Changes in the Sterol Composition of the Plasma Membrane Affect Membrane Potential,Salt Tolerance and the Activity of Multidrug Resistance Pumps in Saccharomyces cerevisiae

We investigated the impact of the deletions of genes from the final steps in the biosynthesis of ergosterol (ERG6, ERG2, ERG3, ERG5, ERG4) on the physiological function of the Saccharomyces cerevisiae plasma membrane by a combination of biological tests and the diS-C₃(3) fluorescence assay. Most of the erg mutants were more sensitive than the wild type to salt stress or cationic drugs, their susceptibilities were proportional to the hyperpolarization of their plasma membranes. The different sterol composition of the plasma membrane played an important role in the short-term and long-term processes that accompanied the exposure of erg strains to a hyperosmotic stress (effect on cell size, pH homeostasis and survival of yeasts), as well as in the resistance of cells to antifungal drugs. The pleiotropic drug-sensitive phenotypes of erg strains were, to a large extent, a result of the reduced efficiency of the Pdr5 efflux pump, which was shown to be more sensitive to the sterol content of the plasma membrane than Snq2p. In summary, the erg4Δ and erg6Δ mutants exhibited the most compromised phenotypes. As Erg6p is not involved in the cholesterol biosynthetic pathway, it may become a target for a new generation of antifungal drugs.     

细胞膜磷脂脂肪酸组成对自絮凝酵母质膜ATP酶响应酒精刺激的影响及其与菌体耐酒精的关系

研究揭示细胞膜磷脂脂肪酸组成与质膜ATP酶在酵母菌耐酒精中的一种新颖关系。实验表明,细胞膜磷脂脂肪酸组成特点对生长于未添加酒精条件下的自絮凝颗粒酵母质膜ATP酶活性没有影响,但却明显影响生长于添加酒精(1％～10％,V／V)条件下的菌体质膜ATP酶对酒精激活的敏感性：预培养于添加0．6mmol／L棕榈酸、亚油酸、或亚麻酸条件下的菌体的质膜ATP酶的最大激活水平分别为各自酶的基态水平(未激活)的3．6、1．5和1．2倍,而对照组(预培养于未添加脂肪酸条件下的菌体)的相应值为2．3倍,酶产生上述最大激活水平时的酒精浓度分别为7％、6％、6％、和7％(V/V)。酶激活后米氏常数Km、最适pH和对钒酸钠(质膜ATP酶特异性抑制剂)的敏感性等性质不变,但最大反应速度υmax明显增加。实验表明,细胞膜磷脂脂肪酸组成特点对提高菌体的耐酒精能力越有利,则其质膜ATP酶被酒精激活的幅度越大,说明菌体耐酒精能力的提高与其质膜ATP酶对酒精激活的敏感性的增加密切相关。细胞膜磷脂脂肪酸组成会影响酵母菌质膜ATP酶对酒精激活的敏感性是观察到的新的实验现象。     

Glycolysis Controls Plasma Membrane Glucose Sensors To Promote Glucose Signaling in Yeasts

Sensing of extracellular glucose is necessary for cells to adapt to glucose variation in their environment. In the respiratory yeast Kluyveromyces lactis, extracellular glucose controls the expression of major glucose permease gene RAG1 through a cascade similar to the Saccharomyces cerevisiae Snf3/Rgt2/Rgt1 glucose signaling pathway. This regulation depends also on intracellular glucose metabolism since we previously showed that glucose induction of the RAG1 gene is abolished in glycolytic mutants. Here we show that glycolysis regulates RAG1 expression through the K. lactis Rgt1 (KlRgt1) glucose signaling pathway by targeting the localization and probably the stability of Rag4, the single Snf3/Rgt2-type glucose sensor of K. lactis. Additionally, the control exerted by glycolysis on glucose signaling seems to be conserved in S. cerevisiae. This retrocontrol might prevent yeasts from unnecessary glucose transport and intracellular glucose accumulation.     

