Investigations on the PGM (phosphoglucomutase) polymorphism by isoelectric focusing inDrosophila melanogaster

Pgm allele frequencies of 383 individuals were determined in a sample ofDrosophila melanogaster from three laboratory Sardinian populations, using the techniques of standard electrophoresis, heat denaturation, and isoelectric focusing. The analysis of the progeny obtained from informative crosses showed that the isoelectric focusing patterns segregate in a Mendelian way. ThePgm ^1.00 andPgm ^0.70 electrophoretic alleles displayed different isoelectric points, whereas thePgm ^1.00,tr andPgm ^1.00,ts isoelectrophoretic alleles could not be differentiated when tested by isoelectric focusing. Moreover, thePgm ^0.70,ts allele was split into two classes, with isoelectric points ofpH 6.4 andpH 6.6.     

Characterisation of the isoenzymes of phosphoglucomutase (PGM) determined by the first (PGM1) and second (PGM2) locus observed by isoelectric focusing

Summary The existence of four alleles of phosphoglucomutase (PGM₁) in human red cell lysates has previously been demonstrated by isoelectric focusing (Bark et al., 1976; Kühnl et al., 1977; Sutton and Burgess, 1978). Experiments are now described in which the position of each of the first-locus (PGM₁) and second-locus (PGM₂) isoenzymes is defined, thus extending and confirming the original proposal made by Bark et al.     

Investigations on the PGM 1 a polymorphism (phosphoglucomutase-EC 2.7.5.1) by isoelectric focusing

Summary The determination of phosphoglucomutase (PGM₁) phenotypes was performed by isoelectric focusing on samples from 1678 unrelated individuals from Hessen, Germany. Ten common phenotypes are considered as gene products of four alleles at the PGM₁ locus with the following frequencies: PGM ₁ ^a1 =0.6305, PGM ₁ ^a2 =0.1844, PGM ₁ ^a3 =0.1320, and PGM ₁ ^a4 =0.0530. Twenty-two different mating types were observed in 113 families with 202 children. The segregation of the phenotypes in the offspring supports the assumed way of autosomal codominant inheritance. The example of a silent allele (PGM ₁ ⁰ ) as well as a rare variant (PGM ₁ ⁷ ) is reported.     

Population genetics of the group specific component (Gc) and phosphoglucomutase (PGM1) studied by isoelectric focusing

For the determination of the group-specific component (Gc) and phosphoglucomutase (PGM1) phenotypes, isoelectric focusing was performed on two samples, one of Jat Sikh of northwest India, the other of northeast English. The subtype frequencies of these two systems do not differentiate the two populations sampled. Synthesis of the existing data shows distinct PGM1 and Gc subtype frequencies in various ethnic and racial groups. The anthropological implication of these subtype frequencies is discussed.     

Population distribution in North and Central America of PGM1 and Gc subtypes as determined by isoelectric focusing (IEF)

Gc subtypes of 20 North and Central American populations and PCM₁ subtypes in 11 populations were analyzed to identify interpopulation variation of the respective gene frequencies for common alleles. A total of 23,304 phenotypings were done. Absolute heterozygosity levels (D) generally increased twofold when phenotyping by isoelectric focusing was compared with conventional electrophoresis. Graphic representation of the Gc subtypes and multivariate analysis to identify genetic affinities of the populations under study reveal genetic clusters consistent with major historical and geographical groupings of man.     

Genetic polymorphism of vitamin B12 binding (R) proteins of human saliva detected by isoelectric focusing

Genetic polymorphism of the vitamin B12 binding (R) proteins of parotid saliva is determined by autosomal inheritance of codominant alleles. This hypothesis is supported by studies in 43 families including 152 children. For randomly collected salivas from 143 whites, 104 blacks, and 75 Chinese, gene frequencies are as follows: for whites, Rs1=0.88, Rs2=0.12; for blacks, Rs1=0.94, Rs2=0.06; for Chinese, Rs1=1.00. This genetic marker is also shared by R proteins of milk, tears, and leukocytes. In the Rs 1--2 salivary type there is less labeling of the protein products of Rs2 v. Rs1 with 57Co B12 as assessed by the intensity of the bands on the autoradiogram. There is no evidence for close linkage (theta less than 0.01) between Rs and TC II (transcobalamin II) or between Rs and salivary protein locus Pr, Db, Gl, or Ps.     

