L'adeninephosphoribosyltransferase des pousses de topinambour Helianthus tuberosus

An extract from Jerusalem artichoke shoots exhibited important adeninephosphoribosyltransferase activity. Only partial purification was possible because of the great instability of the enzyme. Phosphate ions and thiol reducing substances were necessary to stabilize it. The optimal temperature and pH were 40–45° and 5.5 to 6.5. The enzyme showed an absolute requirement for divalent cation: Mn²⁺ > Mg²⁺ = CO²⁺ = Zn²⁺ ? Ca²⁺. Kinetic studies gave K_m values of 6.4 × 10^?1 M for phosphoribosylpyrophosphate (PRPP) and 5.5 × 10^?6 M for adenine. AMP exercised a strong product inhibition, competitive towards PRPP (K_i = 10^?4 M). Inhibition by phosphate and pyrophosphate ions was also observed. The results suggested that the adeninephosphoribosyltransferase of Helianthus tuberosus has a key role in the purine salvage pathway.     

Properties of spinach chloroplast fructose-1,6-diphosphatase

Photosynthetic fructose-1,6-diphosphatase (FDPase) fractions I and II, earlier purified from spinach leaves, show a similar amino acid composition, with the exception of a higher glutamic acid content in the latter. In both fractions glutamic and aspartic acids are the main amino acids. pH activity profiles of fractions I and II are similar, with optima at 8·65–8·70, both showing a high specificity for fructose- 1,6-diphosphate. These two fractions are Mg²⁺-dependent for activity, with an Optimum Mg²⁺ concentration of 10 mM in standard conditions, which shifts to 5 mM when the MG²⁺/EDTA ratio is increased to 10; Mn²⁺ and Co²⁺ are slightly active. EDTA enhances FDPase activity slightly, with an optimum at 0·4–0·8 mM. Cysteine has no activating effect, and acts as an inhibitor above 10 mM. Both I and II have an optimum substrate concentration of 4 mM, and the substrate inhibits at concns above this value. Kinetic velocity curves are sigmoidal, with the concave zone located in the range of physiological substrate concns. (Hill coefficient 1·75 for both). This suggests a strong regulatory role of fructose-1,6-diphosphate. K_m values are 1·4 × 10⁻³ M (fraction I) and 1·1 × 10⁻³ M (fraction II). The highest activity rate occurs at 60°, in accordance with the high thermostability of both fractions; the activation energies are 14·3 kcal/mol (fraction I) and 13·0 kcal/mol (fraction II).     

Purification and properties of agmatine iminohydrolase from groundnut cotyledons

Agmatine iminohydrolase was purified ca 375-fold from groundnut cotyledons. The enzyme exhibited an optimum pH between 5.5 and 8.5 and the energy of activation was 22 kcal/mol. The K_m for agmatine was (7.57 ± 0.77) × 10^?4 M. The enzyme was inhibited by tryptamine, putrescine, cadaverine, spermidine and spermine. Inhibition by cadaverine and spermidine was competitive. The K_i values for cadaverine and spermidine were 4.1 × 10^?3 and 7.5 × 10^?4 M, respectively.     

Three‐dimensional (3‐D) fluorescence spectroscopy analysis of the fluorescent dissolved organic matter released by the marine toxic dinoflagellate Alexandrium catenella exposed to metal stress by zinc or lead

