High Level Of ATPase Activity Research Articles

Waste or die: The price to pay to stay alive.

Microorganisms need to constantly exchange with their habitat to capture nutrients and expel toxic compounds. The ATP-binding cassette (ABC) transporters, a family of membrane proteins especially abundant in microorganisms, are at the core of these processes. Due to their extraordinary ability to expel structurally unrelated compounds, some transporters play a protective role in different organisms. Yet, the downside of these multidrug transporters is their entanglement in the resistance to therapeutic treatments. Intriguingly, some multidrug ABC transporters show a high level of ATPase activity, even in the absence of transported substrates. Although this basal ATPase activity might seem a waste, we surmise that this inherent capacity allows multidrug transporters to promptly translocate any bound drug before it penetrates into the cell.

ATP Hydrolyzing Salivary Enzymes of Caterpillars Suppress Plant Defenses

The oral secretions of herbivores are important recognition cues that can be used by plants to mediate induced defenses. In this study, a degradation of adenosine-5′-triphosphate (ATP) in tomato leaves was detected after treatment with Helicoverpa zea saliva. Correspondingly, a high level of ATPase activity in saliva was detected and three ATP hydrolyzing enzymes: apyrase, ATP synthase and ATPase 13A1 were identified in salivary glands. To determine the functions of these proteins in mediating defenses, they were cloned from H. zea and expressed in Escherichia coli. By applying the purified expressed apyrase, ATP synthase or ATPase 13A1 to wounded tomato leaves, it was determined that these ATP hydrolyzing enzymes suppressed the defensive genes regulated by the jasmonic acid and ethylene pathways in tomato plant. Suppression of glandular trichome production was also observed after treatment. Blood-feeding arthropods employ 5′-nucleotidase family of apyrases to circumvent host responses and the H. zea apyrase, is also a member of this family. The comparatively high degree of sequence similarity of the H. zea salivary apyrase with mosquito apyrases suggests a broader evolutionary role for salivary apyrases than previously envisioned.

Effect of High Temperature During Heading and Early Filling on Grain Yield and Physiological Characteristics in Indica Rice

To disclose the physiological mechanism of heat tolerance in rice ( Oryza sativa L.), this article investigated the pollen development, yield components, and some physiological parameters of indica rice genotypes under high temperature (HT) treatment during heading and early filling stages. Two heat-sensitive genotypes, Shuanggui 1 and T219, and 2 heat-tolerant genotypes, Huanghuazhan and T226, were used in pot experiments with HT treatment (mean temperature during the day above 33ºC) and natural temperature (control) treatments from 0 to 10 d and 10 to 20 d after heading. Compared with the control, the HT treatment significantly reduced the rates of pollen and spikelet fertility in the heat-sensitive genotypes, but had no significant effects on the heat-tolerant genotypes. Seed-setting rate decreased significantly in the heat-sensitive genotypes under HT, leading to a significant reduction in grain yield. Moreover, the reductions of yield of heat-sensitive genotypes were greater in the HT treatment at heading stage (0–10 d after heading) than in the HT treatment at early grain-filling stage (11–20 d after heading). However, the heat-tolerant genotypes were in minor influence under the HT treatment. The activities of enzymes from rice leaves, which were involved in antioxidant system, significantly increased in the heat-tolerant genotypes when treated with HT, whereas they were maintained at relative high levels in the control. The ATPase activity in grains was significantly reduced in the heat-sensitive genotypes, but slightly influenced in the heat-tolerant genotypes. The high temperature increased leaf temperature and malondialdehyde content in leaves, and reduced root activity and photosynthetic rate of flag leaf in all genotypes. However, variation range was much less in heat-tolerant genotypes than in heat-sensitive genotypes. The results suggest that the relative high yield in heat-tolerant genotypes under high-temperature stress is associated with low leaf temperature, high root activity, and the high levels of ATPase activity in grains, photosynthetic rate, and activities of antioxidant enzymes in leaves.

