Effect of high dietary fat content on heat production and lipid and protein deposition in growing immunocastrated male pigs

In immunocastrated (IC) pigs, revaccination (V2) increases lipid deposition (LD) because of increased voluntary feed intake; but little is known on associated effect of diet composition on partitioning of nutrients in IC pigs. Digestibility measurements, N and energy balances in respiration chambers were performed in two subsequent stages in four replicates of two male littermates to determine the changes between 85 (stage 1) and 135 (stage 2) kg live weight due to combined effect of IC, growth and increased feed intake (IC/growth). During stage 1, pigs received a standard low-fat diet (LF diet; 2.5% dry matter (DM) of fat fed at 2.27 MJ metabolizable energy (ME)/kg BW^0.60 per day), whereas during stage 2, feed intake was increased to 2.47 MJ ME/kg BW^0.60 per day and one littermate was fed LF diet whereas the second received a fat-enriched diet (HF diet; 8.9% DM of fat) to determine the effect of increased dietary fat content on energy utilization in IC pigs. Results from N balance and measurements of gas exchanges were used to calculate respiratory quotient (RQ), heat production (HP), nutrient contribution to fat retention, components of HP, protein deposition (PD) and LD. Nutrients and energy apparent digestibility coefficients, methane losses and N retention (P<0.05) increased with IC/growth. Despite higher ME intake, total HP remained similar (1365 kJ/kg of BW^0.60 per day; P=0.47) with IC/growth. Consequently, total retained energy (RE) increased with IC/growth (from 916 to 1078 kJ/kg of BW^0.60 per day; P<0.01) with a higher fat retention (625 to 807 kJ/kg BW^0.60 per day; P<0.01), originating mainly from carbohydrates associated with a higher lipogenesis (536 to 746 kJ/kg BW^0.60 per day; P<0.01) and RQ (1.095 to 1.145; P<0.01). Both PD (from 178 to 217 g/day; P=0.02) and LD (from 227 to 384 g/day; P<0.01) increased due to IC/growth. Feeding HF diet after IC was associated with increased crude fat digestibility (P<0.01) and increased RE as fat (807 to 914 kJ/kg BW^0.60 per day; P=0.03), originating mainly from dietary fat (P<0.01) and resulting in increased LD (384 to 435 g/day; P<0.01) and lower RQ (from 1.145 to 1.073; P<0.01). Altogether, present results indicate that increased fatness of IC pigs is a result of increased daily LD caused by higher energy intake and lower basal metabolic rate. In addition, LD is further enhanced by dietary energy enrichment with fat after V2.     

Quantitative partition of protein,carbohydrate and fat pools in growing pigs

A model combining data from balance experiments with data from oxidation of nutrients demonstrating the pools of protein, carbohydrate and fat and their partition in the body was presented.

Data from more than 200 experiments with growing pigs were used to fill up the “black boxes”; in the model and to discuss the pattern between catabolic and anabolic processes of the different nutrients.

With a ME‐intake of 1.3 MJ/kg^0.75 the proportion of retained protein from the protein pool varied from 50 to 75% depending on the age of animals and the protein quality. At a low protein intake and ME of 0.6 MJ/kg^0.75 the utilization of protein was reduced to about 25% as a substantial part of the protein pool was oxidized.

The carbohydrate group from the protein sources constituted less than 5% of the carbohydrate pool. The oxidation of carbohydrates was between 50–75% of the pool in all experiments, while the rest was used for lipogenesis. Even on a low feeding level the pattern occurred indicating a requirement for specific substances formed in the body.

Lipogenesis constituted 46–96% of the fat pool depending of the amount on digested fat in the different experiments. No oxidation of fat was observed in experiments with pigs (30–90 kg LW) on a high feeding level in spite of the broad variation in the amount of digested fat, indicating a complete storage of the fat pool into body fat. In experiments with piglets (3–9 kg LW) on the same energy intake fat oxidation of about 30% occurred, probably caused by the high energy requirement of the piglets. With a ME‐intake of 0.6 MJ/kg^0.75 the oxidation of fat was higher than the fat pool for pigs between 30–60 kg LW, thereby causing oxidation of body fat to cover the energy requirements.     

