Three catalytic sites in mitochondrial ATPase

Kinetic data obtained after determining the hydrolytic activity of ATPase from rat liver in preparations where the enzyme had been purified, or in mitochondria, strongly suggest the existence of three different catalytic sites with different affinity for the substrate. The results obtained when measuring the ATPase activity at different substrate concentrations, and in the presence of the inhibitors KOCN or KSCN, or of the activators dinitrophenol and bicarbonate, show that the binding of these compounds to a regulatory site or sites affects in a different degree the hydrolytic activity of each catalytic site.     

An NMR Study of a 300-kDa AAA+ Unfoldase

AAA+ ATPases are ubiquitous hexameric unfoldases acting in cellular protein quality control. In complex with proteases, they form protein degradation machinery (the proteasome) in both archaea and eukaryotes. Here, we use solution-state NMR spectroscopy to determine the symmetry properties of the archaeal PAN AAA+ unfoldase and gain insights into its functional mechanism. PAN consists of three folded domains: the coiled-coil (CC), OB and ATPase domains. We find that full-length PAN assembles into a hexamer with C₂ symmetry, and that this symmetry extends over the CC, OB and ATPase domains. The NMR data, collected in the absence of substrate, are incompatible with the spiral staircase structure observed in electron-microscopy studies of archaeal PAN in the presence of substrate and in electron-microscopy studies of eukaryotic unfoldases both in the presence and in the absence of substrate. Based on the C₂ symmetry revealed by NMR spectroscopy in solution, we propose that archaeal ATPases are flexible enzymes, which can adopt distinct conformations in different conditions. This study reaffirms the importance of studying dynamic systems in solution.     

Optimizing the Production of Bacterial Cellulose in Surface Culture: Evaluation of Substrate Mass Transfer Influences on the Bioreaction (Part 1)

The interest in cellulose produced by bacteria from surface cultures has increased steadily in recent years because of its potential for use in medicine and cosmetics. Unfortunately, the low yield of the production process has limited the commercial usefulness of bacterial cellulose. This series of three papers dealing with the production of bacterial cellulose using (batch) surface culture, firstly present a complete and complex analysis of the overall system, which allows a fundamental optimization of the production process to be performed. This material has many applications but the low yield of the process limits its commercial usefulness. In part 1, the effect of the rate of mass transfer of substrate on the microbial process, which is characterized by the growth of the bacteria, product formation, and the utilization of the substrate by the bacteria, is studied. A fundamental model for the diffusion of glucose through the growing cellulose layer is proposed and solved. The model confirmed that the increase in diffusional resistance is indeed significant but other factors will also need to be taken into account.     

Acidic lipids, H-ATPases, and mechanism of oxidative phosphorylation. Physico-chemical ideas 30 years after P. Mitchell's Nobel Prize award

Peter D. Mitchell, who was awarded the Nobel Prize in Chemistry 30 years ago, in 1978, formulated the chemiosmotic theory of oxidative phosphorylation. This review initially analyzes the major aspects of this theory, its unresolved problems, and its modifications. A new physico-chemical mechanism of energy transformation and coupling of oxidation and phosphorylation is then suggested based on recent concepts regarding proteins, including ATPases that work as molecular motors, and acidic lipids that act as hydrogen ion (H⁺) carriers. According to this proposed mechanism, the chemical energy of a redox substrate is transformed into nonequilibrium states of electron-transporting chain (ETC) coupling proteins. This leads to nonequilibrium pumping of H⁺ into the membrane. An acidic lipid, cardiolipin, binds with this H⁺ and carries it to the ATP-synthase along the membrane surface. This transport generates gradients of surface tension or electric field along the membrane surface. Hydrodynamic effects on a nanolevel lead to rotation of ATP-synthase and finally to the release of ATP into aqueous solution. This model also explains the generation of a transmembrane protonmotive force that is used for regulation of transmembrane transport, but is not necessary for the coupling of electron transport and ATP synthesis.     