We investigated the effects of zinc or lead on growth and on exudation of fluorescent dissolved organic matter (FDOM) by the marine toxic dinoflagellate Alexandrium catenella (Whedon & Kofoid) Balech. The species was exposed to increasing free zinc (1.34 × 10^?7 M–3.98 × 10^?6 M) or lead (5.13 × 10^?9 M–1.82 × 10^?7 M) concentra‐tions. Low metal levels ([Zn²⁺] = 1.34 × 10^?7 M; [Pb²⁺] = 5.13 × 10^?9 M) had no effect on cell growth. Toxic effects were observed from higher metal contamination ([Zn²⁺] = 3.98 × 10^?6 M; [Pb²⁺] = 6.54 × 10^?8 M), as a conversion of vegetative cells into cysts. Analysis of the released FDOM by three‐dimensional (3‐D) fluorescence spectroscopy was achieved, using the parallel factor analysis (PARAFAC). The PARAFAC modeling revealed four components associated with two contributions: one related to the biological activity; the other linked to the organic matter decomposition in the culture medium. The C1 component combined a tryptophan peak and characteristics of humic substances, whereas the C2 component was considered as a tryptophan protein fluorophore. The two others C3 and C4 components were associated with marine organic matter production. Relea‐sed fluorescent substances were induced by low ([Zn²⁺]= 1.34 × 10^?7 M; [Pb²⁺] = 5.13 × 10^?9 M) and moderate ([Zn²⁺] = 6.21 × 10^?7 M; [Pb²⁺] = 2.64× 10^?9 M) metal concentrations, suggesting the activation of cellular mechanisms in response to metal stress, to exudate FDOM that could complex metal cations and reduce their toxicity toward A. catenella cells.     

Properties of a repressible alkaline phosphatase secreted by the wild-type strain 74a of neurospora crassa

A repressible extracellular alkaline phosphatase (with activity increasing steadily even up to pH 10.5) was purified from cultures of the wild-type strain 74A of Neurospora crassa, after growth on acetate and under limiting amounts of inorganic phosphate for 72 hr at 30°. The enzyme was homogeneous on polyacrylamide gel electrophoresis (PAGE) with or without sodium dodecyl sulphate (SDS). The MW was ca 172 000 and 82 000 as determined by Sephadex G-200 gel filtration and SDS-PAGE, respectively. The enzyme contained 23.6% neutral sugars, cations were not required for activity, and it was not inactivated by 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) at pH 8. Kinetic data showed Michaelian behaviour for the enzymatic hydrolysis of 4-nitrophenyl disodium orthophosphate (PNP-P) at pH 9 (the K_m value and Hill coefficient were 2.2 × 10^?4 M and 0.95, respectively). It was also shown that, at pH 9, the apparent number of Pi bound per dimer molecule equalled one, with a K_i value of 7.0 × 10^?4 M. The secreted enzyme showed half-lives of 23.5, 49.0 and 23.5 min at, pH 5.4, 7.4 and 9.0, respectively, after thermal inactivation at 60°. At pH 5.4, the half-life value was quite similar, while the others were respectively 2 and 4 times greater than those previously described for the repressible alkaline phosphatase retained by the mycelium at pH 5.6 or secreted by ‘slime’ cells.     

Toxins of the sea anemone Epiactis prolifera

Fe³⁺-EDTA chelates react with the superoxide radical at physiological pH values (k = 1.3 × 10⁶M^?1 s^?1 at pH 7 but is lower at more alkaline pH values) but do not appear to catalyze O₂^? dismutation at a significant rate. Complexes of Fe³⁺ with desferrioxamine, bathophenanthroline, or diethylenetriaminepentaacetic acid react much more slowly, if at all. Fe²⁺ complexes of EDTA, ATP, and diethylenetriaminepentaacetic acid also react with O₂^? at alkaline pH values. The significance of these reactions in the mechanism of the “iron-catalyzed Haber-Weiss reaction” is discussed.     