Levels of copper and zinc in diets for growing and finishing pigs can be reduced without detrimental effects on production and mineral status

One hundred and sixty pigs were used to evaluate dietary copper (Cu) and zinc (Zn) supplementation on performance, fecal mineral levels, body mineral status and carcass and meat quality. Diets differed in mineral form (MF) (Cu and Zn in the form of proteinate amino acid chelate (organic) or sulfate (inorganic)) and inclusion level (IL) (27 mg/kg of total Cu and 65 mg/kg of total Zn ('low') or 156 mg/kg of total Cu and 170 mg/kg of total Zn ('high')) according to a 2 × 2 factorial arrangement of treatments. Pigs were used from 25 to 107 kg body weight (BW) and fed their respective diets ad libitum. Blood and fecal samples were collected on days 14 and 77 of the experiment. Blood was analyzed for concentration of Cu and Zn, hemoglobin (Hb), Cu content of red blood cells (RBC Cu) and alkaline phosphatase (ALP) and feces for Cu and Zn concentration. Hot carcass weight (HCW) and backfat depth were measured at slaughter and indices of meat quality were assessed on a section of longissimus thoracis. Liver, kidney and bone samples were collected immediately after slaughter and liver and kidney were tested for Cu and Zn content, while bone was only tested for Zn. Over the entire experimental period (25 to 107 kg BW) no significant treatment differences in average daily gain (ADG) or average daily feed intake (ADFI) occurred; however, feed conversion ratio (FCR) was improved by the inclusion of proteinate amino acid chelate (P = 0.012). Copper and Zn concentrations in feces were in direct proportion to the IL in the diet. Blood mineral levels were within normal physiological ranges in all treatments and tissue Cu and Zn concentrations increased with dietary IL (P < 0.05). Results indicate that Cu and Zn fecal concentrations were reduced by approximately 6-fold for Cu and by 2.5-fold for Zn by feeding 27 mg/kg Cu and 65 mg/kg Zn, in either the proteinate amino acid chelate or the sulfate form, compared with a diet containing 156 mg/kg Cu and 170 mg/kg Zn. This decrease in total dietary Cu and Zn did not reduce performance or mineral status of pigs.     

Feed ingredients differing in fermentable fibre and indigestible protein content affect fermentation metabolites and faecal nitrogen excretion in growing pigs

To study the fermentation characteristics of different non-conventional dietary fibre (DF) sources with varying levels of indigestible CP content and their effects on the production of fermentation metabolites and on faecal nitrogen (N) excretion, an experiment was conducted with 40 growing pigs (initial BW 23 kg) using wheat bran (WB), pea hulls (PH), pea inner fibres (PIF), sugar beet pulp (SBP) or corn distillers dried grains with solubles (DDGS). The diets also contained soya protein isolate, pea starch and sucrose, and were supplemented with vitamin-mineral premix. Faecal samples were collected for 3 consecutive days from day 10, fed with added indigestible marker (chromic oxide) for 3 days from day 13 and pigs were slaughtered on day 16 from the beginning of the experiment. Digesta from the ileum and colon were collected and analysed for short-chain fatty acids (SCFA) and ammonia (NH3) content. The apparent total tract N digestibility was the lowest (P < 0.001) in diets based on DDGS (74%), medium in diets with WB and SBP (76% each) and highest in those with PIF and PH (79% and 81%, respectively). Expressed per kg fermented non-starch polysaccharides (NSP), faecal N excretion was higher with DDGS and WB diets (130 and 113 g/kg NSP fermented, respectively) and lower with PIF, PH and SBP diets (42, 52 and 55 g/kg NSP fermented, respectively). The PH-based diets had the highest (P < 0.05) SCFA concentrations, both in the ileum and the colon (27 and 122 mMol/kg digesta, respectively). The highest NH3 concentration was also found in the colon of pigs fed with PH (132 mMol/kg digesta). Loading plot of principle component analysis revealed that the CP : NSP ratio was positively related with faecal N excretion and NH3 concentration in colon contents, whereas negatively related with SCFA concentration in colon contents. In conclusion, pea fibres and SBP increased SCFA and reduced NH3 concentration in the pig's intestine and reduced faecal N excretion, which makes pea fibres and SBP an interesting ingredient to use in pig diet to improve the positive effect of DF fermentation on the gastrointestinal tract and reduce faecal N excretion.     

