Active Uptake of Ca++ and Ca++-Activated Mg++ ATPase in Red Cell Membrane Fragments

Isolated human red blood cell membrane fragments (RBCMF) were found to take up Ca⁺⁺ in the presence of ATP.¹ This ATP-dependent Ca⁺⁺ uptake by RBCMF appears to be the manifestation of an active Ca⁺⁺ transport mechanism in the red cell membrane reported previously (Schatzmann, 1966; Lee and Shin, 1969). The influences of altering experimental conditions on Ca⁺⁺-stimulated Mg⁺⁺ ATPase (Ca⁺⁺ ATPase) and Ca⁺⁺ uptake of RBCMF were studied. It was found that pretreatment of RBCMF at 50°C abolished both Ca⁺⁺ ATPase and Ca⁺⁺ uptake. Pretreatment of RBCMF with phospholipases A and C decreased both Ca⁺⁺ ATPase and Ca⁺⁺ uptake, whereas pretreatment with phospholipase D did not significantly alter either Ca⁺⁺ ATPase or Ca⁺⁺ uptake. Both Ca⁺⁺ ATPase and Ca⁺⁺ uptake had ATP specificity, similar optimum pH's, and optimum incubation temperatures. From these results, it was concluded that Ca⁺⁺ uptake is intimately linked to Ca⁺⁺ ATPase.     

Metabolic adaptation to the chronic loss of Ca2+ signaling induced by KO of IP3 receptors or the mitochondrial Ca2+ uniporter

Calcium signaling is essential for regulating many biological processes. Endoplasmic reticulum inositol trisphosphate receptors (IP₃Rs) and the mitochondrial Ca²⁺ uniporter (MCU) are key proteins that regulate intracellular Ca²⁺ concentration. Mitochondrial Ca²⁺ accumulation activates Ca²⁺-sensitive dehydrogenases of the tricarboxylic acid (TCA) cycle that maintain the biosynthetic and bioenergetic needs of both normal and cancer cells. However, the interplay between calcium signaling and metabolism is not well understood. In this study, we used human cancer cell lines (HEK293 and HeLa) with stable KOs of all three IP₃R isoforms (triple KO [TKO]) or MCU to examine metabolic and bioenergetic responses to the chronic loss of cytosolic and/or mitochondrial Ca²⁺ signaling. Our results show that TKO cells (exhibiting total loss of Ca²⁺ signaling) are viable, displaying a lower proliferation and oxygen consumption rate, with no significant changes in ATP levels, even when made to rely solely on the TCA cycle for energy production. MCU KO cells also maintained normal ATP levels but showed increased proliferation, oxygen consumption, and metabolism of both glucose and glutamine. However, MCU KO cells were unable to maintain ATP levels and died when relying solely on the TCA cycle for energy. We conclude that constitutive Ca²⁺ signaling is dispensable for the bioenergetic needs of both IP₃R TKO and MCU KO human cancer cells, likely because of adequate basal glycolytic and TCA cycle flux. However, in MCU KO cells, the higher energy expenditure associated with increased proliferation and oxygen consumption makes these cells more prone to bioenergetic failure under conditions of metabolic stress.     

Metabolism of epidermal tissues, mesophyll cells, and bundle sheath strands resolved from mature nutsedge leaves

