RESUMO
OBJECTIVES: 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. METHODS: 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, nâ=â57) or control formula (nâ=â57). The formulas (Biostime, China) differed only in their SN2 PA proportions. Stool was collected at 6 postnatal weeks. RESULTS: 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, Pâ<â0.05; fat 0.8 g vs 1.2 g, Pâ<â0.05). The lipid component was also significantly lower for the SN2-palmitate group (0.79 g vs 1.19 g, Pâ<â0.05). PA, representing â¼50% 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, Pâ<â0.01). Breast-fed infants had a significantly lower stool dry weight, fat content, and saponified fat excretion compared with formula-fed infants (Pâ<â0.01). CONCLUSIONS: 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.
Assuntos
Dieta , Ácidos Graxos/metabolismo , Fórmulas Infantis/química , Absorção Intestinal/efeitos dos fármacos , Ácido Palmítico/farmacologia , Adulto , Aleitamento Materno , China , Digestão , Esterificação , Fezes/química , Feminino , Humanos , Recém-Nascido , Masculino , Leite Humano/química , Palmitatos , Ácido Palmítico/metabolismo , Gravidez , TriglicerídeosRESUMO
BACKGROUND: Beta-palmitate (sn-2 palmitate) mimics human milk fat, enabling easier digestion.Therefore, we hypothesized that infants consuming high beta-palmitate formula would have more frequent, softer stools and reduced crying compared to infants consuming low beta-palmitate formula. METHODS: Formula-fed infants were randomly assigned to receive either (1) formula with high beta-palmitate (HBP, n = 21) or (2) regular formula with a standard vegetable oil mix (LBP, n = 21). A matched group of breastfed infants served as a reference (BF, n = 21). Crying and stool characteristics data were recorded by the parents for 3 days before the 6- and 12-week visits. RESULTS: We found no significant differences in the stool frequency or consistency between the two formula groups. The percentage of crying infants in the LBP group was significantly higher than that in the HBP and BF groups during the evening at 6 weeks (88.2% vs. 56.3% and 55.6%, p < 0.05) and during the afternoon at 12 weeks (91.7% vs. 50.0% and 40%, p < 0.05). The infants fed HBP had significantly shorter crying durations when compared with infants fed LBP formula (14.90 ± 3.85 vs.63.96 ± 21.76 min/day, respectively; p = 0.047). CONCLUSIONS: Our study indicates that consumption of a high beta-palmitate formula affects infant crying patterns during the first weeks of life. Comparable to breastfeeding, it reduced crying duration and frequency, primarily during the afternoon and evening hours, thereby improving the well-being of formula-fed infants and their parents. TRIAL REGISTRATION: NCT00874068.Registration date March 31, 2009.
Assuntos
Choro , Fórmulas Infantis/administração & dosagem , Fórmulas Infantis/química , Palmitatos/análise , Adulto , Aleitamento Materno , Defecação , Método Duplo-Cego , Feminino , Humanos , Lactente , Recém-Nascido , MasculinoRESUMO
We aimed to compare the effect of 12-week feeding of commercially available infant formulas with different percentages of palmitic acid at sn-2 (beta-palmitate) on anthropometric measures and bone strength of term infants. It was hypothesized that feeding infants with high beta-palmitate (HBP) formula will enhance their bone speed of sound (SOS). Eighty-three infants appropriate for gestational age participated in the study; of these, 58 were formula-fed and 25 breast-fed infants, serving as a reference group. The formula-fed infants were randomly assigned to receive HBP formula (43 % of the palmitic acid is esterified to the middle position of the glycerol backbone, study group; n = 30) or regular formula with low-beta palmitate (LBP, 14 % of the palmitic acid is esterified to the middle position of the glycerol backbone, n = 28). Sixty-six infants completed the 12-week study. Anthropometric and quantitative ultrasound measurements of bone SOS for assessment of bone strength were performed at randomization and at 6 and 12 weeks postnatal age. At randomization, gestational age, birth weight, and bone SOS were comparable between the three groups. At 12 weeks postnatal age, the mean bone SOS of the HBP group was significantly higher than that of the LBP group (2,896 ± 133 vs. 2,825 ± 79 m/s respectively, P = 0.049) and comparable with that of the breast-fed group (2,875 ± 85 m/s). We concluded that infants consuming HBP formula had changes in bone SOS that were comparable to those of infants consuming breast milk and favorable compared to infants consuming LBP formula.