Divergent selection for residual feed intake in group-housed growing pigs: characteristics of physical and behavioural activity according to line and sex

The aim of the study was to assess the impact of selection for residual feed intake (RFI) on the behavioural activity of lines divergently selected for RFI during seven generations. In all, six successive batches from the seventh generation of selection were raised in collective pens equipped with a single-place electronic feeder (SEF) from 10 weeks of age to 100 kg BW. Each batch included four groups of 12 pigs: high RFI (RFI⁺) castrated males, RFI⁺ females, low RFI (RFI⁻) castrated males, RFI⁻ females. At 17 weeks of age, health criteria were evaluated using a gradient scale for increased severity of lameness, body lesions, bursae and tail biting. Individual behavioural activities were recorded by 24-h video tape on the day after health evaluation. The investigative motivation towards unfamiliar objects was quantified at 18 weeks of age. The daily individual feeding patterns were computed from SEF records during the 4 weeks surrounding 12, 17 and 22 weeks of age. All pigs spent significantly most of their time lying in diurnal (80% of total scan) and nocturnal (>89%) periods. The RFI⁻ pigs showed a lower proportion of health problems (P<0.01) than RFI⁺ pigs. The RFI⁻ pigs used the SEF less than the RFI⁺ pigs, in diurnal (5.3% v. 6.4% of video scans, P<0.05) and nocturnal periods (3.6% v. 4.5% of video scans, P<0.05). This was confirmed by a significantly lower daily number and duration of visits to the SEF computed from the SEF data. The feeding activity measured from the video recording was significantly correlated (R>0.34; P<0.05) with feeding patterns computed from the SEF. The RFI⁻ pigs spent less time standing over the 24-h period (9.7% v. 12.2% of scans, i.e. 35 min/day, P<0.05). In terms of energy costs, this amounted to 14% of the line difference in terms of daily metabolizable energy intake. The castrated males used the SEF more than females, especially at night (4.7% v. 3.4% of total scans, P<0.05), whereas females displayed greater investigation of their environment (7.7±0.3% v. 6.6±0.2% of total scans, P<0.05) and the novel objects (10.7% v. 4.9% of total scans, P<0.05). In conclusion, the lower physical activity associated with reduced energy expenditure in RFI⁻ pigs compared with RFI⁺ pigs contributed significantly to their improved efficiency and was not related to worsened health scores.     

Decrease in brain POMC mRNA expression and onset of obesity in guinea pigs exposed to 2-chloroethyl ethyl sulfide, a mustard analogue

The full spectrum of physiological effects resulting from exposure to sulfur mustard and its analogs is currently unknown. In a guinea pig model, initially selected to study the role of an inflammatory cytokine cascade in mustard gas induced lung injury, we observed significant body weight gain in guinea pigs exposed to an intratracheally injected single dose of 2-chloroethyl ethyl sulfide, a mustard analog. The body weight gain was not associated with any apparent change in appetite. To further elucidate a molecular basis for the observed weight gain, we evaluated candidate genes for the obese phenotype by quantitative RT-PCR. We observed a time- and dose-dependent decrease in guinea pig pro-opiomelanocortin (POMC) message following treatment with mustard gas. This reduction in POMC message is consistent with the onset of obesity in the animals. We hypothesize that the POMC melanocortin pathway provides a mechanistic basis for the observed effects of sulfur mustard on body weight.     

