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The β subunit of bovine luteinizing hormone (LH) was crystallized and its structure solved to 3.15 ​Å resolution by molecular replacement using human chorionic gonadotropin (hCG) β subunit as search model. The asymmetric unit contains two copies of the β subunit that are related by a non-crystallographic symmetry (NCS) two-fold axis, both copies of which contain proteolytic cleavages after amino acid 100. It is noteworthy that the oligosaccharide moieties covalently attached at asparagine 13 were particularly pronounced in the electron density, allowing seven sugar residues to be defined. The α subunit of LH, which is common to all glycosylated gonadotropin hormones, was placed by superposition of hCG on the LH beta subunits, thereby yielding a model for the intact hormone., Graphical abstract A model of luteinizing hormone based on the known α subunit and the β subunit determined here by X-ray crystallography.Image 1, Highlights • The X-ray crystallographic structure of the beta subunit of bovine luteinizing hormone (LH) has been determined at 3.15 A resolution • A model for the entire LH has been proposed based on the beta subunit structure reported here and the known structure if the common alpha subunit • A large amount (7 sugar residues on one molecule in the asymmetric unit, 5 on the other) of the oligosaccharide attached at asn 13 was visible in electron density maps • Each of the two molecules in the asymmetric unit, which contains an NCS twofold axis, is associated with two molecules of beta octylglucoside • The beta subunit has a proteolytic cleavage between amino acids 100 and 101, which releases the “seatbelt” peptide, though that peptide remains associated through an S–S bridge to the core.

Objective: The purpose of this study was to compare the effectiveness between total human chorionic gonadotropin (hCG) and free β-hCG in two-marker Down syndrome screening programs during the second trimester in a Taiwanese population. Materials and Methods: From a multicenter collaborative study, we investigated the second-trimester maternal serum levels of total hCG from 67 data, free β-hCG from 72 and alpha-fetoprotein (AFP) from 96 obtained from Taiwanese pregnant women carrying fetuses with Down syndrome. Results: High total hCG and free β-hCG, as well as low AFP levels, were found with median values of 2.06, 2.49 and 0.77 multiples of the median (MoM), respectively. At a 5% false-positive rate, total hCG and free β-hCG could detect 31% and 43% of Down syndrome pregnancies, respectively, whilst AFP alone could detect only 15% of affected cases. When combined with maternal age-specific risk, total hCG could achieve a 52% detection rate, free β-hCG a 54% and AFP a 39%. Combined total hCG and AFP achieved a detection rate of 55%, and combined free β-hCG and AFP achieved a rate of 60%. Conclusion: The measurement of free β-hCG is more beneficial than total hCG in serum screening for Down syndrome during the second trimester of pregnancy.

Ca(V)1.2 (alpha(1c)) is a pore-forming subunit of the voltage-dependent L-type calcium channel and is expressed in many tissues. The beta and alpha(2)/delta subunits are auxiliary subunits that affect the kinetics and the expression of Ca(V)1.2. In addition to the beta and alpha(2)/delta subunits, several molecules have been reported to be involved in the regulation of Ca(V)1.2 current. Calmodulin, CaBP1 (calcium-binding protein-1), CaMKII (calcium/calmodulin-dependent protein kinase II), AKAPs (A-kinase anchoring proteins), phosphatases, Caveolin-3, beta(2)-adrenergic receptor, PDZ domain proteins, sorcin, SNARE proteins, synaptotagmin, CSN5, RGK family, and AHNAK1 have all been reported to interact with Ca(V)1.2 and the beta subunit. This review focuses on the effect of these molecules on Ca(V)1.2 current.

This chapter deals with the primary active transport systems that use metabolic energy directly to pump ions out of cells or organelles. The source of metabolic energy in the different primary active transporters is, in the P-type ATPases, the splitting of adenosine triphosphate (ATP); in the light-driven pumps, the absorption of the energy of a photon; and in the rotary ATPases, the interconversion of the energy present in ATP with that in an electrochemical gradient of protons, which can itself be produced by the flow of electrons in oxidation–reduction reactions. We discuss the kinetics of two of the P-type ATPases (the calcium ATPase of the sarcoplasmic reticulum and the ubiquitous sodium/potassium ATPase) and how this relates to the molecular structural details of these pumps as this has been determined during the last decade and a half. We touch on the evolution of these pumps. The new structural information on the rotary ATPases (especially the F0F1 ATPase) and bacteriorhodopsin is presented and discussed in terms of the proton movements in these systems. The discussion extends to the ABC transporters, including the multidrug resistance protein, P-glycoprotein, that move a great range of substrates across cell membranes, harnessing the energy in ATP to do so.

P-type adenosine triphosphatases (ATPases) are membrane pumps that maintain concentration gradients of ions across biological membranes at the expense of adenosine triphosphate (ATP). Their transport process involves phosphorylation of a conserved residue during the transition between two conformational states with different affinities for counter-transported ions. The overall structure of the catalytical α -subunit of P-type ATPases consists of three cytoplasmic domains and one membrane-bound domain with binding sites for substrate ions. Potassium-transporting ATPases contain an additional smaller β -subunit important, for example, for proper maturation and sorting of the pump. Prominent members of the P-type ATPase family include the sarcoplasmic reticulum calcium pump that is required, for example, for signaling and muscle relaxation, or the sodium–potassium pump and the plasma membrane proton pump that energize membranes in animal cells and in plant and fungi cells, respectively. P-type ATPases are involved in the onset and treatment of various pathological conditions – for example, cardiotonic steroids inhibiting the sodium–potassium pump are used in the treatment of heart failure, gastric ulcers are treated by inhibition of the gastric proton–potassium pump, and the calcium pump has recently been identified as a possible target for prostate cancer drugs.