Equilibrium constants under physiological conditions for the reactions of the nonphosphorylated pathway of L-serine biosynthesis

The observed equilibrium constants (K_obs) for the reactions of d-2-phosphoglycerate phosphatase, d-2-Phosphoglycerate^3? + H₂O → d-glycerate^? + HPO₄^2?; d-glycerate dehydrogenase (EC 1.1.1.29), d-Glycerate^? + NAD⁺ → NADH + hydroxypyruvate^? + H⁺; and l-serine:pyruvate aminotransferase (EC 2.6.1.51), Hydroxypyruvate^? + l-H · alanine^± → pyruvate^? + l-H · serine^±; have been determined, directly and indirectly, at 38 °C and under conditions of physiological ionic strength (0.25 m) and physiological ranges of pH and magnesium concentrations. From these observed constants and the acid dissociation and metal-binding constants of the substrates, an ionic equilibrium constant (K) also has been calculated for each reaction. The value of K for the d-2-phosphoglycerate phosphatase reaction is 4.00 × 10³m [ΔG⁰ = ?21.4 kJ/mol (?5.12 kcal/mol)]([H₂0] = 1). Values of K_obs for this reaction at 38 °C, [K⁺] = 0.2 m, I = 0.25 M, and pH 7.0 include 3.39 × 10³m (free [Mg²⁺] = 0), 3.23 × 10³m (free [Mg²⁺] = 10^?3m), and 2.32 × 10³m (free [Mg²⁺] = 10^?2m). The value of K for the d-glycerate dehydrogenase reaction has been determined to be 4.36 ± 0.13 × 10^?13m (38 °C, I = 0.25 M) [ΔG⁰ = 73.6 kJ/mol (17.6 kcal/mol)]. This constant is relatively insensitive to free magnesium concentrations but is affected by changes in temperature [ΔH⁰ = 46.9 kJ/mol (11.2 kcal/mol)]. The value of K for the serine:pyruvate aminotransferase reaction is 5.41 ± 0.11 [ΔG⁰ = ?4.37 kJ/mol (?1.04 kcal/mol)] at 38 °C (I = 0.25 M) and shows a small temperature effect [ΔH⁰ = 16.3 kJ/ mol (3.9 kcal/mol)]. The constant showed no significant effect of ionic strength (0.06–1.0 m) and a response to the hydrogen ion concentration only above pH 8.5. The value of K_obs is 5.50 ± 0.11 at pH 7.0 (38 °C, [K⁺] = 0.2 m, [Mg²⁺] = 0, I = 0.25 M). The results have also allowed the value of K for the d-glycerate kinase reaction (EC 2.7.1.31), d-Glycerate^? + ATP^4? → d-2-phosphoglycerate^3? + ADP^3? + H⁺, to be calculated to be 32.5 m (38 °C, I = 0.25 M). Values for K_obs for this reaction under these conditions and at pH 7.0 include 236 (free [Mg²⁺] = 0) and 50.8 (free [Mg²⁺] = 10^?3m).     

Purification and Characterization of two Extracellular β-Glucosidases from Trichoderma viride ITCC 1433

T. viride ITCC 1433 synthesizes a two component system for the hydrolysis of cellobiose and cellooligodextrins. 80% of the total activity are solubilized during growth. The large protein (A), mol. weight 98 000 d, is glycosylated and slightly acidic (pH = 6.1). The smaller protein (B), mol. weight 70 000 d, is unglycosylated and neutral (pH = 7.2). Both proteins form a two-step system where β-glucosidase A is active at low substrate concentrations (K_M = 2.3 × 10^?4 M cellobiose) while β-glucosidase B covers the range of 10-fold higher cellobiose concentrations (K_M = 1.8 × 10^?3 M). The enzymes are fairly stable with a residual activity of 70% at 50°C after 24 h.     

Kinetics of reduction of ferrioxamine B by chromium(II), vanadium(II), and dithionite

