Release of enzymes from quiescent fibroblasts during ATP depletion

The association between ATP depletion and enzyme release from cells has been described in two different ways: as a more or less linear dependence, or with a threshold value below which the enzyme release will start. We have investigated the association between ATP depletion caused by various metabolic inhibitors and enzyme release on quiescent fibroblasts. We found that the enzyme release never started before the ATP had decreased to a critical low level. Addition of glucose to cells while ATP was still above this critical level led to a regeneration of ATP and enzyme release did not occur. If ATP was lowered to 35-40% and kept there for 24 h, the enzyme release was minimal. These results support the threshold theory for release of enzymes from cells.     

Kinetic characterization of the unisite catalytic pathway of seven beta-subunit mutant F1-ATPases from Escherichia coli

We have studied the kinetics of "unisite" ATP hydrolysis and synthesis in seven mutant Escherichia coli F1-ATPase enzymes. The seven mutations are distributed over a 105-residue segment of the catalytic nucleotide-binding domain in beta-subunit and are: G142S, K155Q, K155E, E181Q, E192Q, M209I, and R246C. We report forward and reverse rate constants and equilibrium constants in all seven mutant enzymes for the four steps of unisite kinetics, namely (i) ATP binding/release, (ii) ATP hydrolysis/synthesis, (iii) Pi release/binding, and (iv) ADP release/binding. The seven mutant enzymes displayed a wide range of deviations from normal in both rate and equilibrium constants, with no discernible common pattern. Notably, steep reductions in Kd ATP were seen in some cases, the value of Kd Pi was high, and K2 (ATP hydrolysis/synthesis) was relatively unaffected. Significantly, when the data from the seven mutations were combined with previous data from two other E. coli F1-beta-subunit mutations (D242N, D242V), normal E. coli F1, soluble and membranous mitochondrial F1, it was found that linear free energy relationships obtained for both ATP binding/release (log k+1 versus log K1) and ADP binding/release (log k-4 versus log K-4). Two conclusions follow. 1) The seven mutations studied here cause subtle changes in interactions between the catalytic nucleotide-binding domain and substrate ATP or product ADP. 2) The mitochondrial, normal E. coli, and nine total beta-subunit mutant enzymes represent a continuum in which subtle structural differences in the catalytic site resulted in changes in binding energy; therefore insights into the nature of energy coupling during ATP hydrolysis and synthesis by F1-ATPase may be ascertained by detailed studies of this group of enzymes.     

ATP-consuming and ATP-generating enzymes secreted by pancreas

Pancreatic acini release ATP in response to various stimuli, including cholecystokinin octapeptide (CCK-8), as we show in the present study. There were indications that pancreatic juice also contains enzymes that could hydrolyze ATP during its passage through the ductal system. The aim of this study was to determine which ATP-degrading and possibly ATP-generating enzymes were present in pancreatic secretion. For this purpose, pancreatic juice was collected from anesthetized rats stimulated with infusion of CCK-8. Purine-converting activities in juice samples were assayed by TLC using either [gamma-(32)P]ATP or (14)C/(3)H-labeled and unlabeled nucleotides as appropriate substrates. Data show that the juice contains the enzyme ecto-nucleoside triphosphate diphosphohydrolase that can hydrolyze both [(14)C]ATP and [(3)H]ADP about equally well, i.e. CD39. Reverse-phase high-performance liquid chromatography analysis additionally shows that this enzyme has broad substrate specificity toward other nucleotides, UTP, UDP, ITP, and IDP. In addition, secretion contains ecto-5'-nucleotidase, CD73, further converting [(3)H]AMP to adenosine. Along with highly active hydrolytic enzymes, there were also ATP-generating enzymes in pancreatic juice, adenylate kinase, and NDP kinase, capable of sequentially phosphorylating AMP via ADP to ATP. Activities of nonspecific phosphatases, nucleotide pyrophosphatase/phosphodiesterases, and adenosine deaminase were negligible. Taken together, CCK-8 stimulation of pancreas causes release of both ATP-consuming and ATP-generating enzymes into pancreatic juice. This newly discovered richness of secreted enzymes underscores the importance of purine signaling between acini and pancreatic ducts lumen and implies regulation of the purine-converting enzymes release.     