Fluidization of Membrane Lipids Enhances the Tolerance of Saccharomyces cerevisiae to Freezing and Salt Stress

Unsaturated fatty acids play an essential role in the biophysical characteristics of cell membranes and determine the proper function of membrane-attached proteins. Thus, the ability of cells to alter the degree of unsaturation in their membranes is an important factor in cellular acclimatization to environmental conditions. Many eukaryotic organisms can synthesize dienoic fatty acids, but Saccharomyces cerevisiae can introduce only a single double bond at the Δ⁹ position. We expressed two sunflower (Helianthus annuus) oleate Δ¹² desaturases encoded by FAD2-1 and FAD2-3 in yeast cells of the wild-type W303-1A strain (trp1) and analyzed their effects on growth and stress tolerance. Production of the heterologous desaturases increased the content of dienoic fatty acids, especially 18:2Δ^9,12, the unsaturation index, and the fluidity of the yeast membrane. The total fatty acid content remained constant, and the level of monounsaturated fatty acids decreased. Growth at 15°C was reduced in the FAD2 strains, probably due to tryptophan auxotrophy, since the trp1 (TRP1) transformants that produced the sunflower desaturases grew as well as the control strain did. Our results suggest that changes in the fluidity of the lipid bilayer affect tryptophan uptake and/or the correct targeting of tryptophan transporters. The expression of the sunflower desaturases, in either Trp⁺ or Trp⁻ strains, increased NaCl tolerance. Production of dienoic fatty acids increased the tolerance to freezing of wild-type cells preincubated at 30°C or 15°C. Thus, membrane fluidity is an essential determinant of stress resistance in S. cerevisiae, and engineering of membrane lipids has the potential to be a useful tool of increasing the tolerance to freezing in industrial strains.     

Fasting Plasma Glucose (FPG) and the Risk of Impaired Glucose Tolerance in Obese Children and Adolescents

A timely diagnosis of impaired glucose tolerance (IGT) is desirable in obesity. The oral glucose tolerance test (OGTT), the gold standard to diagnose this condition, may not be realistically performed in all patients due to discomfort, labor, and cost. The aim of this study was to assess whether one or more biochemical indexes measured in fasting conditions could be used to identify obese children at risk of IGT. A cohort of 563 white obese children and adolescents (M/F: 315/248; aged 4–17 years) was recruited and underwent anthropometric evaluation and OGTT. Anthropometric parameters, fasting plasma glucose (FPG), fasting serum insulin (FSI), and homeostasis model assessment of insulin resistance (HOMA_IR) were tested in pursuit of a possible threshold to be used as a predictor of IGT. Thirty‐seven children (6.9%) had IGT and one child (0.1%) had type 2 diabetes (T2D). FPG, FSI, and HOMA_IR were all significantly higher in children with IGT than in children without IGT. Receiver‐operating characteristic (ROC) curve analyses run for gender and puberty‐adjusted FPG, FSI, and HOMA_IR were all significant: area under the curve (95% confidence interval) equaled 0.68 (0.59–0.76), 0.66 (0.56–0.76), and 0.68 (0.59–0.78), respectively. The three parameters did not show significantly different sensitivity/specificity in the pooled population or in the gender/puberty subgroups. Thresholds varied among gender/puberty subgroups for FSI and HOMA_IR, but not for FPG, which showed a fixed threshold of 86 mg/dl. A gender/puberty independent cutoff of FPG may be considered a screening tool to narrow clinical indication to OGTT in obese white children and adolescents.     