Mcm Subunits Can Assemble into Two Different Active Unwinding Complexes

The replication fork helicase in eukaryotes is a large complex that is composed of Mcm2-7, Cdc45, and GINS. The Mcm2-7 proteins form a heterohexameric ring that hydrolyzes ATP and provide the motor function for this unwinding complex. A comprehensive study of how individual Mcm subunit biochemical activities relate to unwinding function has not been accomplished. We studied the mechanism of the Mcm4-Mcm6-Mcm7 complex, a useful model system because this complex has helicase activity in vitro. We separately purified each of three Mcm subunits until they were each nuclease-free, and we then examined the biochemical properties of different combinations of Mcm subunits. We found that Mcm4 and Mcm7 form an active unwinding assembly. The addition of Mcm6 to Mcm4/Mcm7 results in the formation of an active Mcm4/Mcm6/Mcm7 helicase assembly. The Mcm4-Mcm7 complex forms a ringed-shaped hexamer that unwinds DNA with 3' to 5' polarity by a steric exclusion mechanism, similar to Mcm4/Mcm6/Mcm7. The Mcm4-Mcm7 complex has a high level of ATPase activity that is further stimulated by DNA. The ability of different Mcm mixtures to form rings or exhibit DNA stimulation of ATPase activity correlates with the ability of these complexes to unwind DNA. The Mcm4/Mcm7 and Mcm4/Mcm6/Mcm7 assemblies can open to load onto circular DNA to initiate unwinding. We conclude that the Mcm subunits are surprisingly flexible and dynamic in their ability to interact with one another to form active unwinding complexes.

Localization of nucleoside triphosphate diphosphohydrolase-1 (NTPDase1) and NTPDase2 in pancreas and salivary gland.

Ectonucleoside triphosphate diphosphohydrolases (NTPDases) are membrane-bound ectoenzymes that hydrolyze extracellular nucleotides. We investigated the distribution of NTPDase1 and NTPDase2 in murine salivary gland and pancreas. Histochemistry and immunostaining (by both light and electron microscopy), combined with functional assays, were used to describe the localization patterns and enzyme activities in the organs of wild-type and NTPDase1/cd39-null mice. Pancreatic acinar cells and salivary gland acinar/myoepithelial cells were positive for NTPDase1 and NTPDase2. Ecto-ATPase activity was slightly higher in salivary glands. Ductal epithelial cells expressed ecto-ATPase activity but NTPDase1 and NTPDase2 expression were weak at best. ATPase activity was found in blood vessels of both tissues and its localization pattern overlapped with NTPDase1 staining. In these structures, NTPDase2 antibodies stained the basolateral aspect of endothelial cells and the supporting cells. Biochemical assays and histochemical staining showed relatively high levels of ATPase activity in both glands of cd39(-/-) mice. Our data therefore support a physiological role for NTPDase2 and other ectonucleotidases in the pancreas and salivary glands. Because NTPDase1 is expressed in non-vascular cell types, this finding suggests that NTPDase1 may have functions in the gastrointestinal tract that differ from those demonstrated in the vascular system.

Methanethiosulfonate Derivatives of Rhodamine and Verapamil Activate Human P-glycoprotein at Different Sites

The human multidrug resistance P-glycoprotein (P-gp, ABCB1) actively extrudes a broad range of potentially cytotoxic compounds out of the cell. Key steps in understanding the transport process are binding of drug substrates in the transmembrane domains, initiation of ATPase activity, and subsequent drug efflux. We used cysteine-scanning mutagenesis of the transmembrane segment residues and reaction with the thiol-reactive drug substrate analog of rhodamine, methane-thiosulfonate-rhodamine (MTS-rhodamine), to test whether P-gp could be trapped in an activated state with high levels of ATPase activity. The presence of such an activated P-gp could be used to further investigate P-gp-drug substrate interactions. Single cysteine mutants (149) were treated with MTS-rhodamine, and ATPase activities were determined after removal of unreacted MTS-rhodamine. One mutant, F343C(TM6), showed a 5.8-fold increase in activity after reaction with MTS-rhodamine. Pre-treatment of mutant F343C with rhodamine B protected it from activation by MTS-rhodamine, indicating that residue Cys-343 contributes to the rhodamine-binding site. The ATPase activity of MTS-rhodamine-treated mutant F343C, however, was not stimulated further by colchicine or calcein-AM. By contrast, verapamil and Hoechst 33342 stimulated and inhibited, respectively, the ATPase activity of the MTS-rhodamine-treated mutant F343C. These results indicate that the MTS-rhodamine binding site overlaps that of colchicine and calcein-AM but not that of verapamil and Hoechst 33342 within the common drug-binding pocket.

The detergent-soluble maltose transporter is activated by maltose binding protein and verapamil.