The proton-translocating ATP synthase is a marvellous example of biological nanotechnology, combining a proton-driven turbine (Fo) and an FI component that is massaged by an eccentric spindle driven by the turbine to generate ATP from ADP and Pi. High-resolution structures are available for nearly all the components, and the molecular mechanism is known in considerable, but not complete, detail. Archaea and a few eubacteria possess distinct but related ATP synthases, while the first molecular rotator motor to be described was that which drives the bacterial flagellum.

P-type ATPases are membrane proteins capable of using the energy rich ATP molecule as substrate to generate, across biological membranes, essential ion gradients which are the basis for such diverse functions as signaling, energy storage and secondary transport processes. They all appear to function by cyclical conversion between two main conformations (E1 and E2) alternately presenting the ion-binding sites on each side of the membrane. In the present chapter, we propose to give the keys to understand the workings of these nano-machines by using the prominent member Ca 2+ -ATPase as a model.

Publisher Summary In 1954 it was discovered that upon illumination suspended thylakoid membranes synthesize ATP from ADP and inorganic phosphate. This process is called photophosphorylation. Further experiments showed that photophosphorylation is coupled to the generation of NADPH. This result was unexpected, as at that time it was generally believed that the synthesis of ATP in chloroplasts, as in mitochondria, was driven by an electron transport from NADPH to oxygen. It soon became apparent, however, that the mechanism of photophosphorylation coupled to photosynthetic electron transport was very similar to that of ATP synthesis coupled to electron transport of mitochondria, termed oxidative phosphorylation. In 1961 Peter Mitchell (Edinburgh) postulated in his chemiosmotic hypothesis that during electron transport a proton gradient is formed, and that it is the proton motive force of this gradient that drives the synthesis of ATP. At first, this revolutionary hypothesis was strongly opposed by many workers in the field, but in the course of time, experimental results of many researchers supported the chemiosmotic hypothesis, which is now fully accepted. The transport of protons across a membrane can have different effects. If the membrane is permeable to counter ions of the proton, the charge of the proton will be compensated, since each transported proton will pull a chloride ion across a membrane. This is how a proton concentration gradient is generated.

Soybean β-conglycinin is a complex protein possessing health-promoting propertiesβ-Conglycinin is a trimeric glycoprotein. Little information related to methods for separation of the individual chains forming β-conglycinin has been so far published and it is of great interest. As a consequence, less data on the bioactivities of α, α' and β subunits of this glycoprotein have been published. The present research aimed to find out new alternative chromatographic conditions to obtain β-conglycinin subunits that are free of contaminating proteins. In the present short communication, we propose the use of a two-step ion exchange chromatographic protocol to achieve this goal. Firstly, β subunit was separated by means of anionic exchange fast protein liquid chromatography. Secondly, α and α' chains were separated from each other by cationic exchange. Our data indicated the feasibility of proposed fractionation protocol to separate soybean β-conglycinin α and α' subunits from other contaminating proteins and to obtain enough amounts of the three individual chains forming this glycoprotein for further characterization and application. The procedure may be easily up-scaled. © 2010 Elsevier B.V.

Most organisms obtain energy for growth from either capture of light or oxidation of suitable reductants. This process involves transfer of electrons between proteins of the cytoplasmic membrane (bacteria and archaea) or the inner mitochondrial membrane (eukaryotes). The unifying feature is that the energy is conserved as a proton (sometimes sodium) electrochemical gradient across the membrane. This gradient then drives uphill reactions, notably synthesis of ATP. There are examples of electron transport-independent generation of the gradient.

Guanylyl cyclases (GC) catalyze the formation of the intracellular second messenger cyclic guanyosine monophosphate (cyclic GMP) from guanosine triphosphate (GTP). They are widely expressed in mammalian and nonmammalian cells. Two families exist, the nitric oxide-sensitive and the peptide-stimulated GCs, which are also known as soluble and membrane-bound GCs. Whereas the membrane-bound GCs are made up of two identical subunits (homodimers), the soluble or cytosolic GCs are heterodimeric and formed by one alpha and one beta subunit. [For review see …

In certain strictly anaerobic bacteria, the energy for growth is derived entirely from a decarboxylation reaction. A prominent example is Propionigenium modestum, which converts the free energy of the decarboxylation of (S)-methylmalonyl-CoA to propionyl-CoA (DeltaG degrees =-20.6 kJ/mol) into an electrochemical Na(+) ion gradient across the membrane. This energy source is used as a driving force for ATP synthesis by a Na(+)-translocating F(1)F(0) ATP synthase. According to bioenergetic considerations, approximately four decarboxylation events are necessary to support the synthesis of one ATP. This unique feature of using Na(+) instead of H(+) as the coupling ion has made this ATP synthase the paradigm to study the ion pathway across the membrane and its relationship to rotational catalysis. The membrane potential (Deltapsi) is the key driving force to convert ion translocation through the F(0) motor components into torque. The resulting rotation elicits conformational changes at the catalytic sites of the peripheral F(1) domain which are instrumental for ATP synthesis. Alkaliphilic bacteria also face the challenge of synthesizing ATP at a low electrochemical potential, but for entirely different reasons. Here, the low potential is not the result of insufficient energy input from substrate degradation, but of an inverse pH gradient. This is a consequence of the high environmental pH where these bacteria grow and the necessity to keep the intracellular pH in the neutral range. In spite of this unfavorable bioenergetic condition, ATP synthesis in alkaliphilic bacteria is coupled to the proton motive force (DeltamuH(+)) and not to the much higher sodium motive force (DeltamuNa(+)). A peculiar feature of the ATP synthases of alkaliphiles is the specific inhibition of their ATP hydrolysis activity. This inhibition appears to be an essential strategy for survival at high external pH: if the enzyme were to operate as an ATPase, protons would be pumped outwards to counteract the low DeltamuH(+), thus wasting valuable ATP and compromising acidification of the cytoplasm at alkaline pH.