Assuntos
Osso e Ossos/efeitos dos fármacos , Ácido Palmítico/uso terapêutico , Antropometria/métodos , Desenvolvimento Ósseo/efeitos dos fármacos , Osso e Ossos/fisiologia , Aleitamento Materno , Método Duplo-Cego , Feminino , Seguimentos , Humanos , Lactente , Alimentos Infantis , Fórmulas Infantis , Recém-Nascido , Masculino , Fatores de Tempo , Ultrassonografia/métodosRESUMO
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 (ß-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 ß-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 ß-palmitate (HBP group, n=14), or low ß-palmitate (LBP group, n=8), where 44% and 14% of the PA was ß-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 1.2×10¹°, 1.2×10¹¹, and 5.6×10¹° CFU/g for LBP, HBP, and BF groups, respectively. Bifidobacteria counts were 5.1×109, 1.2×10¹¹, and 3.9×10¹° 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.
Assuntos
Bifidobacterium/crescimento & desenvolvimento , Fórmulas Infantis/metabolismo , Mucosa Intestinal/microbiologia , Intestinos/microbiologia , Lactobacillus/crescimento & desenvolvimento , Ácido Palmítico/metabolismo , Triglicerídeos/metabolismo , Bifidobacterium/isolamento & purificação , Bifidobacterium/metabolismo , Desenvolvimento Infantil , Estudos de Coortes , Contagem de Colônia Microbiana , Registros de Dieta , Digestão , Método Duplo-Cego , Fezes/microbiologia , Humanos , Fórmulas Infantis/química , Recém-Nascido , Absorção Intestinal , Mucosa Intestinal/metabolismo , Isomerismo , Lactobacillus/isolamento & purificação , Lactobacillus/metabolismo , Leite Humano/química , Leite Humano/metabolismo , Ácido Palmítico/análise , Projetos Piloto , Triglicerídeos/químicaRESUMO
FXYD proteins are a family of seven small regulatory proteins, expressed in a tissue-specific manner, that associate with Na,K-ATPase as subsidiary subunits and modulate kinetic properties. This study describes an additional property of FXYD proteins as stabilizers of Na,K-ATPase. FXYD1 (phospholemman), FXYD2 (γ subunit), and FXYD4 (CHIF) have been expressed in Escherichia coli and purified. These FXYD proteins associate spontaneously in vitro with detergent-soluble purified recombinant human Na,K-ATPase (α1ß1) to form α1ß1FXYD complexes. Compared with the control (α1ß1), all three FXYD proteins strongly protect Na,K-ATPase activity against inactivation by heating or excess detergent (C(12)E(8)), with effectiveness FXYD1 > FXYD2 ≥ FXYD4. Heating also inactivates E(1) â E(2) conformational changes and cation occlusion, and FXYD1 protects strongly. Incubation of α1ß1 or α1ß1FXYD complexes with guanidinium chloride (up to 6 m) causes protein unfolding, detected by changes in protein fluorescence, but FXYD proteins do not protect. Thus, general protein denaturation is not the cause of thermally mediated or detergent-mediated inactivation. By contrast, the experiments show that displacement of specifically bound phosphatidylserine is the primary cause of thermally mediated or detergent-mediated inactivation, and FXYD proteins stabilize phosphatidylserine-Na,K-ATPase interactions. Phosphatidylserine probably binds near trans-membrane segments M9 of the α subunit and the FXYD protein, which are in proximity. FXYD1, FXYD2, and FXYD4 co-expressed in HeLa cells with rat α1 protect strongly against thermal inactivation. Stabilization of Na,K-ATPase by three FXYD proteins in a mammalian cell membrane, as well the purified recombinant Na,K-ATPase, suggests that stabilization is a general property of FXYD proteins, consistent with a significant biological function.
