Inhibition of ATPase activity by vanadate, having K1/2 of 0.5 mM, was demonstrated in the CF1-ATPase. The Ca(2+)-dependent ATPase activity of the isolated enzyme was inhibited in an allosteric manner by vanadate with a Hill coefficient of 3.19 +/- 0.6. Vanadate also inhibited ATPase and Pi-ATP exchange activities of the chloroplast membrane-bound enzyme. Using 51V NMR it was demonstrated that ATP caused partial release of about 1.87 equivalents while ADP caused additional binding of approximately 1.46 equivalents of vanadate, when added to a solution containing CF1 equilibrated with vanadate. The relevance of these results to a possible involvement of a pentacovalent phosphate as transition state intermediate in the hydrolysis of ATP by CF1-ATPase is discussed.
Na+,K+-ATPase (porcine alpha1/His10*beta1 or human alpha1/porcine His10*beta1) has been expressed in Pichia pastoris and purified by Co2+-chelate affinity resin chromatography, yielding about 80% pure, functional, and stable protein in a single step. The protein was eluted in nonionic detergents together with a phosphatidylserine. Size exclusion chromatography showed that the protein eluted in n-dodecyl beta-d-maltoside is an alpha1/beta1 protomer, whereas that in octaethylene glycol dodecyl monoether contains a mixture of alpha1/beta1 protomer and higher order oligomers. The Na+,K+-ATPase activity (8-16 (mumol/min)/mg of protein) is similar in both detergents. Thus, the minimal functional unit is the alpha1/beta1 protomer, and activity is unaffected by the presence of oligomeric forms. Screening of phospholipids for stabilization of the Na+,K+-ATPase activity shows that (a) acid phospholipids are required and phosphatidylserine is somewhat better than phosphatidylinositol and (b) optimal stabilization is achieved with asymmetric phosphatidylserines having saturated (18:0 >or= 16:0) and unsaturated (18:1 > 18:2) side chains at sn-1 an sn-2 positions, respectively. In the presence of phosphatidylserine, cholesterol stabilizes the protein at 37 degrees C, but not at 0 degrees C. Cholesterol also increases the "apparent affinity" of the phosphatidylserine and stabilizes optimally in the presence of phosphatidylserines with a saturated fatty acyl chain at the sn-1 position. Ergosterol is a poor stabilizer. We propose that phosphatidylserine and cholesterol interact specifically with each other near the alpha1/beta1 subunit interface, thus stabilizing the protein. These interactions do not seem to affect Na+,K+-ATPase activity.
Introduction and Objective: sweelin®, a novel sweet protein, ~3,000 sweeter than sugar enables up to 70% sugar reduction in numerous applications without compromising taste as assessed by professional sensory panels. The protein was designed using Agile-Integrative Computational Protein Design (AI-CPD) to improve stability and sensory profile of the monellin protein found in African fruits. It is produced via precision fermentation resulting with highly purified protein. Unlike small-molecule sweeteners, the protein is not expected to exhibit adverse effects, yet its digestibility required elucidation which was conducted via in vitro digestion models recreating gastric and intestinal proteolysis. Methods: Digestibility of sweelin® was studied using a computer controlled in vitro semi-dynamic digestion model that mimics the gastrointestinal conditions of a healthy adult via 2 consecutive steps, a dynamic gastric phase and a small intestinal (duodenal) phase that rely on an adjusted version of the INFOGEST in vitro digestion protocol. Digesta samples were collected at different timepoints of gastric and intestinal digestion and subsequently analyzed by SDS PAGE, with Coomassie Blue stain or Western blotting. Bioaccessible peptides were analyzed by LC-MS/MS. Results: sweelin® was readily digestible under intestinal conditions where bile and pancreatic secretions were added. In fact, the presence of physiological levels of trypsin and chymotrypsin led to the degradation of >95% of sweelin®. LC-MS/MS analysis of bioaccessible peptides reaffirmed that sweelin® is readily digested in the intestine into very short peptides. After 2h of intestinal digestion undigested protein levels were estimated to be <3% of the total ingested dose. Conclusions: sweelin® is a novel hyper-sweet designer protein shown to be effectively digested in the small intestine, much like commonly consumed proteins. Clinical trials are in progress to demonstrate no effect on diabetes-related biomarkers. Disclosure I. Samish: Other Relationship; Amai Proteins. U. Lesmes: None. Y. Lifshitz: Employee; Amai Proteins.
Abstract Calculations of electron inelastic mean free paths and stopping powers for several alkali halides (KF, KCl, KBr, and KI) and metal oxides (BeO, MgO, SiO 2 , and Al 2 O 3 ) have been performed in the 50 eV to 10 keV energy range. The complex dielectric formalism, improved to include the energy gap, was used for estimating the valence part of the transport characteristics, whereas the part related to electron‐core interactions was evaluated according to Gryzinski's theory. An extended comparison of these calculations with the available experimental data as well as with other theoretical predictions is presented. Trends of the energy dependence of the inelastic mean free path and stopping power in alkali halides are studied. The role of the plasmon deexcitation process as a source for low‐energy electrons in secondary electron emission spectra is discussed. The presented data can be used in Monte‐Carlo simulations of electron transport in the considered materials.