膜脂组成与植物抗冷性的关系及其分子生物学研究进展

植物的抗冷性与膜脂的组分和结构密切相关,与质膜中脂肪酸的不饱和度关系更为密切。膜脂不饱和脂肪酸含量越高,膜脂相变温度越低,植物的抗冷性提高。植物体内存在一些降低膜脂脂肪酸饱和程度的酶,如甘油3磷酸酰基转移酶,ω3脂肪酸去饱和酶等,它们能够催化膜中脂肪酸的去饱和反应,生成不饱和脂肪酸,从而提高植物的抗冷性。本文就低温对膜脂的影响、膜脂组成与植物抗寒性的关系及其分子生物学研究进展作一简单综述。     

Isolation and Characterization of Protoplasts from Saccharomyces rouxii

Cells of the osmotolerant yeast Saccharomyces rouxii were transformed to protoplasts in good yield (85%) by digesting cell walls with snail-gut enzyme in the presence of 10 mM dithioerythritol, 0.1 M sodium phosphate buffer (pH 6.8), and 2.0 M KCl. The requirement for 2.0 M KCl compares with that for S. bisporus var. mellis (another osmotolerant species) and contrasts with the 0.3 to 0.8 M KCl concentrations used in the preparation of most yeast protoplasts. Short digestions (60 min or less) produced mostly spheroplasts; longer incubations (90 min or more) yielded mostly protoplasts as judged by electron micrographs. These protoplasts could be transferred to 1.0 M KCl or 2.0 M sorbitol without lysing, but lysis was pronounced in 0.5 M KCl or 1.0 M mannitol and complete in 0.02 M KCl. Protoplasts were separated from isolated cell wall remnants and debris by centrifugation on a linear gradient of Ficoll 400 (35 to 17.5%, wt/vol) containing 2.0 M KCl. Both crude and fractionated protoplast preparations contained vesicles which were identified with the periplasmic bodies of whole cells. Some of the periplasmic bodies were connected to protoplasts by fine pedicels; others appeared free. Independent degeneracy of periplasmic bodies was occasionally observed. beta-Fructofuranosidase (EC 3.2.1.26) activity is cryptic (physically) in cells of S. rouxii in contrast to the expressed enzyme (periplasmic space) of other Saccharomyces species. This enzyme remains cryptic in protoplast preparations of S. rouxii but is expressed upon lysis. The same specific activities were found per unit cell or protoplast. The possible association of the cryptic enzyme with periplasmic bodies is discussed.     

Influence of Glucose Concentration on the Membrane Stability of Human Erythrocytes

The action of glucose as an osmolyte in relation to blood cells is not well-characterized in the literature. This study aimed to study the influence of glucose concentration on the stability of red blood cells. The stability of erythrocytes was evaluated by the half-transition point obtained from the curves of lysis induced by glucose in the absence of salt or by increase in medium hypotonicity in the absence and the presence of different concentrations of glucose. In the presence of 0.9 g/dl NaCl, there was no hemolysis with increasing concentration of glucose from 0 to 10 g/dl. In the absence of NaCl, the dependence of hemolysis with the 0–10 g/dl glucose was described by a decreasing sigmoid, with fully lysed and fully protected cells being encountered in the presence of 0–2 and 4–10 g/dl glucose, respectively. The possible origin of such stabilization effect is discussed with base of what is known about osmostabilization of biological complexes and about the influence of glucose on the rheological properties of erythrocytes.     

Iron Reduction and Trans Plasma Membrane Electron Transfer in the Yeast Saccharomyces cerevisiae

The ferri-reductase activity of whole cells of Saccharomyces cerevisiae (washed free from the growth medium) was markedly increased 3 to 6 h after transferring the cells from a complete growth medium (preculture) to an iron-deficient growth medium (culture). This increase was prevented by the presence of iron, copper, excess oxygen, or other oxidative agents in the culture medium. The cells with increased ferri-reductase activity had a higher reduced glutathione content and a higher capacity to expose exofacial sulfhydryl groups. Plasma membranes purified from those cells exhibited a higher reduced nicotinamide adenine phosphate (NADPH)-dependent ferri-reductase specific activity. However, the intracellular levels of NADPH, NADH, and certain organic acids of the tricarboxylic acids cycle were unchanged, and the activity of NADPH-generating enzymes was not increased. Addition of Fe(III)-EDTA to iron-deprived and iron-rich cells in resting suspension resulted in a decrease in intracellular reduced glutathione in the case of iron-deprived cells and in an increase in organic acids and a sudden oxidation of NADH in both types of cells. The depolarizing effect of Fe³⁺ was more pronounced in iron-rich cells. The metabolic pathways that may be involved in regulating the trans-plasma membrane electron transfer in yeast are discussed.     