The maltose transporter FGK2 complex of Escherichia coli was purified with the aid of a glutathione S-transferase molecular tag. In contrast to the membrane-associated form of the complex, which requires liganded maltose binding protein (MBP) for ATPase activity, the purified detergent-soluble complex exhibited a very high level of ATPase activity. This uncoupled activity was not due to dissociation of the MalK ATPase subunit from the integral membrane protein MalF and MalG subunits. The detergent-soluble ATPase activity of the complex could be further stimulated by wild-type MBP but not by a signaling-defective mutant MBP. Wild-type MBP increased the V(max) of the ATPase 2.7-fold but had no effect on the K(m) of the enzyme for ATP. When the detergent-soluble complex was reconstituted in proteoliposomes, it returned to being dependent on MBP for activation of ATPase, consistent with the idea that the structural changes induced in the complex by detergent that result in activation of the ATPase are reversible. The uncoupled ATPase activity resembled the membrane-bound activity of the complex also with respect to sensitivity to NaN(3), as well as a mercurial, p-chloromercuribenzosulfonic acid. Verapamil, a compound that activates the ATPase activity of the multiple drug resistance P-glycoprotein, activated the maltose transporter ATPase as well. The activation of this bacterial transporter by verapamil suggests that a structural feature that is conserved among both eukaryotic and prokaryotic ATP binding cassette transporters is responsible for this activation.

Characterization and functional reconstitution of the multidrug transporter

P-Glycoprotein, the multidrug transporter, is isolated from the plasma membrane of CHRC5 cells using a selective two-step detergent extraction procedure. The partially purified protein displays a high level of ATPase activity, which has a high KM for ATP, is stimulated by drugs, and can be distinguished from that of other membrane ATPases by its unique inhibition profile. Delipidation completely inactivates ATPase activity, which is restored by the addition of fluid lipid mixtures. P-Glycoprotein was reconstituted into lipid bilayers with retention of both drug transport and ATPase activity. Proteoliposomes containing P-glycoprotein display osmotically sensitive ATP-dependent accumulation of 3H-colchicine in the vesicle lumen. Drug transport is active, generating a stable 5.6-fold concentration gradient, and can be blocked by compounds in the multidrug resistance spectrum. Reconstituted P-glycoprotein also exhibits a high level of ATPase activity which is further stimulated by various drugs. P-Glycoprotein therefore functions as an active drug transporter with constitutive ATPase activity.

Functional reconstitution of drug transport and ATPase activity in proteoliposomes containing partially purified P-glycoprotein.

P-glycoprotein, the multidrug transporter, is proposed to act as an ATP-driven drug efflux pump. We previously reported the partial purification of P-glycoprotein from multidrug-resistant cells (Doige, C. A., Yu, X., and Sharom, F. J. (1992) Biochim. Biophys. Acta 1109, 149-160). We now report the reconstitution of this preparation into phospholipid bilayers using rapid detergent removal by gel filtration chromatography. The resulting proteoliposomes displayed ATP-dependent [3H]colchicine uptake over a time period of 0-4 min. No drug uptake was observed for liposomes of lipid alone, or liposomes reconstituted with a similar extract from drug-sensitive cells. Drug uptake was osmotically sensitive, and abolished by detergent permeabilization, indicating that it represented true transport into the vesicle lumen. Steady-state levels of drug uptake increased with drug concentration, approaching saturation at approximately 150 microM colchicine, with half-maximal accumulation at 50 microM. Drug was accumulated actively against a 5.6-fold concentration gradient. Multidrug resistance spectrum drugs and chemosensitizers inhibited colchicine uptake by P-glycoprotein proteoliposomes, whereas cytosine arabinoside and methotrexate had no effect. Reconstituted liposomes showed high levels of ATPase activity, which was stimulated over 2-fold by verapamil and trifluoperazine. These results suggest that P-glycoprotein functions as an active drug transporter with constitutive ATPase activity.

The relationship between ATPase activity, isometric force, and myosin light-chain phosphorylation and thiophosphorylation in skinned smooth muscle fiber bundles from chicken gizzard.