Methionine oxidation in human IgG2 Fc decreases binding affinities to protein A and FcRn

Susceptibility of methionine residues to oxidation is a significant issue of protein therapeutics. Methionine oxidation may limit the product's clinical efficacy or stability. We have studied kinetics of methionine oxidation in the Fc portion of the human IgG2 and its impact on the interaction with FcRn and Protein A. Our results confirm previously published observations for IgG1 that two analogous solvent‐exposed methionine residues in IgG2, Met 252 and Met 428, oxidize more readily than the other methionine residue, Met 358, which is buried inside the Fc. Met 397, which is not present in IgG1 but in IgG2, oxidizes at similar rate as Met 358. Oxidation of two labile methionines, Met 252 and Met 428, weakens the binding of the intact antibody with Protein A and FcRn, two natural protein binding partners. Both of these binding partners share the same binding site on the Fc. Additionally, our results shows that Protein A may serve as a convenient and inexpensive surrogate for FcRn binding measurements.     

Low sanitary conditions increase energy expenditure for maintenance and decrease incremental protein efficiency in growing pigs

Requirements for energy and particular amino acids (AAs) are known to be influenced by the extent of immune system stimulation. Most studies on this topic use models for immune system stimulation mimicking clinical conditions. Extrapolation to conditions of chronic, low-grade immune system stimulation is difficult. We aimed to quantify differences in maintenance energy requirements and efficiency of energy and protein used for growth (incremental energy and protein efficiency) of pigs kept under low (LSC) or high sanitary conditions (HSC) that were fed either a basal diet or a diet with supplemented AA. Twenty-four groups of six 10-week-old female pigs were kept under either LSC or HSC conditions for 2 weeks and fed a diet supplemented or not with 20% extra methionine, threonine and tryptophan. In week 1, feed was available ad libitum. In week 2, feed supply was restricted to 70% of the realized feed intake (kJ/(kg BW)^0.6 per day) in week 1. After week 2, fasting heat production (FHP) was measured. Energy balances and incremental energy and protein efficiencies were measured and analyzed using a GLM. Low sanitary condition increased FHP of pigs by 55 kJ/(kg BW)^0.6 per day, regardless of diet. Low sanitary condition did not alter the response of faecal energy output to incremental gross energy (GE) intake, but it reduced the incremental response of metabolizable energy intake (12% units), heat production (6% units) and energy retained as protein (6% units) to GE intake, leaving energy retained as fat unaltered. Incremental protein efficiency was reduced in LSC pigs by 20% units. Incremental efficiencies for energy and protein were not affected by dietary AA supplementation. Chronic, low-grade immune stimulation by LSC treatment increases FHP in pigs. Under such conditions, the incremental efficiency of nitrogen utilization for body protein deposition is reduced, but the incremental efficiency of absorbed energy for energy or fat deposition is unaffected.     

Bacterial protein meal in diets for pigs and minks: Comparative studies on protein turnover rate and urinary excretion of purine base derivatives

Abstract

The effect of increasing the dietary content of bacterial protein meal (BPM) on protein turnover rate, and on nucleic acid and creatinine metabolism in growing minks and pigs was investigated in two experiments. In each experiment, 16 animals were allocated to four experimental diets. The diets containing no BPM served as controls, i.e. for minks diet M1, for pigs P1; the experimental diets contained increasing levels of BPM to replace fish meal (minks) or soybean meal (pigs), so that up to 17% (P2), 20% (M2), 35% (P3), 40% (M3), 52% (P4), and 60% (M4) of digestible N was BPM derived. Protein turnover rate was measured by means of the end-product method using [¹⁵N]glycine as tracer and urinary nitrogen as end-product. In minks, protein flux, synthesis, and breakdown increased significantly with increasing dietary BPM. In pigs, diet had no observed effect on protein turnover rate. The intake of nucleic acid nitrogen (NAN) increased from 0.15 g/kg W^0.75 on M1 to 0.26 g/kg W^0.75 on M3 and M4 in the mink experiment, and from 0.08 g/kg W^0.75 on P1 to 0.33 g/kg W^0.75 on P4 in the pig experiment. Increased NAN intake led, in both experiments, to increased allantoin excretion. Analysis of species effects showed that minks excreted 1.72 mmol/kg W^0.75 of allantoin, significantly more than the 0.95 mmol/kg W^0.75 excreted by pigs. In minks, approximately 96% of the excreted purine base derivatives consisted of allantoin, whereas in pigs approximately 93% did. Thus, increasing the dietary content of BPM increased protein turnover rate in minks but not in pigs, and allantoin excretion increased with increasing dietary BPM although it seemed that mink decomposed purine bases to their end-product more completely than pigs did. Collectively these data show that BPM is a suitable protein source for pigs and mink, and recorded differences between species were to a large extent due to differences in protein retention capacity and muscle mass.     