Time course of adaptation to elevated salt by Saccharomyces rouxii

Cells from a 24-h preculture of Saccharomyces rouxii grew without lag on a medium containing yeast extract, neopeptone, and glucose. Additions of 0.3 M KCl or 0.6 M pentaerythritol, which increased the osmolality of the medium 10-fold, still allowed immediate growth, whereas addition of 0.3 M NaCl resulted in a lag of 23 h (after which the growth rate was normal). The time course of growth in this elevated-sodium medium of 0.3 M was studied under defined conditions for inoculum history as well as experimental culture. S. rouxii cells with single exposure to elevated-sodium medium for 23 h exhibited a shorter lag (12 h) on fresh elevated-sodium medium; even with an intervening washing step. Elevated-sodium medium that had received the standard inoculum became conditioned during the lag so that it then supported growth of new, untrained cells with only 2 h lag; even with an intervening filter- or heat-sterilization treatment. The lag period on elevated-sodium medium could also be decreased by (1) using younger cells for inoculum, (2) increasing the amount of the inoculum, or (3) adding extra glucose to the medium. The results indicate that a diffusible, heat-stable factor is synthesized by cells during adaptation to elevated sodium, and that a threshold concentration of the factor is a prerequisite for the normal growth that eventually ensues. A change in retentivity for the factor with cellular age may explain the different growth kinetics with younger or older inocula.     

Structure of Cell Wall and Extracellular Mannans from Saccharomyces rouxii and Their Relationship to a High Concentration of NaCl in the Growth Medium

Cells of Saccharomyces rouxii (a salt-tolerant yeast) were grown in the presence of two levels of NaCl, 0 and 15%. Mannans obtained from both the cell walls and culture filtrates (extracellular) were examined. Yields based on the dry weight of cells demonstrated that the levels of both cell wall and extracellular mannans were lower when cells were grown in the presence of 15% NaCl. However, the carbohydrate and protein contents of the mannan preparations were not altered. The cell wall mannans obtained from the two growth conditions had similar molecular weights, whereas the extracellular mannans had different molecular weight distributions. Structural analyses showed that the cell wall and extracellular mannans had similar structures. Both had an α1-6-linked backbone to which single mannose and mannobiose units were connected as side chains, predominantly by α1-2 linkages. The mannans also contained mannosyloligosaccharides, mannotriose, mannobiose, and mannose attached to protein through an O-glycosidic bond. The molecular structure of the cell wall mannans remained unchanged at both levels of NaCl. However, in the presence of 15% NaCl, the side chains consisting of a mannobiose unit were slightly reduced.     

Filamin-A Increases the Stability and Plasma Membrane Expression of Polycystin-2

Polycystin-2 (PC2), encoded by the PKD2 gene, is mutated in ~15% of autosomal dominant polycystic kidney disease. Filamins are actin-binding proteins implicated in scaffolding and membrane stabilization. Here we studied the effects of filamin on PC2 stability using filamin-deficient human melanoma M2, filamin-A (FLNA)-replete A7, HEK293 and IMCD cells together with FLNA siRNA/shRNA knockdown (KD). We found that the presence of FLNA is associated with higher total and plasma membrane PC2 protein expression. Western blotting analysis in combination with FLNA KD showed that FLNA in A7 cells represses PC2 degradation, prolonging the half-life from 2.3 to 4.4 hours. By co-immunoprecipitation and Far Western blotting we found that the FLNA C-terminus (FLNAC) reduces the FLNA-PC2 binding and PC2 expression, presumably through competing with FLNA for binding PC2. We further found that FLNA mediates PC2 binding with actin through forming complex PC2-FLNA-actin. FLNAC acted as a blocking peptide and disrupted the link of PC2 with actin through disrupting the PC2-FLNA-actin complex. Finally, we demonstrated that the physical interaction of PC2-FLNA is Ca-dependent. Taken together, our current study indicates that FLNA anchors PC2 to the actin cytoskeleton through complex PC2-FLNA-actin to reduce degradation and increase stability, and possibly regulate PC2 function in a Ca-dependent manner.     