Isometric force developed by skinned gizzard muscle fiber bundles and levels of phosphorylation and thiophosphorylation of the 20,000-dalton myosin light chain were determined. These data showed a highly non-linear relationship between isometric force and myosin light-chain phosphorylation. Maximum force was developed at approximately 0.2 mol of phosphate/mol of light chain as reported previously (Hoar, P. E., Kerrick, W. G. L., and Cassidy, P. S. (1979) Science 204, 503-506). In contrast, the relationship between isometric force and myosin light-chain thiophosphorylation was linear, with maximum force occurring at 1.0 mol of thiophosphate/mol of myosin light chain. These observations are consistent with the latch-bridge hypothesis for conditions of varying myosin light-chain phosphatase/myosin light-chain kinase activity ratios as discussed by Hai and Murphy [1988) Am. J. Physiol. 254, C99-C106). To further test the latch-bridge hypothesis, ATPase activity was also measured during isometric force development in these fiber bundles. The relationship between isometric force and ATPase activity was linear whether the myosin light chains were phosphorylated or thiophosphorylated. Thus the number of cycling myosin cross-bridges, as measured by ATPase activity, was directly proportional to the force the muscle developed, not to the level of myosin light-chain phosphorylation. This finding that high levels of tension generated at low levels of light-chain phosphorylation are associated with high levels of ATPase activity is inconsistent with the latch-bridge model (Hai and Murphy, 1988).

Cardiac and skeletal muscle myosin polymorphism.

Skeletal muscles, unlike cardiac tissue, express several myosin isozymes during development which differ in primary structure from adult myosin. Monoclonal antibodies have shown the presence of at least two embryonic myosins, followed by a post-hatch myosin that persists until the appearance of adult myosin in chicken pectoralis muscle. Although the two major cardiac isozymes differ in enzymatic activity, the avian skeletal myosin isozymes all share the same high level of ATPase activity found for adult pectoralis myosin. The functional basis for the extensive myosin polymorphism in skeletal muscles thus remains to be determined.

Stability and substructure of cardiac myosin subfragment 1 and isolation and properties of its heavy-chain subunit.

The substructure and the thermal stability of the subunit interactions of bovine cardiac myosin subfragment 1 (SF1) have been examined. The results are in agreement with previous reports that the cardiac protein is cleaved in a very similar manner [Flink, I. L., & Morkin, E. (1982) Biophys. J. 37, 34; Korner, M., Thiem, N. V., Cardinaud, R., & Lacombe, G. (1983) Biochemistry 22, 5843-5847] but at a much faster rate [Applegate, D., Azarcon, A., & Reisler, E. (1984) Biochemistry 23, 6626-6630] than the skeletal protein. Additionally, it is found that the long-lived, steady-state intermediates formed by these proteins with MgATP at high ionic strength differ in their susceptibilities to tryptic attack especially at the 27K/50K junction of the associated heavy chains, suggesting a different conformation for these intermediates of the cardiac and skeletal SF1's. The thermal stability of the subunit interactions under conditions approaching the physiological state was examined by thermal ion-exchange chromatography of cardiac SF1 at 39.5 degrees C in the presence of MgATP. This results in the separation of part of the protein as the isolated heavy chain which is found to exhibit high levels of ATPase activity in the absence and presence of actin. Tryptic digestion of cardiac SF1 prior to thermal ion-exchange chromatography produces greater dissociation, with the heavy chain in this case being isolated as a complex of 27K, 50K, and 18-20K fragments.(ABSTRACT TRUNCATED AT 250 WORDS)

Myosin isozymes in avian skeletal muscles. I. Sequential expression of myosin isozymes in developing chicken pectoralis muscles

Myosin has been purified from chicken pectoralis muscle at various stages of development, from 10 days' incubation to approximately 10 months after hatching. Embryonic myosin from the earliest stage showed a high level of ATPase activity, similar to that obtained for adult pectoralis myosin. Two-dimensional peptide mapping of partial chymotryptic digests showed, however, that is heavy chain is quite different from that of adult fast myosin. The immunological crossreactivity observed between embryonic myosin and adult fast (pectoralis) myosin is therefore due to shared antigenic determinants rather than the presence of any adult isoforms. In an accompanying paper we will show that embryonic myosin at 10 days' incubation is not a single species, but consists of at least two heavy chain isozymes. The minor fraction binds slow light chains preferentially, and appears to be largely responsible for the observed crossreactivity with slow (ALD) myosin. None of the embryonic myosins is equivalent to the adult forms. Prior to hatching, LC3f is present only in very small amounts (less than 5%), and the adult light chain pattern, containing LC1f and LC3f in equimolar amounts, is not generated until after one week post-hatching. At about that time a new heavy chain population is detected, different from either the embryonic heavy chain or the adult heavy chain. The adult heavy chain peptide pattern appears from about three weeks' post-hatching, but a map indistinguishable from that of adult myosin is not observed until about 26 weeks. None of the observed differences in peptide maps can be related to different strains of chicken; pectoralis myosin from adult White Rock gave an identical map to that from White Leghorn. Unexpectedly, posterior latissimus dorsi (PLD) myosin from White Leghorn appears to be different from pectoralis myosin from the same strain, despite the histochemical and immunocytochemical similarity of the two muscles. We conclude that myosin polymorphism is widespread in muscle tissue, and that the expression of myosin isozymes and their subunits is under developmental regulation.