Kinetic studies of the reduction of ferrioxamine B (Fe(Hdesf)⁺) by Cr(H₂O)₆²⁺, V(H₂O)₆²⁺, and dithionite have been performed. For Cr(H₂O)₆²⁺ and V(H₂O)₆²⁺, the rate is ?d[Fe(Hdesf)⁺]/dt = k[Fe(Hdesf)⁺][M²⁺]. For Cr(H₂O)₆²⁺, k = 1.19 × 10⁴ M^?1 sec^?1 at 25°C and μ = 0.4 M, and k is independent of pH from 2.6 to 3.5. For V(H₂O)₆²⁺, k = 6.30 × 10² M^?1 sec^?1 at 25°C, μ = 1.0 M, and pH = 2.2. The rate is nearly independent of pH from 2.2 to 4.0. For Cr(H₂O)₆²⁺ and V(H₂O)₆²⁺, the activation parameters are ΔH^≠ = 8.2 kcal mol^?1, ΔS^≠ ?12 eu and ΔH^≠ = 1.7 kcal mol^?1, ΔS^≠ = ?40 eu (at pH 2.2) respectively. Reduction by Cr(H₂O)₆²⁺ is inner-sphere, while reduction by V(H₂O)₆²⁺ is outer-sphere. Reduction by dithionite follows the rate law ?d[Fe(Hdesf)⁺]/dt =kK^{$\text{1}\text{2}$}[Fe(Hdesf)⁺][S₂O₄^2?]^{$\text{1}\text{2}$} where K is the equilibrium constant for dissociation of S₂O₄^2? into SO₂^? radicals. The value of k at 25°C and μ = 0.5 is 2.7 × 10³ M^?1 sec^?1 at pH 5.8, 3.5 × 10³ M^?1 sec^?1 at pH 6.8, and 4.6 × 10³ M^?1 sec^?1 at pH 7.8, and ΔH^≠ = 6.8 kcal mol^?1 and ΔS^≠ = ?19 eu at pH 7.8.     

Catalytic properties of three lactate dehydrogenases from potato tubers (Solanum tuberosum)

Two l-lactate dehydrogenase isoenzymes and one dl-lactate dehydrogenase could be separated from potato tubers by polyacrylamide-gel electrophoresis. The enzymes are specific for lactate, while β-hydroxybutyric acid, glycolic acid, and glyoxylic acid are not oxidized. Their pH optima are pH 6.9 for the oxidation and 8.0 for the reduction reaction.The K_m values for l-lactate for the two isoenzymes are 2.00 × 10^?2 and 1.82 × 10^?2, m. In the reverse reaction the affinities for pyruvate are 3.24 × 10^?4 and 3.34 × 10^?4, m. Both enzymes have similar affinities for NAD and NADH (3.00 × 10^?4; 4.00 × 10^?4, and 8.35 × 10^?4; 5.25 × 10^?4, m).The dl-lactate oxidoreductase may transfer electrons either to NAD or N-methyl-phenazinemethosulfate. The K_m values of this enzyme for l-lactate are 4.5 × 10^?2, m and for d-lactate 3.34 × 10^?2, m. Its affinity for pyruvate is 4.75 × 10^?4, m. The enzyme is inhibited by excess NAD (K_m = 1.54 × 10^?4, M) and has an affinity toward NADH (K_m = 5.00 × 10^?3, M) which is about one tenth of that of the two isoenzymes of l-lactate dehydrogenase.     

Soybean alcohol dehydrogenase

Alcohol dehydrogenase was prepared from germinating soybean seeds. Specific activity was increased from 511 to 31316 units. The coenzyme is NAD with a K_m of 10^?4M. Allyl alcohol is oxidized faster than ethanol; with the latter substrate, the K_m is 1.3 × 10^?2M, and the pH optimum 8.7. The enzyme catalyses acetaldehyde reduction, with a K_m of 10^?2M and a pH opt of 7.1. The MW is 53(±5) × 10^?3.     

Properties of fructose-1,6-diphosphate phosphatase and fructose-1,6-diphosphate aldolase fromPseudomonas putida

The fructose-1,6-P₂ (FDP) phosphatase, (FDPase) and FDP aldolase fromPseudomonas putida were partially purified by a combination of (NH₄)₂SO₄ fractionation and DEAE-Sephadex column chromatography. Michaelis-Menten kinetics were observed with, respect to FDP in both FDPase and FDP aldolase. TheK _m for FDP at pH 8.0 was 1.2×10⁻⁵M for FDPase and 3.0×10⁻⁵M for FDP aldolase. The specific activities of these two enzymes (assayed under optimal conditions in cell-free extracts ofP. putida grown ond-fructose), as well as their kinetic properties, are consistent with the suggestion that during growth ond-fructose most, of the FDP generated is converted to fructose-6-P (F-6-P), which is subsequently utilized via the Entner-Doudoroff pathway (EDP).     