A Contrast of the Plasma Membrane Lipid Composition of Oat and Rye Leaves in Relation to Freezing Tolerance

The lipid composition of the plasma membrane isolated from leaves of spring oat (Avena sativa L. cv Ogle) was vastly different from that of winter rye (Secale cereale L. cv Puma). The plasma membrane of spring oat contained large proportions of phospholipids (28.8 mol% of the total lipids), cerebrosides (27.2 mol%), and acylated sterylglucosides (27.3 mol%) with lesser proportions of free sterols (8.4 mol%) and sterylglucosides (5.6 mol%). In contrast, the plasma membrane of winter rye contained a greater proportion of phospholipids (36.6 mol%), and there was a lower proportion of cerebrosides (16.4 mol%); free sterols (38.1 mol%) were the predominant sterols, with lesser proportions of sterylglucosides (5.6 mol%) and acylated sterylglucosides (2.9 mol%). Although the relative proportions of individual phospholipids, primarily phosphatidylcholine and phosphatidylethanolamine, and the molecular species of these two phospholipids were similar in oat and rye, the relative proportions of di-unsaturated species of these two phospholipids were substantially lower in oat than in rye. The relative proportions of sterol species in oat were different from those in rye; the molecular species of cerebrosides were similar in oat and rye, with only slight differences in the proportions of the individual species. After 4 weeks of cold acclimation, the proportion of phospholipids increased significantly in both oat (from 28.8 to 36.8 mol%) and rye (from 36.6 to 43.3 mol%) as a result of increases in the proportions of phosphatidylcholine and phosphatidylethanolamine. For both oat and rye, the relative proportions of di-unsaturated species increased after cold acclimation, but the increase was greater in rye than in oat. In both oat and rye, this increase occurred largely during the first week of cold acclimation. During the 4 weeks of cold acclimation, there was a progressive decrease in the proportion of cerebrosides in the plasma membrane of rye (from 16.4 to 10.5 mol%), but there was only a small decrease in oat (from 27.2 to 24.2 mol%). In both oat and rye, there were only small changes in the proportions of free sterols and sterol derivatives during cold acclimation. Consequently, the proportions of both acylated sterylglucosides and cerebrosides remained substantially higher in oat than in rye after cold acclimation. The relationship between these differences in the plasma membrane lipid composition of oat and rye and their freezing tolerance is presented.     

Involvement of the Cdc42 Pathway in CFTR Post-Translational Turnover and in Its Plasma Membrane Stability in Airway Epithelial Cells

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is expressed on the apical plasma membrane (PM) of epithelial cells. The most common deleterious allele encodes a trafficking-defective mutant protein undergoing endoplasmic reticulum-associated degradation (ERAD) and presenting lower PM stability. In this study, we investigated the involvement of the Cdc42 pathway in CFTR turnover and trafficking in a human bronchiolar epithelial cell line (CFBE41o-) expressing wild-type CFTR. Cdc42 is a small GTPase of the Rho family that fulfils numerous cell functions, one of which is endocytosis and recycling process via actin cytoskeleton remodelling. When we treated cells with chemical inhibitors such as ML141 against Cdc42 and wiskostatin against the downstream effector N-WASP, we observed that CFTR channel activity was inhibited, in correlation with a decrease in CFTR amount at the cell surface and an increase in dynamin-dependent CFTR endocytosis. Anchoring of CFTR to the cortical cytoskeleton was then presumably impaired by actin disorganization. When we performed siRNA-mediated depletion of Cdc42, actin polymerization was not impacted, but we observed actin-independent consequences upon CFTR. Total and PM CFTR amounts were increased, resulting in greater activation of CFTR. Pulse-chase experiments showed that while CFTR degradation was slowed, CFTR maturation through the Golgi apparatus remained unaffected. In addition, we observed increased stability of CFTR in PM and reduction of its endocytosis. This study highlights the involvement of the Cdc42 pathway at several levels of CFTR biogenesis and trafficking: (i) Cdc42 is implicated in the first steps of CFTR biosynthesis and processing; (ii) it contributes to the stability of CFTR in PM via its anchoring to cortical actin; (iii) it promotes CFTR endocytosis and presumably its sorting toward lysosomal degradation.     

