Solubilization of adenylate cyclase from Ceratitis capitata neural membranes and incorporation into liposomes

Solubilization of the adenylate cyclase from neural membranes of the dipterous Ceratitis capitata, by using several detergents, and regulatory characteristics of the solubilized enzyme were examined. Triton X-100 is the most effective detergent in solubilizing this enzyme activity. The adenylate cyclase in Triton X-100-solubilized preparations (105,000 g supernatant) does not respond to either guanine nucleotides or fluoride and it apparently seems to be devoid of a functional regulatory component. When this preparation is centrifuged again at 300,000 g for 30 min no enzyme activity is detectable in the supernatant, however only 8% of total activity is recovered in the pellet. The activation pattern for the enzyme in the 300,000 g pellet is similar to that observed for the enzyme in the 105,000 g supernatant. Incorporation of solubilized enzyme into dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC) or cholesterol-enriched DOPC liposomes increases the 300,000 g pellet adenylate cyclase activity in a similar extension; thus, this increase in enzyme activity appears to be independent not only on the phospholipid composition but also on the liposome fluidity.     

Differential effects of non-ionic detergents on microsomal and sarcolemmal adenylate cyclase in cardiac muscle

1. About 4 and 23% of the homogenate adenylate cyclase activity was recovered in the microsomal and sarcolemmal fractions isolated from guinea-pig heart ventricles. 2. Cardiac microsomal adenylate cyclase activity [basal as well as p[NH]ppG (guanyl-5′-yl imidodiphosphate)- and NaF-stimulated] was increased over 2-fold in the presence of Lubrol-PX (0.01–0.1%). 3. The sarcolemmal enzyme, however, showed concentration-dependent inhibition caused by the detergent under all assay conditions, except when p[NH]ppG was included in the assay. In the latter case, the detergent (0.01–0.02%) caused a modest increase (30–45%) in enzyme activity. 4. Another non-ionic detergent, Triton X-100, also stimulated the microsomal cyclase and inhibited the sarcolemmal enzyme. 5. With either membrane fraction, Lubrol-PX solubilized the enzyme when the detergent/membrane protein ratio was 2.5 (μmol of detergent/mg of protein). 6. The findings with homogenate and a washed particulate fraction resembled those obtained with sarcolemma, and those with isolated sarcoplasmic reticulum resembled those with microsomal preparations. 7. p[NH]ppG, and to some extent NaF, protected the detergent-induced inactivation of the enzyme observed at higher detergent concentrations (0.5% Lubrol-PX and 0.05–0.5% Triton X-100). 8. In the absence of detergents, p[NH]ppG increased the basal enzyme activity about 2-fold in microsomal fractions, but did not appreciably stimulate the sarcolemmal enzyme. Isoproterenol, on the other hand, increased the sarcolemmal enzyme activity (>2-fold) in the presence of p[NH]ppG and caused only moderate stimulation (31%) of the microsomal enzyme under these conditions. 9. These findings support the view that, although the bulk of adenylate cyclase resides in heart sarcolemma (plasma membrane), the microsomal activity cannot be accounted for solely by contamination of the microsomal fraction with sarcolemma, as has been suggested by others [Besch, Jones & Watanabe (1976) Circ. Res. 39, 586–595; Engelhard, Plut & Storm (1976) Biochim. Biophys. Acta 451, 48–61]. Further, the results of this study show that cardiac sarcoplasmic-reticulum membranes possess this enzyme.     