Effect of the protonmotive force on ATP-linked processes and mobilization of the bound natural ATPase inhibitor in beef heart submitochondrial particles

In an attempt to determine whether the natural ATPase inhibitor (IF₁) plays a role in oxidative phosphorylation, the time course of ATP synthesis and ATP hydrolysis in inside-out submitochondrial particles from beef heart mitochondria either possessing IF₁ (Mg-ATP particles) or devoid of IF₁ (AS particles) was investigated and compared to movements of IF₁, as assessed by an isotopic assay. The responses of the above reactions to preincubation of the particles in aerobiosis with NADH or succinate were as follows: (1) The few seconds lag that preceded the steady-rate phase of ATP synthesis was shortened and even abolished both in Mg-ATP particles and AS particles. The rate of ATP synthesis in the steady state was independent of the length of the lag. (2) ATPase was slowly activated, maximal activation being obtained after a 50-min preincubation; there was no direct link between the development of the protonmotive force (maximal within 1 sec) and ATPase activation. (3) Bound IF₁ was slowly released; the release of bound IF₁ as a function of the preincubation period was parallel to the enhancement of ATPase activity; the maximal amount of IF₁ released was a small fraction of the total IF₁ bound to the particles (less than 20%). (4) The double reciprocal plots of the rates of ATP and ITP hydrolysis vs. substrate concentrations that were curvilinear in the absence of preincubation with a respiratory substrate became linear after aerobic preincubation with the substrate. The data conclusively show that only ATPase activity in submitochondrial particles is correlated with the release of IF₁, and that the total extent of IF₁ release induced by respiration is limited. On the other hand, the kinetics of ATPase in control and activated particles are consistent with the existence of two conformations of the membrane-bound F₁-ATPase, directed to ATP synthesis or ATP hydrolysis and distinguishable by their affinity for IF₁.     

A kinetic model for product formation of microbial and mammalian cells

Growth of microbial and mammalian cells can be classified into substrate-limited and substrate-sufficient growth according to the relative availability of the substrate (carbon and energy source) and other nutrients. It has been observed for a number of microbial and mammalian cells that the consumption rate of substrate and energy (ATP) is generally higher under substratesufficient conditions than under substrate limitation. Accordingly, the product formation under substrate excess often exhibits different patterns from those under substrate limitation. The extent of increase or decrease in product formation may depend not only on the nature of limitation and cell growth rate but also on the residual substrate concentration in a relatively wide range. The product formation kinetic models existing in literature cannot describe these effects. In this study, the Luedeking-Piret kinetic is extended to include a term describing the effect of residual substrate concentration. The extended model has a similar structure to the kinetic model for substrate and energy consumption rate recently proposed by Zeng and Deckwer. The applicability of the extended model is demonstrated with three microbial cultures for the production of primary metabolites and three hybridoma cell cultures for the production of ammonia and lactic acid over a wide range of substrate concentration. The model describes the product formation in all these cultures satisfactorily. Using this model, the range of residual substrate concentration, in which the product formation is affected, can be quantitatively assessed. (c) 1995 John Wiley & Sons, Inc.     

In vivo activation of the yeast plasma membrane ATPase during nitrogen starvation Identification of the regulatory domain that controls activation

Yeast plasma membrane ATPase is activated during nitrogen starvation when a fermentable substrate is present. This activation is due to changes in the V_max and it is irreversible, independent of protein synthesis and apparently triggered by a decrease in the intracellular pH. It is shown that the ATPase regulatory domain implicated in the activation by fermentable carbon sources is also implicated in activation by nitrogen starvation and by external acidification.     

