Physiological responses of the adult male collared peccary, Tayassu tajacu (Tayassuidae), to severe dietary restriction

Metabolic and hormonal responses of eight adult male collared peccaries (Tayassu tajacu) to an ad libitum diet intake, or 25% of an ad libitum intake, were examined. Blood samples for hematological, serum-biochemical and hormonal profiles were collected at three week intervals during the nine week experiment starting 4 August 1983. Males fed on the restricted diet lost an average of 26% of their body weight during the trial, compared to a slight weight gain for those fed ad libitum. Characteristics of the red and white blood cell populations were not influenced by diet intake, with the exception of mean corpuscular volume, which was consistently lower amongst males fed on the restricted diet. Restricted food intake resulted in significantly elevated serum values for urea nitrogen, urea nitrogen:creatinine, urea index, alpha globulin:beta globulin, gamma globulin:albumin, nonesterified fatty acids, alkaline phosphatase and lactate dehydrogenase isozymes (LD1 and LD2). Restricted food intake resulted in significantly lowered serum values for total alpha globulin, alpha-1 globulin, total beta globulin, beta-1 globulin, beta-2 globulin, glucose, triglycerides, calcium, magnesium, sodium, chloride, copper and triiodothyronine. Serum levels of creatinine, total protein, albumin, alpha-2 globulin, uric acid, total bilirubin, cholesterol, aspartate aminotransferase, alanine aminotransferase, gamma glutamyltransferase, lactate dehydrogenase, phosphorus, calcium:phosphorus, potassium, iron, zinc and thyroxine were unaffected by diet intake level. Semen evaluation indicated spermatogenesis was not affected by dietary restriction despite reductions in scrotal circumference and ejaculate gel volume. Serum testosterone levels were significantly lower among males fed on the restricted diet after nine weeks. These data suggest male libido might be depressed during poor range conditions, while maintenance of spermatogenesis might permit them to take immediate advantage of improved range conditions. Blood analysis of metabolic and hormonal function can provide useful information for predicting the adult male's nutritional and reproductive condition.     

Serum and urine biochemical indicators of nutritional status in adult female collared peccaries,Tayassu tajacu (tayassuidae)

• 1.1. Physiological responses of 13 adult female collared peccaries (Tayassu tajacu) to high quality and low quality diets, fed for 15 weeks, were examined. The low quality diet simulated energy and protein intake of peccaries during poor range conditions resulting from drought. Blood samples were collected after 10 and 15 weeks of dietary treatment; urine samples were collected after 15 weeks of treatment.
• 2.2. Females receiving the low quality diet for 15 weeks lost 27.4% of their original body weight, compared to no weight change among high quality-fed females.
• 3.3. Red blood cell counts, hematocrits, and hemoglobin concentrations were significantly greater among females fed a high quality diet compared to those receiving a low quality diet. High quality-fed females also had a higher mean corpuscular hemoglobin concentration. Plasma fibrinogen concentration was nearly twice as great among females receiving the low quality diet compared to the high quality group.
• 4.4. Consumption of the low quality diet resulted in significantly elevated serum levels of nonesterified fatty acids, alkaline phosphatase, phosphorus, alpha-2 globulin and alpha globulin: beta globulin ratio.
• 5.5. Consumption of the low quality diet resulted in significantly lowered serum levels of urea nitrogen, calcium, zinc, calcium: phosphorus, urea index, beta-1 flobulin, beta globulin: albumin ratio, thyroxine and triiodothyronine.
• 6.6. Serum levels ofcreatinine, total bilirubin, glucose, cholesterol, gamma glutamyltransferase, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, potassium, copper, magnesium, sodium chloride, total protein and gamma globulin were unaffected by diet quality.
• 7.7. Urine chemistry results suggested pH, osmolarity, albumin, creatinine phosphokinase, calcium and phosphorus concentrations might be useful indices for assessing nutritional status in female peccaries.

     

Equation for osmotic pressure of serum protein (fractions).