Influence of the Linear Alkylbenzene Sulfonate (LAS) on hematological and biochemical parameters of Nile Tilapia,Oreochromis niloticus

The acute toxicity of household detergent (Ariel) on blood parameters and histology of Oreochromis niloticus was investigated using static bioassay for 96 h. Linear alkylbenzene sulfonate (LAS) is an anionic surfactant widely used in detergents and cleaners, both in industrial and household applications. LAS contaminating aquatic ecosystems as a potential toxic pollutant, was investigated in the present study for acute toxicity. The fish samples were divided into six groups, including 20 fish in each group. Normal feed was given to control group without detergents treatment. Hematological parameters (RBC count, Hb, Ht and platelets) were significantly declined, while WBC count showed a highly significant increase. Compared with the control group, significant elevation of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) was recorded in fish treated with different concentrations of detergent. Catalase (CAT), Superoxide dismutase (SOD) activities and Reduced Glutathione (GSH) concentration showed a highly significant reduction. Total proteins showed significant decrease, while total lipids, cholesterol and triglycerides significantly increased. The mean lethal concentration (LC₅₀) for 96 h of Ariel was at concentration 10 mg/L. Relative percentage of detergent residues in fish muscles was increased with higher detergent concentrations. In conclusion, exposure to detergents resulted in great alterations in the histological structure of liver and gills.     

The functional unit of calcium-plus-magnesium-ion-dependent adenosine triphosphatase from sarcoplasmic reticulum. The aggregational state of the deoxycholate-solubilized protein in an enzymically active form

Vesicles consisting of (Ca²⁺+Mg²⁺)-dependent ATPase (adenosine triphosphatase), and lipid were prepared from sarcoplasmic reticulum of rabbit skeletal muscle. As with non-ionic detergents [le Maire, Møller & Tanford (1976) Biochemistry 15, 2336–2342] the (Ca²⁺+Mg²⁺)-dependent ATPase after solubilization by deoxycholate showed a pronounced tendency to form oligomers in gel-chromatographic experiments, when eluted in the presence of deoxycholate and phosphatidylcholine. To evaluate the functional significance of oligomer formation the properties of enzymically active preparations of ATPase, solubilized by deoxycholate, were studied. Such preparations were obtained at a protein concentration of 2.5mg/ml in the presence of a high salt concentration (0.4m-KCl) and sucrose (0.3m) in the solubilization medium. Analytical ultracentrifugation of solubilized ATPase showed one protein boundary moving at the same rate as gel-chromatographically prepared monomeric ATPase (s_20,w=6.0S). From simultaneous measurements of the diffusion coefficient an apparent molecular weight of 133000 was calculated, consistent with solubilization of ATPase in predominantly monomeric form. The enzymic activity of deoxycholate-solubilized ATPase when measured directly in the solubilization medium at optimal Ca²⁺ and MgATP concentrations was about 35–50% of that of vesicular ATPase. The dependence of enzymic activity on MgATP concentration indicated that the solubilized ATPase retained high-affinity binding of MgATP, but the presence of high concentrations of the nucleotide did not stimulate activity further, in contrast with that of vesicular ATPase. The dependence of enzymic activity on the free Ca²⁺ concentration was essentially the same for both solubilized and vesicular forms, indicating that interaction of ATPase with more than one molecule of Ca²⁺ is required for enzyme activity. Solubilized enzyme at 20°C was phosphorylated to about the same degree as vesicular ATPase. It is concluded that the catalytic activity of monomeric ATPase retains most of the features of vesicular ATPase and that extensive oligomer formation in gel-chromatographic experiments in the presence of deoxycholate probably reflects processes taking place during inactivation and delipidation of the protein.     

Multiple sub-unit structure and microheterogeneity of α-galactosidase I from vicia faba seeds

Tetrameric α-galactosidase I from Vicia faba seeds is dissociated with urea (2.5-5.0 M) into active sub-unit forms. At low urea concentrations dissociation is only apparent if methyl α-D-mannoside is present which may be indicative of the involvement of lectin interactions in subunit aggregation. Chromatofocusing of α-galactosidase I yields multiple tetrameric forms with pI values ranging from 8.75 to 7.35. It is suggested that native α-galactosidase I is a closely related mixture of tetramers resulting from post-translational changes in the enzyme protein.     