The sulfonylurea receptor (SUR) serves as the regulatory subunit of the ATP-sensitive potassium (K ATP ) channel, endowing it with the ability to respond to changes in cell metabolism. The K ATP channel is a hetero-octameric complex of four Kir6.x and four SUR subunits. Kir6.x belongs to the family of inwardly rectifying K channels and assembles into tetramers to form the channel. Binding of ATP to the intracellular domains of Kir6.x produces channel inhibition. Associated with each Kir6.x subunit is a regulatory subunit, the sulfonylurea receptor (SUR). SUR has two large intracellular domains, containing consensus sequences for nucleotide binding and hydrolysis, which are known as the nucleotide binding domains (NBDs). Interaction of Mg-nucleotides with these NBDs mediates activation of the K ATP channel. The SUR subunit also binds therapeutic drugs, such as the sulfonyl ureas, which inhibit K ATP channel activity, and the K ATP channel openers that stimulate channel activity. Kir6.2 serves as the pore-forming subunit, but it associates with different SUR subunits: for example, SUR1 in pancreas and brain, SUR2A in heart, and skeletal muscle and SUR2B in a variety of tissues including brain and smooth muscle. In some smooth muscles, the K ATP channel comprises Kir6.1 in association with SUR2B. This chapter focuses on the physiological role of the K ATP channel and how this is impaired in disease. It then discusses the molecular composition of the K ATP channel, and details its regulation by nucleotides, metabolism and other signaling molecules. Finally, what is known of the pharmacological properties of the channel is summarized.

Publisher Summary ATP synthase is highly conserved; it is present in mitochondria, chloroplasts, both aerobic and photosynthetic bacteria, and even those bacteria that lack a functional respiratory chain as well as where the enzyme generates Δp (protonmotive force) at the expense of hydrolysing ATP produced in glycolysis. The function of the ATP synthase is to utilize Δp to maintain the mass-action ratio for the ATPase reaction 7–10 orders of magnitude away from equilibrium, or in the case of fermentative bacteriato utilize ATP to maintain Δp for the purpose of transport. The ATP synthase can be visualized under the electron microscope in preparations of submitochondrial particles (SMPs) that have been negatively stained with phosphotungstate. The complexes appear as roughly spherical knobs projecting from the original matrix side of the membrane.

Publisher Summary This chapter discusses (1) the experimental approaches that have documented subunit rotation in F 1 and tentatively in F 0 F 1 and (2) the studies of the way the structural organization and interactions of subunits within the adenosine triphosphate (ATP) synthase may fit as functional parts of a molecular motor. ATP has a central role in cellular metabolism with cleavage of its high-energy phosphoanhydride bond(s) serving as a common “currency” to drive a variety of energy-requiring processes. These include mechanical transport processes catalyzed by molecular motors, such as muscle contraction by myosin ATPase, the transport of vesicles and organelles along microtubules by dynein and kinesin ATPases, and the vectorial unwinding of double-stranded (ds)DNA by DNA helicases. Most cellular ATP is synthesized from adenosine disphosphate (ADP) and P i by a ubiquitous coupling enzyme, the H + - transporting ATP synthase, in the terminal step of oxidative- and photo-phosphorylation. The ATP synthase is found embedded in the inner membrane of mitochondria, the thylakoid membrane of chloroplasts, and the plasma membrane of eubacteria. It is referred to as “F 0 F 1 ATP synthase,” because, experimentally, it can be separated into two complexes that retain the distinct partial functions of the intact synthase. The F 0 portion is embedded in the membrane and functions in proton transport; in the absence of F 1 , F 0 catalyzes the passive flux of protons across the bilayer. F 1 can be released from F 0 as a water-soluble complex that contains the catalytic sites for ATP synthesis but, uncoupled from a proton current, soluble F 1 can catalyze only the net hydrolysis of ATP. Thus, it is often referred to as “F 1 -ATPase.”

ATP is synthesized on the disk membrane isolated from rod outer segments of the bovine retina. Together with a slow component which accounted for a constant rate of about 22 nmol ATP/min/mg of protein and which was due to the adenylate kinase activity, a fast component with a maximal activity of about 58 nmol ATP/min/mg of protein was measured at physiological calcium concentrations. This fast activity disappeared in the presence of the Ca2+ ionophore A23187, was inhibited by vanadate or thapsigargin but not by oligomycin, suggesting that this ATP synthesis is due to the reversal functioning of the Ca2+-ATPase previously found on the disk membranes.

Publisher Summary This chapter discusses structure and function of adenosine triphosphate (ATP)-sensitive potassium (K ATP ) channels. Molecular biological studies have revealed that the K ATP channel is a complex of two subunits: a member of the ATP-binding cassette (ABC) transporter superfamily sulfonylurea receptor (SUR), and a member of the inward rectifying K + channel family, (Kir6.2). While the Kir6.2 subunit forms the K ATp channel pore and determines ion selectivity and the rectification properties of the channel, the SUR subunit confers ATP sensitivity and various pharmacological properties. However, ATP sensitivity might also be endowed by the Kir6.2 subunit, so the precise domain conferring ATP sensitivity is still inconclusive. Clarification of the molecular basis of K ATp channels should provide a better understanding of the relationship of the function of the channel to its structure as well as of its roles in physiological functions and pathophysiological states.