Parameters affecting incorporation and by-product formation during the production of structured phospholipids by lipase-catalyzed acidolysis in solvent-free system

By-product formation is a serious problem in the lipase-catalyzed acyl exchange of phospholipids (PL). By-products are formed due to parallel hydrolysis reactions and acyl migration in the reaction system. A clear elucidation of these side reactions is important for practical operation in order to minimize by-products during reaction. In the present study we examined the lipozyme RM IM-catalyzed acidolysis for the production of structured phospholipids between phosphatidylcholine (PC) and caprylic acid in the solvent-free system. A five-factor response surface design was used to evaluate the influence of major factors and their relationships on a number of responses reflecting the turnover of main reactions as well as side reactions. The five factors, including enzyme dosage, reaction time, reaction temperature, substrate ratio (mol/mol caprylic acid/PC) and water addition, were varied at three levels with two star points. All parameters besides water addition had an effect on the incorporation of caprylic acid into PC and lysophosphatidylcholine (LPC). Reaction time and enzyme dosage showed increased effect on incorporation into PC, while substrate ratio and reaction temperature showed opposite effect. The PC content decreased with increase of all parameters except for substrate ratio. Optimal conditions are recommended as enzyme dosage 40%, reaction temperature 55 °C, water addition 1%, reaction time 70 h, and substrate ratio 6 mol/mol caprylic acid/PC. Under these conditions an incorporation of 46% with PC accounting for 53% of the PL fraction can be obtained. Regiospecific analysis of the product revealed that the caprylic acid was mainly incorporated into the sn-1 position accounting for 80% of the fatty acids incorporated.     

Interaction of the chaperone BiP with an antibody domain: implications for the chaperone cycle

BiP is an Hsp70 homologue found in the endoplasmic reticulum of eukaryotic cells. Like other Hsp70 chaperones, BiP interacts with its substrate proteins in an ATP-dependent manner. The functional analysis has so far been performed mainly with short, synthetic peptides. Here, we present an experimental system that allows to study the partial reactions of the BiP chaperone cycle for a natural substrate protein domain in its soluble, stably unfolded conformation. This unfolded antibody domain forms a binary complex with BiP in the absence of ATP. The dissociation of the BiP dimer seems to be the rate-limiting step in this reaction. The BiP-C(H)3 complexes dissociate rapidly in the presence of ATP. The affinity for BiP-binding peptides and the non-native antibody domain was determined to be similar, suggesting that only the peptide binding site is involved in these interactions. Furthermore, these results imply that, also in the context of the antibody domain, an extended peptide sequence is recognized. However, the accessibility of the BiP-binding site in the non-native protein seems to influence the kinetics of complex formation.     

Common patterns and unique features of P-type ATPases: a comparative view on the KdpFABC complex from Escherichia coli (Review)

P-type ATPases are ubiquitously abundant primary ion pumps, which are capable of transporting cations across the cell membrane at the expense of ATP. Since these ions comprise a large variety of vital biochemical functions, nature has developed rather sophisticated transport machineries in all kingdoms of life. Due to the importance of these enzymes, representatives of both eu- and prokaryotic as well as archaeal P-type ATPases have been studied intensively, resulting in detailed structural and functional information on their mode of action. During catalysis, P-type ATPases cycle between the so-called E1 and E2 states, each of which comprising different structural properties together with different binding affinities for both ATP and the transport substrate. Crucial for catalysis is the reversible phosphorylation of a conserved aspartate, which is the main trigger for the conformational changes within the protein. In contrast to the well-studied and closely related eukaryotic P-type ATPases, much less is known about their homologues in Bacteria. Whereas in Eukarya there is predominantly only one subunit, which builds up the transport system, in Bacteria there are multiple polypeptides involved in the formation of the active enzyme. Such a rather unusal prokaryotic P-type ATPase is the KdpFABC complex of the enterobacterium Escherichia coli, which serves as a highly specific K⁺ transporter. A unique feature of this member of P-type ATPases is that catalytic activity and substrate transport are located on two different polypeptides. This review compares generic features of P-type ATPases with the rather unique KdpFABC complex and gives a comprehensive overview of common principles of catalysis as well as of special aspects connected to distinct enzyme functions.     