Invertases of Lilium Pollen : Characterization and Activity during In Vitro Germination

Two different forms of invertase are found in pollen of lily (Lilium auratum). One form is cytoplasmic (Invertase 1) and the other is bound to the pollen wall (Invertase 2). Invertase 1 has been partially purified and is a glycoprotein (apparent molecular weight, 450 kilodaltons) with a K_m of 0.65 millimolar for sucrose. The two invertases differ in pH optimum and thermal stability. Invertases of lily pollen are β-fructofuranosidases which can hydrolyze sucrose but not melizitose. The mature pollen grains have enzyme activity in both cytoplasmic and wall fractions, and no increase in the activity of either occurs during germination. The wall-bound enzyme could not be released by treatments with detergents or high salt concentrations.     

The fully-active and structurally-stable form of the mitochondrial ATP synthase of Polytomella sp. is dimeric

Mitochondrial F₁F_O-ATP synthase of chlorophycean algae is a stable dimeric complex of 1,600 kDa. It lacks the classic subunits that constitute the peripheral stator-stalk and the orthodox polypeptides involved in the dimerization of the complex. Instead, it contains nine polypeptides of unknown evolutionary origin named ASA1 to ASA9. The isolated enzyme exhibited a very low ATPase activity (0.03 Units/mg), that increased upon heat treatment, due to the release of the F₁ sector. Oligomycin was found to stabilize the dimeric structure of the enzyme, providing partial resistance to heat dissociation. Incubation in the presence of low concentrations of several non-ionic detergents increased the oligomycin-sensitive ATPase activity up to 7.0–9.0 Units/mg. Incubation with 3% (w/v) taurodeoxycholate monomerized the enzyme. The monomeric form of the enzyme exhibited diminished activity in the presence of detergents and diminished oligomycin sensitivity. Cross-linking experiments carried out with the dimeric and monomeric forms of the ATP synthase suggested the participation of the ASA6 subunit in the dimerization of the enzyme. The dimeric enzyme was more resistant to heat treatment, high hydrostatic pressures, and protease digestion than the monomeric enzyme, which was readily disrupted by these treatments. We conclude that the fully-active algal mitochondrial ATP synthase is a stable catalytically active dimer; the monomeric form is less active and less stable. Monomer-monomer interactions could be mediated by the membrane-bound subunits ASA6 and ASA9, and may be further stabilized by other polypeptides such as ASA1 and ASA5. Alexa Villavicencio-Queijeiro and Miriam Vázquez-Acevedo have contributed equally to this work.     

β-Galactosidases of Lilium pollen

Lily (Lilium auratum) pollen contains very high levels of β-galactosidase. There are three forms: β-galactosidase I and II differ in M_r, while β-galactosidase III is firmly bound in the pollen wall. The two cytoplasmic forms were separated and partially purified using a combination of chromatography on DEAE-cellulose, Sephadex G-200 and Sepharose 6B. Forms I and II appear to be glycoprotein in nature as shown by binding to Con A-Sepharose. The three enzymes were optimally active near pH 4, and all were inhibited by galactose and galactonolactone. The wall-bound enzyme, β-galactosidase III effectively hydrolysed nitrophenyl β-galactosidase but not lactose, and could not be released from the wall polysaccharide matrix by high salt concentrations or detergents. The total β-galactosidase activity of lily pollen remained constant during in vitro germination. A possible role for this enzyme may be in degradation of stylar arabinogalactans providing a carbon source for pollen tube nutrition.     

Regulation of glutamine synthetase adenylylation and deadenylylation by the enzymatic uridylylation and deuridylylation of the PII regulatory protein

