more efficiently, as compared with unoxidized fatty acids, thereby increasing there absorption. In an in vitro assay at concentrations of 1, 5, and 10 mM, oxidized linoleic acid (ox-18:2) increased the solubilization of cholesterol (3.06, 8.16, and 15.46 nmol/ml) in a dose dependent manner compared with a 10 mM unoxidized linoleic acid (unox-18:2 at 0.97 nmol/ml). The uptake of cholesterol solubilized in the presence of ox-18:2 by Caco-2 cells and everted rat intestinal sacs was greater (1.78 and 1.95 nmol/ml respectively) as compared with the cholesterol solubilized in the presence of unox-18:2 (0.29 and 0.61 nmol/ml; P = 0.05). In addition, when LDL receptor deficient mice were fed a high fat diet along with ox-18:2 their plasma cholesterol levels were greater than animals fed the high fat diet alone (1290 mg/dl vs. 1549 mg/dl, P = 0.013). From these results, we suggest that ox-FA, by enhancing the solubilization of luminal cholesterol, increases the uptake of cholesterol that might lead to hypercholesterolemia and atherosclerosis.     

Rapid labeling of lipoproteins in plasma with radioactive cholesterol. Application for measurement of plasma cholesterol esterification

In order to efficiently and rapidly label lipoproteins in plasma with [3H]cholesterol, micelles consisting of lysophosphatidylcholine (lysoPC) and [3H]cholesterol (molar ratio, 50:1) were prepared. When trace amounts of these micelles were injected into plasma, [3H]cholesterol rapidly equilibrated among the plasma lipoproteins, as compared to [3H]cholesterol from an albumin-stabilized emulsion. The distributions of both [3H]cholesterol and unlabeled free cholesterol in plasma lipoproteins were similar in labeled plasma samples. This method of labeling can be used for the measurement of cholesterol esterification, or lecithin:cholesterol acyltransferase activity, in small amounts (20-40 microliters) of plasma samples.     

Use of 13C in biosynthetic studies. The labelling pattern in tenellin enriched from isotope-labelled acetate, methionine, and phenylalanine

The biogenetic origin of the carbon atoms in tenellin has been established by adding 13C-enriched compounds to cultures of Beauveria bassiana, and determining the isotopic distribution in the metabolite by 13C nuclear magnetic resonance spectrometry. Tenellin is formed by condensation of an acetate-derived polyketide chain with a phenylpropanoid unit that may be phenylalanine. Alternate carbon atoms of the polyketide chain were labelled with sodium [1(-13C)]- and [2-(13C]-acetate; sodium [1,2-(13C)]acetate was incorporated as intact two-carbon units, the presence of which in tenellin was apparent from coupling between adjacent 13C-enriched carbons. Substituent methyl groups of the polyketide-derived alkenyl chain were labelled with L-[Me-13C]methionine. The labelling patterns from DL-[carboxy-13C]phenylalanine and DL-[alpha-13C]phenylalanine indicated a rearrangement of the propanoid component at some stage in the synthesis. The mass spectrum of tenellin from cultures administered L-[15N]phenylalanine showed isotopic enrichment similar to that obtained with 13C- or 14C-labelled phenylalanine. During incorporation of L-[carboxy-14C, beta-3H]phenylalanine 96% of the tritium label was lost, discounting the possibility of a 1,2-hydride shift during biosynthesis of the metabolite.     

13C n.m.r. studies of gluconeogenesis in rat liver suspensions and perfused mouse livers

Our early 31P n.m.r. studies of compartmentation in suspensions of rat liver cells have been extended by following fructose-1-phosphate peaks, known to be in the cytosol, which gave the same pH as the Pi peak previously assigned to the cytosol. Gluconeogenesis have been followed from [13C]glycerol labelled at C1,3 or at C2 and from labelled [3-13C]alanine. With the glycerol substrate it was possible to follow the label into alpha-glycerophosphate and to determine its distribution in the glucose formed. To a first approximation (i.e. 90%) the glucose level could be followed from its original glycerol position, e.g. [1,3-13C]glycerol to strongly labelled positions 1, 3, 4 and 6 of glucose. Slightly more than 10% of the label was scrambled (i.e. 10% movement of C2 to C1 and ca. 10% of C1 was lost, the remainder being unchanged). These are consistent with a flux through the pentose shunt, dominated by the transketolase pathway. With [3-13C]alanine, about 14 resonances are assigned to different carbons of the intermediates beta-hydroxybutyrate, acetoacetate, lactate, pyruvate, glutamate, glutamine, asparate, as well as C2-alanine, while another 7 resonances are observed from the different anomeric carbons of glucose. The effects of thyroid hormone treatment of the rats upon numerous in vivo rates are clearly observed and will be illustrated.     

13C NMR studies of glycogen turnover in the perfused rat liver

To assess whether hepatic glycogen is actively turning over under conditions which promote net glycogen synthesis we perfused livers from 24-h fasted rats with 20 mM D-[1-13C]glucose, 10 mM L-[3-13C]alanine, 10 mM L-[3-13C]lactate, and 1 microM insulin for 90 min followed by a 75-min "chase" period with perfusate of the same composition containing either 13C-enriched or unlabeled substrates. The peak height of the C-1 resonance of the glucosyl subunits in glycogen was monitored, in real time, using 13C NMR techniques. During the initial 90 min the peak height of the C-1 resonance of glycogen increased at almost a constant rate reflecting a near linear increase in net glycogen synthesis, which persisted for a further 75 min if 13C-enriched substrates were present during the "chase" period. However, when the perfusate was switched to the unenriched substrates, the peak height of the C-1 resonance of glycogen declined in a nearly linear manner reflecting active glycogenolysis during a time of net glycogen synthesis. By comparing the slopes of the curve describing the time course of the net [1-13C] glucose incorporation into glycogen with the rate of net loss of 13C label from the C-1 resonance of glycogen during the "chase" period we estimated the relative rate of glycogen breakdown to be 60% of the net glycogen synthetic rate. Whether this same phenomenon occurs to such an appreciable extent in vivo remains to be determined.