Cowpea ribonuclease: properties and effect of NaCl-salinity on its activation during seed germination and seedling establishment

Pitiúba cowpea [Vigna unguiculata (L.) Walp] seeds were germinated in distilled water (control treatment) or in 100 mM NaCl solution (salt treatment), and RNase was purified from different parts of the seedlings. Seedling growth was reduced by the NaCl treatment. RNase activity was low in cotyledons of quiescent seeds, but the enzyme was activated during germination and seedling establishment. Salinity reduced cotyledon RNase activity, and this effect appeared to be due to a delay in its activation. The RNases from roots, stems, and leaves were immunologically identical to that found in cotyledons. Partially purified RNase fractions from the different parts of the seedling showed some activity with DNA as substrate. However, this DNA hydrolyzing activity was much lower than that of RNA hydrolyzing activity. The DNA hydrolyzing activity was strongly inhibited by Cu²⁺, Hg²⁺, and Zn²⁺ ions, stimulated by MgCl₂, and slowly inhibited by EDTA. This activity from the most purified fraction was inhibited by increasing concentrations of RNA in the reaction medium. It is suggested that the major biological role of this cotyledon RNase would be to hydrolyze seed storage RNA during germination and seedling establishment, and it was discussed that it might have a protective role against abiotic stress during later part of seedling establishment.     

Effects of cholesterol on (Na+,K+)-ATPase ATP hydrolyzing activity in bovine kidney

The (Na+,K+)-ATPase ATP hydrolyzing activity from rabbit kidney medulla basolateral membrane vesicles was studied as a function of the cholesterol content of the basolateral membranes. The cholesterol content of the membranes was modified by incubation with phospholipid vesicles. When the cholesterol content was increased above that found in the native membrane, the (Na+,K+)-ATPase ATP hydrolyzing activity was inhibited. When the cholesterol content was decreased from that found in the native membranes, the (Na+,K+)-ATPase ATP hydrolyzing activity was inhibited. Analogous effects were found with the K+-activated phosphatase activity of the same membrane vesicles. Therefore, at low cholesterol contents, cholesterol was stimulatory, and at high cholesterol contents, cholesterol was inhibitory. The structural specificity of this effect was tested by introducing lanosterol and ergosterol as 50% of the membrane sterol. Ergosterol was the least effective at supporting (Na+,K+)-ATPase ATP hydrolyzing activity, while lanosterol was more effective, but still not as effective as cholesterol.     

Membrane-bound ATPase of a thermoacidophilic archaebacterium, Sulfolobus acidocaldarius

The membranes of Sulfolobus, a thermoacidophilic archaebacterium showed two types of ATP hydrolyzing activity. One was that of a neutral ATPase at an optimum pH around 6.5. This enzyme was activated by 10 mM sulfate with a shift of optimum pH to 5. In these respects, the enzyme was similar to membrane-bound ATPase of Thermoplasma, another thermoacidophilic archaebacterium, reported by Searcy and Whatley [1982) Zbl. Bakt. Hyg., I. Abt. Orig. C3, 245-257). The enzyme hydrolyzed ATP and other NTPs, but not ADP or AMP. It was highly thermostable, but irreversibly inactivated in 0.1 M HCl. The other activity was that of an acidic apyrase at an optimum pH around 2.5. This enzyme was extremely stable toward high temperature and acid and inhibited by sulfate. Both of these ATP hydrolyzing enzymes were resistant to N,N'-dicyclohexylcarbodiimide (DCCD), azide, oligomycin, N'-ethylmaleimide, p-chloromercuribenzoate, orthovanadate, or ouabain. Sulfolobus ATPases differ from F1 and other transport ATPases so far described.     

Cadmium stress affects seed germination and seedling growth in <Emphasis Type="Italic">Sorghum bicolor</Emphasis> (L.) Moench by changing the activities of hydrolyzing enzymes