Regulation of glutamine synthetase activity in Escherichia coli is mediated by covalent attachment and detachment of an adenylyl group to each subunit of the enzyme [Kingdon, H. S. et al., Proc. Nat. Acad. Sci., 58, 1703, (1967); Wulff, K. D. et al., Biochem. Biophys. Res. Commun.28, 740, (1967)]. Adenylylation and deadenylylation of the enzyme are both catalyzed by a single adenylyltransferase (ATase) whose activity is modulated by various metabolites and by a regulatory protein, P_II [Shapiro, B. M., Biochemistry; Anderson, W. B. et al., Proc. Nat. Acad. Sci.67, 1761 (1970)].The present study confirms preliminary results [Brown, M. S. et al., Proc. Nat. Acad. Sci.68, 2949 (1971)] showing that: (1) the regulatory protein (P_II) exists in two interconvertible forms, P_IIA and P_IID, which, respectively, stimulate adenylylation and deadenylylation activity of ATase; (2) conversion of P_IIA to P_IID requires the presence of UTP, 2-oxoglutarate, ATP, and either Mg²⁺ or Mn²⁺; (3) this conversion involves covalent attachment of a uridine derivative to P_IIA. It is further established that the covalently bound uridine derivative is UMP which is derived from UTP in a reaction catalyzed by a specific uridylyltransferase (UTase). Removal of the covalently bound UMP from P_IID is catalyzed by a separate enzyme, referred to as the uridylyl-removing enzyme (UR-enzyme). This enzyme has an obligatory requirement for Mn²⁺.Regulation of glutamine synthetase activity in E. coli is thus facilitated by a highly sophisticated cascade system of proteins, consisting of an ATase, the regulatory protein (P_II), UTase, and the UR-enzyme. The activities of these various components is rigorously controlled by various metabolites, including glutamine, 2-oxoglutarate, ATP, P_i, UTP, and the divalent cations, Mn²⁺ and Mg²⁺.     

Synthesis,properties, and reactions of α- and β-D-glucopyranosyl esters of some tripeptides

The 2,3,4,6-tetra-O-benzyl-1-O-(N-benzyloxycarbonyltripeptidyl)-D-glucopyranoses 1, 8, and 13 were synthesised from 2,3,4,6-tetra-O-benzyl-α-D-glucopyranose and the active esters of the appropriate N-protected tripeptides (Gly-Gly-Gly-, L-Phe-Gly-Gly-, and Gly-Gly-L-Phe-) in the presence of imidazole; the anomeric mixtures were resolved and the α and β anomers characterised. The β anomer of 13, containing the L and D enantiomers (ratio ≈ 3:1) of Gly-Gly-Phe- as the aglycon, could be resolved by column chromatography into the pure isomeric forms. Catalytic hydrogenolysis of the β anomers, in the presence and absence of a strong acid, yielded the free 1-esters 2β, 9β, and 14β, which were characterised as the monooxalate or trifluoroacetate salts and as free bases. Similarly, the α anomers afforded 2α, 9α, and 14α, whereas omission of the strong acid led to accompanying 1→2 acyl migration, to give the 2-O-acyl derivatives. All of the compounds prepared were converted into the N-acetyl and/or peracetylated derivatives. The 1-esters 2β and 9β, both in the charged and uncharged form, and the trifluoroacetate salt of 14β, are susceptible to cleavage by β-D-glucosidase; the enzyme had no effect on the uncharged form of 14β. This difference between 14β and its salt is discussed in conformational terms.     

5-Substituted 3-chlorokenpaullone derivatives are potent inhibitors of Trypanosoma brucei bloodstream forms

Trypanothione synthetase is an essential enzyme for kinetoplastid parasites which cause highly disabling and fatal diseases in humans and animals. Inspired by the observation that N(5)-substituted paullones inhibit the trypanothione synthetase from the related parasite Leishmania infantum, we designed and synthesized a series of new derivatives. Although none of the new compounds displayed strong inhibition of Trypanosoma brucei trypanothione synthetase, several of them caused a remarkable growth inhibition of cultivated Trypanosoma brucei bloodstream forms. The most potent congener 3a showed antitrypanosomal activity in double digit nanomolar concentrations and a selectivity index of three orders of magnitude versus murine macrophage cells.     

A simplified method for analysis of inorganic phosphate in the presence of interfering substances.