The colloid or protein osmotic pressure (Pi) is a function of protein molarity (linear) and of Donnan and other effects. Albumin is the major osmotic protein, but also globulins influence Pi. Equations based on concentrations of albumin and nonalbumin (globulin concentration + fibrinogen concentration) protein approximate Pi better than albumin alone. Globulins have a wide range of molecular weights, and a 1956 diagram indicated that Pi of globulin fractions decreased in the order alpha1-, alpha2-, beta-, and gamma-globulin. The molecular weight of the serum protein fractions had been extrapolated, so van't Hoff's law and nonlinear regression analysis of the curves permitted expression of the diagram as an equation: product Pi(s,Ott,2 degrees C,cmH2O)=x(alb)(0.338C(tot)+0.00339C(tot)(2))+x(alpha1)(0.518C(tot)+0.0107C(tot)(2))+x(alpha2)(0.203C(tot)+0.00155C(tot)(2))+x(beta)(0.187C(tot)+0.000577C(tot)(2))+x(gamma)(0.161C(tot)+0.000223C(tot)(2)), where Pi(s,Ott,2 degrees C,cmH2O) is Pi of serum at 2 degrees C (in cmH2O) computed from the 1956 diagram, C(tot) is the concentration (g/l) of total protein in serum, and x(alb), x(alpha1), x(alpha2), x(beta), and x(gamma) are the fractions of albumin, alpha1-, alpha2-, beta-, and gamma-globulin, respectively. At one and the same concentration of fractions, Pi("Ott") decreases in the order alpha1-globulin, albumin, alpha2-globulin, beta-globulin, and gamma-globulin.     

Effect of melatonin on 24h changes in plasma protein levels during the preclinical phase of Freund's adjuvant arthritis in rats

The 24h rhythms in plasma protein concentration were examined in rats on the third day after injection of Freund's complete adjuvant or adjuvant's vehicle, performed 3h after light on. In rats treated with adjuvant's vehicle, peak values of albumin and gamma globulin occurred during the nocturnal activity span (P < .02 and P < .0001, respectively), while those of alpha-1, alpha-2, and beta globulins were found late during the rest span (P < .002, P < .0001, and P < .0004, respectively). Freund's adjuvant administration abolished temporal changes in plasma albumin and beta globulin levels. It also decreased the amplitude of daily changes in alpha-1 and alpha-2 globulin (P < .05) and diminished mean values of alpha-2 globulin (P < .01). Pretreatment of rats with melatonin (30 microg daily) for 11 days, 11h after light on, counteracted mycobacterial adjuvant-induced suppression of the 24h rhythms in albumin and alpha-1, alpha-2, and beta globulins. The results further support the existence of preventive properties of a pharmacological dose of melatonin in situations in which a lost of circadian rhythmicity is expected.     

Serum Protein Electrophoresis in Tuberculosis

Serial serum protein electrophoretic determinations at two-month intervals were carried out on 84 tuberculous patients undergoing treatment in a sanatorium. An attempt was made to correlate changes in the albumin/alpha-2 globulin ratios and gamma globulin levels with clinical and roentgenographic status. It was observed that before treatment of the tuberculous process the albumin/alpha-2 globulin ratios were low and the gamma globulin levels were increased. As improvement occurred, albumin/alpha-2 globulin ratios increased and gamma globulin values decreased. A poor prognosis was indicated by decreasing albumin/alpha-2 globulin ratios. Electrophoretograms are of particular value in assessing the effect of surgery, especially when roentgenographic studies are not informative. They are also a valuable guide in deciding upon the duration of therapy necessary for individual cases.     