Assuntos
Proteínas de Membrana/química , Complexos Multiproteicos/química , Fosfatidilserinas/química , Fosfoproteínas/química , Dobramento de Proteína , ATPase Trocadora de Sódio-Potássio/química , Animais , Células HeLa , Temperatura Alta , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Fosfatidilserinas/genética , Fosfatidilserinas/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ratos , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
The human α(1)/His(10)-ß(1) isoform of the Na,K-ATPase has been expressed in Pichia pastoris, solubilized in n-dodecyl-ß-maltoside, and purified by metal chelate chromatography. The α(1)ß(1) complex spontaneously associates in vitro with the detergent-solubilized purified human FXYD1 (phospholemman) expressed in Escherichia coli. It has been confirmed that FXYD1 spontaneously associates in vitro with the α(1)/His(10)-ß(1) complex and stabilizes it in an active mode. The functional properties of the α(1)/His(10)-ß(1) and α(1)/His(10)-ß(1)/FXYD1 complexes have been investigated by fluorescence methods. The electrochromic dye RH421 which monitors binding to and release of ions from the binding sites has been applied in equilibrium titration experiments to determine ion binding affinities and revealed that FXYD1 induces an â¼30% increase of the Na(+)-binding affinity in both the E(1) and P-E(2) conformations. By contrast, it does not affect the affinities for K(+) and Rb(+) ions. Phosphorylation induced partial reactions of the enzyme have been studied as backdoor phosphorylation by inorganic phosphate and in kinetic experiments with caged ATP in order to evaluate the ATP-binding affinity and the time constant of the conformational transition, Na(3)E(1)-P â P-E(2)Na(3). No significant differences with or without FXYD1 could be detected. Rate constants of the conformational transitions Rb(2)E(1) â E(2)(Rb(2)) and E(2)(Rb(2)) â Na(3)E(1), investigated with fluorescein-labeled Na,K-ATPase, showed only minor or no effects of FXYD1, respectively. The conclusion from all these experiments is that FXYD1 raises the binding affinity of α(1)ß(1) for Na ions, presumably at the third Na-selective binding site. In whole cell expression studies FXYD1 reduces the apparent affinity for Na ions. Possible reasons for the difference from this study using the purified recombinant Na,K-ATPase are discussed.
Assuntos
Regulação Enzimológica da Expressão Gênica , Proteínas de Membrana/fisiologia , Fosfoproteínas/fisiologia , ATPase Trocadora de Sódio-Potássio/química , Sódio/química , Trifosfato de Adenosina/química , Animais , Sítios de Ligação , Bovinos , Relação Dose-Resposta a Droga , Humanos , Íons , Cinética , Proteínas de Membrana/química , Fosfoproteínas/química , Pichia/metabolismo , Isoformas de Proteínas , Proteínas Recombinantes/química , Albumina Sérica/química , Espectrometria de Fluorescência/métodosRESUMO
There are four isoforms of the alpha subunit (alpha1-4) and three isoforms of the beta subunit (beta1-3) of Na,K-ATPase, with distinct tissue-specific distribution and physiological functions. alpha2 is thought to play a key role in cardiac and smooth muscle contraction and be an important target of cardiac glycosides. An alpha2-selective cardiac glycoside could provide important insights into physiological and pharmacological properties of alpha2. The isoform selectivity of a large number of cardiac glycosides has been assessed utilizing alpha1beta1, alpha2beta1, and alpha3beta1 isoforms of human Na,K-ATPase expressed in Pichia pastoris and the purified detergent-soluble isoform proteins. Binding affinities of the digitalis glycosides, digoxin, beta-methyl digoxin, and digitoxin show moderate but highly significant selectivity (up to 4-fold) for alpha2/alpha3 over alpha1 (K(D) alpha1 > alpha2 = alpha3). By contrast, ouabain shows moderate selectivity ( approximately 2.5-fold) for alpha1 over alpha2 (K(D) alpha1 Assuntos
Glicosídeos Cardíacos/metabolismo
, Glicosídeos Digitálicos/metabolismo
, ATPase Trocadora de Sódio-Potássio/química
, Membrana Celular/metabolismo
, Humanos
, Íons
, Cinética
, Modelos Biológicos
, Modelos Estatísticos
, Miocárdio/metabolismo
, Pichia/metabolismo
, Potássio/química