Effects of Polyenes, Detergents, and Other Potential Membrane Perturbants on an Osmotolerant Yeast, Saccharomyces rouxii

The osmotolerance of Saccharomyces rouxii 48-28 was confirmed with both NaCl- and KCl-fortified growth media, with more tolerance being exhibited for the potassium salt. Washed and buffered cells from unfortified medium were challenged with a variety of compounds (and also with physical treatments) that potentially would elicit membrane perturbations. The efficacy of these brief treatments was judged primarily by monitoring subsequent viability. Change in the degree of expression of beta-fructofuranosidase (EC 3.2.1.26), which is cryptic in young cells of S. rouxii, was a second criterion. There was a linear correlation between cell death and enzyme expression for treatments with polyenes, detergents, some organic solvents which did not denature the enzyme, and various freeze-thaw regimens in graded amounts of glycerol. The species is relatively insensitive to polyene antimycotics, the order of decreasing effect being filipin, nystatin, and amphotericin B. S. rouxii was found to be less sensitive to osmotic shock than is Saccharomyces cerevisiae, but in neither species is beta-fructofuranosidase released to the medium. The sensitivity of S. rouxii to ionic detergents, but not to nonionic detergents, was rationalized as being due to cell wall discrimination against larger micelles for the nonionic examples. This was confirmed by showing that protoplasts were sensitive to both classes. In cultures older than 5 days the normal agreement between colony-forming units and methylene blue exclusion (another test of viability) no longer held. Delayed fermentation of sucrose by S. rouxii, which is a diagnostic feature of the species, is explained by death of some cells, expression of their beta-fructofuranosidase, and utilization of the monosaccharides by the surviving cells.     

Regulation of aspartate-derived amino-acid metabolism in Zygosaccharomyces rouxii compared to Saccharomyces cerevisiae

To elucidate the growth inhibitory effect of threonine, the regulation of the aspartate-derived amino-acid metabolism in Zygosaccharomyces rouxii, an important yeast for the flavor development in soy sauce, was investigated. It was shown that threonine inhibited the growth of Z. rouxii by blocking the methionine synthesis. It seemed that threonine blocked this synthesis by inhibiting the conversion of aspartate. In addition, it was shown that the growth of Z. rouxii, unlike that of Saccharomyces cerevisiae, was not inhibited by the herbicide sulfometuron methyl (SMM). From enzyme assays, it was concluded that the acetohydroxy acid synthase in Z. rouxii, unlike that in S. cerevisiae, was not sensitive to SMM. Furthermore, the enzyme assays demonstrated that the activity of threonine deaminase in Z. rouxii, like in S. cerevisiae, was strongly inhibited by isoleucine and stimulated by valine. From this work, it is clear that the aspartate-derived amino-acid metabolism in Z. rouxii only partly resembles that in S. cerevisiae.     