Publisher Summary P-type ATPases comprise a family of ATP-dependent ion pumps that are related through structural homology and catalytic mechanism. Higher animal pumps have a high affinity for ATP and are regulated through a low-affinity ATP binding site, whereas bacterial, plant, and lower animal pumps exhibit a low affinity for ATP and are not regulated. The substrate specificity of the former group is low and large substitutions on the ATP molecule are tolerated. This may be because the principal mode of regulation of the cycle is by nucleotide binding close to or at the active site. Analysis of introdexon boundaries of the large cytoplasmic loop of the genes of plant and animal ATPases suggests a five-domain structure interupted by antigenic elaborations in the larger pumps. Labeling and mutagenesis studies identify active site residues and lead to a speculative arrangement of residues binding ATP and tertiary structural model of the extramembranous portion of the protein. Probes located at the active site and the reactivity of active site residues have yielded information of changes in active site conformation that seem to reflect local disturbances rather than fundamental structural movements, such as hinge bending or opening and closing of the site.

Publisher Summary This chapter highlights the molecular features of the ATP synthase and discusses the way its complex structure is integrated to link catalysis to transport. F 0 F 1 ATP synthase is a multi-subunit complex, which is largely conserved throughout evolution, is found in the inner membranes of mitochondria, chloroplast, and bacteria, where it couples the downhill movement of protons to the synthesis of ATP from ADP and inorganic phosphate. The ATP synthase can be dissociated into two sectors: the water soluble F 1 sector, which contains the catalytic sites, and the hydrophobic F 0 sector, which conducts protons through the membrane. The experiments described in the chapter make clear that interactions among subunits are an essential part of catalytic cooperativity, ion translocation, and coupling between catalysis and transport. Future work will concentrate on revealing the structure of the complex at atomic resolution, and at the same time, mutagenesis and protein labeling experiments will identify functionally important amino acids. These two approaches will be used in concert to work toward an understanding of the way the F 0 F 1 carries out coupled transport.

Publisher Summary This chapter focuses on F-type H + ATPase that is a proton pump coupling H + transport and the chemical reaction, “adenosine triphosphate (ATP) ↔ adenosine diphosphate (ADP) + Pi,” an enzyme that is conserved among eukaryotic and prokaryotic cells and synthesizes ATP in most cases. The higher ordered structure of the catalytic sector α 3 β 3 γ complex is established by X-ray diffraction. The catalytic site and proton pathway are studied extensively by means of mutagenesis and chemical approaches. The roles of the γ and other subunits forming a stalk region in energy coupling are discussed. The γ subunit seems to play a key role in the coupling. The chapter describes the structure of the β subunit with all side chains. The crystallization of F 0 F 1 and/or F 0 is the important step for elucidation of the mechanism and structure of the proton pathway. The energy coupling between the transport and chemical reaction is also discussed; “successive conformation transmission through subunit rotation” is a fascinating model of energy coupling. The conformational transmission between different subunits during catalysis is also summarized. The β → α → β → α subunit conformational transmission is required for the co-operativity of the multiple catalytic sites. The β subunit interacts with the γ and ɛ subunits for catalysis and energy coupling. The γ and ɛ subunits interact with the β subunit for energy coupling. During ATP synthesis, proton transport may cause F 0 subunit conformational changes that are transmitted to the γ and ɛ subunits, followed by the β subunit.

Summary The synthesis of ATP by mitochondrial oxidative phosphorylation is described. The outer membrane encloses an intermembrane space and is relatively permeable to small molecules. The inner membrane encloses the matrix which contains the enzymed necessary for the oxidation of pyruvate by the citrate cycle and of the β-oxidation of fatty acids. It has good electrical insulating properties and contains specific transporters for polar compounds including substrates, ADP/ATP, and phosphate. Electrons from substrate oxidations are transferred to dioxygen by the electron-transport chain in the inner membrane. The electron-transport chain contains four multimeric complexes containing redox centers (flavins, cytochromes, iron-cytochrome c, and ubiquinone. Complexes I, III and IV use the free energy of electron transport to pump protons out of the matrix to generate a membrane potential and pH gradient across the inner membrane (the proton motive force or ΔP). ΔP drives protons (probably three) through complex V (ATP synthase) in the inner membrane, and this uses the energy of ΔP to convert ADP + Pi to ATP on its matrix face. The ADP/ATP exchange across the inner membrane also requires the uptake of a proton. The control of the rate of ATP synthesis is shared by several processes including the demand for ATP, the rate of ADP/ATP exchange, the activities of the enzymes of substrate oxidation, substrate supply, and the activity of complex V, when their relative contributions depend on the rate. When the rate of ATP synthesis is slow ΔP is higher and more protons leak back into the matrix by-passing complex V. The exact stoichiometry of ATP synthesis and extent of the proton leak varies with the metabolic state.

Summary ATP is a kinetically stable molecule with a high free energy of hydrolysis/high phosphate transfer potential. This means it can act as a common unit of exchange of energy between a variety of highly exergonic catabolic processes and energy requiring reactions within the aqueous medium of a cell. The chemical nature of the ATP molecule means that it can drive a wide variety of such reactions, including movement of ions and proteins, dehydrations (in macromolecule biosyntheses), activation of small molecules, and imparting a negative charge to sugars and proteins. The relative contributions of these processes to the energy demand of a cell depends on the tissue; muscle expends some 70% of its ATP turnover on movements of actomyosin, brain about 40% of its ATP on Na + transport, and exocrine cells about 50% on biosyntheses. The molecular basis for the coupling of ATP hydrolysis to non-chemical processes (e.g., muscle contraction, ion pumping) is not precisely known. A model is presented in which the transducing enzyme can manipulate the stages in the release of energy from ATP to link binding energy changes to conformational changes. This rationale unifies existing schemes for the function of these enzymes, and also for the functioning of the mitochondrial ATP synthase, which couples transmembrane proton flow to ATP synthesis. In humans, the bulk of ATP synthesis occurs via oxidative phosphorylation in mitochondria, although the fuel oxidized is dependent on tissue. In some tissues (and in rapidly growing tumors), however, considerable amounts of ATP are made by glycolytic conversion of glucose to lactate. This reflects an adaptation of these tissues to specific functions; for example, sustained contraction (and hence restricted O 2 supply) in white muscle or a high requirement for biosynthetic precursors (e.g., in lymphocytes). ATP levels inside cells are maintained very precisely (around 8 mM) under all physiological conditions by increasing the rate of ATP synthesis to match demand. In contrast, rates of ATP synthesis can vary greatly (5–100-fold) with the energy demands of the tissue. Together, these facts indicate that ATP itself cannot regulate its own synthesis. Cytoplasmic (glycolytic) ATP synthesis is regulated internally by AMP, changes in whose concentration amplify the changes in [ATP]; other regulators are Ca 2+ and cAMP, which signal an actual or potential increased work rate by the tissue. Mitochondrial (oxidative) ATP synthesis is regulated by cytoplasmic ADP (entering via the adenine nucleotide translocase) and/or by cytoplasmic Ca 2+ (entering via the Ca 2+ uniport), depending on the tissue. Prolonged ATP depletion leads to cell death, largely due to the development of ionic and osmotic imbalance. Such depletion occurs in a variety of clinical conditions. There may be deficiencies in the enzymes of ATP production (e.g., pyruvate kinase, pyruvate dehydrogenase, mitochondrial dehydrogenases) or conditions which lead to abnormal ATP utilization (e.g., fructose intolerance, malignant hyperpyrexia, ischemia/reperfusion). The resulting symptoms vary considerably, depending on the tissue most susceptible to the defect.