Mesophyll cells and bundle sheath strands were isolated from Cyperus rotundus L. leaf sections infiltrated with a mixture of cellulase and pectinase followed by a gentle mortar and pestle grind. The leaf suspension was filtered through a filter assembly and mesophyll cells and bundle sheath strands were collected on 20-μm and 80-μm nylon nets, respectively. For the isolation of leaf epidermal strips longer leaf cross sections were incubated with the enzymes and gently ground as above. Loosely attached epidermal strips were peeled off with forceps. The upper epidermis, which lacks stomata, could be clearly distinguished from the lower epidermis which contains stomata. Microscopic evidence for identification and assessment of purity is provided for each isolated tissue.Enzymes related to the C₄-dicarboxylic acid cycle such as phosphoenolpyruvate carboxylase, malate dehydrogenase (NADP⁺), pyruvate, P_i dikinase were found to be localized, ≥98%, in mesophyll cells. Enzymes related to operating the reductive pentose phosphate cycle such as RuDP carboxylase, phosphoribulose kinase, and malic enzyme are distributed, ≥99%, in bundle sheath strands. Other photosynthetic enzymes such as aspartate aminotransferase, pyrophosphatase, adenylate kinase, and glyceraldehyde 3-P dehydrogenase (NADP⁺) are quite active in both mesophyll and bundle sheath tissues.Enzymes involved in photorespiration such as RuDP oxygenase, catalase, glycolate oxidase, hydroxypyruvate reductase (NAD⁺), and phosphoglycolate phosphatase are preferentially localized, ≥84%, in bundle sheath strands.Nitrate and nitrite reductase can be found only in mesophyll cells, while glutamate dehydrogenase is present, ≥96%, in bundle sheath strands.Starch- and sucrose-synthesizing enzymes are about equally distributed between the mesophyll and bundle sheath tissues, except that the less active phosphorylase was found mainly in bundle sheath strands. Fructose-1,6-diP aldolase, which is a key enzyme in photosynthesis and glycolysis leading to sucrose and starch synthesis, is localized, ≥90%, in bundle sheath strands. The glycolytic enzymes, phosphoglyceromutase and enolase, have the highest activity in mesophyll cells, while the mitochondrial enzyme, cytochrome c oxidase, is more active in bundle sheath strands.The distribution of total nutsedge leaf chlorophyll, protein, and PEP carboxylase activity, using the resolved leaf components, is presented. ¹⁴CO₂ Fixation experiments with the intact nutsedge leaves and isolated mesophyll and bundle sheath tissues show that complete C₄ photosynthesis is compartmentalized into mesophyll CO₂ fixation via PEP carboxylase and bundle sheath CO₂ fixation via RuDP carboxylase. These results were used to support the proposed pathway of carbon assimilation in C₄-dicarboxylic acid photosynthesis and to discuss the individual metabolic characteristics of intact mesophyll cells, bundle sheath cells, and epidermal tissues.     

The Enzymes of Carbon Metabolism in the Thermotolerant Bacillar Strain K1

To determine enzymatic activities in the thermotolerant strain K1 (formerly Sulfobacillus thermosulfidooxidans subsp. thermotolerans), it was grown in a mineral medium with (1) thiosulfate and Fe²⁺ or pyrite (autotrophic conditions), (2) Fe²⁺, thiosulfate, and yeast extract or glucose (mixotrophic conditions), and (3) yeast extract (heterotrophic conditions). Cells grown mixo-, hetero-, and autotrophically were found to contain enzymes of the tricarboxylic acid (TCA) cycle, as well as malate synthase, an enzyme of the glyoxylate cycle. Cells grown organotrophically in a medium with yeast extract exhibited the activity of the key enzymes of the Embden–Meyerhof–Parnas and Entner–Doudoroff pathways. The increased content of carbon dioxide (up to 5 vol %) in the auto- and mixotrophic media enhanced the activity of the enzymes involved in the terminal reactions of the TCA cycle and the enzymes of the pentose phosphate pathway. Carbon dioxide is fixed in the Calvin cycle. The highest activity of ribulose bisphosphate carboxylase was detected in cells grown autotrophically at the atmospheric content of CO₂ in the air used for aeration of the growth medium. The activities of pyruvate carboxylase, phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase, and phospho-enolpyruvate carboxytransphosphorylase decreased with increasing content of CO₂ in the medium.     

