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PubMed2004

Stoichiometry of the T-cell receptor-CD3 complex and key intermediates assembled in the endoplasmic reticulum.

The T-cell receptor (TCR)-CD3 complex is critical for T-cell development and function, and represents one of the most complex transmembrane receptors. Models of different stoichiometry and valency have been proposed based on cellular experiments and these have important implications for the mechanisms of receptor triggering. Since determination of receptor stoichiometry in T-cells is not possible due to the presence of previously synthesized, unlabeled receptor components with different half-lives, we examined the stoichiometry of the receptor assembled in endoplasmic reticulum (ER) microsomes of B-cell origin. The stoichiometric relationship among all subunits was directly determined using intact radiolabeled TCR-CD3 complexes that were isolated with a sequential, non-denaturing immunoprecipitation method, and identical results were obtained with two detergents belonging to different structural classes. The results firmly establish that the alphabeta TCR-CD3 complex assembled in the ER is monovalent and composed of one copy of the TCRalphabeta, CD3deltaepsilon, CD3gammaepsilon and zeta-zeta dimers.

Amino Acid Sequence↗
PubMed1988

The intrinsic as opposed to the apparent stoichiometry of the glycine-proton symport of the yeast Saccharomyces carlsbergensis.

1. Various ways of computing the proton stoichiometry of glycine absorption were examined in relation to the problem of distinguishing the proton flow (i) through the symport from the basal proton flow (ii) outside it. By depolarizing the plasma membrane, i will tend to inhibit ii. 2. A series of 23 yeast (Saccharomyces carlsbergensis) preparations grown with proline or glutamate were used, some of which were starved in the presence of glucose. Consequently, after ATP depletion, the rate of glycine uptake from a 0.2 mM solution varied through the series from 3 to 14 nmol.min-1.mg-1. Basal proton uptake in the absence of glycine was fairly constant at 3-4 nmol.min-1.mg-1. 3. After addition of glycine, the number of extra equivalents of protons entering the yeast with each amino acid equivalent in 30 s was 0.5 at the lowest rate of glycine absorption and 1.8 equivalents at the fastest rate. However, total proton absorption in 30 s increased in direct proportion to the amount of glycine absorbed. The proportionality factor, indicative of the carrier stoichiometry, was 2.25 +/- 0.13 (23) S.E.M. The effective basal proton uptake was negligibly small. 4. Progress of proton and glycine absorption by each yeast preparation in the period up to 180 s fitted the mathematical model described in the preceding paper by Eddy, Hopkins & Johnson [(1988) Biochem. J. 251, 111-114]. The analysis led to two estimates of the constant ratio of the inflow of protons to the inflow of glycine that would apply when the basal proton flow vanished. These further estimates of the carrier stoichiometry were also near 2, being 2.07 +/- 0.24 (6) and 2.22 +/- 0.07 (17).

Absorption↗
PubMed1989

Altered stoichiometry of the human leucocyte Na+/H+ antiport with decreasing pH.

The Na+/H+ antiport is an important regulator of cellular volume, pH and Na+ concentration in mammalian cells. The stoichiometry of this antiporter has previously been shown to be a 1:1 exchange of internal H+ for external Na+. We have investigated this stoichiometry in human leucocytes by using a novel intracellular pH-clamping technique and measuring 22Na+ influx and H+ efflux in the same cells. As internal pH was lowered, the stoichiometry of H+/Na+ exchange rose to a mean +/- S.D. of 2.23 +/- 0.69. This mechanism allows a higher H+ efflux in the face of intracellular acid stress without causing excessive intracellular Na+ overload.

Carrier Proteins↗
PubMed1986

Variable stoichiometry of phosphate-linked anion exchange in Streptococcus lactis: implications for the mechanism of sugar phosphate transport by bacteria.