Seed germination, one of the most important phases in the life cycle of a plant, is highly responsive to existing environment. Hydrolyzing enzymes play a major role in the mobilization of food reserves by hydrolyzing carbohydrates, proteins and fats. This paper reports on the effect of Cd toxicity on seed germination and the activities of hydrolyzing enzymes, like acid phosphatases (ACPs), proteases and α-amylases in Sorghum bicolor (L.) Moench. The metal uptake by embryonic axes and seeds was quantified. We found that sorghum could tolerate up to 0.5 mM Cd. At concentrations above 3.0 mM, seed germination was adversely affected with a complete cessation of seedling growth. All investigated hydrolyzing enzymes exhibited a significant decrease in activity with increasing Cd concentrations. The isozyme profiles indicated the loss of one or two isozymes of ACP, induction of a new isozyme for total protease (at 3.0 mM Cd) and a decline in the intensity of α-amylase isozymes. SEM studies revealed that Cd affected a change in root hair density. SEM investigations also confirmed the assay results of the inhibition of starch mobilization from endosperm. This suggested an inhibition of the hydrolysis of reserve carbohydrates and translocation of hydrolyzed sugars, ultimately resulting in decreased germination and disruption of seedling growth. Because sorghum is an important dryland crop, its response to the presence of Cd in agro-ecosystems and Cd-induced phytotoxicity during seed germination and seedling growth needs critical investigation.     

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.     

Mitochondrial F1-ATPase will bind and cleave ATP but only slowly release ADP after N,N'-dicyclohexylcarbodiimide or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole derivatization

The ATPase from the inner mitochondrial membrane is known to be inhibited by modification of one of the three catalytic subunits with N,N'-dicyclohexylcarbodiimide (DCCD) or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. An experimental approach described in this paper shows that most of the residual ATPase activity observed after the usual DCCD or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole modification is due to the presence of unmodified enzyme, although the large fraction of modified enzyme retains a weak catalytic activity. This weak catalytic activity can be stimulated by methanol or dimethyl sulfoxide. When the modified enzymes are exposed to Mg2+ and [3H]ATP, about equal amounts of [3H]ATP and [3H]ADP appear at catalytic sites. The turnover rate for these enzymes is less than 1/1000 that of the native enzyme when it is calculated from the rate at which the enzyme becomes labeled at the catalytic sites with [3H]ATP and [3H]ADP during steady state hydrolysis. In addition, a higher ATP concentration is required for steady state turnover and, after ATP binding, the principal rate-limiting step is the capacity of the derivatized enzyme to undergo the binding changes necessary for the release of ADP and Pi. When the modified enzymes are not hydrolyzing ATP, they convert to form(s) that show a distinct lag in the replacement of bound nucleotides at catalytic sites. The replacement of bound nucleotides is still promoted by MgATP, even though the enzymes have been converted to sluggish forms. Contrary to a recent suggestion based on the study of the DCCD-modified enzyme (Soong, K.S., and Wang, J.H. (1984) Biochemistry 23, 136-141), our data provide evidence for the existence of catalytic cooperatively between at least two alternating sites in the modified enzyme and are consistent with continued sequential participation of all three sites.     

The action of two classes of pyrethroids on the inhibition of brain Na-Ca and Ca + Mg ATP hydrolyzing activities of the American cockroach

Ca2+-stimulated ATP hydrolyzing activities (i.e. Na-Ca ATP hydrolysis and Ca + Mg ATP hydrolysis) measured in cockroach brain tissue were highly sensitive to the action of pyrethroid insecticides under in vitro conditions. Non-cyano-containing pyrethroids inhibited Na-Ca ATP hydrolysis to a greater extent than their cyano-containing counterparts. The reverse is true for pyrethroid action on Ca + Mg ATP hydrolysis. Nonmitochondrial Ca + Mg ATP hydrolysis of disrupted synaptosomes was the most sensitive activity examined. Ca2+-stimulated ATP hydrolyzing activities were inhibited in cockroaches poisoned by permethrin in vivo. In vivo poisoning occurred in the presence of a similar amount of bound [14C]permethrin which had been determined to cause a substantial amount of inhibition to Ca2+-stimulated ATP hydrolyzing activities in vitro.     

Novel Microbial Inhibitors of Angiotensin-converting Enzyme,Aspergillomarasmines A and B

Brush border membrane vesicles (BBMV) from the midgut epithelial cells of silkworm larvae were prepared. ATP hydrolyzing activity (ATPase activity) was associated with the BBMV. ATPase activity without Mg^{2 +} was not observed at pH 7 but substantial ATP hydrolyzing activity was observed at pH 7 with Mg^{2 +}. The enzyme required Mn^{2 +}, Mg^{2 +}, or Ca²⁺ ions. The enzyme also hydrolyzed ITP and GTP but not p-NPP, ADP, or AMP. KNO₃ and NEM strongly inhibited the ATPase activity. Behaviours of the ATPase against inhibitors suggested that it resembled vacuolar type ATPase.     