A new phosphate analysis method is described which is designed primarily for use with routine phosphatase assays. The method is modified extensively from that of Fiske and SubbaRow [(1925) J. Biol. Chem.66, 375] by decreasing 10-fold the level of reducing agent, ANSA, increasing the concentration of ammonium molybdate to 4 mm, utilizing HCl as the only acid present, and incorporating SDS. The method is insensitive to pH, ATP, proteins, and detergents at concentrations normally encountered in enzyme purification and analysis experiments. The color developed is fivefold more stable than that from the original Fiske-SubbaRow method, and the standard curve retains linearity for at least 24 hr with acid concentrations between 0.6 and 1.0 n HCl and for wavelengths between 500 and 900 nm. The simplicity, stability, and general applicability of the method allow for convenient and rapid analysis of large numbers of samples under a wide variety of conditions.     

Biochemical and Electron Microscopic Studies of the Streptomyces reticuli Cellulase (Avicelase) in Its Mycelium-Associated and Extracellular Forms

Streptomyces reticuli is able to grow efficiently with crystalline cellulose (Avicel) as the sole carbon source. Cultivation in the presence of the nonionic detergent Tween 80 at a concentration of 0.1% led to a 10-fold increase in extracellular cellulolytic activity. Under these conditions, one single 82-kDa cellulase (Avicelase) capable of degrading crystalline and soluble cellulose as well as cellodextrins and p-nitrophenylcellobioside was purified to apparent homogeneity by a procedure which consisted of two consecutive anion-exchange chromatographies followed by chromatofocusing. Aggregation, which was a major problem during protein purification, could be avoided by including Triton X-100 at a concentration of 0.1% in every chromatographic step. The Avicelase was identified in extracellular and mycelium-associated forms, the latter of which could be released efficiently by nonionic detergents. In addition, a 42-kDa truncated form retaining cellulolytic activity was identified which had been generated from the 82-kDa enzyme by a protease. Antibodies raised against the mycelium-associated Avicelase reacted with the 42-kDa derivative and the extracellular form. The mycelial association of the enzyme was confirmed by immunofluorescence and immunoelectron microscopies.     

Maintenance of respiratory control by beef heart mitochondria incubated at 25 degrees C: response to protective agents and to protective agents and to prior stress.

Lysophospholipase D (EC 3.1.4.-) activity was demonstrated in rat kidneys, intestines, lungs, testes, and liver. The liver enzyme was studied in greatest detail and its labeled products were identified by chemical and Chromatographic techniques. This enzyme hydrolyzes 1-[1-¹⁴C]hexadecyl-sn-glycero-3-phosphoethanolamine and 1-[1-¹⁴C]hexadecyl-sn-glycero-3-phosphocholine to yield 1-[1-¹⁴C]hexadecyl-sn-glycero-3-phosphate; the initial product is subsequently dephosphorylated by a phosphohydrolase in microsomes to form 1-[1-¹⁴C]hexadecyl-sn-glycerol. The possibility that phospholipase C and a phosphotransferase were responsible for the formation of 1-[1-¹⁴C]hexadecyl-sn-glycero-3-phosphate was ruled out. Neither 1-[1-¹⁴C]hexadecyl-2-acyl-sn-glycero-3-phosphoethanolamine nor 1-[1-¹⁴C]hexadecyl-2-acyl-sn-glycero-3-phosphocholine was hydrolyzed. The enzyme requires Mg²⁺, is inhibited by Ca²⁺, and is stimulated by high salt concentrations; it is localized in the microsomal fraction and has a pH optimum between 7.0 and 7.6. Inhibition by sulfhydryl reagents and protection by glutathione and dithiothreitol suggest that a sulfhydryl group is required for activity. The enzyme is inhibited by detergents and by organic solvent extraction. It appears to be tightly bound to the microsomes, since repeated freeze-thawing or sonication did not release the activity, and trypsin digestion (either in the presence or in the absence of 0.04% deoxycholate) did not destroy the activity. Lysophospholipase D was previously known to occur only in brain (R. L. Wykle and J. M. Schremmer, 1974, J. Biol. Chem., 249, 1742–1746).     