Adenosine triphosphatase activity of cutaneous nerve fibers

Summary The histochemical study of Mg⁺⁺-activated adenosine triphosphatase (Mg⁺⁺-ATPase) activity was carried out on the peripheral nerves of mouse digital skin by light and electron microscopy. Under the light microscope, the ATPase activity was clearly demonstrated on the nerve fibers as a fine network in the subepidermal regions. Under the electron microscope, the reaction product of enzyme activity was located in the interspace between axolemma and the surrounding Schwann cells of the unmyelinated nerve fibers. No reaction product was observed in the space between the axolemma and the Schwann cells associated with myelinated nerve fibers. Demonstrable activity was absent at the nodes of Ranvier as well as on the para- and internodal regions of these myelinated axons. The part of the axolemma lacking a Schwann cell sheath failed to show a reaction product. The perineural epithelial cells surrounding the nerve fibers displayed reaction product in the caveolae. These results suggest a functional difference in the axon-Schwann interface of myelinated as compared to unmyelinated nerve fibers. The function of the perineural epithelial cell would be expected to be a regulatory one in transferring materials across the epithelium to keep the proper humoral environment around nerve fibers.     

The effect of anti-ATPase antibodies upon the Ca++ transport of sarcoplasmic reticulum

Sheep or guinea pig antisera against the purified Ca⁺⁺ transport ATPase of sarcoplasmic reticulum inhibit Ca⁺⁺ transport due to a complement-dependent damage of the membrane, which causes massive leakage of Ca⁺⁺. The Ca⁺⁺-activated ATPase activity is only slightly affected even at ten times higher antibody concentration than that required for inhibition of Ca⁺⁺ transport. Antibodies prepared against the Ca⁺⁺ binding protein (C₁ protein) have no influence upon either ATPase activity or Ca⁺⁺ transport and ferritin-labeled anti-C₁ antibodies do not bind to microsomes.     

Metabolism via the pentose phosphate pathway in rat pheochromocytoma PC12 cells: Effects of nerve growth factor and 6-aminonicotinamide

Exposure of rat pheochromocytoma PC12 cells to 0.1 mM 6-aminonicotinamide (6AN) for 24 hours resulted in a 500-fold increase in 6-phosphogluconate indicating active metabolism of glucose via the oxidative enzymes of the pentose phosphate pathway. Amounts of 6-phosphogluconate that accumulated in 6AN-treated cells at 24 hours were significantly increased by treatment of the cells with nerve growth factor (NGF) (100 ng 7S/ml) suggesting that metabolism of glucose via the pentose pathway at this time was enhanced by NGF. This stimulation of metabolism via the pentose pathway is probably a late response to NGF because initial rates of 6-phosphogluconate accumulation in 6AN-treated cells were the same in the presence and absence of NGF. Moreover, amounts of¹⁴CO₂ generated from 1-[¹⁴CO₂]glucose during the initial six hour incubation period were the same in control and NGF-treated cells. Specific activities of hexose phosphates labeled from 1-[¹⁴CO₂]glucose were also the same in control and NGF-treated cells. The observation that 6AN inhibited metabolism via the pentose phosphate pathway but failed to inhibit NGF-stimulated neurite outgrowth suggests that NADPH required for lipid biosynthesis accompanying NGF-stimulated neurite outgrowth from PC12 cells can be derived from sources other than, or in addition to, the oxidative enzymes of the pentose phosphate pathway.Special Issue dedicated to Dr. O. H. Lowry.     

Effect of lipid on the access of ATP and calcium to the delipidated Ca++-ATPase of intestinal brush border membrane.

The delipidated Ca⁺⁺-ATPase prepared from intestinal brush border membranes showed a higher activity of Ca⁺⁺-independent ATPase, a lower Km value for ATP and a higher Km value for Ca⁺⁺ than its original membrane Ca⁺⁺-ATPase. The addition of phosphatidylcholine re-activated the delipidated Ca⁺⁺-ATPase to approximately 89 % of its original membrane Ca⁺⁺-ATPase activity but did not restore the affinity for Ca⁺⁺. This phospholipid raised the Km value for ATP but had little effect on the Km value for Ca⁺⁺. Palmitic acid elevated the Km value for Ca⁺⁺ but did not change the Km value for ATP. Kinetic analyses of these data suggest that the hydrocarbon chain of phosphatidylcholine is an important rate-limiting factor for the access of Ca⁺⁺ to the enzyme and the polar head groups of phosphorylcholine and ester bond may be the factor for the access of ATP.     