细胞质膜蛋白与肿瘤的相关性研究进展

肿瘤相关研究一直是科研领域的重点与难点。肿瘤的发生、发展和转移与细胞质膜蛋白关系密切,质膜蛋白的过量表达、缺失或修饰,使细胞的信号转导、物质运输、黏附作用、免疫原性等发生改变,从而影响了肿瘤细胞的上述过程。简要综述了相关领域的研究进展。     

Plasma Adiponectin Levels in High Risk African‐Americans with Normal Glucose Tolerance,Impaired Glucose Tolerance,and Type 2 Diabetes**

Objective: We studied plasma adiponectin, insulin sensitivity, and insulin secretion before and after oral glucose challenge in normal glucose tolerant, impaired glucose tolerant, and type 2 diabetic first degree relatives of African‐American patients with type 2 diabetes. Research Methods and Procedures: We studied 19 subjects with normal glucose tolerance (NGT), 8 with impaired glucose tolerance (IGT), and 14 with type 2 diabetes. Serum glucose, insulin, C‐peptide, and plasma adiponectin levels were measured before and 2 hours after oral glucose tolerance test. Homeostasis model assessment‐insulin resistance index (HOMA‐IR) and HOMA‐β cell function were calculated in each subject using HOMA. We empirically defined insulin sensitivity as HOMA‐IR < 2.68 and insulin resistance as HOMA‐IR > 2.68. Results: Subjects with IGT and type 2 diabetes were more insulin resistant (as assessed by HOMA‐IR) when compared with NGT subjects. Mean plasma fasting adiponectin levels were significantly lower in the type 2 diabetes group when compared with NGT and IGT groups. Plasma adiponectin levels were 2‐fold greater (11.09 ± 4.98 vs. 6.42 ± 3.3811 μg/mL) in insulin‐sensitive (HOMA‐IR, 1.74 ± 0.65) than in insulin‐resistant (HOMA‐IR, 5.12 ± 2.14) NGT subjects. Mean plasma adiponectin levels were significantly lower in the glucose tolerant, insulin‐resistant subjects than in the insulin sensitive NGT subjects and were comparable with those of the patients with newly diagnosed type 2 diabetes. We found significant inverse relationships of adiponectin with HOMA‐IR (r = ?0.502, p = 0.046) and with HOMA‐β cell function (r = ?0.498, p = 0.042) but not with the percentage body fat (r = ?0.368, p = 0.063), serum glucose, BMI, age, and glycosylated hemoglobin A1C (%A1C). Discussion: In summary, we found that plasma adiponectin levels were significantly lower in insulin‐resistant, non‐diabetic first degree relatives of African‐American patients with type 2 diabetes and in those with newly diagnosed type 2 diabetes. We conclude that a decreased plasma adiponectin and insulin resistance coexist in a genetically prone subset of first degree African‐American relatives before development of IGT and type 2 diabetes.     

Thermal Stability of the Plasma Membrane Calcium Pump. Quantitative Analysis of Its Dependence on Lipid-Protein Interactions

Thermal stability of plasma membrane Ca²⁺ pump was systematically studied in three micellar systems of different composition, and related with the interactions amphiphile-protein measured by fluorescence resonance energy transfer. Thermal denaturation was characterized as an irreversible process that is well described by a first order kinetic with an activation energy of 222 ± 12 kJ/mol in the range 33–45°C. Upon increasing the mole fraction of phospholipid in the mixed micelles where the Ca²⁺ pump was reconstituted, the kinetic coefficient for the inactivation process diminished until it reached a constant value, different for each phospholipid species. We propose a model in which thermal stability of the pump depends on the composition of the amphiphile monolayer directly in contact with the transmembrane protein surface. Application of this model shows that the maximal pump stability is attained when 80% of this surface is covered by phospholipids. This analysis provides an indirect measure of the relative affinity phospholipid/detergent for the hydrophobic transmembrane surface of the protein (K _LD ) showing that those phospholipids with higher affinity provide greater stability to the Ca²⁺ pump. We developed a method for directly measure K _LD by using fluorescence resonance energy transfer from the membrane protein tryptophan residues to a pyrene-labeled phospholipid. K _LD values obtained by this procedure agree with those obtained from the model, providing a strong evidence to support its validity. Received: 5 August 1999/Revised: 20 October 1999