, Ligação Proteica
, Isoformas de Proteínas
, Fatores de Tempo
RESUMO
PURPOSE: The aim of the current study was to evaluate the therapeutic effects of omega-3 plant sterol esters (n-3-PSE) on lipid profile and other coronary heart disease risk factors in subjects with mixed hyperlipidemia. METHODS: Ninety-one patients with mixed hyperlipidemia were randomized in a double blind fashion to receive either placebo (corn oil) or n-3-PSE. Twenty four patients dropped out or were excluded from the efficacy analysis due to protocol violation. The primary efficacy endpoint was mean change in plasma low-density lipoprotein cholesterol (LDL-C) levels after 12 weeks of treatment. Other efficacy measures included plasma lipids, lipoproteins, and high-sensitivity C-reactive protein (hsCRP) levels. Participants who completed the double-blind study were given the option to continue into an open-label, 12-weeks follow up phase. RESULTS: n-3-PSE treatment did not result in a significant change in LDL-C levels. Triglyceride levels were reduced significantly by 19% (51 mg/dL, p < 0.0001) in the n-3-PSE group in comparison with the placebo group (p = 0.025). Diastolic blood pressure and hsCRP were reduced by 7% (5.9 mmHg) and 7.8% (0.6 mg/L), respectively, and were significantly different from the placebo group (p = 0.036 and p = 0.018, respectively). CONCLUSIONS: In patients with mixed hyperlipidemia, n-3-PSE treatment may offer a safe and effective therapy for triglyceride level reduction while avoiding the typical increase in LDL-C levels associated with n-3 fatty acid treatment. The observed reduction in blood pressure and inflammation markers warrants further evaluation. The positive effect of n-3-PSE treatment was preserved at the end of the follow up phase.
Assuntos
Ácidos Graxos Ômega-3/uso terapêutico , Hipercolesterolemia/sangue , Hipercolesterolemia/tratamento farmacológico , Fitosteróis/uso terapêutico , Pressão Sanguínea/efeitos dos fármacos , Proteína C-Reativa/metabolismo , LDL-Colesterol/sangue , Método Duplo-Cego , Ésteres/efeitos adversos , Ésteres/uso terapêutico , Ácidos Graxos Ômega-3/efeitos adversos , Feminino , Humanos , Hipercolesterolemia/fisiopatologia , Lipídeos/sangue , Lipoproteínas/sangue , Masculino , Pessoa de Meia-Idade , Fitosteróis/efeitos adversos , Placebos , Triglicerídeos/sangueRESUMO
The amount, composition, and sources of nutrition support provided to preterm infants is critical for normal growth and development, and particularly for structural and functional neurodevelopment. Although omega-3 long chain polyunsaturated fatty acids (LC-PUFA), and particularly docosahexanoic acid (DHA), are considered of particular importance, results from clinical trials with preterm infants have been inconclusive because of ethical limitations and confounding variables. A translational large animal model is needed to understand the structural and functional responses to DHA. Neurodevelopment of preterm pigs was evaluated in response to feeding formulas to term-equivalent age supplemented with DHA attached to phosphatidylserine (PS-DHA) or sunflower oil as the placebo. Newborn term pigs were used as a control for normal in utero neurodevelopment. Supplementing formula with PS-DHA increased weight of the brain, and particularly the cerebellum, at term-equivalent age compared with placebo preterm pigs (P's < 0.10 and 0.05 respectively), with a higher degree of myelination in all regions of the brain examined (all p < 0.06). Brains of pigs provided PS-DHA were similar in weight to newborn term pigs. Event-related brain potentials and performance in a novel object recognition test indicated the PS-DHA supplement accelerated development of sensory pathways and recognition memory compared with placebo preterm pigs. The PS-DHA did not increase weight gain, but was associated with higher survival. The benefits of PS-DHA include improving neurodevelopment and possibly improvement of survival, and justify further studies to define dose-response relations, compare benefits associated with other sources of DHA, and understand the mechanisms underlying the benefits and influences on the development of other tissues and organ systems.