A highly ion-sensitive ATP-phosphorylation system in lobster nerve

The transfer of -phosphate from ³²P labeled adenosine-triphosphate (ATP) at low concentrations (10^?10 to 10^?7M) into the peripheral nerve of the lobster was found to be highly sensitive to external ionic environments. The phosphorylation process is inhibited at conditions similar to extracellular environments (high Na⁺, Ca⁺⁺ and pH) and stimulated by those close to intracellular medium (high K⁺, Mg⁺⁺ and low pH). This system is not related to NaK ATPase (pump ATPase) which is highly sensitive to ouabain and is active only at higher ATP concentrations (>10^?6M). The system is membrane bound and sensitive to a variety of neuro-active agents which are known to interfere with ionic conductance changes in axons.     

Studies on ATP hydrolysis in medium for histochemical demonstration of ATPase activity

Summary Nonenzymatic ATP hydrolysis in medium of Wachstein and Meisel for histochemical demonstration of ATPase activity was investigated. In this medium considerable amounts of phosphorus are released without the participation of the enzyme. ATP hydrolysis in Wachstein-Meisel's medium increase with the concentration of Pb⁺⁺ and decrease at its small concentrations. The degree of ATP hydrolysis appeared to increase with increase both temperature and pH. At high concentration of ATP (5.76 mM) the degree of ATP hydrolysis in Wachstein-Meisel's medium is lower than at 1.44 mM ATP. 10.0 mM Ca⁺⁺ or 3.6 mM Fe⁺⁺ speed up ATP hydrolysis after 30- and 60-minute incubation. In the presence of 3.6 mM Co⁺⁺ or 2.6 mM Cu⁺⁺ ATP hydrolysis in Wachstein-Meisel's medium increased throughout the whole period examined. On the contrary, 3.6 mM Fe⁺⁺⁺ decreases ATP hydrolysis in this medium.10.0 mM F^– raises the degree of ATP hydrolysis which is, however, lowered in the presence of 2.5 mM pCMB or 3.6 mM KCN. 2.0 mM cysteine highly inhibits the process of nonenzymatic ATP hydrolysis in Wachstein-Meisel's medium.These data show that the histochemical reaction for ATPase activity in Wachstein-Meisel's medium does not originate exclusively from the hydrolysis of ATP in the presence of Pb⁺⁺, but take rise, above all, as a result of an enzymatic reaction.     

Achromobacter denitrificans Strain YD35 Pyruvate Dehydrogenase Controls NADH Production To Allow Tolerance to Extremely High Nitrite Levels

We identified the extremely nitrite-tolerant bacterium Achromobacter denitrificans YD35 that can grow in complex medium containing 100 mM nitrite (NO₂⁻) under aerobic conditions. Nitrite induced global proteomic changes and upregulated tricarboxylate (TCA) cycle enzymes as well as antioxidant proteins in YD35. Transposon mutagenesis generated NO₂⁻-hypersensitive mutants of YD35 that had mutations at genes for aconitate hydratase and α-ketoglutarate dehydrogenase in the TCA cycle and a pyruvate dehydrogenase (Pdh) E1 component, indicating the importance of TCA cycle metabolism to NO₂⁻ tolerance. A mutant in which the pdh gene cluster was disrupted (Δpdh mutant) could not grow in the presence of 100 mM NO₂⁻. Nitrite decreased the cellular NADH/NAD⁺ ratio and the cellular ATP level. These defects were more severe in the Δpdh mutant, indicating that Pdh contributes to upregulating cellular NADH and ATP and NO₂⁻-tolerant growth. Exogenous acetate, which generates acetyl coenzyme A and then is metabolized by the TCA cycle, compensated for these defects caused by disruption of the pdh gene cluster and those caused by NO₂⁻. These findings demonstrate a link between NO₂⁻ tolerance and pyruvate/acetate metabolism through the TCA cycle. The TCA cycle mechanism in YD35 enhances NADH production, and we consider that this contributes to a novel NO₂⁻-tolerating mechanism in this strain.     