A simple micro-assay for inorganic phosphate

Modification of two assay procedures (Van Belle, H. (1970) Anal. Biochem.33, 132–142 and Itaya, K., and Ui, M. (1966) Clin. Chim. Acta14, 361) has allowed the development of a manual assay for inorganic phosphate of high simplicity and sensitivity. Total analysis requires only three reagents and is accomplished in less than 5 min, and smaples containing less than 1 μg/ml of inorganic phosphate may be detected. This assay retains a unique principle of the former two, complexation (instead of reduction) of the phosphomolybdate heteropoly complex with an appropriate triphenylmethane dye (malachite green, methyl green). Use of detergents has been eliminated and some further properties of the dyes, the assay, and the latter's applicability to a coupled enzyme system for phosphomonoester and phosphodiester analysis are discussed. Consideration is also given to the associated phenomena of transphosphorylation. (Dayan, J., and Wilson, I. B. (1964) Biochim. Biophys. Acta81, 620–623).     

Choline acetyltransferase from a temperature-sensitive mutant of caenorhabditis elegans

A temperature dependent paralytic mutant of C. elegans was isolated and mapped to be an allele of the cha-1 gene that has been shown to be the structural gene for acetyl-CoA: choline-O-acetyltransferase (EC 2.3.1.6; ChAT) (Hosono et al., J. Exp. Zool.235, 409–421, 1985; Rand and Russell, Genetics106, 227–248, 1984). In crude extracts from the mutant, ChAT activity was present when assayed at a permissive temperature but not detectable at a temperature that provoked abnormal phenotypes. The mutant ChAT was purified to a specific activity of 2.9 nmol of product min ^-1 per mg of protein at 10°C and its enzymatic properties were studied by comparison with the wild-type enzyme. The temperaturesensitivity of the mutant ChAT was so remarkable that no activity was detected over 20°C. This inactivation at higher temperature appeared to be partly reversible. The Km values of the mutant enzyme for choline and acetyl-CoA were about twice of those in the wild-type enzyme, but increased 10- to 20-fold in the presence of high salt concentrations. The mutant enzyme was also more sensitive to sulfhydryl reagents. These findings indicate that depending upon changes in the physical environment, the mutant ChAT may lose the normal-conformation leading to inactivation.     

The phospholipid fatty acids of Porphyridium purpureum cultured in the presence of triton x-100 and sodium desoxycholate

The phospholipid fatty acid composition of Porphyridium purpureum grown on a solid medium was studied in the presence of Triton X-100 (TX) and sodium desoxycholate (SDC). The most common fatty acids in PC and PE were palmitic (16:0), stearic (18:0), linoleic (18:2ω6), arachidonic (20:4ω6) and eicosapentaenoic (20:5ω3) acids, 20:4ω6 being very abundant. In PG the most common acids were 16:0, trans-hexaenoic acid (tr16:1ω3), oleic acid (18:1) and 20:4ω6. Both detergents caused an increase in the saturation of PC and, to a lesser extent, of PE. The relative amounts of short chain fatty acids increased. Both detergents increased the amounts of 16:0 and, correspondingly, decreased the amounts of 20:4ω6. In PG the amounts of both 16:0 and tr 16:1ω3 increased and the amounts of 18:0, 18:2ω6 and 20:4ω6 decreased in the presence of detergents. The changes were always greatest at the concentrations of 5–10 ppm TX or SDC. At 20 ppm the fatty acid compositions, especially with SDC, were very similar to the controls, which suggests a change in the detergent effect between 10–20 ppm. The normal PC/PE ratio was 5.6 and the (PC+ PE)/PG ratio 39.0. Both detergents caused a marked decrease in these ratios. Because the detergent effects are not linear, it seems that even very low detergent concentrations have an important influence on algae in polluted waters.     