The Metabolism of Serum Proteins in Neonatal Rabbits

1. Incorporation of S³⁵-labeled amino acids into serum proteins has been studied in neonatal and developing rabbits. It was found that, per unit weight, neonatal rabbits synthesized only about 1/36 of the gamma globulin, 1/7 of the beta globulin, ½ of the alpha globulin, and ⅛ of the albumin that an adult synthesized. The growing rabbit developed the ability to synthesize various serum proteins at different times. 2. Plasma volumes and serum protein concentrations were determined at different times during the growth period of the rabbit. Plasma volumes were found to be 1 and ½ times larger in newborn animals than in adults, with a gradual decline to the adult level. The total serum protein concentration at birth was about 60 to 65 per cent of the adult value and gradually increased with growth as the plasma volume decreased. 3. Half-lives of homologous albumin and gamma globulin were studied. The half-life of albumin in neonates was nearly twice as long as the half-life in adults, the latter value being reached at 1 month of age. The half-life of gamma globulin in neonates was more than twice as long as the half-life in adults and reached adult values at 2 to 3 months. 4. Attempts were made to alter serum protein metabolism. Gamma globulin synthesis early in life was augmented with antigen injections.     

A differential scanning calorimetric study of the bovine lens crystallins

Differential scanning calorimetry was performed on the five major lens crystallin fractions [HM-alpha, alpha, beta H, beta L, and (beta s + gamma)] of the bovine lens as well as on more purified forms of alpha- and gamma-crystallins. All were found to be relatively thermally stable although the alpha-crystallin were found to at least partially unfold at an approximately 10 degrees C lower temperature than the beta and gamma fractions. Increasing protein concentration had little effect on gamma-crystallin thermograms but had marked effects on those of the alpha- and beta-crystallins. Increases in the thermal stability with increasing protein concentration for the beta-crystallins can be explained most simply by the known beta L/beta H equilibrium, but, in the case of the alpha-crystallins, excluded volume effects may be an important factor. In both cases, the increased stability at high concentrations could be of physiological relevance. As well as the expected endothermic unfolding transitions, all of the lens crystallins revealed exothermic peaks that correlate with protein precipitation. Interestingly, this phenomenon occurs only after extensive structural alteration in the case of the alpha-crystallins but is present very early in the initial stages of structural perturbation of the beta- and gamma-crystallins.     

Experimental infection with Ancylostoma caninum larvae in mice. IV. Serum protein pattern during primary infection.

Serum protein of Swiss albino mice infected with Ancylostoma caninum larvae, were analysed electrophoretically. Twenty mice were infected with a dose of 1000 larvae and the alterations in serum protein during an infection period of 30 days were recorded and compared with uninfected controls. The result showed a significant decrease in albumin and gamma globulin with an increase in beta globulin. There were no significant changes in the alpha-1 and alpha-2 globulins. The total globulins increased and A/G ratio decreased. These changes appeared to be remarkable on the 9th day after infection.     

The development of serum proteins in pig foetuses during prenatal period

The present work describes data dealing with the quantitative relationship between individual serum proteins in pig foetuses of different age (38–112 days). Using immuncelectrophoresis in agar gel, six different fractions were determined (prealbumin, albumin, fetuin, alpha_1-, alpha_2-, and beta globulin). No gamma globulin was found. Using a simple radial immunodiffusion method, an average level of 70 gamma/ml of IgG was found in 112-day-old foetuses. Neither IgM nor IgA could be detected.     

The horses of Giara: variation of the thyroid activity and certain blood parameters between winter and summer

The changes of some parameters in the blood serum of six Giara horses (3 males and 3 females) were checked weekly, in relation to the environmental temperature throughout a one winter and summer. T3, total lipids, triglycerides, urea nitrogen, creatinine, total protein, albumin, beta and gamma globulin showed significant difference between winter and summer and, with the exception of triglycerides, urea nitrogen and total protein were correlated to the environmental temperature. T4, glucose, uric acid and alpha globulin showed no difference between the two seasons and no correlation with the temperature. The results indicate that the seasonal variations in thyroid activity affected the T3 only, and that the blood lipids were transported from the beta globulin and, perhaps, in a smaller quantity, from the albumin, while the alpha globulin was not affected.     