GTPase and transglutaminase are associated in the secretion of the rat anterior prostate.

We have found that in the secretion of rat anterior prostate, a hydrolyzing activity on GTP is present with a high affinity for the substrate; ATP, GDP, and ADP are not substrates for enzymatic activity. At the same time we have shown that GTP is a negative modulator for the well-known type IV transglutaminase activity present in the prostatic secretion. The hydrolyzing activity on GTP appears to be due to two molecular species: a high-molecular-weight GTPase, having electrophoretical mobility higher than 100 kDa, and a low-molecular-weight GTPase, of about 30 kDa. The two enzymatic activities are associated in the prostatic secretion with the transglutaminase (type IV). We describe an experimental procedure to separate them.     

Submitochondrial localization and function of alkaline inorganic pyrophosphatase in maize seedlings.

Alkaline inorganic pyrophosphatase and Mg-ATPase are localized within the mitoplast of maize seeding mitochondria. NaF inhibited the PPase activity, whereas oligomycin and dicyclohexylcarbodiimide inhibited the Mg-ATPase activity. The mitoplast preparation synthesized PPi from Pi under conditions excluding hydrolysis of endogenous ATP. PPi synthesis was inhibited by ADP, antimycin A, NaCN and 2,4- dinitrophenol but not by oligomycin. It is suggested that PPi synthesis in the maize seedling mitochondria proceeds at the expense of the energy of electron transport chain and is independent of the ATP synthesis.     

Characteristics of the nucleotide phosphohydrolases of hamster embryo cells

The characteristics and distribution of nucleotide phosphohydrolases of hamster embryo cells were examined. The hydrolysis of ATP and ADP by monolayers of hamster embryo cells was stimulated by Mg²⁺ and Ca²⁺ ions. In contrast, the hydrolysis of AMP was not increased by these ions. These observations suggest that the enzyme hydrolyzing AMP (ecto-AMPase) is different from the enzymes hydrolyzing ATP and ADP. About 70–90% of the total activity of the nucleotide phosphohydrolases of hamster embryo cells was localized on the surface of the cell membrane. These ecto-enzymes hydrolyzed a variety of nucleotides at rates comparable to those observed for the hydrolysis of AMP, ADP, and ATP. These findings indicated that the ecto-enzymes of hamster embryo cells have a low substrate specificity. The activities of the ecto-enzymes of tumor cells induced in the hamster by Rous sarcoma virus were greatly diminished or abolished in comparison to those of normal hamster embryo cells. This suggests that the ecto-nucleotide phosphohydrolases of hamster embryo cells may be suitable biochemical markers of transformation.     

Active vanadate-sensitive H+ translocation in corn roots membrane vesicles and proteoliposomes

A member fraction from corn roots which contains a vanadate-sensitive ATPase activity has been prepared. The specific activity at 38°C is between 3 and mol 12 μmol · min⁻¹ · mg⁻¹, depending on the age of roots. Addition of ATP promotes a very rapid quenching of the fluorescence of 9-amino-6-chloro-3-methoxy-acridin (ACMA). Proton pumping exhibits a delayed sensitivity to vanadate but is strongly and instantaneously inhibited by the new inhibitor SW 26. Both proton pumping, measured by the initial quenching rate, and ATP hydrolysis show maximum activities at ATP concentrations in the millimolar range, but the apparent K_m-value for hydrolysis is higher than that observed for proton pumping. This is interpreted as being due to the presence of two populations of ATPases, one of them hydrolyzing ATP without creating a pH-gradient. The vanadate-sensitive ATP hydrolysis and H⁺-pumping activity may be solubilized with lysolecithin and reconstituted into liposomes either by a freeze-thawing-sonication or an octylglucoside dilution procedure. Both methods yield proteoliposomes exhibiting very effecient proton pumping, which is more sensitive to vanadate (I₅₀ = 2 μM) or to SW 26 (I₅₀ = 0.5 μM) than that of the original membrane fractions.     

Hydrolysis of a phosphonate ester catalyzed by an enzyme from Dictyostelium discoideum.