Modulation of nucleotide specificity of thermophilic F(o)F(1)-ATP Synthase by epsilon-subunit

The C-terminal two α-helices of the ε-subunit of thermophilic Bacillus F(o)F(1)-ATP synthase (TF(o)F(1)) adopt two conformations: an extended long arm ("up-state") and a retracted hairpin ("down-state"). As ATP becomes poor, ε changes the conformation from the down-state to the up-state and suppresses further ATP hydrolysis. Using TF(o)F(1) expressed in Escherichia coli, we compared TF(o)F(1) with up- and down-state ε in the NTP (ATP, GTP, UTP, and CTP) synthesis reactions. TF(o)F(1) with the up-state ε was achieved by inclusion of hexokinase in the assay and TF(o)F(1) with the down-state ε was represented by εΔc-TF(o)F(1), in which ε lacks C-terminal helices and hence cannot adopt the up-state under any conditions. The results indicate that TF(o)F(1) with the down-state ε synthesizes GTP at the same rate of ATP, whereas TF(o)F(1) with the up-state ε synthesizes GTP at a half-rate. Though rates are slow, TF(o)F(1) with the down-state ε even catalyzes UTP and CTP synthesis. Authentic TF(o)F(1) from Bacillus cells also synthesizes ATP and GTP at the same rate in the presence of adenosine 5'-(β,γ-imino)triphosphate (AMP-PNP), an ATP analogue that has been known to stabilize the down-state. NTP hydrolysis and NTP-driven proton pumping activity of εΔc-TF(o)F(1) suggests similar modulation of nucleotide specificity in NTP hydrolysis. Thus, depending on its conformation, ε-subunit modulates substrate specificity of TF(o)F(1).     

Subtyping of human red cell phosphoglucomutase locus 1 (PGM1) polymorphism: a third PGM1(1) allele common among Twa Pygmies from North Rwanda.

Seventy-eight Twa Pygmies from North Rwanda have been subtyped by acid starch gel electrophoresis for the polymorphism at the phosphoglucomutase locus 1 (PGM1). A third common PGM1(1) allele that has been named PGM1(1Twa) was detected in heterozygous association with both PGM1(1S) and PGM1(1F) alleles. The PGM1(1Twa) product is faster than those of the other two PGM1(1) alleles and has the same electrophoretic mobility as the rare PGM1(6) enzyme. The frequency of PGM1(1Twa) was found to be 0.45.     

A normal mode analysis of structural plasticity in the biomolecular motor F(1)-ATPase

Normal modes have been used to explore the inherent flexibility of the alpha, beta and gamma subunits of F(1)-ATPase in isolation and as part of the alpha(3)beta(3)gamma complex. It was found that the structural plasticity of the gamma and beta subunits, in particular, correlates with their functions. The N and C-terminal helices forming the coiled-coil domain of the gamma subunit are highly flexible in the isolated subunit, but more rigid in the alpha(3)beta(3)gamma complex due to interactions with other subunits. The globular domain of the gamma subunit is structurally relatively rigid when isolated and in the alpha(3)beta(3)gamma complex; this is important for its functional role in coupling the F(0) and F(1) complex of ATP synthase and in inducing the conformational changes of the beta subunits in synthesis. Most important, the character of the lowest-frequency modes of the beta(E) subunit is highly correlated with the large beta(E) --> beta(TP) transition. This holds for the C-terminal domain and the nucleotide-binding domain, which undergo significant conformational transitions in the functional cycle of F(1)-ATPase. This is most evident in the ligand-free beta(E) subunit; the flexibility in the nucleotide-binding domain is reduced somewhat in the beta(TP) subunit in the presence of Mg(2+).ATP. The low-frequency modes of the alpha(3)beta(3)gamma complex show that the motions of the globular domain of the gamma subunit and of the C-terminal and nucleotide binding domains of the beta(E) subunits are coupled, in accord with their function. Overall, the normal mode analysis reveals that F(1)-ATPase, like other macromolecular assemblies, has the intrinsic structural flexibility required for its function encoded in its sequence and three-dimensional structure. This inherent plasticity is an essential aspect of assuring a small free energy cost for the large-scale conformational transition that occurs in molecular motors.     

Low resolution structure of subunit b (b (22-156)) of Escherichia coli F(1)F(O) ATP synthase in solution and the b-delta assembly

The first low resolution solution structure of the soluble domain of subunit b (b _22–156) of the Escherichia coli F₁F_O ATPsynthase was determined from small-angle X-ray scattering data. The dimeric protein has a boomerang-like shape with a total length of 16.2 ± 0.3 nm. Fluorescence correlation spectroscopy (FCS) shows that the protein binds effectively to the subunit δ, confirming their described neighborhood. Using the recombinant C-terminal domain (δ_91–177) of subunit δ and the C-terminal peptides of subunit b, b _120–140 and b _140–156, FCS titration experiments were performed to assign the segments involved in δ–b assembly. These data identify the very C-terminal tail b _140–156 to interact with δ_91–177. The novel 3D structure of this peptide has been determined by NMR spectroscopy. The molecule adopts a stable helix formation in solution with a flexible tail between amino acid 140 to 145.     