Effects of anabolic steroids on acute phase responses in intra-abdominal sepsis

The acute phase response is an important adaptive response to sepsis and injury. As anabolic steroids increase protein synthesis we postulated that these agents might also increase hepatic acute phase protein synthesis. Male Wistar rats were pretreated with testosterone or danazol for 48 h prior to caecal ligation and puncture (CLP). Thirty-six h following surgery the animals were killed and blood taken for full blood count, total protein, albumin, alpha, beta and gamma globulin fractions on serum electrophoresis, complement C(3) and transferrin levels. Danazol increased the alpha1, alpha2 and beta1 globulin serum protein fractions in comparison with no surgery and CLP alone groups. These results indicate that danazol increases plasma acute phase proteins, as measured by electrophoresis, in this model of intra-abdominal sepsis.     

A study of the uterine protein variations during the estrus cycle in the cow: a comparison with the serum proteins

To investigate uterine protein changes during the estrus cycle in the bovine, 115 pluriparous genital tracts and blood samples were collected from the abattoir in Urmia. Genital tracts were considered healthy based on gross examination of the uterus and uterine histopathological findings. The phase of the estrus cycle was determined by the examination of the structures present on the ovaries and the uterine tonicity. Of the collected samples, 24 were pro-estrus, 21 estrus, 24 met-estrus and 46 diestrus. The uterus was incised and uterine fluid was collected by gentle scraping of the uterine mucosa with a curette. The total protein concentration, protein profiles (on agarose gel electrophoresis) in the uterine fluid were evaluated and compared with those of the serum. Total protein, alpha1, alpha2, beta1 and beta2 globulin values in the uterus were significantly higher than those of the serum (P<0.05), while, the albumin, gamma1 and gamma2 globulin values in the serum were higher than those of the uterus throughout the cycle. During pro-estrus, uterine fluid beta2 (1.96 g/dl) and serum gamma1 (1.07 g/dl) and gamma2 (1.27 g/dl) globulins were higher than those in the other phases of the cycle. During estrus, serum total protein was lower than the other phases (4.92 g/dl), which was considered to be due to a reduction in serum alpha1 (0.25 g/dl), gamma1 (0.65 g/dl) and gamma2 (0.64 g/dl) globulins in this phase. In met-estrus uterine fluid beta1 globulin was in the lowest (1.19 g/dl) and serum gamma2 globulin at a high level (1.24 g/dl). It was concluded that uterine proteins as well as serum proteins fluctuate during the estrus cycle and, except for the albumin and gamma globulins, its protein content is higher than the serum. During the follicular phase of the cycle uterine alpha globulins are higher than those in other phases, with an elevation in beta1 and a reduction in beta2 and gamma globulin values during estrus, which may reflect the preparation of the uterus for receiving spermatozoa for the conception in this phase.     

Electrophoretic protein analysis in the conditioned captive wild coyote, Canis latrans

Total protein, albumin and serum protein values were determined on 19 male and 14 female captive, vaccinated, wild coyotes. Male coyotes had significantly higher total protein, alpha 1 and alpha 2 globulin levels than female coyotes. Captive, wild coyotes had lower values for total protein, albumin and beta globulins, and higher values for alpha 2 and gamma globulins than similar values for laboratory dogs. Albumin values determined by bromcresol green were slightly higher than values derived by electrophoresis. This difference was non-significant.     

Electrophoretic and immunoelectrophoretic studies of sera from normal, tuberculous, and noninfected tuberculin-sensitive guinea pigs