Phosphorylation of heavy sarcoplasmic reticulum vesicles: identification and characterization of three phosphorylated proteins.

Heavy sarcoplasmic reticulum vesicles derived from the terminal cisternae of the sarcoplasmic reticulum have been shown to contain endogenous protein kinase activity and associated substrate proteins. Heavy vesicles were phosphorylated at room temperature in 5mm MgCl2, 1mm EGTA, 10mm HEPES (pH 7.4) and 10 μm γ-32P-ATP.32P-phosphoproteins were determined by sodium dodecyl sulphate gel electrophoresis and autoradiography. In the absence of ethylene glycol bis (β-aminoethyl ether) N,N,N′,N′-tetraacetic acid (EGTA), there was little phosphorylation due to the high level of ATPase activity. Phosphorylation of three proteins of 64,000 daltons (E1), 42,000 daltons (E2), and 20,000 daltons (E3) was observed in the presence of 1mm EGTA. Phosphorylation of these proteins wascAMP-independent, hydroxylamine-resistant, and was seen without the addition of protein kinase. In the presence of HgCl2 (2.5mm) or sodium deoxycholate (1%) no protein phosphorylation was observed. ProteinE1 was heavily phosphorylated in the presence of 200mm KCl, while its phosphorylation was inhibited by 20 μm sodium dantrolene, an inhibitor of Ca2+ release. PhosphoproteinE3 was found in light and heavy sarcoplasmic reticulum vesicles whileE1 andE2 were found only in heavy vesicles. The phosphoproteinE2 had the properties of an intrinsic membrane protein while the proteinE1 bejaved as an extrinsic membrane protein. ProteinsE2 andE3 corresponded in mobility to minor sarcoplasmic reticulum proteins whileE1 had the same mobility as calsequestrin. The presence of high calcium (5mm) during electrophoresis caused calsequestrin to run at a lower molecular weight (≈56,000 instead of 64,000 daltons), and correspondingly the phosphoproteinE1 ran at a lower molecular weight. Finally, calsequestrin purified by a double gel electrophoresis method has been shown to be phosphorylated.

Ultrastructure du parenchyme sécréteur de la glande postanale du cælacanthe, <i>Latimeria chalumnae</i> Smith

Latimeria chalumnae Smith, the only surviving crossopterygian, possesses a well-developed, unpaired, postanal gland whose function is as yet undocumented. The availability of tissues suitable for ultrastructural and histochemical studies from the recent International Comores Expedition has permitted a reappraisal of the comparative morphology and functional hypotheses relating to the postanal gland. The richly vascularized tubuloacinar postanal gland is composed of cells which show the characteristic specializations of active ion transport. These include complex and extensive development of basal and lateral cell membrane infolding (cisternae) which are closely associated with numerous large, dense mitochondria. Histochemical studies have shown a high level of ATPase activity within the gland cells. These features of the postanal gland correlate well with those of the rectal glands of elasmobranchs and suggest a similar role in ion excretion and osmoregulation.

Bacterial and Bacteroid Properties of Mutants of Rhizobium Trifolii Strain T1 Uncoupled in Oxidative Phosphorylation

Using the neomycin-resistance selection technique, mutants uncoupled in oxidative phosphorylation were isolated in R. tri/olii strain Tl. These mutants had no detectable ATP-synthesizing activity and between 8 and 20% of the ATP-degrading activity of their parent strain in the bacterial form. The mutants formed nitrogen-fixing nodules on red, white, and subterranean clovers, and bacteroids isolated from white clover nodules had a high level of ATPase activity of about 70% of that found in bacteroids of their parental strain. The possibility that a new ATPase complex is synthesized when the bacteria differentiate to form bacteroids within the nodule is discussed.