Publisher Summary Adenosine triphosphate (ATP) is the central compound of energy metabolism in all living cells. As the membrane of these cells is typically impermeable to ATP, the ATP molecule is permanently synthesized within the cell from adenosine diphosphate (ADP) and inorganic phosphate with the consumption of an energy source, such as light or chemical energy. Most of the ATP synthesis in aerobic cells is performed by F-type or F 1 F 0 ATP synthases. These enzymes are located in the inner membrane of mitochondria, in the thylakoid membrane of chloroplasts or in the cytoplasmic membrane of bacteria. The membrane-bound location of the F 1 F 0 ATPases is essential for the energy converting function of these catalysts. In photophosphorylation or oxidative phosphorylation light, chemical energy is converted into an electrochemical proton gradient (ΔμH + ), which is exploited by the ATPase to drive the endergonic synthesis of ATP from ADP and inorganic phosphate. Despite the wide distribution of the F 1 F 0 ATPases in nature, structure and function of these catalysts are highly conserved. The usual physiological function of these enzymes is to synthesize ATP under the consumption of ΔμH + .

Studies of the control of cardiac respiration using isolated heart mitochondria and submitochondrial particles are reviewed and compared with studies of cardiac respiration in perfused hearts and in intact organisms. The studies of respiratory control in isolated mitochondria indicate that the rate of ATP synthesis is controlled by the substrates of the reaction, phosphate and ADP, and by the electrochemical potential gradient of protons across the mitochondrial inner membrane. The controversy about whether the ATP synthase itself or the adenine nucleotide translocase provides kinetic limitation to flux is discussed from the point of view that the more important kinetic control is provided by the synthase. When rates of ATP synthesis are varied in intact organs other than the adult mammalian heart, NMR measurements suggest that free ADPalso controls respiration and ATP synthesis in vivo . However, in the adult heart in vivo there appears to be an additional control mechanism which permits ATP synthesis to vary over a 5- to 6-fold range in the absence of appropriate increases in substrate level or increases in the electrochemical potential gradient of protons, a value recently measured in working hearts. Possible mechanisms are discussed in light of reports of allosteric control of the ATP synthase gathered using submitochondrial particles.

Publisher Summary This chapter discusses the structure and assembly of ATP synthase. The ATP synthase or F 0 F 1 -ATPase catalyses the formation of ATP from ADP and P i in oxidative or photosynthetic phosphorylation. This membrane-spanning enzyme is located in mitochondria, chloroplasts or the cytoplasmic membrane of bacteria and is structurally conserved. The multiple functional aspects of ATP Synthases are shared amongst many subunits. It is possible to discriminate two major types of enzyme complex organization. The first is the more simple bacterial and chloroplast type, which has a clearly demarcated F 1 sector carrying nucleotide binding and catalytic properties, and a membrane-embedded F 0 sector which contains the proton channel and, in conjunction with F 1 , the energy-transducing or coupling functions of the complex. The biogenesis of mitochondrial ATP synthase requires contributions from both the mitochondrial and nuclear genomes. At least two subunits are encoded in mitochondrial DNA of all organisms so far examined.

Publisher Summary Among biological membranes, one of the most highly structured and physiologically complex is that of the chloroplast thylakoid, wherein at least five distinct ensembles of molecular events can be identified and studied: light capture, redox reactions, transmembrane charge separation, ionic transport, and ATP synthesis. All these are organized for the central purpose of converting light energy to the currency most convenient for biological functions: the energy of redox equivalents and the energy of phosphate anhydride bonds in ATP. This chapter focuses on the process of ATP synthesis in the thylakoid membrane, and-in keeping with the general subject of this volume-will treat especially those properties of the membrane ATPase and ATP synthetase involved in the utilization of electric fields and proton gradients to accomplish ATP synthesis. Despite structural differences among membrane ATPases-the general mechanisms of ATP hydrolysis and synthesis seem to be similar in a variety of biological membranes.

Publisher Summary This chapter describes a procedure to obtain a preparation of H + transporting ATP synthase from pig heart mitochondria as pure and as active for ATP synthesis as possible. The basic approach of the procedure is to extract the H + -transporting ATP synthase from the mitochondrial inner membrane after elimination of as many contaminating proteins as possible from either face of the membrane. The rate of ATP synthesis is estimated by the ATP-P i exchange based on the incorporation of 32 P i into the terminal phosphoryl group of ATP, modified by the addition of excess ADP to obtain linear rates of ATP synthesis even in preparations with low rates of ATPase activity. The vesicular preparation of H + -transporting ATP synthase is non-permeant and inverted. It yields a very efficient enzyme with high ATP synthase activity. It is a good model to study the properties of H + -transporting ATP synthase.