Dardas, T. J. (Michigan State University, East Lansing), and V. H. Mallmann. Electrophoretic and immunoelectrophoretic studies of sera from normal, tuberculous, and noninfected tuberculin-sensitive guinea pigs. J. Bacteriol. 92:76-81. 1966.-Normal guinea pig serum was separated into seven fractions by electrophoresis on cellulose acetate membranes. Thirty antigens were found by immunoelectrophoresis: albumin, 6 alpha(1) globulins, 11 alpha(2) globulins, 6 beta(1) globulins, 5 beta(2) globulins, and gamma globulin. Hyper-alpha(2)-globulinemia was detected in sera from guinea pigs 14 days after inoculation with viable virulent Mycobacterium bovis. An additional alpha(2) globulin, not demonstrable prior to infection, was detected concomitantly with the hyper-alpha(2)-globulinemia by immunoelectrophoresis. The additional alpha(2) globulin was tentatively named alpha(2)-T. It persisted until the death of the guinea pigs. Neither hyper-alpha-globulinemia nor the alpha(2)-T antigen was detected by cellulose acetate electrophoresis and immunoelectrophoresis of sera from guinea pigs sensitized with heat-killed M. bovis. Both changes were due to the disease, not to delayed sensitivity alone.     

Determination of the Optimal Ammonium Sulfate Concentration for the Fractionation of Rabbit, Sheep, Horse, and Goat Antisera

Various ammonium sulfate concentrations and reaction conditions were employed in the fractionation of sera from rabbits, sheep, horses, and goats. Precipitates and supernatant fluids were analyzed by electrophoresis to study the effects of the controlled variables. At room temperature, the third precipitate in 35% saturated (NH(4))(2)SO(4) was the best fraction from both rabbit and sheep sera; 80 to 90% of the gamma globulins were recovered. The second and third precipitates of horse sera proteins in 30% saturated (NH(4))(2)SO(4) were both satisfactory, but only 44% of the gamma globulin was recovered after three precipitations. Goat sera yielded a very satisfactory fraction; 80% of the gamma globulin was recovered after two precipitations-the first in 30% and the second in 45% saturated (NH(4))(2)SO(4). The composition of these fractions was not influenced by the pH of the sulfate solutions (pH 5.8 and 7.2), by a range of normal room temperatures (20 to 30 C), or by diluting the sera before fractionation. Crude globulins and fluorescein isothiocyanate-labeled globulins were successfully refractionated by one precipitation in the optimal sulfate concentration for the appropriate animal species. The refractionated products contained considerably less beta and alpha globulins than did the original crude fractions and little or no albumin.     