Phosphofructokinase in the plerocercoids of Schistocephalus solidus (Cestoda:Pseudophyllidea)

Beis I. and Theophilidis G. 1982. Phosphofructokinase in the plerocercoids of Schistocephatus solidus (Cestoda: Pseudophyllidea). International Journal for Parasitology12: 389–393. The Phosphofructokinase from the plerocercoids of Schistocephalus solidus was found to be inhibited by ATP. AMP relieves the ATP inhibition and activates the enzyme. In contrast to mammalian phosphofructokinase, the plerocercoid enzyme does not appear to be sensitive to inhibition by citrate at physiological ATP concentrations. Except for AMP and 3'–5' cyclic AMP no other monophosphate nucleotides were found to activate the enzyme. Succinate, α-ketoglutarate, malate, isocitrate, β-hydroxybutyrate, butyrate, acetoacetate and CoA all inhibit the plerocercoid enzyme at the concentrations tested. The significance of these results in the regulation of glycolysis and the tricarboxylic acid cycle in this parasite is discussed.     

Equilibria of the anomeric and ring forms of ammonium 3-deoxy-d-manno-octulosonate in deuterium oxide

The tautomeric composition of a solution of ammonium 3-deoxy-d-manno-octulosonate (KDO, 1a) in D₂O at 28° was assessed by means of ¹³ C-F.t.-n.m.r. spectroscopy. The results revealed the presence of 6?0 and 11 % of the α and β anomers of the pyranose, and 20 and 9 % of the two furanoses, and suggested, but did not unequivocally prove, that the major furanose form is the α anomer. To facilitate interpretation of the spectral results for 1, ammonium 3,5-dideoxy-d-arabino(or ribo)-octulosonate (3a) was prepared by the reaction of 5-deoxy-d-erythro-pentose with sodium oxalacetate at pH 11. A chromatographically homogeneous, noncrystalline sample of 3 was obtained by lyophilization, and characterized as its (4-nitrophenyl)hydrazone (m.p. 162-163°). The ¹³C-n.m.r. spectrum of a solution of 3a in D₂O revealed it to be substantially all in the α-pyranose form. No signals were obtained for the possible 1,4-lactone of 3. As the 1,5-lactone and furanose forms are impossible for 3, it exhibited no signals analogous to those attributed to furanoid 1. On the basis of these results for 3, the two lactone forms of 1 were excluded from consideration, and the three pairs of ¹³C-n.m.r. signals observed at ≈45, 86, and 104 p.p.m. were assigned to the furanose forms of 1.     

The mode of activation by potassium of potassium-dependent and ouabain-sensitive phosphatase activity.

A new simple procedure has been developed for the purification of plasma membranes from rabbit kidney microsomes which yields a three- to fourfold increase in the specific activity of Na⁺-K⁺-adenosine triphosphatase (ATPase). The procedure differs from previous methods with deoxycholate or other detergents and does not change the molecular activity of the ATPase. The K⁺-dependent p-nitrophenylphosphatase activity of the native Na⁺-K⁺-ATPase is controlled more effectively by Mg²⁺ in the presence of K⁺ at concentrations higher than that of Mg²⁺, and by K⁺ in the presence of Mg²⁺ at concentrations higher than that of K⁺. The enzyme in its Mg²⁺-regulating state, which shows K⁺-saturation curves with a Hill coefficient of 1, is less sensitive to ouabain (I_0.5 = 90 μM) and corresponds to the enzyme conformation reported previously which is inhibited by the concurrent presence of Na⁺ and ATP or of Na⁺ and oligomycin (I_0.5 is the midpoint of the saturation curve). The enzyme in its K⁺-regulating state, which shows K⁺-saturation curves with a Hill coefficient of 2, is more sensitive to ouabain inhibition (I₀₅ = 8 μM) and corresponds to the enzyme conformation which is stimulated by the concurrent presence of Na⁺ and ATP or of Na⁺ and oligomycin. There appear to be two conformations of the enzyme that are regulated by Mg²⁺ binding on the inhibitory sites of the enzyme.