The isolation of pig liver monoamine oxidase

Monoamine oxidase from pig liver has been isolated and purified approximately three hundred-fold. This enzyme has a molecular weight of 1,200,000, is highly polymeric, and contains subunits of molecular weight 146,000, as determined by Sephadex chromatography. The apparent K_m at 25°C is 1.28 × 10^?6 M at pH 9.0 (0.05 M glycine) and 1.74 × 10^?5 M at pH 7.2 (0.2 M phosphate) using benzylamine as a substrate. This enzyme contains approximately 8 copper(II) ions per 1,200,000 molecular weight.     

An inducible hydrolase from Mortierella isabellina (basidiomycetes). The deacylation of (+)-usnic acid

An inducible enzyme catalysing the hydrolysis of (+)-usnic acid to (+)-2-desacetylusnic acid and acetic acid has been purified 150-fold from the mycelium of Mortierella isabellina grown in the presence of (+)-usnic acid. Purification was achieved by treatment with protamine sulfate, (NH₄)₂SO₄ fractionation, negative adsorption on alumina Cγ gel and hydroxylapatite followed by chromatography on DEAE-cellulose and Sephadex G-200. The elution pattern from a Sephadex G-200 column indicated a MW of ca 7.6 × 10⁴ for the enzyme. The apparent K_m value for (+)-usnic acid at the pH optimum (pH 7) was 4.0 × 10^?5 M. The enzyme was specific for (+)-usnic acid and inactive towards (?)-usnic acid, (+)-isousnic acid or certain phloracetophenone derivatives. Its activity was enhanced in the presence of divalent metal ions such as Co²⁺, Ni²⁺, Mn²⁺, Mg²⁺ and Zn²⁺.     

Partial purification of nitrilase from Chinese cabbage

Nitrilase was purified ca 28-fold from Chinese cabbage seedlings. K_m values of 5.2 × 10^?4 and 2.6 × 10^?3 M were obtained for indoleacetonitrile (IAN) and 3-cyanopyridine (3-CP) as substrates. For hydrolysis of 3-CP, the maximal velocity was 44 times higher than for the natural substrate IAN. The pH optimum is at 7.5. IAA concentrations from 10^?6 to 10^?3 M did not inhibit the partially purified enzyme. Nitrilase activity was investigated during development of seedlings grown under continuous light. Roots with hypocotyls exhibited only slightly lower activity than cotyledons based on fresh weight, although their specific activity was ca 5 times higher. Etiolated seedlings showed a very similar distribution of nitrilase activity. The significance of the results for IAA biosynthesis is discussed.     

pH effects on growth and lipid accumulation of the biofuel microalgae Nannochloropsis salina and invading organisms