Assuntos
Encéfalo/efeitos dos fármacos , Suplementos Nutricionais , Ácidos Docosa-Hexaenoicos/administração & dosagem , Neurogênese/efeitos dos fármacos , Fosfatidilserinas/administração & dosagem , Nascimento Prematuro , Fatores Etários , Ração Animal , Fenômenos Fisiológicos da Nutrição Animal , Animais , Comportamento Animal/efeitos dos fármacos , Encéfalo/diagnóstico por imagem , Encéfalo/crescimento & desenvolvimento , Ácidos Docosa-Hexaenoicos/metabolismo , Potenciais Evocados/efeitos dos fármacos , Idade Gestacional , Imageamento por Ressonância Magnética , Fosfatidilserinas/metabolismo , Reconhecimento Psicológico/efeitos dos fármacos , Células Receptoras Sensoriais/efeitos dos fármacos , Sus scrofa , Aumento de PesoRESUMO
Human milk provides the optimal balanced nutrition for the growing infant in the first months after birth. The human mammary gland has evolved with unusual pathways, resulting in a specific positioning of fatty acids at the outer sn-1 and sn-3, and center sn-2 of the triacylglyceride, which is different from the triglycerides in other human tissues and plasma. The development of structured triglycerides enables mimicking the composition as well as structure of human milk fat in infant formulas. Studies conducted two decades ago, together with very recent studies, have provided increasing evidence that this unusual positioning of 16:0 in human milk triglycerides has a significant role for infant health in different directions, such as fat and calcium absorption, bone health, intestinal flora and infant comfort. This review aims to unravel the relevance of human milk triglyceride sn-2 16:0 for intestinal health and inflammatory pathways and for other post-absorption effects.
Assuntos
Ácidos Palmíticos/metabolismo , Triglicerídeos/fisiologia , Animais , Desenvolvimento Ósseo , Cálcio/metabolismo , Gorduras na Dieta/metabolismo , Humanos , Lactente , Bem-Estar do Lactente , Absorção Intestinal , Leite Humano/fisiologiaRESUMO
OBJECTIVE: To evaluate the efficacy and safety of soybean-derived phosphatidylserine (SB-PS) (300 mg/day) in improving cognitive performance in elderly with memory complaints, following a short duration of 12 weeks' SB-PS administration. METHODS: SB-PS was administered daily for 12 weeks to 30 elderly volunteers with memory complaints (age range 50-90 years). Cognitive performance was determined by a computerized test battery and by the Rey Auditory Verbal Learning Test (Rey-AVLT). Physical examination and blood safety parameters were part of the extensive safety analysis of PS that was performed. RESULTS: The computerized test results showed that SB-PS supplementation significantly increased the following cognitive parameters: memory recognition (P = 0.004), memory recall (P = 0.006), executive functions (P = 0.004), and mental flexibility (P = 0.01). The Rey-AVLT indicated that, following SB-PS administration, total learning and immediate recall improved significantly (P = 0.013 and P = 0.007, respectively). Unexpected results from the safety tests suggested that SB-PS significantly reduces both systolic (P = 0.043) and diastolic (P = 0.003) blood pressure. SB-PS consumption was well tolerated and no serious adverse events were reported during the study. CONCLUSION: This exploratory study demonstrates that SB-PS may have favorable effects on cognitive function in elderly with memory complaints. In addition, the study suggests that SB-PS is safe for human consumption and may serve as a safe alternative to phosphatidylserine extracted from bovine cortex. These results encourage further extended studies in order to establish the safety and efficacy of SB-PS treatment.