Phosphate/2-deoxyglucose 6-phosphate antiport in Streptococcus lactis showed an exchange stoichiometry that varied over a 2-fold range when assay pH was shifted between pH 8.2 and pH 5.2. At pH 7.0 and above, 2 mol of phosphate moved per mol of sugar phosphate; at pH 6.1 the ratio was 1.5:1, while at pH 5.2 the overall stoichiometry fell to 1.1:1. This pattern was not affected by valinomycin in potassium-based media, nor could variable stoichiometry be attributed to altered hydrolysis of the sugar phosphate substrate. In kinetic studies at pH 7.0 or pH 5.2, sugar 6-phosphate was a competitive inhibitor of phosphate transport, indicating operation of a single system. Parallel tests showed that the affinity of antiport for its sugar 6-phosphate substrate was insensitive to pH in this range. Overall, such results suggest a neutral exchange that has specificity for monovalent phosphate but that selects randomly among the available mono- and divalent sugar 6-phosphates. A simple model that shows this behavior suggests a mechanistic role for anion exchange in bacterial transport of sugar phosphate or other organic anions.

Anions↗
PubMed1996

Pentameric subunit stoichiometry of a neuronal glutamate receptor.

Ionotropic glutamate receptors, neurotransmitter-activated ion channels that mediate excitatory synaptic transmission in the central nervous system, are oligomeric membrane proteins of unknown subunit stoichiometry. To determine the subunit stoichiometry we have used a functional assay based on the blockade of two alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate/kainate receptor subunit 1 (GluR1) mutant subunits selectively engineered to exhibit differential sensitivity to the open channel blockers phencyclidine and dizolcipine (MK-801). Coinjection into amphibian oocytes of weakly sensitive with highly sensitive subunit complementary RNAs produces functional heteromeric channels with mixed blocker sensitivities. Increasing the fraction of the highly sensitive subunit augmented the proportion of drug-sensitive receptors. Analysis of the data using a model based on random aggregation of receptor subunits allowed us to determine a pentameric stoichiometry for GluR1. This finding supports the view that a pentameric subunit organization underlies the structure of the neuronal ionotropic glutamate receptor gene family.

Animals↗
PubMed1996

Stoichiometry of 2',5'-oligoadenylate-induced dimerization of ribonuclease L. A sedimentation equilibrium study.

Ribonuclease L is an endoribonuclease that is activated by binding of 2',5'-linked oligoadenylates. Activation of ribonuclease L also induces dimerization. Here, we demonstrate using equilibrium sedimentation that dimerization requires the binding of one 5'-monophosphate 2',5'-(adenosine)3 molecule per ribonuclease L monomer. No dimerization was observed in the absence of activator up to a protein concentration of 18 microM, indicating that unliganded enzyme is unable to dimerize or the association is very weak. In parallel with dimerization, enzymatic activity is also maximized at a 1:1 activator: ribonuclease L stoichiometry. The same stoichiometry for dimerization is observed using a nonphosphorylated activator 2'-5'-(adenosine)3. Adenosine triphosphate or RNA oligonucleotide substrates do not induce dimerization. The observed stoichiometry supports a model for ribonuclease L dimerization in which activator binds to monomer, which subsequently dimerizes.

Adenine Nucleotides↗
PubMed1998

MinK-KvLQT1 fusion proteins, evidence for multiple stoichiometries of the assembled IsK channel.

IsK, a slowly activating delayed rectifier K+ current through channels formed by the assembly of two channel proteins KvLQT1 and MinK, modulates the repolarization of cardiac action potentials. Mutations that map to the KvLQT1 and minK genes account for more than 50% of an inherited cardiac disorder, the Long QT syndrome (Splawski, I., Tristani-Firouzi, M., Lehmann, M. H., Sanguinetti, M. C., and Keating, M. T. (1997) Nat. Genet. 17, 338-340). Despite the importance of these channels to human cardiac function, the molecular basis of their uniquely slow gating properties as well as the stoichiometry and interaction sites of these two subunits are still unclear. We have constructed several fusion channel proteins to begin investigating the stoichiometry of these two subunits and the role of voltage-dependent subunit assembly in channel gating. Functional properties of these constructs were measured using whole cell patch clamp recordings of transiently transfected Chinese hamster ovary cells. The constructs we tested are as follows: MK24 (C terminus of MinK linked to N terminus of KvLQT1); KK40 (a tandem homodimer of KvLQT1); and MKK44 (C terminus of MinK linked to N terminus of KK40). In control experiments (no DNA, control DNA, or only MinK), no time-dependent K+ current was observed. Expression of KvLQT1 or KK40 produced currents that activate and inactivate in a voltage-dependent manner as reported by others for KvLQT1. In contrast, expression of MK24 and MKK44 elicited current with activation kinetics and voltage dependence very similar to native IsK and identical to currents expressed by cells co-transfected with independent MinK and KvLQT1 cDNA. Expression of MK24 plus additional MinK significantly slows current kinetics. Our data raise the possibility 1) of multiple MinK/KvLQT1 stoichiometries and 2) indicate that uniquely slow kinetics of IsK channels is due to voltage-dependent conformational changes of the channel protein and not to assembly of channel subunits.