Biofuels derived from non-crop sources, such as microalgae, offer their own advantages and limitations. Despite high growth rates and lipid accumulation, microalgae cultivation still requires more energy than it produces. Furthermore, invading organisms can lower efficiency of algae production. Simple environmental changes might be able to increase algae productivity while minimizing undesired organisms like competitive algae or predatory algae grazers. Microalgae are susceptible to pH changes. In many production systems, pH is kept below 8 by CO₂ addition. Here, we uncouple the effects of pH and CO₂ input, by using chemical pH buffers and investigate how pH influences Nannochloropsis salina growth and lipid accumulation as well as invading organisms. We used a wide range of pH levels (5, 6, 7, 8, 9, and 10). N. salina showed highest growth rates at pH 8 and 9 (0.19?±?0.008 and 0.19?±?0.011, respectively; mean ± SD). Maximum cell densities in these treatments were reached around 21 days into the experiment (95.6?×?10⁶?±?9?×?10⁶ cells mL^?1 for pH 8 and 92.8?×?10⁶?±?24?×?10⁶ cells mL^?1 for pH 9). Lipid accumulation of unbuffered controls were 21.8?±?5.8 % fatty acid methyl esters content by mass, and we were unable to trigger additional significant lipid accumulation by manipulating pH levels at the beginning of stationary phase. Ciliates (grazing predators) occurred in significant higher densities at pH 6 (56.9?±?39.6?×?10⁴ organisms mL^?1) than higher pH treatments (0.1–6.8?×?10⁴ organisms mL^?1). Furthermore, the addition of buffers themselves seemed to negatively impact diatoms (algal competitors). They were more abundant in an unbuffered control (12.7?±?5.1?×?10⁴ organisms mL^?1) than any of the pH treatments (3.6–4.7?×?10⁴ organisms mL^?1). In general, pH values of 8 to 9 might be most conducive to increasing algae production and minimizing invading organisms. CO₂ addition seems more valuable to algae as an inorganic carbon source and not as an essential mechanism to reduce pH.     

The influence of Al3+ ion on porcine pepsin activity in vitro

The in vitro effect of Al³⁺ ions in the concentration range 1.7·10^{? 6} M–8.7·10^{? 3} M on pepsin activity at pH 2, via kinetic parameters and its electrophoretic mobility was evaluated. Kinetic study demonstrated the existence of an activation effect of Al³⁺ at pH 2 on pepsin molecule. Kinetic analysis with respect to concentrations of haemoglobin showed that Al³⁺ ions increase the maximal velocity (V_max) and k_cat values rather than apparent affinity for substrate (K_S) implying the non-competitive nature of activation which indicated that aluminium was a non-essential activator of partial non-competitive type. The values of the equilibrium constants K_S and K_mA for dissociation of corresponding complexes were evaluated as 0.904 ± 0.083 mM and 8.56 ± 0.51 μM, respectively. Dissociation constant K_A, of activator from enzyme-activator complex calculated via kinetic and direct measurement of Al³⁺ binding data, as well as activation constant A₅₀, the activator concentration that gives a rate equal to half at a saturating concentration of activator, were found to be 8.82 ± 0.90 μM, 8.39 ± 0.76 μM, and 8.05 ± 0.48 μM respectively. Native PAGE electrophoresis shows the decrease in electrophoretic mobility of pepsin and confirms modification of the electric charge and conformational changes of pepsin caused by bound Al³⁺ on the pepsin molecule. Al³⁺ induced conformational changes of pepsin were verified by UV-VIS and IR spectra. Moreover, the absence of conformational changes in the haemoglobin molecule in the presence of Al³⁺ ions confirms that the obtained activation is a consequence of conformational changes caused only in the pepsin molecule.     

The p-nitrophenyl phosphatase activity of muscle carbonic anhydrase

Carbonic anhydrase III from rabbit muscle, a newly discovered major isoenzyme of carbonic anhydrase, has been found to be also a p-nitrophenyl phosphatase, an activity which is not associated with carbonic anhydrases I and II. The p-nitrophenyl phosphatase activity has been shown to chromatograph with the CO₂ hydratase activity; both activities are associated with each of its sulfhydryl oxidation subforms; and both activities follow the same pattern of pH stability. This phosphomonoesterase activity of carbonic anhydrase III has an acidic pH optimum (<5.3); its true substrate appears to be the phosphomonoanion with a K_m of 2.8 mm. It is competitively inhibited by the typical acid phosphatase inhibitors phosphate (K_i = 1.22 × 10^?3M), arsenate (K_i = 1.17 × 10^?3M), and molybdate (K_i = 1.34 × 10^?7M), with these inhibitors having no effect on the CO₂ hydratase or the p-nitrophenyl acetate esterase activities of carbonic anhydrase III. The p-nitrophenyl acetate esterase activity of carbonic anhydrase III, on the other hand, has the sigmoidal pH profile with an inflection at neutral pH, typical of carbonic anhydrases for all of their substrates, and is inhibitable by acetazolamide (a highly specific carbonic anhydrase inhibitor) to the same degree as the CO₂ hydratase activity. The acid phosphatase-like activity of carbonic anhydrase III is slightly inhibited by acetazolamide at acidic pH, and inhibited to nearly the same degree at neutral pH. These data are taken to suggest that the phosphatase activity follows a mechanism different from that of the CO₂ hydratase and p-nitrophenyl acetate esterase activities and that there is some overlap of the binding sites.     