Publisher Summary This chapter discusses characterization and reconstitution of proton pumping of proton ATPases in Golgi Vesicles and endoplasmic reticulum. The discovery that endocytic vesicles are acidified by a proton-translocating ATPase in urinary epithelia and elsewhere, and that lysosomes also contain them led to search for these proton ATPases in other intracellular organelles, such as Golgi vesicles and endoplasmic reticulum. The methods of preparation of these organelles are essentially those used by others with some minor modification. The reconstituted proton-pumping activity has all the characteristics of that in the native vesicles. The transport is electrogenic, inhibited by N-ethylmaleimide, but not by oligomycin or vanadate. The proton pump of these vesicles generates a pH difference and a membrane potential. In an open-circuited system, the proton pump generates an electrochemical gradient, sufficient to bring net H + transport to zero. In the absence of H + leaks, the proton pump can shut itself off.

Publisher Summary This chapter focuses on the coupling between proton movements and the synthesis of ATP, a reaction catalyzed by the protontranslocating ATPase of bacteria. It discusses that in some cells, such as the streptococci, the ATPase normally couples ATP hydrolysis to the extrusion of protons, establishing both the membrane potential and pH gradient needed for other work functions. In all the experiments, the strategy examines the coupling between proton entry and ATP formation when an electrochemical proton gradient is artificially imposed, for in this way the effects of membrane potentials and pH gradients of known size are studied directly. For several reasons, it has been done using intact cells of Streptococcus lactis , an anaerobe. Such cells are easily depleted of metabolizable reserves, and in washed cells, the electrochemical proton gradient falls to nearly zero. In addition, this gram-positive organism is sensitive to ionophores without special pretreatment, so that the size of the membrane potential is manipulated by varying the ratio of internal to external potassium in the presence of the ionophore valinomycin. Finally, internal buffering power of the intact cell is high, so that net proton fluxes are readily observed in response to applied electrical or chemical gradients.

Publisher Summary This chapter describes the modifiers of F1-ATPases and the associated peptides. The chemical modifiers of proteins can be divided into two classes—the group-directed reagents (chemical reagents capable of binding to side-chain residues in the investigated protein); and the active site-directed reagents (affinity and photoaffinity labels). The mitochondrial, bacterial, and chloroplast ATPases are H + -dependent ATPases (ATP synthases), as they catalyze both ATP hydrolysis and synthesis. Dicyclohexylcarbodiimide (DCCD) is used as a chemical reagent to block the proton conduction in the F 0 channel of the ATPase complex at the level of the DCCD-binding protein. Intermolecular cross-linking by random collisions between independent F 1 molecules is not predominant at the protein concentrations used for chemical modification. Intramolecular cross-linking can establish bridges in a single subunit (hairpin formation) or between two adjacent subunits but the cross-linking requires higher concentrations of reagents than inactivation.

Publisher Summary The release of endogenous mitochondrial K + caused by the ionophore valinomycin can be coupled to net adenosine triphosphate (ATP) synthesis. Thus, the free energy of a K + concentration gradient can drive an endergonic process, such as phosphorylation; preloading mitochondria with K+ augments the yield of ATP considerably. The use of ionophores to generate a cation diffusion potential to drive various endergonic processes has been applied to a broad spectrum of energy transductions. Exergonic valinomycin-induced K + transport has been shown to drive (1) ATP synthesis by chloroplasts, (2) ATP synthesis by submitochondrial particles, (3) reversed mitochondrial electron transport, (4) Ca 2+ transport by mitochondria, (5) energy-linked spectral changes in bacterial carotenoids, and (6) energy-dependent fluorescence shifts of various membrane probes. These and related phenomena are explained in terms of membrane potential generation coupled to H + transport. The reproducible demonstration of ATP synthesis via mitochondrial K + gradients and includes a description of related processes. Factors important for investigating the relationships between ion gradients and various energy transformations are discussed in the chapter.

Publisher Summary This chapter describes the structure and function of ATP synthetase. The structure of the ATP synthase has been known longer than its function. It was first described as a tripartite unit in electron micrographs of bovine-heart mitochondria after negative staining. The tripartie unit, consisting of a base-piece, a stalk and a knob was first interpreted by Green to represent the four multi-subunit enzymes required for the oxidation of NADH and succinate by oxygen. The base-piece is identified with the membrane moiety, FO. The stalk is identical with a protein OSCP (oligomycin-sensitivity conferring protein) that is required for the attachment of F1 to FO and is also required to make the ATPase activity of F1 sensitive to oligomycin that does not bind to F1 itself, but to FO. The knob F1 is quite hydrophilic and has been crystallized. The function of the ATP synthase in aerobic organisms is to utilize energetic protons originating from electron transfer in the respiratory chain for the synthesis of ATP from ADP and phosphate. In some anaerobic organisms, it has the converse function, to couple the hydrolysis of ATP to the supply of energetic protons.

Publisher Summary The active transport of Na + through cellular membranes catalyzed by the sodium pump is considered to start in the ATP-binding site of Na, K-ATPase. The K + transport ends there. Because of the close relationship of ATP hydrolysis and active Na + transport, studies with ATP analogs might be helpful for (1) the determination of essential amino acids involved in the recognition of ATP, (2) the titration of the number of ATP-binding sites of the membrane-bound enzyme, (3) the detection of structural changes of the ATP-binding site caused by the transport substrates and divalent cations, and (4) the retardation of the turn­over of the enzyme to facilitate the study of the transport of ions. Using Na, K-ATPase from pig kidney, the properties of the following ATP affinity labels are studied: the disulfide of thioinosine triphosphate (sinoPPP)2 and 3’-o-[3-(4-azido-2-nitrophenyl)propionyl]-ATP (N 3 -ATP), and the MgATP complex analogs Cr(III)-ATP and Co(NH 3 )4-ATP. A study of the dependence of this process on the nature of the MgATP-complex analog revealed that all tested analogs inactivate the enzyme.