应用实时定量RT-PCR技术检测b地中海贫血 珠蛋白基因的表达

为了定量检测 b 地中海贫血(b 地贫)的 a、b 和γ珠蛋白基因表达水平, 提取正常成人对照组、正常胎儿对照组和b 地贫患者组组成的样本 DNA,采用反向点杂交法（RDB）分析b 地贫各种突变类型;提取样本RNA用于进行针对a、b 和γ珠蛋白基因的荧光实时定量RT-PCR（FQ RT-PCR）。根据FQ RT-PCR原理,设计合成分别对应于a、b 和γ珠蛋白基因的3对引物和3条荧光探针,FQ RT-PCR在ABI 7700系统进行。用SPSS 10.0对实验数据进行统计学分析,分别计算正常对照组 (bA/bA,aa/aa),脐带血组(bA/bA,aa/aa),轻型b 地贫组(bT/bA,aa/aa),重型b地贫组(bT/bT,aa/aa)的a、b 和γmRNA比值,其中a/b分别为4.62±1.20、7.81±2.89、13.51±5.12、188.24±374.04;a/(b +γ)分别为4.43±1.17、0.56±0.49、9.62±4.37、2.14±1.58;γ/(b+γ) 分别为0.04±0.03、0.92±0.06、0.28±0.18、0.95±0.04。由于组与组之间均值变异范围较大,将其进行对数转换后再进行方差分析。结果表明： a/b与a/(b+γ)在所有组与组之间均有显著性差异。γ/(b+γ)除了在脐带血组和重型b地贫组之间无显著性差异外,在其他组与组之间均有显著性差异。实验说明,人类b珠蛋白基因的表达水平从正常对照组到重型b地贫组急剧下降且以重型b地贫组为最低;相反γ珠蛋白基因表达却明显升高,以重型b地贫组为最高。与正常成人对照组相比,胎儿期b mRNA水平较低但γmRNA 水平较高。因此,正常个体不同时期和不同类型b 地贫之间a、b与γ珠蛋白基因表达不同而且互相影响。 Abstract：whole blood samples were collected from 100 normal healthy adults, from umbilical cord of 33 newborn infants, 111 individuals with b-thalassemia minor (bT/bA,aa/aa) and 39 with b-thalassemia major (bT/bT,aa/aa). Prior to quantitative analysis of globin gene expression, DNA was extracted from all blood samples and used for b-thalassemia genotype analysis. Different types of b globin gene mutations were analyzed using reverse dot blotting (RDB) method. Total RNA were extracted and subjected to real-time RT-PCR for quantitative measurement of a, b andγglobin mRNA using three sets of primers and fluorescent-labeled probes, designed according to the sequences of a, b andγhuman globin gene. Real-time RT-PCR was performed in ABI 7700 system. Following the real-time RT-PCR, the mean values of a, b andγglobin mRNA were calculated and the ratios of a/b, a/(b + γ) andγ/(b + γ) were determined to characterize the relative expression levels of different globin genes among normal adult, infant, b-thalassemia minor and b-thalassemia major patients. The resultant data were analyzed using SPSS 10.0 software to determine statistical significance of human globin gene expression among normal controls and b-thalassemia patients. Due to vast variations of the mean globin gene mRNA levels among different groups, log conversion of a/b + 1, a/(b + γ) + 1 andγ/(b + γ) +1 was used for statistical analyses and intergroup comparison. The a/b globin gene mRNA ratios were determined to be 4.62±1.20, 7.81±2.89, 13.51±5.12, and 188.24±374.04 for normal healthy adult (bA/bA,aa/aa), infant (bA/bA,aa/aa), b- thalassemia minor (bT/bA,aa/aa) and b-thalassemia major(bT/bT,aa/aa) respectively. The a/(b+γ) ratios were 4.43±1.17, 0.56±0.49, 9.62±4.37, and 2.14±1.58 for normal healthy adult (bA/bA,aa/aa), infant (bA/bA,aa/aa), b- thalassemia minor (bT/bA,aa/aa) and b- thalassemia major(bT/bT,aa/aa) respectively. Theγ/(b+γ) ratios were 0.04±0.03, 0.92±0.06, 0.28±0.18, and 0.95±0.04 for normal healthy adult (bA/bA,aa/aa), infant (bA/bA,aa/aa), b- thalassemia minor (bT/bA,aa/aa) and b- thalassemia major(bT/bT,aa/aa) respectively. Following statistical analyses, the a/b and a/(b+γ) globin gene mRNA ratios were significantly different among four different groups (normal adult, normal infant, b- thalassemia minor and b- thalassemia major). The γ/(b + γ) globin gene mRNA ratio was significantly different among all groups except for between infant and b- thalassemia major patients. Human b globin gene mRNA levels decrease progressively and dramatically from normal adults to b-thalassemia patients with b-thalassemia major having the lowest levels. On the other hand, the γglobin gene mRNA levels increase progressively from normal adult to b-thalassemia patients with b-thalassemia major having the highest levels. Infants have relatively lower levels of b but higher levels of γglobin gene mRNA as compared to those in normal adults. Thus, the relative expression levels of a, b or γglobin genes varied but inter-related among different ages of normal individuals and different b-thalassemia genotypes.     

Various functional characteristics of enolase isoenzymes in mammals

Considerable stability of beta beta- (muscular) and gamma gamma- (neurospecific) forms to the denaturing influence of neutral salts of alkali metals is shown on purified isoenzymes (alpha alpha-, beta beta-, gamma gamma-forms) of beef brain and rabbit muscle. The rate of influence of these salts on the enzymic activity of the mentioned isoenzymes corresponds to the "lyotropic" series. Km of alpha alpha- and gamma gamma-isoenzymes are determined for 2-phosphoglycerate and phosphoenolpyruvate. It is found that alpha alpha-isoenzyme has larger affinity to phosphoenolpyruvate than gamma gamma-isoenzyme and it appears to play an essential role in participation of the given isoenzymes in the glycolysis and glyconeogenesis processes.     