Palmitic acid (PA) comprises 17% to 25% of human milk fatty acids, of which 70% to 75% are esterified to the SN2 position of the triglyceride (SN2-palmitate). In vegetable oils, which are commonly used in infant formulas, palmitate is primarily esterified to other positions, resulting in reduced calcium and fat absorption and hard stools. The aim of this study was to elucidate the effects of SN2-palmitate on nutrient excretion.In total, 171 Chinese infants were included (within 14 days of birth) in this multicenter study. Formula-fed infants were randomly assigned to receive either SN2-palmitate formula (INFAT, n = 57) or control formula (n = 57). The formulas (Biostime, China) differed only in their SN2 PA proportions. Stool was collected at 6 postnatal weeks.The stool dry weight and fat content of the SN2-palmitate group were lower compared with the control group (dry weight 4.25 g vs 7.28 g, P < 0.05; fat 0.8 g vs 1.2 g, P < 0.05). The lipid component was also significantly lower for the SN2-palmitate group (0.79 g vs 1.19 g, P < 0.05). PA, representing ∼50% of the saponified fatty acids, was significantly lower in the SN2-palmitate group compared with the control group (0.3 g vs 0.7 g, P < 0.01). Breast-fed infants had a significantly lower stool dry weight, fat content, and saponified fat excretion compared with formula-fed infants (P < 0.01).Similar to breast milk, the SN2-palmitate infant formula primarily reduced calcium-saponified fat excretion. The results of this study further emphasize the nutritional importance of SN2-palmitate structured fat for infants.
ABSTRACT Objectives: Palmitic acid (PA) constitutes 17% to 25% of the human milk fatty acids, and ∼70% is esterified in the sn‐2 position of triglycerides (β‐palmitate). In the sn‐2 position, PA is not hydrolyzed and thus is efficiently absorbed. The PA in palm oils, commonly used in infant formulas, is esterified in the sn‐1 and sn‐3 positions. In these positions, PA is hydrolyzed and forms poorly absorbed calcium complexes. The present study assessed whether high β‐palmitate in infant formulas affects the intestinal flora. Methods: Thirty‐six term infants were enrolled: 14 breast‐fed (BF group) and 22 formula‐fed infants who were randomly assigned to receive formula containing high β‐palmitate (HBP group, n = 14), or low β‐palmitate (LBP group, n = 8), where 44% and 14% of the PA was β‐palmitate, respectively. The total amount of PA in the formulas was 19% and 22% in the LBP and HBP groups, respectively. Neither formula contained pre‐ or probiotics. Stool samples were collected at enrollment and at 6 weeks for the quantification of bacteria. Results: At 6 weeks, the HBP and BF groups had higher Lactobacillus and bifidobacteria counts than the LBP group ( P < 0.01). The Lactobacillus counts at 6 weeks were not significantly different between the HBP and BF groups. Lactobacillus counts were 1.2 × 10 10 , 1.2 × 10 11 , and 5.6 × 10 10 CFU/g for LBP, HBP, and BF groups, respectively. Bifidobacteria counts were 5.1 × 10 9 , 1.2 × 10 11 , and 3.9 × 10 10 CFU/g for LBP, HBP, and BF groups, respectively. Conclusions: HBP formula beneficially affected infant gut microbiota by increasing the Lactobacillus and bifidobacteria counts in fecal stools.
Background Palmitic-acid esterified to the sn-1,3 positions of the glycerol backbone (alpha, alpha'-palmitate), the predominant palmitate conformation in regular infant formula fat, is poorly absorbed and might cause abdominal discomfort. In contrast, palmitic-acid esterified to the sn-2 position (beta-palmitate), the main palmitate conformation in human milk fat, is well absorbed. The aim of the present study was to examine the influence of high alpha, alpha'-palmitate fat (HAPF) diet and high beta-palmitate fat (HBPF) diet on colitis development in Muc2 deficient (Muc2−/−) mice, a well-described animal model for spontaneous enterocolitis due to the lack of a protective mucus layer. Methods Muc2−/− mice received AIN-93G reference diet, HAPF diet or HBPF diet for 5 weeks after weaning. Clinical symptoms, intestinal morphology and inflammation in the distal colon were analyzed. Results Both HBPF diet and AIN-93G diet limited the extent of intestinal erosions and morphological damage in Muc2−/− mice compared with HAPF diet. In addition, the immunosuppressive regulatory T (Treg) cell response as demonstrated by the up-regulation of Foxp3, Tgfb1 and Ebi3 gene expression levels was enhanced by HBPF diet compared with AIN-93G and HAPF diets. HBPF diet also increased the gene expression of Pparg and enzymatic antioxidants (Sod1, Sod3 and Gpx1), genes all reported to be involved in promoting an immunosuppressive Treg cell response and to protect against colitis. Conclusions This study shows for the first time that HBPF diet limits the intestinal mucosal damage and controls the inflammatory response in Muc2−/− mice by inducing an immunosuppressive Treg cell response.