Animals↗
PubMed1999

Subcellular localization, stoichiometry, and protein levels of 26 S proteasome subunits in yeast.

The 26 S proteasome of eukaryotes is responsible for the degradation of proteins targeted for proteolysis by the ubiquitin system. Yeast has been an important model organism for understanding eukaryotic proteasome structure and function. Toward a quantitative characterization of the proteasome, we have determined the localization, cellular levels, and stoichiometry of proteasome subunits. The subcellular localization of two ATPase components of the regulatory complex of the proteasome, Sug2/Rpt4 and Sug1/Rpt6, and a subunit of the 20 S proteasome, Pre1, were determined by immunofluorescence. In contrast to findings in multicellular organisms, these proteins are localized almost exclusively to the nucleus throughout the cell cycle. We have also determined the cellular abundance and stoichiometry of these proteasome subunits. Sug1/Rpt6, Sug2/Rpt4, and Pre1 are present in roughly equal stoichiometry with an abundance of 15,000-30,000 molecules/cell, corresponding to a concentration of 13-26 microM in the nucleus. Also, in contrast to mammalian cells, we find no evidence of a p27-containing "modulator" of the proteasome in yeast. This information will be useful in comparing and contrasting the yeast and mammalian proteasomes and should contribute to a mechanistic understanding of how this complex functions.

Amino Acid Sequence↗
PubMed1999

Stoichiometry and kinetics of the high-affinity H+-coupled peptide transporter PepT2.

Proton-coupled peptide transporters mediate the absorption of a large variety of di- and tripeptides as well as peptide-like pharmacologically active compounds. We report a kinetic analysis of the rat kidney high-affinity peptide transporter PepT2 expressed in Xenopus oocytes. By use of simultaneous radioactive uptake and current measurements under voltage-clamp condition, the charge to substrate uptake ratio was found to be close to 2 for both D-Phe-L-Ala and D-Phe-L-Glu, indicating that the H+:substrate stoichiometry is 2:1 and 3:1 for neutral and anionic dipeptides, respectively. The higher stoichiometry for anionic peptides suggests that they are transported in the protonated form. For D-Phe-L-Lys, the charge:uptake ratio averaged 2.4 from pooled experiments, suggesting that Phe-Lys crosses the membrane via PepT2 either in its deprotonated (neutral) or its positively charged form, averaging a H+:Phe-Lys stoichiometry of 1.4:1. These findings led to the overall conclusion that PepT2 couples transport of one peptide molecule to two H+. This is in contrast to the low-affinity transporter PepT1 that couples transport of one peptide to one H+. Quinapril inhibited PepT2-mediated currents in presence or in absence of external substrates. Oocytes expressing PepT2 exhibited quinapril-sensitive outward currents. In the absence of external substrate, a quinapril-sensitive proton inward current (proton leak) was also observed which, together with the observed pH-dependent PepT2-specific presteady-state currents (Ipss), indicates that at least one H+ binds to the transporter prior to substrate. PepT2 exhibited Ipss in response to hyperpolarization at pH 6.5-8.0. However, contrary to previous observations on various transporters, 1) no significant currents were observed corresponding to voltage jumps returning from hyperpolarization, and 2) at reduced extracellular pH, no significant Ipss were observed in either direction. Together with observed lower substrate affinities and decreased PepT2-mediated currents at hyperpolarized Vm, our data are consistent with the concept that hyperpolarization exerts inactivation effects on the transporter which are enhanced by low pH. Our studies revealed distinct properties of PepT2, compared with PepT1 and other ion-coupled transporters.