The nucleotide-independent Fe(III)-binding site is located on beta subunit of the mitochondrial F(1)-ATPase

The need for methods to identify disease biomarkers is underscored by the survival-rate of patients diagnosed at early stages of cancer progression. Surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) is a novel approach to biomarker discovery that combines two powerful techniques: chromatography and mass spectrometry. One of the key features of SELDI-TOF MS is its ability to provide a rapid protein expression profile from a variety of biological and clinical samples. It has been used for biomarker identification as well as the study of protein-protein, and protein-DNA interaction. The versatility of SELDI-TOF MS has allowed its use in projects ranging from the identification of potential diagnostic markers for prostate, bladder, breast, and ovarian cancers and Alzheimer's disease, to the study of biomolecular interactions and the characterization of posttranslational modifications. In this minireview we discuss the application of SELDI-TOF MS to protein biomarker discovery and profiling.     

Genetic structure of the Sardinian population at the D1S80 VNTR locus and population diversity

We typed the Sardinian population at the D1S80 VNTR locus. Nineteen alleles were detected in a sample of 92 unrelated individuals, allele frequency distribution showing a modal pattern mostly in agreement with other Caucasoid populations. A high degree of heterozygosity (observed value=80.4%) was present. Goodness-of-fit tests demonstrated no departure from Hardy-Weinberg expectations. Data regarding heterozygosity, number of alleles and singletons appeared in accordance with the IAM mutation-drift equilibrium model and showed no evidence of hidden substructuring. Allele 34 exhibited in Sardinians the highest frequency never observed in Caucasians. Nonetheless, the comparison with other European populations did not disclose Sardinian genetic peculiarity. Indeed, measures of genetic divergence among Europeans demonstrated definitely smaller values at the D1S80 locus in comparison with those calculated over a high number of (pre-DNA) polymorphic loci. High mutation rate and selective neutrality typical of VNTRs could account for the observed moderate genetic divergence. Isolation and genetic drift, on the other hand, may have determined certain deviations in allele frequency distribution, as occurred to allele 34 in the Sardinian population.     

Some genetic implications of isoelectric focusing of human red cell phosphoglucomutase (PGM1) and serum protein group specific component (Gc): genetic diversity in the populations of Himachal Pradesh,India

For the study of group specific component (Gc) and phosphoglucomutase (PGM1) polymorphism, isoelectric focusing was performed on eleven tribal and non-tribal populations of Himachal Pradesh, India. They were chosen to illustrate interregional and intraregional variations. The subtype frequencies of these two systems showed clear differences in the genetic constitution of these populations of Himachal at both levels. There is a large increase in the mean heterozygosity (H) for each system by isoelectric focusing over that shown by electrophoresis. Discriminant and distance analyses both suggest that the subtype frequencies provide greater potential for the study of genetic diversity among populations. The data on these additional alleles found by isoelectric focusing are examined for some of their genetic and anthropological implications.     

The oligomycin sensitivity conferring protein (OSCP) of beef heart mitochondria: Studies of its binding to F1 and its function

The binding of oligomycin sensitivity conferring protein (OSCP) to soluble beef-heart mitochondrial ATPase (F₁) has been investigated. OSCP forms a stable complex with F₁, and the F₁ · OSCP complex is capable of restoring oligomycin- and DCCD-sensitive ATPase activity to F₁- and OSCP-depleted submitochondrial particles. The F₁ · OSCP complex retains 50% of its ATPase activity upon cold exposure while free F₁ is inactivated by 90% or more. Both free F₁ and the F₁ · OSCP complex release upon cold exposure a part—probably 1 out of 3—of their subunits; whether subunits are also lost is uncertain. The cold-treated F₁ · OSCP complex is still capable of restoring oligomycin- and DCCD-sensitive ATPase activity to F₁- and OSCP-depleted particles. OSCP also protects F₁ against modification of its subunit by mild trypsin treatment. This finding together with the earlier demonstration that trypsin-modified F₁ cannot bind OSCP indicates that OSCP binds to the subunit of F₁ and that F₁ contains three binding sites for OSCP. The results are discussed in relation to the possible role of OSCP in the interaction of F₁ with the membrane sector of the mitochondrial ATPase system.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - OSCP oligomycin sensitivity conferring protein - SDS sodium dodecylsulfate This paper is dedicated to the memory of David E. Green—scholar, pioneer, visionary.     