Action of adenosine hosphates on the release of intracellular lactate dehydrogenase from human and rat lymphocytes

Differences in the action of adenosine phosphates on the release of intracellular enzymes from human and rat lymphocytes have been studied. The protective effect of ATP on the human cells was found to be less than on the rat cells. The greatest discrepancy was exhibited by AMP which exerted a protective effect on human lymphocytes, but increased enzyme efflux from rat lymphocytes. The activities of adenosine kinase, adenylate kinase, phosphoribosyl-pyrophosphate synthetase, pyruvate kinase, phosphoglycerate kinase and creatine kinase were compared in the cells of both species. Although significant differences were observed, they were too small to suggest the presence of a mechanism for the conversion of AMP into ATP in human lymphocytes not found in the rat cells. It seems therefore that the protective effect exerted by AMP on the human cells is not mediated by its conversion into ATP, and hence that some factor other than the intracellular energy content is concerned in controlling the release of intracellular enzymes from human cells.     

Structural dissection of ATP turnover in the prototypical GHL ATPase TopoVI

GHL proteins are functionally diverse enzymes defined by the presence of a conserved ATPase domain that self-associates to trap substrate upon nucleotide binding. The structural states adopted by these enzymes during nucleotide hydrolysis and product release, and their consequences for enzyme catalysis, have remained unclear. Here, we have determined a complete structural map of the ATP turnover cycle for topoVI-B, the ATPase subunit of the archaeal GHL enzyme topoisomerase VI. With this ensemble of structures, we show that significant conformational changes in the subunit occur first upon ATP binding, and subsequently upon release of hydrolyzed P(i). Together, these data provide a structural framework for understanding the role of ATP hydrolysis in the type II topoisomerase reaction. Our results also suggest that the GHL ATPase module is a molecular switch in which ATP hydrolysis serves as a prerequisite but not a driving force for substrate-dependent structural transitions in the enzyme.     

The defective proton-ATPase of uncD mutants of Escherichia coli. Two mutations which affect the catalytic mechanism

The catalytic characteristics of F1-ATPases from uncD412 and uncD484 mutant strains of Escherichia coli were studied in order to understand how these beta-subunit mutations cause defective catalysis. Both mutant enzymes showed reduced affinity for ATP at the first catalytic site. While uncD412 F1 was similar to normal in other aspects of single site catalysis, uncD484 F1 showed a Keq of bound reactants greatly biased toward bound substrate ATP and an abnormally fast rate of Pi release. Impairment of productive catalytic cooperativity was the major cause of the reduced steady state ("multisite") catalytic rate in both mutant enzymes. Addition of excess ATP to saturate second and/or third catalytic sites did promote ATP hydrolysis and product release at the first catalytic site of uncD412 F1, but the multisite turnover rate was significantly slower than normal. In contrast, with uncD484 F1, addition of excess ATP induced rapid release of ATP from the first catalytic site and so productive catalytic cooperativity was almost completely absent. The results show that both mutations affect properties of the catalytic site and catalytic site cooperativity and further that the relatively more severe uncD484 mutation affects a residue which acts as a determinant of the fate of bound substrate ATP during promotion of catalysis. Taken together with previous studies of uncA mutant F1-ATPases (Wise, J. G., Latchney, L. R., Ferguson, A. M., and Senior, A. E. (1984) Biochemistry 23, 1426-1432) the results indicate that catalytic site cooperativity in F1-ATPases involves concerted beta-alpha-beta intersubunit communication between catalytic sites on the beta-subunits.     

Subcellular distribution of enzymes determined by rapid digitonin fractionation of isolated hepatocytes

Conditions were determined for rapid separation of cytosolic and mitochondrial compartments by digitonin fractionation of rat hepatocytes. The minimum time required for separation of mitochondrial and cytosolic enzyme markers decreased rapidly with increasing temperature. Kyro EOB, a non-ionic detergent, increases the release of cytosolic enzymes, particularly at lower temperatures. Experimental procedures are described for greater than 90% release of cytosolic enzymes and less than 2% release of mitochondrial enzymes in 3s. By using appropriate concentrations of digitonin and Kyro EOB in a fractionation medium maintained at 1°C and a minimum time of exposure to the medium, nearly separate patterns of release were obtained for enzyme markers for the cytosol, mitochondrial matrix and mitochondrial intermembrane space. The distribution of enzymes that exist in more than one of these compartments was quantified by comparing their rates of release with those of marker enzymes. The cytosol/mitochondrial-matrix distributions for such enzymes in hepatocytes from starved rats were 16%/84% for aspartate aminotransferase, 34%/66% for fumarase and 77%/23% for ATP citrate lyase. In hepatocytes from rats that were induced to synthesize ATP citrate lyase by starvation and re-feeding, the ratio had increased to 95%/5%. The maximum cytosol/intermembrane-space ratio for adenylate kinase was 8%/92%. A procedure is also described for treating commercial digitonin that increases its solubility in water from about 1mg/ml to more than 800mg/ml.     