Emergent insights from the synthesis of conceptual frameworks for biological invasions

A general understanding of biological invasions will provide insights into fundamental ecological and evolutionary problems and contribute to more efficient and effective prediction, prevention and control of invasions. We review recent papers that have proposed conceptual frameworks for invasion biology. These papers offer important advances and signal a maturation of the field, but a broad synthesis is still lacking. Conceptual frameworks for invasion do not require invocation of unique concepts, but rather should reflect the unifying principles of ecology and evolutionary biology. A conceptual framework should incorporate multicausality, include interactions between causal factors and account for lags between various stages. We emphasize the centrality of demography in invasions, and distinguish between explaining three of the most important characteristics by which we recognize invasions: rapid local population increase, monocultures or community dominance, and range expansion. As a contribution towards developing a conceptual synthesis of invasions based on these criteria, we outline a framework that explicitly incorporates consideration of the fundamental ecological and evolutionary processes involved. The development of a more inclusive and mechanistic conceptual framework for invasion should facilitate quantitative and testable evaluation of causal factors, and can potentially lead to a better understanding of the biology of invasions.     

Transport ATPases in biological systems and relationship to human disease: a brief overview

Interest in the field of transport ATPases has grown dramatically during the past 20 years and gained considerable visibility for several reasons. First, it was shown that most transport ATPases can be lumped into only a few categories designated simply as P, V, F, and ABC types, the latter consisting of a large superfamily. Second, it has been shown that many transport ATPases have a clear relevance to human disease. Third, the field of transport ATPases has become rather advanced in the study of the reaction mechanisms and structure–function relationships associated with several of these enzymes. Finally, the Nobel committee recently recognized major accomplishments in this field of research. Here, the author provides a brief discussion of transport ATPases that are present in biological systems and their relevance or possible relevance to human disease.     

Interaction of insecticides with mammalian P-glycoprotein and their effect on its transport function

We studied the effects of four commonly used insecticides (methylparathion, endosulfan, cypermethrin and fenvalerate) on P-glycoprotein isolated from multidrug-resistant cells. All the pesticides stimulated P-glycoprotein ATPase activity, with maximum stimulation of up to 213% in a detergent-solubilized preparation, and up to 227% in reconstituted liposomes. The ATPase stimulation profiles were biphasic, displaying lower stimulation, and in the case of methylparathion, inhibition of activity, at higher insecticide concentrations. Quenching of the intrinsic Trp fluorescence of purified P-glycoprotein was used to quantitate insecticide binding; the estimated K_d values fell in the range 4-6 μM. Transport of the fluorescent substrate tetramethylrosamine (TMR) into proteoliposomes containing P-glycoprotein was monitored in real time. The TMR concentration gradient generated by the transporter was collapsed by the addition of insecticides, and prior addition of these compounds prevented its formation. The rate of TMR transport was inhibited in a saturable fashion by all the compounds, indicating that they compete with the substrate for membrane translocation. Taken together, these data suggest that the insecticides bind to Pgp with high affinity and effectively block drug transport. Inhibition of Pgp by pesticides may compromise its ability to clear xenobiotics from the body, leading to a higher risk of toxicity.     

The development of transient SV40 based shuttle vectors for mutagenesis studies: problems and solutions

Shuttle-vector plasmids would appear to provide a powerful technology for studying mutagenesis in mammalian (including human) cells. Recently, as described in this and other papers in this volume, several shuttle-vector systems have been described and applied. The development of the first shuttle vectors for these purposes was hindered by two major problems. The first of these was the 'poison' sequence present in many pBR322 based vectors. The second was the problem of spontaneous mutagenesis associated with transfection of the plasmids into mammalian cells. Effective solutions for both problems have been devised, and it is now possible to experimentally address a variety of questions concerning mutagenesis and repair in mammalian cells.     