In vitro prediction of standardised ileal crude protein and amino acid digestibilities in grain legumes for growing pigs

The study was conducted to validate in vitro prediction of standardised ileal digestibilities (SID) of crude protein (CP) and amino acids (AA) in grain legumes for growing pigs using six different cultivars of faba beans (Vicia faba), six different cultivars of field peas (Pisum sativum), and five different cultivars of lupins (Lupinus spp.). The SID for CP and AA were predicted from in vitro analysis by means of a two-step enzymatic method using pepsin and pancreatin incubations. In vitro predicted SID values of CP and AA were generally higher than the corresponding SID values measured in vivo. There were strong linear relationships (r2 = 0.73 for Lys to r2 = 0.91 for Cys and Trp) between in vivo and in vitro predicted SID values in the assay feed ingredients if grain legume species (i.e. faba beans, field peas and lupins) was included as a covariate in multiple linear regression analysis. However, to rapidly and accurately predict SID of CP and AA in individual batches of various feed ingredients, further studies are warranted.     

Colonisation of wheat by VA-mycorrhizal fungi was found to be higher on a farm managed in an organic manner than on a conventional neighbour

Levels of colonisation by vesicular-arbuscular (VA)-mycorrhizal fungi were compared between adjacent farms, one operated in a conventional manner and the other run according to organic farming principles. Wheat grown on the organic farm was found to have VA-mycorrhizal colonisation levels consistently 2 to 3 times higher than wheat on the conventional farm. Glasshouse and field trials indicated that the lower colonisation levels on the conventional farm were due to continual use of fertiliser containing soluble phosphorus (P). The fertiliser appeared to have an immediate negative effect on the rate of colonisation, and also appeared to have a long term negative effect through maintaining higher levels of soluble P in the soil, and by decreasing inoculum levels. Use of the relatively insoluble reactive rock phosphate fertiliser on the organic farm did not decrease levels of VA-mycorrhizas. Colonisation levels did not vary between wheat varieties, and herbicides and seed dressings were also not found to be having any significant effect on levels of colonisation.     

Performance and total-tract digestibility responses to exogenous xylanase and phytase in diets for growing pigs

Sixty individually housed male pigs (Large White X Landrace, average weight 19.7±0.56 kg) were used in a completely randomised block design having five wheat-based dietary treatments (n=12 pigs per treatment) to study the interactive effects between added xylanase (XYL) and phytase (PHY), either alone or in combination, and days on trial on coefficient of total-tract digestibility (CTTAD) and performance. The diets used were: (i) positive control (PC); (ii) negative control with reduced energy and mineral content (NC); (iii) NC with added XYL (NCX); (iv) NC with added PHY (NCP); and (v) NC with added XYL plus PHY (NCXP). The performance response and CTTAD of dietary components were measured 3 and 7 weeks after commencement of feeding, with the experiment finishing when pigs reached approximately 65 kg live weight.Pigs fed the NC diet decreased average daily gain (ADG) by 13% (P<0.01) and increased feed conversion ratio (FCR) by 11% (P<0.01) compared to pigs fed the PC diet. Addition of PHY in the NC diet increased ADG by 9% (P<0.05) and decreased FCR by 5% (P=0.065), whilst addition of XYL in the NC diet had no effect on the performance indices. Combined addition of XYL and PHY in the NC diet did not improve performance of pigs for the first 21 days but improved FCR by 8% (P=0.008) during the days 22–49. Digestibility measurements showed that combined use of XYL and PHY in the NC diet improved CTTAD of dry matter (DM, P<0.05), gross energy (GE, P<0.05) and crude protein (CP, P<0.01) only during the days 22–49. Independent supplementation of PHY in the NC diet tended to improve CTTAD of GE (P=0.09) only during the days 22–49. In contrast, supplementation of PHY immediately improved CTTAD of P and maintained it to similar levels at 49 days. Results suggested that supplementation of PHY plus XYL in combination in the NC diet improved FCR and CTTAD of DM, GE, CP and P but the beneficial effects other than CTTAD of P were derived mainly during days 22–49.     