胶质细胞的细胞间通讯

星型胶质细胞虽然没有动作电位,但是可以表达多种受体和离子通道,并且以细胞内钙波传递的方式来响应各类刺激。星型胶质细胞同样可以释放多种信号分子来介导细胞间的通讯。尤为特别的是,星型胶质细胞的钙波传播和突触功能的反馈调节都需要其释放ATP才得以完成。然而,星型胶质细胞释放ATP的途径和机理还有待研究。尽管人们已经在星型胶质细胞中发现了小囊泡和大致密核心囊泡的标记物,可是用以胞吐的囊泡究竟是什么还并不清楚。作者等近期的研究成果表明,FM染料——一种被成功应用于研究神经元和其他分泌型细胞囊泡循环的染料,可以特异地标记星型胶质细胞的溶酶体,并依不同程度的刺激表现出两种不同模式的钙离子依赖性胞吐：在较低强度刺激下（ATP,谷氨酸）发生部分胞吐,而在高强度刺激下（氰化钾）则发生完全胞吐。进一步研究表明,溶酶体中含有大量ATP,并且在部分胞吐时少量释放ATP,完全胞吐时大量释放ATP,同时释放溶酶体酶。选择性地裂解星型胶质细胞的溶酶体,发现ATP释放和钙波传播都消失了。总之,星型胶质细胞的溶酶体可以通过调节性胞吐对生理和病理条件下的细胞间信号传递产生重要意义。     

Asymmetric interactions of ATP with the AAA+ ClpX6 unfoldase: allosteric control of a protein machine

ATP hydrolysis by AAA+ ClpX hexamers powers protein unfolding and translocation during ClpXP degradation. Although ClpX is a homohexamer, positive and negative allosteric interactions partition six potential nucleotide binding sites into three classes with asymmetric properties. Some sites release ATP rapidly, others release ATP slowly, and at least two sites remain nucleotide free. Recognition of the degradation tag of protein substrates requires ATP binding to one set of sites and ATP or ADP binding to a second set of sites, suggesting a mechanism that allows repeated unfolding attempts without substrate release over multiple ATPase cycles. Our results rule out concerted hydrolysis models involving ClpX(6)*ATP(6) or ClpX(6)*ADP(6) and highlight structures of hexameric AAA+ machines with three or four nucleotides as likely functional states. These studies further emphasize commonalities between distant AAA+ family members, including protein and DNA translocases, helicases, motor proteins, clamp loaders, and other ATP-dependent enzymes.     

CR1-mediated ATP Release by Human Red Blood Cells Promotes CR1 Clustering and Modulates the Immune Transfer Process

Humans and other higher primates are unique among mammals in using complement receptor 1 (CR1, CD35) on red blood cells (RBC) to ligate complement-tagged inflammatory particles (immune complexes, apoptotic/necrotic debris, and microbes) in the circulation for quiet transport to the sinusoids of spleen and liver where resident macrophages remove the particles, but allow the RBC to return unharmed to the circulation. This process is called immune-adherence clearance. In this study we found using luminometric- and fluorescence-based methods that ligation of CR1 on human RBC promotes ATP release. Our data show that CR1-mediated ATP release does not depend on Ca²⁺ or enzymes previously shown to mediate an increase in membrane deformability promoted by CR1 ligation. Furthermore, ATP release following CR1 ligation increases the mobility of the lipid fraction of RBC membranes, which in turn facilitates CR1 clustering, and thereby enhances the binding avidity of complement-opsonized particles to the RBC CR1. Finally, we have found that RBC-derived ATP has a stimulatory effect on phagocytosis of immune-adherent immune complexes.     

Effect of extracellular Ca2+ and Mg2+ on enzyme release from quiescent fibroblasts during various exposures

Release of enzymes from human fibroblasts, as a result of either ATP depletion caused by metabolic inhibitors or a direct cell membrane damage, was increased in the absence of extracellular Ca2+. The lack of extracellular Mg2+ resulted only in a small nonsignificant increase of enzyme release during ATP depletion. However, in a Ca2+-free medium the enzyme release was very much enhanced during ATP depletion if Mg2+ was absent concomitantly. The data show that extracellular calcium protects the cells from cell damage, and that the presence of extracellular magnesium in a calcium-free medium protects the cell against energy-dependent cell damage.     