Characteristics of an adenosine triphosphatase in erythrocyte membranes stimulated by 2,4-dinitrophenol

Studies were made of the stimulation by 2,4-dinitrophenol (DNP) of an adenosine triphosphatase (ATPase) in stromata of human erythrocytes. Activation by 2,4-dinitrophenol occurs in the range 10^?5 to 10^?3 M and was seen in whole cells, ghosts reconstituted with Mg and ATP, and in osmotic ghosts prepared at a low ratio of cells to water. Phloretin and phloridzin also activated the DNP sensitive system but inhibited it at higher concentrations. DNP increased the K_m and V_max values of the enzyme equally. The DNP sensitive and Na⁺ + K⁺ sensitive enzymes of the stromata were compared. The activities of the two ATPases are additive, require the presence of Mg⁺⁺ and require that the substrate be located at the inner surface of the membrane. The two enzymes differ in their substrate specificity, in their sensitivity to inhibition by ouabain and phloretin and in their sensitivity to some factor in hemolysates. The possible roles of this system in the erythrocyte were discussed.     

Photoheterotrophic Fluxome in Synechocystis sp. Strain PCC 6803 and Its Implications for Cyanobacterial Bioenergetics

This study investigated metabolic responses in Synechocystis sp. strain PCC 6803 to photosynthetic impairment. We used 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU; a photosystem II inhibitor) to block O₂ evolution and ATP/NADPH generation by linear electron flow. Based on ¹³C-metabolic flux analysis (¹³C-MFA) and RNA sequencing, we have found that Synechocystis sp. PCC 6803 employs a unique photoheterotrophic metabolism. First, glucose catabolism forms a cyclic route that includes the oxidative pentose phosphate (OPP) pathway and the glucose-6-phosphate isomerase (PGI) reaction. Glucose-6-phosphate is extensively degraded by the OPP pathway for NADPH production and is replenished by the reversed PGI reaction. Second, the Calvin cycle is not fully functional, but RubisCO continues to fix CO₂ and synthesize 3-phosphoglycerate. Third, the relative flux through the complete tricarboxylic acid (TCA) cycle and succinate dehydrogenase is small under heterotrophic conditions, indicating that the newly discovered cyanobacterial TCA cycle (via the γ-aminobutyric acid pathway or α-ketoglutarate decarboxylase/succinic semialdehyde dehydrogenase) plays a minimal role in energy metabolism. Fourth, NAD(P)H oxidation and the cyclic electron flow (CEF) around photosystem I are the two main ATP sources, and the CEF accounts for at least 40% of total ATP generation from photoheterotrophic metabolism (without considering maintenance loss). This study not only demonstrates a new topology for carbohydrate oxidation but also provides quantitative insights into metabolic bioenergetics in cyanobacteria.     

Evidence implicating a membrane ATPase in the control of passive permeability of excitable cells

The temperature sensitivity of the ATPase enzyme systems in a muscle microsomal preparation from the crayfish, Astacus pallipes, was studied. Preincubation of the enzyme preparation in the range 33–36°C produced a marked inactivation of the ATPases; the Mg⁺⁺-dependent ATPase was very much more sensitive to this treatment than the Na⁺-K⁺-Mg⁺⁺-dependent ATPase. Thus, the Arrhenius μ for the inactivation of the Mg⁺⁺-dependent ATPase produced by eight minute preincubation is > 100 Kcals. These results are compared with the changes that are observed during the heat death of the whole animal, where exposure to 35°C produces a dramatic change in Na⁺ permeability within five minutes. Arrhenius μ for heat death is also > 100 Kcals and operates over the identical critical temperature range. It is suggested that the Mg⁺⁺-dependent ATPase controls passive permeability in these excitable cells and the results also confirm the view that Mg⁺⁺ and Na⁺-K⁺-Mg⁺⁺ ATPases are separate enzymes.     