Increasing levels of rapeseed expeller meal in diets for pigs: effects on protein and energy metabolism

The heavy reliance on imported soybean meal (SBM) as a protein source makes it necessary for the European pig industry to search for alternatives and to develop pigs that perform efficiently when fed such ingredients. Digestion and metabolism are major physiological processes contributing to variation in feed efficiency. Therefore, an experiment was conducted to assess the effects of replacing SBM with increasing levels of rapeseed meal (RSM) in diets for young pigs on apparent total tract digestibility (ATTD) of energy and nutrients, nitrogen (N) balance, energy metabolism and carbohydrate, protein and fat oxidation. Four diets were fed to 32 pigs (22.7±4.1 kg initial BW) for three weeks. The diets consisted of a control cereal grain-SBM basal diet and three test diets where SBM and wheat were partially replaced with 10%, 20%, and 30% of expeller RSM. Increasing level of RSM in the diets linearly reduced ATTD of organic matter, CP, total carbohydrates, dietary fiber and energy. Utilization of digested nitrogen (DN) for N retention and total N excretion were not affected by RSM inclusion, however, RSM inclusion induced a shift in N excretion from urine to feces. Despite a linear increase in liver to metabolic BW ratio, heat production and utilization of metabolizable energy (ME) for retention were not affected by increasing RSM inclusion. In conclusion, replacing SBM with up to 30% of expeller RSM in nutritionally balanced diets for young pigs reduced the ATTD of most nutrients and energy, but did not affect N and energy retention in the body or efficiency of utilization of DN or ME for retention.     

Development of a quantitative Real‐Time TaqMan PCR assay for determination of the minimal dose of Mycoplasma hyopneumoniae strain 116 required to induce pneumonia in SPF pigs

Aims: A triplex real‐time PCR assay to quantify Mycoplasma hyopneumoniae in specimens from live and dead pigs was developed and validated. The minimal dose of Myc. hyopneumoniae required to induce pneumonia in specific pathogen‐free pigs was determined. Methods and Results: This TaqMan test simultaneously detected three genes encoding the proteins P46, P97 and P102. All Myc. hyopneumoniae strains analysed were detected, including strains isolated in three countries (France, England and Switzerland) and from several pig farms (n = 33), and the test was specific. The estimated detection thresholds were 1·3 genome equivalents (μl^?1) for the targets defined in p97 and p102 genes and 13 genome equivalents (μl^?1) for the segment defined in the p46 gene. This test was used to quantify Myc. hyopneumoniae in specimens sampled from experimentally infected pigs. In live pigs, c. 10⁷, 10⁸ and 10¹⁰ genome equivalents (ml^?1) of Myc. hyopneumoniae were detected in the nasal cavities, tonsils and trachea samples, respectively. In dead pigs, 10⁸–10¹⁰ genome equivalents (ml^?1) of Myc. hyopneumoniae were detected in the lung tissue with pneumonia. The estimated minimal dose of Myc. hyopneumoniae required to induce pneumonia was 10⁵ colour‐changing units (CCU) per pig (corresponding to 10⁸ mycoplasmas). Conclusion: The triplex RT‐PCR test was validated and can be used for testing samples taken on the pig farms. Significance and Impact of the Study: This test should be a very useful tool in pig herds to control enzootic pneumonia or healthy carrier pigs and to study the dynamics of Myc. hyopneumoniae infections.     