Animals↗
PubMed2001

Stoichiometry of DnaA and DnaB protein in initiation at the Escherichia coli chromosomal origin.

Initiation of DNA replication at the Escherichia coli chromosomal origin, oriC, occurs through an ordered series of events that depend first on the binding of DnaA protein, the replication initiator, to DnaA box sequences within oriC followed by unwinding of an AT-rich region near the left border. The prepriming complex then forms, involving the binding of DnaB helicase at oriC so that it is properly positioned at each replication fork. We assembled and isolated the prepriming complexes on an oriC plasmid, then determined the stoichiometries of proteins in these complexes by quantitative immunoblot analysis. DnaA protein alone binds to oriC with a stoichiometry of 4-5 monomers per oriC DNA. In the prepriming complex, the stoichiometries are 10 DnaA monomers and 2 DnaB hexamers per oriC plasmid. That only two DnaB hexamers are bound, one for each replication fork, suggests that the binding of additional molecules of DnaA in forming the prepriming complex restricts the loading of additional DnaB hexamers that can bind at oriC.

Adenosine Triphosphate↗
PubMed2002

Stoichiometry of active smad-transcription factor complexes on DNA.

Transforming growth factor (TGF)-beta signals through a heteromeric complex of serine/threonine kinase receptors. The type I receptor phosphorylates and activates the receptor-regulated Smads (R-Smads), Smad2 and Smad3, which form hetero-oligomeric complexes with the co-Smad, Smad4, and translocate to the nucleus. Smad3 and Smad4 can bind directly to consensus DNA-binding elements in the promoters of target genes, whereas Smad2/Smad4 complexes are targeted to DNA by interacting with sequence-specific DNA-binding transcription factors that contain a well-defined Smad interaction motif (SIM). The exact stoichiometry of Smad homo- and hetero-oligomers both before and after ligand stimulation is controversial. Here we determine the stoichiometry of TGF-beta-induced Smad-transcription factor complexes on DNA. We show that complexes of Smad2/Smad4 with the transcription factors Fast-1 or Fast-3 contain one Fast, two Smad2s, and one Smad4. In contrast, Smad3/Smad4 complexes that bind the Smad-binding element from the c-jun promoter, are heterodimers. Furthermore, these Smad3/Smad4 complexes contain at least two additional components essential for complex formation, one of which contains a SIM. Our data suggest that the R-Smads can form heterodimers or heterotrimers with Smad4, and we propose that the exact stoichiometries of active Smad complexes on DNA may be determined by the transcription factors with which they associate.

3T3 Cells↗
PubMed2004

Protein stoichiometry of a multiprotein complex, the human spliceosomal U1 small nuclear ribonucleoprotein: absolute quantification using isotope-coded tags and mass spectrometry.

The human U1 snRNP (small nuclear ribonucleoprotein), which is a part of the spliceosome, consists of U1 snRNA and ten different proteins: seven Sm proteins B/B', D1, D2, D3, E, F, and G and the three U1-specific proteins U1-70 K, U1-A, U1-C. To determine the stoichiometry of all ten proteins, the complex was denatured, digested completely with an endoproteinase and labeled with an amine-specific tag. Corresponding peptides were synthesized and labeled with the same tag containing heavier isotopes. The digest was then spiked with defined amounts of the synthetic peptides, and the resulting isotopic peptide pairs were analyzed quantitatively by mass spectrometry. The mass spectra provided information about the absolute amount of each component in the starting protein mixture. The use of the isotope-coded, amine-specific reagents propionyl-N-oxysuccinimide and nicotinoyl-N-oxysuccinimide was evaluated for stoichiometry determination; the nicotinoyl reagent was found to be advantageous because of its greater mass spectrometric sensitivity. Absolute quantities of all ten proteins were measured, showing equal numbers of all ten proteins in the U1 spliceosomal snRNP. These data demonstrate that quantitative mass spectrometry has great potential for the determination of the stoichiometry of multiprotein complexes.