Predominant localization of mitochondria enriched with glycine-decarboxylating enzymes in bundle sheath cells of Alternanthera tenella, a C3–C4 intermediate species

Mesophyll protoplasts and bundle sheath cells were prepared by enzymatic digestion of leaves of Alternanthera tenella, a C₃-C₄ intermediate species. The intercellular distribution of selected photosynthetic, photorespiratory and respiratory (mitochondrial) enzymes in these meso-phyll and bundle sheath cells was studied. The activity levels of photosynthetic enzymes such as PEP carboxylase (EC 4.1.1.31) or NAD-malic enzyme (EC 1.1.1.39) and photorespiratory enzymes such as glycolate oxidase (EC 1.1.3.1) or NADH-hydroxypyruvate reductase (EC 1.1.1.29) were similar in the two cell types. The activity levels of mitochondrial TCA cycle enzymes such as citrate synthase (EC 4.1.3.7) or fumarase (EC 4.2.1.2) were 2- to 3-fold higher in bundle sheath cells. On the other hand, the activity levels of mitochondrial photorespiratory enzymes, namely glycine decarboxylase (EC 2.1.2.10) and serine hydroxymethyltransferase (EC 2.1.2.1), were 6-9-fold higher in bundle sheath cells than in mesophyll protoplasts. Such preferential localization of mitochondria enriched with the glycine-decarboxylating system in the inner bundle sheath cells would result in efficient refixa-tion of CO₂ from not only photorespiration but also dark respiration before its exit from the leaf. We propose that predominant localization of mitochondria specialized in glycine decarboxylation in bundle sheath cells may form the basis of reduced photorespiration in this C₃-C₄ intermediate species.     

Separate effects of mercurial compounds on the ionophoric and hydrolytic functions of the (Ca+++Mg++)-ATPase of sarcoplasmic reticulum

Summary We have shown that a Ca⁺⁺-ionophore activity is present in the (Ca⁺⁺+Mg⁺⁺)-ATPase of rabbit skeletal muscle sarcoplasmic reticulum (A.E. Shamoo & D.H. MacLennan, 1974.Proc. Nat. Acad. Sci. USA 71:3522). Methylmercuric chloride inhibited the (Ca⁺⁺+Mg⁺⁺)-ATPase and Ca⁺⁺ transport, but had no effect on the activity of the Ca⁺⁺ ionophore. Mercuric chloride inhibited ATPase, transport and ionophore activity. The ATPase and transport functions were more sensitive to methylmercuric chloride than to mercuric chloride. The two functions were inhibited concomitantly by methylmercuric chloride but slightly lower concentrations of mercuric chloride were required to inhibit Ca⁺⁺ transport than were required to inhibit ATPase. Methylmercuric chloride and mercuric chloride probably inhibited ATPase and Ca⁺⁺ transport by blocking essential-SH groups. However, it appears that there are no essential-SH groups in the Ca⁺⁺ ionophore and that mercuric chloride inhibited the Ca⁺⁺ ionophore activity by competition with Ca⁺⁺ for the ionophoric site. Blockage of Ca⁺⁺ transport by mercuric chloride probably occurs both at sites of essential-SH groups and at sites of ionophoric activity. These data suggest the separate identity of the sites of ATP hydrolysis and of Ca⁺⁺ ionophoric activity.     

Adenosine triphosphatase from rat liver mitochondria: separate sites involved in ATP hydrolysis and in the reversible, high affinity binding of ADP.

Homogeneous ATPase from rat liver mitochondria binds one mole of ADP per mole of enzyme reversibly, and with high affinity (K_D = 1–2 μM). The high affinity binding site is highly specific for ADP and dADP. AMP does not bind. Agents which inhibit ATP hydrolysis have little inhibitory effect on the high affinity binding of ADP. These agents include adenylyl imidodiphosphate (AMP-PNP), azide, sucrose, and the divalent cation Mg⁺⁺. AMP-PNP inhibits ATPase activity in phosphorylating membrane preparations of rat liver mitochondria by about 90 percent, but is without effect on ATP synthesis. These results are consistent with the view that the purified soluble, and the membrane-bound ATPase of rat liver mitochondria contain separate sites involved in ATP hydrolysis and in the reversible, high affinity binding of ADP.     