Assuntos
Glycine max/química , Transtornos da Memória/tratamento farmacológico , Fosfatidilserinas/uso terapêutico , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Feminino , Avaliação Geriátrica , Humanos , Masculino , Pessoa de Meia-Idade , Testes Neuropsicológicos , Projetos Piloto , Estatísticas não Paramétricas , Resultado do TratamentoRESUMO
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.
Assuntos
Colite/prevenção & controle , Dieta Hiperlipídica/métodos , Regulação da Expressão Gênica/imunologia , Mucina-2/deficiência , Palmitatos/farmacologia , Animais , Colite/genética , Colite/imunologia , Primers do DNA/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas Histológicas , Imuno-Histoquímica , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estatísticas não Paramétricas , Linfócitos T Reguladores/imunologiaAssuntos
Proteínas de Membrana/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Sítios de Ligação , Células HeLa , Humanos , Proteínas de Membrana/química , Subunidades Proteicas , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , ATPase Trocadora de Sódio-Potássio/química , TransfecçãoRESUMO
Human alpha1 and alpha2 isoforms of Na,K-ATPase have been expressed with porcine 10*Histidine-tagged beta1 subunit in Pichia pastoris. Methanol-induced expression of alpha2 is optimal at 20 degrees C, whereas at 25 degrees C, which is optimal for expression of alpha1, alpha2 is not expressed. Detergent-soluble alpha2beta1 and alpha1beta1 complexes have been purified in a stable and functional state. alpha2beta1 shows a somewhat lower Na,K-ATPase activity and higher K0.5K compared to alpha1beta1, while values of K0.5Na and KmATP are similar. Ouabain inhibits both alpha1beta1 (K0.5 24.6 +/- 6 nM) and alpha2beta1 (K0.5 102 +/- 14 nM) with high affinity. A striking difference between the isoforms is that alpha2beta1 is unstable. Both alpha1beta1 and alpha2beta1 complexes, prepared in C12E8 with an added phosphatidyl serine, are active, but alpha2beta1 is rapidly inactivated at 0 degrees C. Addition of low concentrations of cholesterol with 1-stearoyl-2-oleoyl-sn-glycero-3-[phospho-l-serine] (SOPS) stabilizes strongly, maintaining alpha2beta1 active up to two weeks at 0 degrees C. By contrast, alpha1beta1 is stable at 0 degrees C without added cholesterol. Both alpha1beta1 and alpha2beta1 complexes are stabilized by cholesterol at 37 degrees C. Human FXYD1 spontaneously associates in vitro with either alpha1beta1 or alpha2beta1, to form alpha1beta1/FXYD1 and alpha2beta1/FXYD1 complexes. The reconstituted FXYD1 protects both alpha1beta1 and alpha2beta1 very strongly against thermal inactivation. Instability of alpha2 is attributable to suboptimal phophatidylserine-protein interactions. Residues within TM8, TM9 and TM10, near the alphabeta subunit interface, may play an important role in differential interactions of lipid with alpha1 and alpha2, and affect isoform stability. Possible physiological implications of isoform interactions with phospholipids and FXYD1 are discussed.
Assuntos
Colesterol/farmacologia , Expressão Gênica , Proteínas de Membrana/metabolismo , Fosfatidilserinas/farmacologia , Fosfoproteínas/metabolismo , Pichia/metabolismo , ATPase Trocadora de Sódio-Potássio/isolamento & purificação , ATPase Trocadora de Sódio-Potássio/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Cinética , Proteínas de Membrana/genética , Modelos Moleculares , Fosfoproteínas/genética , Pichia/genética , Ligação Proteica , Desnaturação Proteica , Estrutura Quaternária de Proteína , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/genética , TemperaturaRESUMO
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.