Extracellular ATP induces P2X7-dependent nicotinamide phosphoribosyltransferase release in LPS-activated human monocytes

Nicotinamide phosphoribosyltransferase (NAMPT), an enzyme involved in NAD biosynthesis, has recently been identified as a novel mediator of innate immunity. In the present study, we report that treatment of LPS-primed monocytes with ATP greatly enhanced the secretion of NAMPT in a time- and concentration-dependent manner without displaying any cytotoxic effect. NAMPT release was suppressed by pretreatment with the P2X(7) receptor (P2X(7)R) inhibitors oxidized ATP (oxATP) and KN-62, indicating the engagement of P2X(7)Rs. Furthermore, P2X(7)R was found to be involved in mediating cell permeability caused by the addition of ATP. To define a role of endogenous ATP in NAMPT secretion, LPS-primed monocytes were incubated in the presence of oxATP and KN-62, as well as the ATP-hydrolyzing enzymes apyrase and hexokinase. With the exception of oxATP, neither substance led to a decrease in NAMPT release, suggesting that autocrine/paracrine ATP is unlikely to be responsible for the LPS-induced release of NAMPT. In conclusion, the enhanced release of NAMPT by extracellular ATP described here indicates the requirement of a second stimulus for the efficient secretion of NAMPT. This mode of secretion, which also applies to IL-1β, might represent a general mechanism for the release of leaderless secretory proteins at locally restricted sites.     

Rlp24 activates the AAA-ATPase Drg1 to initiate cytoplasmic pre-60S maturation

Formation of eukaryotic ribosomes is driven by energy-consuming enzymes. The AAA-ATPase Drg1 is essential for the release of several shuttling proteins from cytoplasmic pre-60S particles and the loading of late joining proteins. However, its exact role in ribosome biogenesis has been unknown. Here we show that the shuttling protein Rlp24 recruited Drg1 to pre-60S particles and stimulated its ATPase activity. ATP hydrolysis in the second AAA domain of Drg1 was required to release shuttling proteins. In vitro, Drg1 specifically and exclusively extracted Rlp24 from purified pre-60S particles. Rlp24 release required ATP and was promoted by the interaction of Drg1 with the nucleoporin Nup116. Subsequent ATP hydrolysis in the first AAA domain dissociated Drg1 from Rlp24, liberating both proteins for consecutive cycles of activity. Our results show that release of Rlp24 by Drg1 defines a key event in large subunit formation that is a prerequisite for progression of cytoplasmic pre-60S maturation.     

Cobalt(III) labeling of methionyl-tRNA synthetase from Escherichia coli.

Native and trypsin-modified methionyl-tRNA synthetases from Escherichia coli were found to be inactivated by incubation in the presence of Co(III) complexes of ATP, stabilized either by imidazole or phenanthroline, or by oxidation in situ to Co(III) of the substrate ATP-Co(II). It has been shown that the inactivation proceeds by specific labeling of the catalytic ATP-Mg(II) site of the synthetases. The enzymes are completely inactivated by the incorporation of one cobalt atom and one ATP molecule per active site. The inactivated enzymes may be stored for a long period without significant reactivation or removal of the cobalt label. In the presence of dithiothreitol or 2-mercaptoethanol, the labeled enzymes recover full activity with concomittant release of the bound label molecules.     

Thermodynamic analyses of the catalytic pathway of F1-ATPase from Escherichia coli. Implications regarding the nature of energy coupling by F1-ATPases

Thermodynamic properties of 12 different F1-ATPase enzymes were analyzed in order to gain insights into the catalytic mechanism and the nature of energy coupling to delta mu H+. The enzymes were normal soluble Escherichia coli F1, a group of nine beta-subunit mutant soluble E. coli F1 enzymes (G142S, K155Q, K155E, E181Q, E192Q, M209I, D242N, D242V, R246C), and both soluble and membrane-bound bovine heart mitochondrial F1. Unisite activity was studied by use of Gibbs free energy diagrams, difference energy diagrams, and derivation of linear free energy relationships. This allowed construction of binding energy diagrams for both the unisite ATP hydrolysis and ATP synthesis reaction pathways, which were in agreement. The binding energy diagrams showed that the step of Pi binding is a major energy-requiring step in ATP synthesis, as is the step of ATP release. It is suggested that there are two major catalytic enzyme conformations, and ATP- and an ADP-binding conformation. The effects of the mutations on the rate-limiting steps of multisite as compared to unisite activity were correlated, suggesting a direct link between the rate-limiting steps of the two types of activity. Multisite activity was analyzed by Arrhenius plots and by study of relative promotion from unisite to multisite rate. Changes in binding energy due to mutation were seen to have direct effects on multisite catalysis. From all the data, a model is derived to describe the mechanism of ATP synthesis. ATP hydrolysis, and energy coupling to delta mu H+ in F1F0-ATPases.     