The ATPase cycle mechanism of the DEAD-box rRNA helicase, DbpA

DEAD-box proteins are ATPase enzymes that destabilize and unwind duplex RNA. Quantitative knowledge of the ATPase cycle parameters is critical for developing models of helicase activity. However, limited information regarding the rate and equilibrium constants defining the ATPase cycle of RNA helicases is available, including the distribution of populated biochemical intermediates, the catalytic step(s) that limits the enzymatic reaction cycle, and how ATP utilization and RNA interactions are linked. We present a quantitative kinetic and equilibrium characterization of the ribosomal RNA (rRNA)-activated ATPase cycle mechanism of DbpA, a DEAD-box rRNA helicase implicated in ribosome biogenesis. rRNA activates the ATPase activity of DbpA by promoting a conformational change after ATP binding that is associated with hydrolysis. Chemical cleavage of bound ATP is reversible and occurs via a γ-phosphate attack mechanism. ADP-P_i and RNA binding display strong thermodynamic coupling, which causes DbpA-ADP-P_i to bind rRNA with > 10-fold higher affinity than with bound ATP, ADP or in the absence of nucleotide. The rRNA-activated steady-state ATPase cycle of DbpA is limited both by ATP hydrolysis and by P_i release, which occur with comparable rates. Consequently, the predominantly populated biochemical states during steady-state cycling are the ATP- and ADP-P_i-bound intermediates. Thermodynamic linkage analysis of the ATPase cycle transitions favors a model in which rRNA duplex destabilization is linked to strong rRNA and nucleotide binding. The presented analysis of the DbpA ATPase cycle reaction mechanism provides a rigorous kinetic and thermodynamic foundation for developing testable hypotheses regarding the functions and molecular mechanisms of DEAD-box helicases.     

Flagellar movement and adenosine triphosphatase activity in sea urchin sperm extracted with triton X-100

Extraction with 0 04% (w/v) Triton X-100 removes the flagellar membrane from sea urchin sperm while leaving the motile apparatus apparently intact When reactivated in a suitable medium containing exogenous adenosine triphosphate (ATP), nearly 100% of the sperm are motile and they swim in a manner resembling that of live sperm. Under standard conditions, with 1 mM ATP at 25°C, the reactivated sperm had an average frequency of 32 beats/sec and progressed forward a distance of 2.4 µm/beat; comparable figures for live sperm in seawater were 46 beats/sec and 3 9 µm/beat. The adenosine triphosphatase (ATPase) activity of the reactivated sperm was measured with a pH-stat in the presence of oligomycin to inhibit residual mitochondrial ATPase. The motile sperm had an ATPase activity of 0.16 µmole P_i/(min x mg protein), while sperm that had been rendered non-motile by homogenizing had an activity of 0 045 µmole P_i/(min x mg protein). The difference between the ATPase activities of the motile and nonmotile sperm was tentatively interpreted as the amount of activity coupled to movement, and under optimal conditions it amounted to about 72% of the total ATPase activity Under some conditions the movement-coupled ATPase activity was proportional to the beat frequency, but it was possibly also affected by other wave parameters. The coupled ATPase activity decreased to almost zero when movement was prevented by raising the viscosity, or by changing the pH or salt concentration. The motility of reactivated sperm was wholly dependent on the presence of ATP; other nucleotides gave very low phosphatase activity and no movement. The requirement for a divalent cation was best satisfied with Mg⁺⁺, although some motility was also obtained with Mn⁺⁺ and Ca⁺⁺. The coupled ATPase activity had a Michaelis constant (K_m) of 0.15 mM. The beat frequency of the reactivated sperm varied with the ATP concentration, with an effective "K_m" of 0.2 mM.     

Effect of exogenous ATP on the volume of TA3 ascites tumor cells

When exogenous ATP is added to suspensions of TA₃ ascites tumor cells suspended in Ca⁺⁺ and Mg⁺⁺ free media, a significant increase in cell volume can be measured. This increase is reversible upon addition of Ca⁺⁺ and/or Mg⁺⁺ back to the media. The enlargement of these cells is temperature sensitive and specific for ATP; no other nucleotides, EDTA or ouabain were effective. The evidence suggest that this phenomena may be due to an alteration in membrane permeability and that the regulation of membrane permeability is an energy dependent process.