Inheritance of alleles for glutelin alpha-2 subunit genes in rice and identification of their corresponding cDNA clone

Rice glutelins consist of acidic (alpha) and basic (beta) subunits which are further separated into three polypeptide components assigned as alpha-1, alpha-2, and alpha-3 subunit components and beta-1, beta-2 and beta-3 subunit components. Nine rice mutant lines with a decreased amount of the glutelin alpha-2 subunit component (alpha-2L) were obtained by screening about 6,800 potential mutant lines derived from the fertilized egg treatment with N-methyl-N-nitrosourea (MNU) using the SDS-PAGE method. The mutants were classified into three types of the increased alpha-1 subunit (alpha-1H/alpha-2L), the decreased beta-2 subunit (beta-2L/alpha-2L) and the increased alpha-3 subunit (alpha-3H/alpha-2L) represented by EM278, CM1707 and EM659, respectively. Iso-electric focus (IEF) analysis revealed that all of the mutants had an extremely low amount of a polypeptide with a 6.71 pI value, whereas a polypeptide with either a 6.50 pI value or with a 6.90 pI value increased significantly in alpha-1H/alpha-2L mutants or in alpha-3H/alpha-2L mutants, respectively. The beta-2L/alpha-2L mutants had a decreased amount of a basic polypeptide with a 8.74 pI value. Genetic analysis revealed that the three types of mutants were controlled by a single incomplete dominant gene respectively, and the three are alleles. The gene was temporarily named glu4, which was found to be located on chromosome 1 linked with the eg and spl6 genes. Two-dimensional electrophoresis analysis revealed that the glu4 encoded polypeptides of pI 6.71/alpha-2 and pI 8.74/beta-2. Amino acid sequence analysis suggested that the mutated acidic polypeptide was the product of a GluA subfamily gene. Northern and RT-PCR analyses revealed that glu4 corresponded to the GluA-1 gene.     

Stoichiometry of a ligand-gated ion channel determined by fluorescence energy transfer

We have developed a method to determine the stoichiometry of subunits within an oligomeric cell surface receptor using fluorescently tagged antibodies to the individual subunits and measuring energy transfer between them. Anti-c-Myc monoclonal antibody (mAb 9-E10) derivatized with a fluorophore (europium cryptate, EuK) was used to individually label c-Myc-tagged alpha1-, beta2-, or gamma2-subunits of the hetero-oligomeric gamma-aminobutyric acid (GABAA) receptor in intact cells. The maximal fluorescent signal derived from the alpha1(c-Myc)beta2gamma2 and the alpha1beta2(c-Myc)gamma2 receptors was twice that obtained with alpha1beta2gamma2(c-Myc), suggesting that there are 2x alpha-, 2x beta-, and 1x gamma-subunits in a receptor monomer. This observation was extended using fluorescence energy transfer. Receptors were half-maximally saturated with EuK-anti-c-Myc mAb, and the remaining alpha1(c-Myc) subunits were labeled with excess anti-c-Myc mAb derivatized with the fluorescence energy acceptor, XL665. On exposure to laser light, energy transfer from EuK to XL665 occurred with alpha1(c-Myc)beta2gamma2 and alpha1beta2(c-Myc)gamma2, but no significant energy transfer was observed with alpha1beta2gamma2(c-Myc) receptors, indicating the absence of a second gamma-subunit in a receptor monomer. We confirm that the GABAA receptor subtype, alpha1beta2gamma2, is composed of two copies each of the alpha- and beta-subunits and one copy of the gamma-subunit (i.e. (alpha1)2(beta2)2(gamma2)1) and conclude that this method would have general applicability to other multisubunit cell surface proteins.