Chromatography, Liquid↗
PubMed2006

Stoichiometry of the murine gammadelta T cell receptor.

The T cell receptor for antigen (TCR) complex is organized into two functional domains: the antigen-binding clonotypic heterodimer and the signal-transducing invariant CD3 and TCRzeta chains. In most vertebrates, there are two different clonotypic heterodimers (TCRalphabeta and TCRgammadelta) that define the alphabeta and gammadelta T cell lineages, respectively. alphabeta- and gammadeltaTCRs also differ in their invariant chain subunit composition, in that alphabetaTCRs contain CD3gammaepsilon and CD3deltaepsilon dimers, whereas gammadeltaTCRs contain only CD3gammaepsilon dimers. This difference in subunit composition of the alphabeta- and gammadeltaTCRs raises the question of whether the stoichiometries of these receptor complexes are different. As the stoichiometry of the murine gammadeltaTCR has not been previously investigated, we used two quantitative immunofluorescent approaches to determine the valency of TCRgammadelta heterodimers and CD3gammaepsilon dimers in surface murine gammadeltaTCR complexes. Our results support a model of murine gammadeltaTCR stoichiometry in which there are two CD3gammaepsilon dimers for every TCRgammadelta heterodimer.

Animals↗
PubMed1986

Na+ and K+ transport at basolateral membranes of epithelial cells. I. Stoichiometry of the Na,K-ATPase.

The stoichiometry of pump-mediated Na/K exchange was studied in isolated epithelial sheets of frog skin. 42K influx across basolateral membranes was measured with tissues in a steady state and incubated in either beakers or in chambers. The short-circuit current provided estimates of Na+ influx at the apical membranes of the cells. 42K influx of tissues bathed in Cl- or SO4-Ringer solution averaged approximately 8 microA/cm2. Ouabain inhibited 94% of the 42K influx. Furosemide was without effect on pre-ouabain-treated tissues but inhibited a ouabain-induced and Cl--dependent component of 42K influx. After taking into account the contribution of the Na+ load to the pump by way of basolateral membrane recycling of Na+, the stoichiometry was found to increase from approximately 2 to 6 as the pump-mediated Na+ transport rate increased from 10 to 70 microA/cm2. Extrapolation of the data to low rates of Na+ transport (less than 10 microA/cm2) indicated that the stoichiometry would be in the vicinity of 3:2. As pump-mediated K+ influx saturates with increasing rates of Na+ transport, Na+ efflux cannot be obligatorily coupled to K+ influx at all rates of transepithelial Na+ transport. These results are similar to those of Mullins and Brinley (1969. Journal of General Physiology. 53:504-740) in studies of the squid axon.

Animals↗
PubMed1977

Phosphorylated protamines. I. Binding stoichiometry and thermal stability of complexes in DNA.

To decipher on a molecular level the role of protamine phosphorylation in spermiogenesis, clupeine Z species containing one, two or three serine phosphates were prepared utilizing a recently developed chemical procedure. The melting of complexes with calf thymus DNA showed that thermal stability decreases with increasing degree of phosphorylation. The stoichiometry of the nucleoprotamine complexes was investigated analyzing the melting curves and using the fluorescamine assay recently described. Phosphorylation significantly reduces binding stoichiometry defined as DNA-nucleotides covered by a protamine molecule. Thus, phosphorylated protamines are more densely packed along DNA; the implications on processes occurring in spermiogenesis as i. e. histone replacement, are discussed. A general discussion on the variability in protein-DNA stoichiometry values obtained by different procedures is included.

Chemical Phenomena↗
PubMed2001

Physiological significance of the regulation of photosystem stoichiometry upon high light acclimation of Synechocystis sp. PCC 6803.