Cerebral microvessels contain a beta 2-adrenergic receptor

Cerebral microvessels isolated from cat forebrain contain a specific β-adrenergic-sensitive adenylate cyclase. Among various compounds tested, the most potent activator of enzyme activity is isoproterenol (k_a = 1.4 × 10^?7M), followed in order by epinephrine (k_a= 1.5 × 10^?6M), norepinephrine (k_a= 1.4 × 10^?5M) and phenylephrine (k_a> 3 × 10^?4M). Isoproterenol-stimulated enzyme activity is blocked by propranolol (k_i= 2.4 × 10^?9M, IPS 339 (k_i= 4 × 10^?9M), H35/25 (k_i = 1.2 × 10^?7M), atenolol (k_i= 5.9 × 10^?6M) and practolol (k_i= 1.8 × 10^?5M). These agonist and antagonist properties are quite similar to those demonstrated by β₂-adrenergic receptors and β₂-stimulated adenylate cyclase present in other tissues and indicate that the majority of adenylate cyclase-associated adrenergic receptors in cerebral microvessels are β₂. The findings are relevant to physiological studies of cerebral blood flow and vascular permeability.     

Tricyclohexyltin hydroxide effects on cationic and substrate activation kinetics of beta-adrenergic–stimulated cardiac Ca2+-ATPase

Previous studies from this laboratory have indicated that tricyclohexyltin hydroxide (Plictran) is a potent inhibitor of both basal- and isoproterenol-stimulated cardiac sarcoplasmic reticulum (SR) Ca²⁺-ATPase, with an estimated IC-50 of 2.5 × 10^?8M. The present studies were initiated to evaluate the mechanism of inhibition of Ca²⁺-ATPase by Plictran. Data on substrate and cationic activation kinetics of Ca²⁺-ATPase indicated alteration of V_max and K_m by Plictran (1 and 5×10^?8M), suggesting a mixed type of inhibition. The beta-adrenergic agonist isoproterenol increased V_max of both ATP- and Ca²⁺-dependent enzyme activities. However, the K_m of enzyme was decreased only for Ca²⁺ Plictran inhibited isoproterenol-stimulated Ca²⁺-ATPase activity by altering both and V_max and K_m of ATP as well as Ca²⁺-dependent enzyme activities, suggesting that after binding to a single independent site, Plictran inhibits enzyme catalysis by decreasing the affinity of enzyme for ATP as well as for Ca²⁺ Preincubation of enzyme with 15 μM cAMP or the addition of 2mM ATP to the reaction mixture resulted in slight activation of Plictran-inhibited enzyme. Pretreatment of SR with 5 × 10^?7M propranolol and 5 × 10^?8M Plictran resulted in inhibition of basal activity in addition to the loss of stimulated activity. Preincubation of heart SR preparation with 5 × 10^?5M coenzyme A in combination with 5 × 10^?8M Plictran partly restored the beta-adrenergic stimulation. These results suggest that some critical sites common to both basal- and beta-adrenergic-stimulated Ca²⁺-ATPase are sensitive to binding by Plictran, and the resultant conformational change may lead to inhibition of beta-adrenergic stimulation.