Publisher Summary Although a great deal of experimental information on the elementary steps of the hydrolysis of ATP catalyzed by the Na, K–ATPase is available, few attempts have been made to see to what extent reaction schemes based on results from experiments on partial reactions are able to predict the steady-state kinetic behavior of the enzyme. This chapter reviews the experiments of the interactions of ATP, a nonhydrolyzable analog of ATP adenylylmethylene diphosphonate (AMPPCP), Na + , and K with the Na, K–ATPase and confronted the results with the predictions of the current schemes for the hydrolysis of ATP by the Na, K–ATPase. Na, K–ATPase was prepared from dog kidney red outer medulla by the simpler of the two procedures described by Jorgensen. ATPase activity was measured by the release of [ 32 P]P i from [γ- 32 P]ATP at 37°C and at pH 7.4 and 150 m M total salt concentration. AMPPCP acts as a competitive inhibitor of ATP at the high-affinity component of the substrate curve of the ATPase. The high-affinity component of the substrate curve expresses the combination of ATP at a catalytic site and that the low-affinity component expresses the combination of ATP at a site from which the nucleotide activates the ATPase without undergoing hydrolysis.

Publisher Summary This chapter describes the structure and function of ATP synthase. ATP is synthesized through oxidative phosphorylations by an ATP-synthase complex coupled to the respiratory chain. All ATP-synthase complexes producing great amounts of ATP in living organisms are coupled to chains of electron transporters integrated in membranes or in lipid–protein lamellar structures, which are named transducing membranes. In each case, the transporter chain is reduced at one end by a flow of electrons and protons, and the ATP synthesis occurs during the reoxidation process of the chain, using the liberated redox energy. It has been stressed in various studies that ATP-synthases exhibit very striking similarities of structure and properties. By the use of various techniques, ATP-synthase has been either isolated as a partially purified oligomycine-sensitive ATPase (OS-ATPase) or progressively dissociated in its various sectors. A small protein inhibitor has been found associated to ATP-synthases. It can be dissociated from the membrane by an alkali treatment followed by a Sephadex G-50 chromatography.

Publisher Summary The Na/K pump in human red cells is fueled by ATP synthesized during glycolysis. In human red cells, it has been suggested that two glycolytic enzymes, glyceraldehydes-3-phosphate dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK), bound to the red cell membrane synthesize ATP and deposit it in a membrane-associated compartment from which it is used by the Na/K pump. Active Na transport into inside-out vesicles (IOVs) is promoted by addition of the substrates for GAPDH and PGK, without added ATP. The GAPDH–PGK complex synthesizes ATP that remains associated with the red cell membrane. The membrane-associated ATP must be compartmentalized because it is inaccessible to degradation catalyzed by hexokinase. The membrane-associated ATP fuels the pump because its level is reduced by the addition of Na and this reduction is inhibited by strophanthidin.

Publisher Summary This chapter describes the preparation of membrane fractions highly enriched for each of the ATPases, and the assay procedures used to quantitate the amount of activity specifically due to each enzyme. Three H + -translocating ATPases have been described in membranes of Neurospora crassa . The mitochondrial ATPase catalyzes the synthesis of ATP and has a complex structure consisting of 11 different polypetides, typical of the family of F 0 F 1 -ATPases. The plasma membrane ATPase serves as an electrogenic pump, hydrolyzing ATP to generate an electrochemical proton gradient, which can be used to drive a series of H + cotransport systems. The vacuolar ATPase hydrolyzes ATP and generates a pH gradient (and possibly a membrane potential) that is coupled to the uptake of basic amino acids into the vacuolar interior. The primary purpose in isolating membranes from Neurospora has been to characterize the H + -translocating ATPases. Therefore, it has been important to obtain homogeneous preparations with a single type of ATPase. After solubilization, the ATPases tend to copurify, making mixed-membrane preparations unsuitable for enzyme purification. The most straightforward procedure for distinguishing among the three proton pumps is to measure their ATPase activities. The three membrane ATPases are characterized by different pH optima and specific inhibitor sensitivities.

Information available at present documents the existence of three well-defined classes of guanine nucleotide binding proteins functioning as signal transducers: Gs and Gi which stimulate and inhibit adenylate cyclase, respectively, and transducin which transmits and amplifies the signal from light-activated rhodopsin to cGMP-dependent phosphodiesterase in ROS membranes. Go is a fourth member of this family. Its function is the least known among GTP binding signal transducing proteins. The family of G proteins has a number of properties in common. All are heterotrimers consisting of three subunits, alpha, beta, and gamma. Each of the subunits may be heterogeneous depending on species and tissue of origin and may be posttranslationally modified covalently. The alpha subunits vary in size from 39 to 52 kDa. The sequences for Gs alpha and transducin alpha have 42% overall homology and those of Gi alpha and Gs alpha 43%, whereas those of Gi alpha and transducin alpha have a higher degree (68%) of homology. All alpha subunits bind guanine nucleotides and are ADP-ribosylated by either pertussis toxin (Gi, transducin, Go) or cholera toxin (Gs, Gi, transducin). Thus, transducin and Gi, which have the highest degree of sequence homology, are also ADP-ribosylated by both toxins. The beta subunits have molecular weights of 36 and 35 kDa, respectively. While Gs, Gi, and Go contain a mixture of both, transducin contains only the larger (36-kDa) beta-polypeptide. The relationship of the 36- and the 35-kDa beta subunits is not defined. Although the complete sequence of the 36-kDa beta subunit of transducin has been deduced from the cDNA sequence, complete sequences of other beta subunits are not yet available so that detailed comparisons cannot be made at present. However, the proteolytic profiles of each class of the beta subunits of different G proteins are indistinguishable. The gamma subunit of bovine transducin has been completely sequenced. It has a Mr of 8400. Again complete sequences of other gamma subunits are not yet available. While the gamma subunits of Gs, Gi, and Go have identical electrophoretic mobility in SDS gels, they differ significantly in this respect from the gamma subunit of transducin. Moreover, crossover experiments point to functional differences between gamma subunits from G protein and transducin complexes. In addition, a role for beta, gamma in anchoring guanine nucleotide binding proteins to membranes has been postulated.(ABSTRACT TRUNCATED AT 400 WORDS)