The CbbQO-type rubisco activases encoded in carboxysome gene clusters can activate carboxysomal form IA rubiscos

The CO₂-fixing enzyme rubisco is responsible for almost all carbon fixation. This process frequently requires rubisco activase (Rca) machinery, which couples ATP hydrolysis to the removal of inhibitory sugar phosphates, including the rubisco substrate ribulose 1,5-bisphosphate (RuBP). Rubisco is sometimes compartmentalized in carboxysomes, bacterial microcompartments that enable a carbon dioxide concentrating mechanism (CCM). Characterized carboxysomal rubiscos, however, are not prone to inhibition, and often no activase machinery is associated with these enzymes. Here, we characterize two carboxysomal rubiscos of the form IA^C clade that are associated with CbbQO-type Rcas. These enzymes release RuBP at a much lower rate than the canonical carboxysomal rubisco from Synechococcus PCC6301. We found that CbbQO-type Rcas encoded in carboxysome gene clusters can remove RuBP and the tight-binding transition state analog carboxy-arabinitol 1,5-bisphosphate from cognate rubiscos. The Acidithiobacillus ferrooxidans genome encodes two form IA rubiscos associated with two sets of cbbQ and cbbO genes. We show that the two CbbQO activase systems display specificity for the rubisco enzyme encoded in the same gene cluster, and this property can be switched by substituting the C-terminal three residues of the large subunit. Our findings indicate that the kinetic and inhibitory properties of proteobacterial form IA rubiscos are diverse and predict that Rcas may be necessary for some α-carboxysomal CCMs. These findings will have implications for efforts aiming to introduce biophysical CCMs into plants and other hosts for improvement of carbon fixation of crops.     

Transport ATPases into the year 2008: a brief overview related to types, structures, functions and roles in health and disease

Transport ATPases can be lumped into four distinct types, P, F, V, and ABC, with the first three designated 20 years ago (Pedersen, P.L. and Carafoli, E., Trends Biochem. Sci. 12, 146–150, 1987) and the ABC type included more recently. The mini-reviews (>20) that comprise this volume of the Journal of Bioenergetics and Biomembranes describe work presented at the 2007 FASEB Conference (6th) on Transport ATPases (Kathleen Sweadner, Chair; Rajini Rao, Co-Chair). Since these conferences began in 1997, the “transport ATPase field” has seen tremendous progress. Advances include a much better understanding of the structure, mechanism, and regulation of each of the four major ATPase types as well as their physiological and medical relevance. In fact, the transport ATPase field has entered a new era in which work on these enzymes is likely to contribute to new therapies for multiple diseases that affect both people and animals. Among these are cancer and heart disease, mitochondrial diseases, osteoporosis, macromolecular degeneration, immune deficiency, cystic fibrosis, diabetes, ulcers, nephro-toxicity, hearing loss, skin disorders, lupus, and malaria. In addition, as several members of the transport ATPase family include those involved in drug resistance their study may help alleviate this recurring problem in drug development. Finally, the transport ATPase field is also paving the way for nanotechnology focused on nano-motors with work on the F-type ATPases (F₀F₁) leading the way. These ATPases driven in reverse by a proton gradient have the capacity to interconvert electrochemical energy into mechanical energy and finally into chemical energy conserved in the terminal bond of ATP. In mammalian mitochondria these events occur on a larger complex or “nano-machine” called the “ATP synthasome” that consists of the ATP synthase in complex formation with carriers for P_i and ADP/ATP.     

Studies on the Arylsulphatase and phenol sulphotransferase activities of Aspergillus oryzae.

1. Crude extracts of Aspergillus oryzae grown under conditions of sulphur limitation possess high arylsulphatase activity. 2. This activity can be greatly enhanced by the inclusion of tyramine or a number of other phenols in the assay medium. 3. The arylsulphatase activity of these extracts can be resolved into three distinct fractions by chromatography on DEAE-cellulose. 4. The effect of tyramine is restricted to one of these fractions only. 5. Evidence is presented which indicates that this effect is the consequence of a phenol sulphotransferase activity, which shows no requirement for 3'-phosphoadenosine 5'-phosphate as a cofactor, and which will not transfer sulphate from 3'-phosphoadenosine 5'-sulphatophosphate to potential phenolic acceptors. 6. The three enzymes differ also in their molecular weights and substrate specificities.     