An activity capable of hydrolyzing the phosphonate ester, 4-nitrophenyl phenylphosphonate has been detected in the eucaryotic microorganism Dictyostelium discoideum. This activity (4-nitrophenyl phenylphosphonate-esterase) was found in both membrane-enriched and soluble fractions. Two other activities present in this fraction, namely cyclic AMP-diesterase and an ATP-hydrolase, did not copurify with the 4-nitrophenyl phenylphosphonate-esterase. Also while ATP and cAMP inhibited the hydrolysis of 4-nitrophenyl phenlyphosphonate, they did so in a noncompetitive manner and, furthermore, saturating concentrations of 4-nitrophenyl phenylphosphonate had no effect on the hydrolysis of either cyclic AMP or ATP. The partially purified preparation was more active at pH 7 than at either pH 5 or 9, more active with acetate than chloride, and more active at 22 °C than at either 12 or 30 °C. The activity is inhibited by AMP in a competitive manner, but is not affected by levamisole, an inhibitor specific for alkaline phosphatase activity.     

Ecto-nucleotidases of ectonucleoside triphosphate diphosphohydrolase family: structure, localization and functional significance

Ecto-nucleotidases are enzymes of hydrolase class. They split extracellular nucleoside tri- and diphosphate. In this review a short history of these enzymes investigation, classification, structure, and functional significance of ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDase) has been presented. These enzymes are glycoproteins anchored in membranes. They do not form phosphorylated enzyme's form during catalytic circle, and (by analogy with membrane-bound ATPases) form homooligomeric ensembles. Activity of these enzymes depends on bivalent ions, in particular Ca2+ and Mg2+. E-NTPDases function in the composition of ecto-nucleotidase cascade that contains other nucleotide-hydrolyzing enzymes. They regulate P2-receptors by hydrolyzing its ligand specifically ATP. Both modern information and results of our investigation about influence of different endo- and exogenous factors on activity of these enzymes has been presented.     

Binding of an intrinsic ATPase inhibitor to the F(1)FoATPase in phosphorylating conditions of yeast mitochondria

Yeast mitochondrial ATP synthase has three regulatory proteins; ATPase inhibitor, 9K protein, and 15K protein. A mutant yeast lacking these three regulatory factors was constructed by gene disruption. Rates of ATP synthesis of both wild-type and the mutant yeast mitochondria decreased with decrease of respiration, while their membrane potential was maintained at 170-160 mV under various respiration rates. When mitochondrial respiration was blocked by antimycin A, the membrane potential of both types of mitochondria was maintained at about 160 mV by ATP hydrolysis. ATP hydrolyzing activity of F(1)FoATPase solubilized from normal mitochondria decreased in proportion to the rate of ATP synthesis, while the activity of the mutant F(1)FoATPase was constant regardless of changes in the rate of phosphorylation. These observations strongly suggest that F(1)FoATPase in the phosphorylating mitochondria is a mixture of two types of enzyme, phosphorylating and non-phosphorylating enzymes, whose ratio is determined by the rate of respiration and that the ATPase inhibitor binds preferentially to the non-phosphorylating enzyme.     

萌发绿豆子叶衰老期间的呼吸作用与线粒体功能的改变

暗中培养的绿豆幼苗子叶在萌发后3—4天时,外观出现衰老征状,6天后子叶凋落。随子叶日龄的增加,子叶的呼吸强度一直下降,呼吸商始终小于1。当外加L—苹果酸、a—酮戊二酸、琥珀酸和NADH为底物测定离体线粒体氧化活性时,衰老子叶的线粒体对上述四种底物的氧化活性有不同程度的增加;抗氰呼吸也有所升高。子叶衰老时,线粒体的ADP／O和呼吸控制(RC值均降低);线粒体ATPase水解ATP的活性升高。衰老绿豆子叶线粒体氧化磷酸化偶联效率的降低和ATPase水解活性的增强是与线粒体结构改变相联系的一种功能变化,它导致能量亏缺,并进一步加速了衰老的恶化进程。     

Characterization of Starch-Debranching Enzymes in Pea Embryos

Two distinct types of debranching enzymes have been identified in developing pea (Pisum sativum L.) embryos using native gel analysis and tests of substrate preference on purified or partially purified activities. An isoamylase-like activity capable of hydrolyzing amylopectin and glycogen but not pullulan is present throughout development and is largely or entirely confined to the plastid. Activities capable of hydrolyzing pullulan are present both inside and outside of the plastid, and extraplastidial activity increases relative to the plastidial activity during development. Both types of debranching enzyme are also present in germinating embryos. We argue that debranching enzymes are likely to have a role in starch metabolism in the plastid of the developing embryo and in starch degradation during germination.     