We characterized the photosynthetic properties of the pmgA mutant of Synechocystis PCC 6803, which cannot change its photosystem stoichiometry under a high-light condition (200 micromol x m(-2) x s(-1)), in order to clarify the physiological significance of the regulation of photosystem stoichiometry. We found that (1) PSII activity was inhibited more in wild-type cells on the first day under the high-light conditions than in mutant cells. (2) The growth of the mutants following the initial imposition of high light was faster than that of wild-type cells. (3) However, growth was severely inhibited in the mutants after the third day of exposure to high light. (4) The growth inhibition in the mutants under the extended high-light conditions was reversed by the addition of sublethal concentrations of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), which seemed to mimic photoinhibition of PSII. These results suggest that the main role of adjusting the photosystem stoichiometry with respect to light intensity is not to maintain efficient photosynthesis, but to down regulate electron transfer. Failure to down regulate electron flow leads to cell death under prolonged exposure to high light in this cyanobacterium.

Cyanobacteria↗
PubMed1993

Stoichiometry of a recombinant GABAA receptor deduced from mutation-induced rectification.

Ligand-gated ion channels generally display a heterooligomeric subunit structure. The present report describes an electrophysiological method that provides criteria indicating the subunit stoichiometry of a recombinant GABAA receptor composed of alpha 3, beta 2 and gamma 2 subunits. Our results exclude the stoichiometries 3 alpha 1 beta 1 gamma, 1 alpha 3 beta 1 gamma, 1 alpha 1 beta 3 gamma and suggest that the possible subunit stoichiometries for this receptor are 2 alpha 1 beta 2 gamma, 2 alpha 2 beta 1 gamma or 1 alpha 2 beta 2 gamma, of which the alpha subunit composition 2 alpha 1 beta 2 gamma may be favoured. The method is based on the quantification of the outward rectification of the GABA-evoked current induced by point mutation of charged amino acids located near the ion channel pore.

Amino Acid Sequence↗
PubMed2002

Stoichiometry of the photosynthetic apparatus and phycobilisome structure of the cyanobacterium Plectonema boryanum UTEX 485 are regulated by both light and temperature.

The role of growth temperature and growth irradiance on the regulation of the stoichiometry and function of the photosynthetic apparatus was examined in the cyanobacterium Plectonema boryanum UTEX 485 by comparing mid-log phase cultures grown at either 29 degrees C/150 micromol m(-2) s(-1), 29 degrees C/750 micromol m(-2) s(-1), 15 degrees C/150 micromol m(-2) s(-1), or 15 degrees C/10 micromol m(-2) s(-1). Cultures grown at 29 degrees C/750 micromol m(-2) s(-1) were structurally and functionally similar to those grown at 15 degrees C/150 micromol m(-2) s(-1), whereas cultures grown at 29 degrees C/150 micromol m(-2) s(-1) were structurally and functionally similar to those grown at 15 degrees C/10 micromol m(-2) s(-1). The stoichiometry of specific components of the photosynthetic apparatus, such as the ratio of photosystem (PS) I to PSII, phycobilisome size and the relative abundance of the cytochrome b(6)/f complex, the plastoquinone pool size, and the NAD(P)H dehydrogenase complex were regulated by both growth temperature and growth irradiance in a similar manner. This indicates that temperature and irradiance may share a common sensing/signaling pathway to regulate the stoichiometry and function of the photosynthetic apparatus in P. boryanum. In contrast, the accumulation of neither the D1 polypeptide of PSII, the large subunit of Rubisco, nor the CF(1) alpha-subunit appeared to be regulated by the same mechanism. Measurements of P700 photooxidation in vivo in the presence and absence of inhibitors of photosynthetic electron transport coupled with immunoblots of the NAD(P)H dehydrogenase complex in cells grown at either 29 degrees C/750 micromol m(-2) s(-1) or 15 degrees C/150 micromol m(-2) s(-1) are consistent with an increased flow of respiratory electrons into the photosynthetic intersystem electron transport chain maintaining P700 in a reduced state relative to cells grown at either 29 degrees C/150 micromol m(-2) s(-1) or 15 degrees C/10 micromol m(-2) s(-1). These results are discussed in terms of acclimation to excitation pressure imposed by either low growth temperature or high growth irradiance.

Acclimatization↗