Vesicles derived from the sarcoplasmic reticulum (SR) retain a membrane bound ATPase that can catalyze both the hydrolysis and the synthesis of adenosine triphosphate (ATP). When suspended in a medium containing ATP and Mg , these vesicles can reduce the calcium (Ca) concentration of the medium from 10 -4 to less than 10 -7 M. In this process, the ATPase uses the chemical energy derived from the hydrolysis of ATP to pump Ca 2+ into the vesicles and to maintain a steep Ca 2+ concentration gradient that is formed across the vesicle membrane· In 1971, it was shown that the entire process of Ca 2+ transport can be reversed. When a Ca 2+ concentration gradient is formed across the vesicle membrane, depending on the conditions used, the ATPase can catalyze either the synthesis of ATP or a rapid ATP⇆ P i exchange. During ATP ⇆ P i exchange, the ATPase operates simultaneously forward (ATP hydrolysis) and backwards (ATP synthesis from ADP and P i) . When vesicles previously loaded with calcium are incubated in a medium containing EGTA, Ca 2+ flows out of the vesicles at a slow rate due to the low Ca 2+ permeability of the membrane.

Although granulosa cells may be isolated by simple expression from Graafian follicles, the viability and functional activities of the resulting cells have been markedly enhanced by incubating ovaries sequentially in EGTA and hypertonic sucrose prior to expression. It is believed that this treatment gently dissociates interconnecting gap junctions and thereby reduces the extent of cellular disruption that usually results from expression. By incubating the cells with [35S]-methionine at various times after injecting human chorionic gonadotropin (hCG), and then subjecting cellular homogenates to two dimensional electrophoresis, it was found that hCG-induced luteinization is accompanied by changes in synthesis of specific proteins, and that these changes occur prior to the onset of morphologic signs of luteinization. To obtain information on the fate of hCG bound to the granulosa cells, we labeled the non-identical subunits with different radioisotopes of iodine, and demonstrated that the dual labeled hormone was biologically active. The particulate fraction of granulosa cells showed a preferential retention of the beta subunit-associated radioactivity that was not seen in any other target or non-target control tissues. We conclude that a portion of the beta subunit of hCG is selectively retained by luteinizing granulosa cells. Whether or not this retention is causally related to the onset of luteinization remains to be determined.

Publisher Summary This chapter presents a study in which the amounts of Ca2+ and ATP bound to the enzyme in various enzyme states were measured using the double membrane filtration method devised by Yamaguchi and Tonomura. The studies on the binding of Ca2+ to SR ATPase showed that (1) 2 mol of Ca2+ binds to 1 mol of the ATPase with high affinity, (2) Ca2+ bound to the enzyme takes an occluded form when E1P is formed, and (3) Ca2+ is released by the conversion of E1P into E2P. Furthermore, the measurements of ATP binding indicated two classes of ATP binding sites on the ATPase, that is, a catalytic site with high affinity and a regulatory site with low affinity. Upon the addition of Ca2+, the ATP bound at the catalytic site was converted to EP + adenosine diphosphate (ADP) , which was in equilibrium with a slowly exchanging enzyme-ATP complex, E*ATP. The equilibrium was shifted toward EP by the ATP binding to the regulatory site.

Publisher Summary This chapter reviews two outstanding facts about transport ATPases. First, plasma membrane ATPases and mitochondrial ATPases differ greatly in both structure and reaction mechanism. Second, even among plasma membrane ATPases, reaction mechanisms appear to differ with regard to the degree of internal charge coupling-that is, the degree of electrogenicity. The phosphate group may be regarded as central to the transport mechanisms of these proteins, whether or not it is actually covalently bound. In all cases, the phosphate group determines the affinity transitions during transport and determines the sidedness of the transport “channels.” The chapter reveals that Na + ,K + - ATPase, the plant membrane H + -ATPases, and mitochondrial-type ATPases all generate ion gradients that are subsequently used for coupled transport processes. Although the chemical gradients of these ions may vary widely under physiological conditions, the electrogenic component in the pump reaction should allow the total electrochemical gradient to be invariant, or at least large, during wide swings of the concentration gradient.

Publisher Summary This chapter discusses the reconstitution of adenosine triphosphate (ATP) synthetase on a collagen membrane. This method can be used to synthesize ATP using a pH gradient and has been demonstrated with a number of energy-transducing membranes, such as chloroplasts, mitochondria, submitochondrial particles, whole bacteria, bacterial membrane vesicles, and vesicles reconstituted from purified ATPase and phospholipids of a thermophilic bacterium. In all cases, the proton gradient is created between the interior of the vesicles or the liposomes and the external medium, the pH inside the vesicles being determined by the use of fluorescent or colored probes. It is necessary to include valinomycin and a KCl gradient to obtain a significant ATP synthesis. The procedure of reconstitution comprises three steps: (1) extraction of the membrane factor and F 1 ATPase from pig heart mitochondria, (2) covalent grafting on a collagen film of the membrane factor, and (3) separation of the compartments in a cell.