Assuntos
Membrana Celular/enzimologia , Lipídeos de Membrana/metabolismo , Pichia/enzimologia , ATPase Trocadora de Sódio-Potássio/isolamento & purificação , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Colesterol/farmacologia , Detergentes/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Expressão Gênica , Humanos , Lipídeos de Membrana/química , Modelos Biológicos , Fosfolipídeos/metabolismo , Pichia/genética , Pichia/ultraestrutura , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/isolamento & purificação , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/genética , SuínosRESUMO
Human FXYD1 (phospholemman, PLM) has been expressed in Pichia pastoris with porcine alpha1/His10-beta1 subunits of Na+,K+-ATPase or alone. Dodecyl-beta-maltoside-soluble complexes of alpha1/beta1/PLM have been purified by metal chelate chromatography, either from membranes co-expressing alpha1,His10-beta1, and PLM or by in vitro reconstitution of PLM with alpha1/His10-beta1 subunits. Comparison of functional properties of purified alpha1/His10-beta1 and alpha1/His10-beta1/PLM complexes show that PLM lowered K0.5 for Na+ ions moderately (approximately 30%) but did not affect the turnover rate or Km of ATP for activating Na+,K+-ATPase activity. PLM also stabilized the alpha1/His10-beta1 complex. In addition, PLM markedly (>3-fold) reduced the K0.5 of Na+ ions for activating Na+-ATPase activity. In membranes co-expressing alpha1/His10-beta1 with PLM the K0.5 of Na+ ions was also reduced, compared with the control, excluding the possibility that detergent or lipid in purified complexes compromise functional interactions. When expressed in HeLa cells with rat alpha1, rat PLM significantly raised the K0.5 of Na+ ions, whereas for a chimeric molecule consisting of transmembranes segments of PLM and extramembrane segments of FXYD4, the K0.5 of Na+ ions was significantly reduced, compared with the control. The opposite functional effects in P. pastoris and HeLa cells are correlated with endogenous phosphorylation of PLM at Ser68 or unphosphorylated PLM, respectively, as detected with antibodies, which recognize PLM phosphorylated at Ser68 (protein kinase A site) or unphosphorylated PLM. We hypothesize that PLM interacts with alpha1/His10-beta1 subunits at multiple locations, the different functional effects depending on the degree of phosphorylation at Ser68. We discuss the role of PLM in regulation of Na+,K+-ATPase in cardiac or skeletal muscle cells.
Assuntos
Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Pichia/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Western Blotting , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Células HeLa , Humanos , Imunoprecipitação , Cinética , Proteínas de Membrana/isolamento & purificação , Fosfoproteínas/isolamento & purificação , Fosforilação , ATPase Trocadora de Sódio-Potássio/isolamento & purificaçãoRESUMO
Corticosteroid hormone-induced factor (CHIF) and the gamma subunit of the Na,K-ATPase (gamma) are two members of the FXYD family whose function has been elucidated recently. CHIF and gamma interact with the Na+ pump and alter its kinetic properties, in different ways, which appear to serve their specific physiological roles. Although functional interactions with the Na,K-ATPase have been clearly demonstrated, it is not known which domains and which residues interact with the alpha and/or beta subunits and affect the pump kinetics. The current study provides the first systematic analysis of structure-function relations of CHIF and gamma. It is demonstrated that the stability of detergent-solubilized complexes of CHIF and gamma with alpha and/or beta subunits is determined by the trans-membrane segments, especially three residues that may be involved in hydrophobic interactions. The transmembrane segments also determine the opposite effects of CHIF and gamma on the Na+ affinity of the pump, but the amino acids involved in this functional effect are different from those responsible for stable interactions with alpha.