Protective Effects of Pyridoxal Phosphate Against Glucose Deprivation-Induced Damage in Cultured Hippocampal Neurons

Abstract: When hippocampal cultures were deprived of glucose, massive release of lactate dehydrogenase (LDH), an indicator of neuronal death, occurred via NMDA receptor activation. Addition of pyridoxal phosphate (PLP; 1 and 10 µ M ) inhibited this LDH release in a concentration-dependent manner. Prior exposure to PLP evoked more potent inhibitory effects on LDH release compared with those treated at the onset of glucose deprivation. Furthermore, PLP inhibited the reduction of intracellular content of pyruvate induced by glucose deprivation, which was accompanied by the reversal of intracellular ATP depletion. A noteworthy elevation of extracellular glutamate in response to glucose deprivation was completely reversed by addition of PLP. Aminooxyacetic acid, a potent inhibitor of PLP-dependent enzymes, antagonized the effects of PLP on LDH release, pyruvate production, and ATP formation. These results suggest that PLP protects neurons from glucose deprivation-induced damage by enhancing the formation of energy-yielding products and relieving extracellular load of glutamate. The observed phenomena further indicate that PLP might be used prophylactically against neuronal death induced by metabolic disorders.     

Kinesin and myosin ATPases: variations on a theme.

The enzymes kinesin and myosin are examples of molecular motors which couple ATP hydrolysis to directed movement of biological structures. Myosin has been extensively studied and its structure and mechanism of coupling are known in detail. Much less is known about kinesin, but many of its major properties are similar to those of myosin. Both enzymes have two catalytic head groups at the end of a long alpha-helical rod. The head groups contain the sites for ATP hydrolysis and interaction with their respective partners for movement (microtubules or F-actin). In each case the binding and hydrolysis of ATP is rapid and the steady state ATPase rate is limited by a slow step in the region of product release. This slow release of product is accelerated by interaction with actin or microtubules coupled to changes in binding affinity. As there is no evidence for a close evolutionary link between kinesin and myosin, these and other similarities may represent convergence to set of common functional properties which are constrained by the requirements of protein structure and the use of ATP hydrolysis as a source of energy. It will be of particular interest to determine if these common properties are also shared by the large number of divergent proteins which have recently been discovered to possess a domain which is homologous to the head group of kinesin.     

Association of ATP citrate lyase with mitochondria

(1) The association of ATP citrate lyase with mitochondria was studied with isolated rat hepatocytes and mitochondria. (2) When hepatocytes were treated with digitonin, about 25% of the lyase activity was released like a mitochondrial enzyme. (3) The effect of temperature on release of lyase from hepatocytes was different from that on the release of other cytosolic or mitochondrial enzymes. (4) The fraction of total hepatic lyase in mitochondrial preparations made with exogenous MgCl₂ was 30 times greater than that for a cytosolic marker enzyme, phosphoglycerate kinase. (5) Lyase substrates enhanced the release of the enzyme both from hepatocytes and from isolated mitochondria. (6) The metabolic significance of association of ATP citrate lyase with mitochondria is discussed. (7) Data obtained in the course of these experiments indicate that less than 3% of adenylate kinase is cytosolic.     

Purinergic signalling links mechanical breath profile and alveolar mechanics with the pro-inflammatory innate immune response causing ventilation-induced lung injury

Severe pulmonary infection or vigorous cyclic deformation of the alveolar epithelial type I (AT I) cells by mechanical ventilation leads to massive extracellular ATP release. High levels of extracellular ATP saturate the ATP hydrolysis enzymes CD39 and CD73 resulting in persistent high ATP levels despite the conversion to adenosine. Above a certain level, extracellular ATP molecules act as danger-associated molecular patterns (DAMPs) and activate the pro-inflammatory response of the innate immunity through purinergic receptors on the surface of the immune cells. This results in lung tissue inflammation, capillary leakage, interstitial and alveolar oedema and lung injury reducing the production of surfactant by the damaged AT II cells and deactivating the surfactant function by the concomitant extravasated serum proteins through capillary leakage followed by a substantial increase in alveolar surface tension and alveolar collapse. The resulting inhomogeneous ventilation of the lungs is an important mechanism in the development of ventilation-induced lung injury. The high levels of extracellular ATP and the upregulation of ecto-enzymes and soluble enzymes that hydrolyse ATP to adenosine (CD39 and CD73) increase the extracellular adenosine levels that inhibit the innate and adaptive immune responses rendering the host susceptible to infection by invading microorganisms. Moreover, high levels of extracellular adenosine increase the expression, the production and the activation of pro-fibrotic proteins (such as TGF-β, α-SMA, etc.) followed by the establishment of lung fibrosis.