Extraction of an actomyosin-like pootein from amoebae of Dictyostelium discoideum

An actomyosin-like protein has been extracted from amoebae of Dictyostelium discoideum, V-12. The purified protein exhibited a reversible change in viscosity upon addition of ATP, indicating an ATP sensitivity of 75–85% and a specific viscosity of 0.1. At low ionic strength in the presence of Mg⁺⁺ and ATP the amoeba protein displayed the phenomenon of superprecipitation. The protein extract was found to be an adenosinetriphosphatase (ATP'ase) hydrolyzing ATP to ADP and inorganic phosphate. Both Mg⁺⁺ and Ca⁺⁺ at low ionic strength accelerated the ATP ase activity whereas at high ionic strength only Ca⁺⁺ stimulated ATP hydrolysis. The ATP'ase activity was inhibited by ethylene-diamine-tetraacetic-acid, Mersayl and p-chloromercuribenzoate. The physico-chemical and enzymatic properties of the extracted amoeba protein are qualitatively comparable to those of muscle actomyosin, and very similar in quantitative properties to smooth muscle actomyosin and the actomyosin-like proteins of blood platelets, leucocytes and slime mold plasmodia. The significance of the presence of this actomyosin-like protein in Dictyostelium amoebae is discussed in relation to amoeboid form and movement.     

Trophic interactions in the system host (eelpout Lota lota)—Parasite (Eubothrium rugosum)—Symbiotic microflora at hydrolysis of carbohydrate food components

It is established that bacteria with the ability to produce enzymes hydrolyzing carbohydrates of different complexity degrees are associated with the intestine digestive-transport surfaces of the eelpout Lota lota (L.) and of cestoids Eubothrium rugosum parasitizing in it. The release by bacteria of enzymes hydrolyzing not only complex carbohydrates, but also disaccharides, decreases the energy expenditures of the host and parasite for synthesis of their own hydrolases and increases the glucose concentration near the transport surfaces, which allows all members of this formed community to use glucose. The total amylolytic activity (TAA) of enzymes and the α-amylase activity of the studied bacteria are realized in a wide range of pH values. The levels of the enzyme general amylolytic activity and of bacterial α-amylase activity under the experiment conditions are comparable with similar characteristics of enzymes desorbed from the studied surfaces and participating in the membrane digestion processes in the host and parasite, which can suggest a significant contribution of the symbiotic microflora enzymes to digestion of the host and parasite.     

Relation of phosphatidylinositol metabolism to glycolytic pathway in skeletal muscle membranes

Skeletal muscle triads are possessing the whole set of enzymes of the phosphatidylinositol (PI)-linked signal generating pathway, PI-kinase, PI(4)P-kinase, and PI(4,5)P₂-phospholipase C (PLC). The activities of these enzymes are comparable to those found in other cell types for which a functional role of the PI-pathway in intracellular signal transduction has been established. For skeletal muscle an unequivocal function and an initiating signal for Ins(1,4,5)P₃-liberation is still unknown. However, the observed Ca-dependency of PLC activity suggests that here Ins(1,4,5)P₃ production is a consequence rather than a cause of increasing cytosolic Ca²⁺. Recently, the glycolytic enzyme aldolase, whose activity can be modulated by inositol polyphosphates, has been localized in the triadic structure. The enzyme which has a high affinity to Ins(1,4)P₂, Ins(1,4,5)P₃ and Ins(1,3,4,5)P₄, seems to be compartmentalized to the junctional foot structure from which it is released upon binding of these molecules. This phenomenon could reflect a capability for regulation of the glycolytic flux even for aldolase, especially if a non steady-state situation in the junctional gap is considered. Meanwhile we have accumulated evidence for the operation of a partial glycolytic sequence in the junctional region established by the enzymes aldolase, glyceraldehyde-3-P (GAP) dehydrogenase and phosphoglycerate kinase. This system is able to produce ATP upon oxidation of GAP and could be, because of the inositol polyphosphate-sensing abilities of aldolase, a target for the membrane associated PI-pathway. The ATP production is however transient which indicates the coupling to an ATP hydrolyzing reaction. Thus, it appears that the ATP produced by the membrane associated system is effectively utilized by an ATP consuming membrane localized system like PI-metabolism or protein kinases. There are indications that exogeneously added ATP does not equilibrate with the ATP synthesized in the junctional region which suggests an effective structural or kinetical compartmentalization of this system. Therefore it is hypothesized that the ATP synthesized by the membrane associated glycolytic sequence is utilized in membrane localized reactions.