Subcellular distribution of phytin in the endosperm of developing castor bean: a possibility for its synthesis in the cytoplasm prior to deposition within protein bodies

Studies using light and electron microscopy, and energy-dispersive X-ray analysis have allowed us to identify phytin particles within the cytoplasm of the developing endosperm of castor bean (Ricinus communis L.). These particles are present at the time of the formation of globoid particles within the protein bodies, but they are absent from mature tissue with fully formed protein bodies. We suggest that phytin is formed initially in the cytoplasm (perhaps in association with the cisternal endoplasmic reticulum) before being transported to the protein bodies, wherein it condenses to form the globoid.Abbreviations AMBB alcoholicmercuric bromophenol blue - ATBO acidic toluidine blue O - CER cisternal endoplasmic reticulum - DAP days after pollination - EDX energy-dispersive X-ray     

Acetate oxidation to CO2 via a citric acid cycle involving an ATP-citrate lyase: a mechanism for the synthesis of ATP via substrate level phosphorylation in Desulfobacter postgatei growing on acetate and sulfate

Desulfobacter postgatei is an acetate-oxidizing, sulfate-reducing bacterium that metabolizes acetate via the citric acid cycle. The organism has been reported to contain a si-citrate synthase (EC 4.1.3.7) which is activated by AMP and inorganic phosphate. It is show now, that the enzyme mediating citrate formation is an ATP-citrate lyase (EC 4.1.3.8) rather than a citrate synthase. Cell extracts (160,000xg supernatant) catalyzed the conversion of oxaloacetate (apparent K _m=0.2 mM), acetyl-CoA (app. K _m=0.1 mM), ADP (app. K _m=0.06 mM) and phosphate (app. K _m=0.7 mM) to citrate, CoA and ATP with a specific activity of 0.3 mol·min^-1·mg^-1 protein. Per mol citrate formed 1 mol of ATP was generated. Cleavage of citrate (app. K _m=0.05 mM; V _max=1.2 mol · min^-1 · mg^-1 protein) was dependent on ATP (app. K _m=0.4 mM) and CoA (app. K _m=0.05 mM) and yielded oxaloacetate, acetyl-CoA, ADP, and phosphate as products in a stoichiometry of citrate:CoA:oxaloacetate:ADP=1:1:1:1. The use of an ATP-citrate lyase in the citric acid cycle enables D. postgatei to couple the oxidation of acetate to 2 CO₂ with the net synthesis of ATP via substrate level phosphorylation.     

Intestinal fatty acid binding protein: a specific residue in one turn appears to stabilize the native structure and be responsible for slow refolding.

The intestinal fatty acid binding protein is one of a class of proteins that are primarily beta-sheet and contain a large interior cavity into which ligands bind. A highly conserved region of the protein exists between two adjacent antiparallel strands (denoted as D and E in the structure) that are not within hydrogen bonding distance. A series of single, double, and triple mutations have been constructed in the turn between these two strands. In the wild-type protein, this region has the sequence Leu 64/Gly 65/Val 66. Replacing Leu 64 with either Ala or Gly decreases the stability and the midpoint of the denaturation curve somewhat, whereas mutations at Gly 65 affect the stability slightly, but the protein folds at a rate similar to wild-type and binds oleate. Val 66 appears not to play an important role in maintaining stability. All double or triple mutations that include mutation of Leu 64 result in a large and almost identical loss of stability from the wild-type. As an example of the triple mutants, we investigated the properties of the Leu 64 Ser/Gly 65 Ala/Val 66 Asn mutant. As measured by the change in intrinsic fluorescence, this mutant (and similar triple mutants lacking leucine at position 64) folds much more rapidly than wild-type. The mutant, and others that lack Leu 64, have far-UV CD spectra similar to wild-type, but a different near-UV CD spectrum. The folded form of the protein binds oleate, although less tightly than wild-type. Hydrogen/deuterium exchange studies using electrospray mass spectrometry indicate many more rapidly exchangeable amide protons in the Leu 64 Ser/Gly 65 Ala/Val 66 Asn mutant. We propose that there is a loss of defined structure in the region of the protein near the turn defined by the D and E strands and that the interaction of Leu 64 with other hydrophobic residues located nearby may be responsible for (1) the slow step in the refolding process and (2) the final stabilization of the structure. We suggest the possibility that this region of the protein may be involved in both an early and late step in refolding.