Isoelectric focusing of red cell phosphoglucomutase (E.C.:2.7.5.1) at the PGM1 locus in a French-Canadian population

Summary Phosphoglucomutase₁ (PGM₁) polymorphism was studied in a French-Canadian population of Québec city, Canada by means of a low voltage (max 500 V) isoelectric focusing (IEF) procedure on vertical polyacrylamide gel slabs. Frequencies of the four common PGM₁ genes estimated from the phenotype distribution in 308 unrelated individuals were PGM ₁ ¹⁺ , 0.61 (±0.02); PGM ₁ ^1- , 0.13 (±0.01); PGM ₁ ¹⁺ , 0.61 (±0.02); PGM ₁ ^1- , 0.18 (±0.02); and PGM ₁ ¹⁺ , 0.61 (±0.02); PGM ₁ ^1- , 0.08 (±0.01). The segregation patterns observed in 154 families, which included 31 different mating types and 353 children, confirmed a Mendelian inheritance of four autosomal genes. The distribution of the PGM₁ phenotypes observed or expected in a Hardy-Weinberg equilibrium was compared with that of other populations. A significant (P<0.001) difference was found between the Québec population and a Black population from Keneba, Gambia, West-Africa.     

The Inhibitor Protein (IF1) of the F1F0-ATPase Modulates Human Osteosarcoma Cell Bioenergetics

The bioenergetics of IF₁ transiently silenced cancer cells has been extensively investigated, but the role of IF₁ (the natural inhibitor protein of F₁F₀-ATPase) in cancer cell metabolism is still uncertain. To shed light on this issue, we established a method to prepare stably IF₁-silenced human osteosarcoma clones and explored the bioenergetics of IF₁ null cancer cells. We showed that IF₁-silenced cells proliferate normally, consume glucose, and release lactate as controls do, and contain a normal steady-state ATP level. However, IF₁-silenced cells displayed an enhanced steady-state mitochondrial membrane potential and consistently showed a reduced ADP-stimulated respiration rate. In the parental cells (i.e. control cells containing IF₁) the inhibitor protein was found to be associated with the dimeric form of the ATP synthase complex, therefore we propose that the interaction of IF₁ with the complex either directly, by increasing the catalytic activity of the enzyme, or indirectly, by improving the structure of mitochondrial cristae, can increase the oxidative phosphorylation rate in osteosarcoma cells grown under normoxic conditions.     

Predicting the potential of open-pollinating populations for the production of superior F1 hybrids

Summary The distributive properties of a single population or of a population resulting from a cross between two populations are reproduced when inbreds randomly extracted from the population itself or from the two parental populations are randomly paired. Hence, population parameters that are usually obtained during a breeding programme can be used to predict the performance of the F₁ hybrids that can be derived from them at that stage. Multiple allelism, epistasis and deviations from Hardy-Weinberg equilibria should not cause biasis to the predictions. While in theory genotype x environment interaction and linkage disequilibrium may disturb the predictions, in practice they are unlikely to create problems that cannot be accommodated. Genotypic and phenotypic predictions of the proportions of the F₁ hybrid distribution scoring above or below a given standard are made and analysed for three characters, weight of the ears, plant height and height of the ear, in two populations of maize per se and their interpopulational cross. Because no random inbred lines from the experimental populations are presently available we cannot check our predictions. However, genotypic and phenotypic predictions and observations of F₁ hybrids obtained from populations created by computer simulation are provided to illustrate our procedures.J. F. F. de Toledo is a geneticist on leave from Centro National de Pesquisa de Soja-EMBRAPA, Caixa Postal 1061, 86100-Londrina-Pr, BrazilJ. B. de Miranda Filho is Professor of Genetics at Escola Superior de Agriculture Luiz de Queiroz-ESALQ, Caixa Postal 83, 13400-Piracicaba-SP, Brazil     

Proton ATPase of rat liver mitochondria: A rapid procedure for purification of a stable, reconstitutively active F1 preparation using a modified chloroform method

A method is described for the purification of rat liver F1-ATPase by a modification of the chloroform extraction procedure originally described by Beechey et al. (Biochem. J. (1975) 148, 533). Purified liver membrane vesicles are extracted with chloroform in the presence of ATP and EDTA. The procedure yields pure F1 in only 2-3 h without the necessity of ion-exchange chromatography. The enzyme exhibits the alpha, beta, gamma, delta, and epsilon bands characteristic of F1-ATPase. It has a high ATPase specific activity, and is reconstitutively active, catalyzing high rates of ATP synthesis. Significantly, it can be readily crystallized. If desired, the enzyme can be passed over a gel filtration column to place it in a stabilizing phosphate-EDTA buffer, lyophilized and stored indefinitely at -20 degrees C.