NaCl及KCl对叶绿体膜上偶联因子腺三磷酶活力的调节

在不外加Mg~(2 )的条件下,激活液中加入30 mMNaCl或15 mM KCl,能促进叶绿体膜上偶联因子的腺三磷酶活力。其促进程度与叶绿素浓度、反应底物ATP浓度及DTT的存在有关。但此促进现象可被加入低浓度的EDTA所消除,NaCl及KCl可将叶绿体内含有的内源或结合态的Mg~(2 )释放出来,有活化腺三磷酶的作用。     

Characterization of 3-methoxy flavones for their interaction with ABCG2 as suggested by ATPase activity

Breast Cancer Resistance Protein (BCRP/ABCG2) belongs to the superfamily of ATP binding cassette (ABC) transporters. Characteristic of some of these transporter proteins is the transport of a variety of structurally unrelated substances against a concentration gradient by using the energy of ATP hydrolysis. ABCG2 has been found to confer multidrug resistance (MDR) in cancer cells. Several anticancer drugs have been identified as ABCG2 substrates including mitoxantrone, etoposide and topotecan. As inhibition of the transporter is one of the strategies to overcome MDR, we have synthesized and tested several 3-methoxy flavones and investigated them for their ABCG2 inhibition. Among these, pentamethyl quercetin (compound 4) and pentamethyl morin (compound 5) were found to be fluorescent and hence screened for their possible transport by ABCG2 using confocal microscopy. This study showed that pentamethyl quercetin was far less accumulated in ABCG2 overexpressing MDCK BCRP cells as compared to MDCK sensitive cells, suggesting possible efflux of this compound by ABCG2. Pentamethyl morin showed no visible difference in both cell lines. Based on this observation, we studied several other fluorescent 3-methoxy flavones for their accumulation in ABCG2 overexpressing cells. To confirm the substrate or inhibitor nature of the tested compounds, these compounds were further investigated by ATPase assay. If stimulation of the transporter ATPase activity is detected, one can conclude that the compound is probably a transported substrate. All compounds except pentamethyl morin (compound 5) and tetramethyl quercetin (compound 6) were found to stimulate ATPase activity pointing to possible substrates despite being potent inhibitors of ABCG2.     

A state of the art literature review on anaerobic digestion of food waste: influential operating parameters on methane yield

A thorough literature review was conducted to investigate the behaviour of food waste in anaerobic digestion experiments. The main goal of this literature review was to study the effect of several operating parameters on methane yields and to develop a simplified regression equation to predict methane generation. Using a data prospection methodology, all the papers published within 2013–2015 that contained selected keywords were included in this study (a total of 613 papers). After screening, 167 papers were finally retrieved using the search engines and our methodology. From these papers, data from 231 experiments were recorded and evaluated. The parameters recorded in each paper were: operation mode (batch or continuous), temperature (mesophilic or thermophilic), moisture content (wet or dry), presence or absence of pretreatment, reactor scale (laboratory, bench, pilot, demonstration/full scale), presence or absence of co-substrates (co- or mono-digestion), organic loading rate, hydraulic retention time (HRT) and methane yield. The novelty of the work is that it employed various statistical tools to examine the effect of the above-mentioned factors on food waste methane generation. Most of the experiments were performed at mesophilic temperatures, at a wet system without substrate pretreatment. An equal number of papers described mono-digestion and co-digestion studies, and an equal number of papers described batch and continuous reactor experiments. The mean HRT for the continuous processes was 36.7 days. Statistical analysis indicated that the parameters that significantly affected methane yields were the “operation mode” and “pretreatment”. A best reduced regression model was fitted to the methane yield data to describe the above effects. As a general conclusion, with this methodology, that involved the analysis of a large number of studies (with different conditions and set-ups, heterogeneous waste, etc.), correlations between some typical operating parameters of anaerobic digestion and methane yields were not obvious.