Characterization of an octopamine-sensitive adenylate cyclase from insect brain (Mamestra configurata Wlk.)5

1979 ◽  
Vol 57 (3) ◽  
pp. 226-232 ◽  
Author(s):  
Robert P. Bodnaryk
Keyword(s):  
Enzyme Activity ◽  
Adenylate Cyclase ◽  
Metal Ion ◽  
Cyclase Activity ◽  
Mammalian Brain ◽  
Insect Brain ◽  
Ph Optimum ◽  
Mamestra Configurata ◽  
Specificity Effect

An adenylate cyclase present in the brain of the moth Mamestra configurata Wlk. that is stimulated selectively by low (micromolar) concentrations of octopamine has been characterized with respect to several properties. The optimum pH, optimum ATP:Mg2+ ratio, the concentration of ATP required for half-maximal and maximal reaction velocity, metal ion specificity, effect of NaF, and effects of GTP and 5′-guanylylimidodiphosphate were in general similar to those of catecholamine-sensitive adenylate cyclases from various regions of mammalian brain. However, ethylene glycol bis-(β-aminoethyl ether)-N,N-tetraacetic acid (EGTA), a calcium chelator, stimulated both basal and octopamine-sensitive enzyme activity in the insect brain, whereas in mammalian brain EGTA is usually observed to inhibit basal activity but not catecholamine-stimulated activity.Adenylate cyclase activity of the 47 000 g particulate fraction of the insect brain was almost undetectable in the absence of added GTP. Addition of saturating concentrations (100 μM) of GTP to the particles restored about 30% of the basal and octopamine-sensitive enzyme activity present in the homogenate. Addition of 100 000 g supernatant to the particles doubled both basal and octopamine-sensitive enzyme activity in the presence of saturating concentrations of GTP, indicating that in addition to GTP, a cytosolic factor(s) is necessary for enhanced adenylate cyclase activity.

scholarly journals CHARACTERIZATION OF CRUDE EXTRACT POLYPHENOLOXIDASE ENZYME FROM BLACK TIGER SHRIMP (Penaeus monodon)

2013 ◽  
Vol 5 (2) ◽  
Author(s):  
Made Suhandana ◽  
Tati Nurhayati ◽  
Laksmi Ambarsari
Keyword(s):  
Enzyme Activity ◽  
Metal Ion ◽  
Penaeus Monodon ◽  
Extraction Condition ◽  
Ph Optimum ◽  
Km Value ◽  
Shrimp Industry ◽  
Optimum Extraction ◽  
Inhibited Enzyme

<p>Shrimp is a  very important export  commodity with  high market value world wide. However, it is still facing problem related to the waste and deterioration quality as main issues for the shrimp industry. In this experiment, polyphenoloxidase from the carapace of Penaeus monodon was extracted and characterized. The research was carried out to obtain the optimum extraction condition and to evaluate the properties of enzyme i.e., pH, optimum temperature for activating enzyme, kinetic enzyme, and chelating on metal ion. The best method for PPO enzyme extraction used buffer with 1:3  proportion.  The optimum activity of enzyme was at pH 7 and temperature of 35°C. The kinematic enzyme (Km) value and the maximum substrate concentration were 5.42 mM and 7.5 mM, respectively.  Na<sup>+</sup>, Ca<sup>2+</sup>, Zn<sup>2+</sup>, and EDTA with concentration 5 and 10 mM inhibited enzyme activity.  Cu<sup>2+</sup>at concentration of 10 mM and Mn<sup>2+</sup> at concentration 5 mM also inhibited enzyme activity</p> <p>Keywords: carapace, characterization, polyphenoloxidase, shrimp</p>

scholarly journals Calmodulin activation of adenylate cyclase in the mouse B16 melanoma

1984 ◽  
Vol 224 (2) ◽  
pp. 453-460 ◽  
Author(s):  
S Mac Neil ◽  
S W Walker ◽  
H J Senior ◽  
A Pollock ◽  
B L Brown ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Adenylate Cyclase ◽  
Metal Ion ◽  
Cultured Cells ◽  
Marked Change ◽  
Earth Metal ◽  
B16 Melanoma ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Maximal Stimulation

Calmodulin antagonists inhibited hormone-stimulated cyclic AMP accumulation in both cultured cells and cell lysates of mouse B16 melanoma. Particulate preparations of B16 melanoma contained 34-45% of total cell calmodulin, which could not be dissociated by extensive washing irrespective of the presence of EGTA in the buffer. The adenylate cyclase activity in such preparations was unaffected by the addition of exogenous calmodulin. However, the rare-earth-metal ion La3+, which can mimic or replace Ca2+ in many systems, produced an immediate inhibition of agonist-stimulated adenylate cyclase activity and preincubation of particulate preparations was La3+ followed by washing with La3+-free buffer dissociated calmodulin (96% loss) from particulate preparations. The loss of calmodulin from particulate preparations was associated with a decrease in agonist responsiveness (74%) and a marked change in the Ca2+-sensitivity of the enzyme, low concentrations of calcium (approx. 10 nM) now failing to stimulate enzyme activity, high concentrations of calcium (greater than or equal to 100 nM) producing greater-than-normal inhibition of enzyme activity. Direct activation of adenylate cyclase by the addition of pure calmodulin was now demonstrable in such calmodulin-depleted particulate preparations. Half-maximal stimulation of agonist-responsive adenylate cyclase occurred at 80 nM-calmodulin in the presence of 10 microM free Ca2+. Maximal stimulation by calmodulin (at 300-600 nM) restored enzyme activity to 89 +/- 5% (mean +/- S.E.M., n = 7) of the activity in untreated, calmodulin-intact, preparations.

scholarly journals CHARACTERIZATION OF CRUDE EXTRACT POLYPHENOLOXIDASE ENZYME FROM BLACK TIGER SHRIMP (Penaeus monodon)

2013 ◽  
Vol 5 (2) ◽  
Author(s):  
Made Suhandana ◽  
Tati Nurhayati ◽  
Laksmi Ambarsari
Keyword(s):  
Enzyme Activity ◽  
Metal Ion ◽  
Penaeus Monodon ◽  
Extraction Condition ◽  
Ph Optimum ◽  
Km Value ◽  
Shrimp Industry ◽  
Optimum Extraction ◽  
Inhibited Enzyme

Shrimp is a  very important export  commodity with  high market value world wide. However, it is still facing problem related to the waste and deterioration quality as main issues for the shrimp industry. In this experiment, polyphenoloxidase from the carapace of Penaeus monodon was extracted and characterized. The research was carried out to obtain the optimum extraction condition and to evaluate the properties of enzyme i.e., pH, optimum temperature for activating enzyme, kinetic enzyme, and chelating on metal ion. The best method for PPO enzyme extraction used buffer with 1:3  proportion.  The optimum activity of enzyme was at pH 7 and temperature of 35°C. The kinematic enzyme (Km) value and the maximum substrate concentration were 5.42 mM and 7.5 mM, respectively.  Na+, Ca2+, Zn2+, and EDTA with concentration 5 and 10 mM inhibited enzyme activity.  Cu2+at concentration of 10 mM and Mn2+ at concentration 5 mM also inhibited enzyme activity Keywords: carapace, characterization, polyphenoloxidase, shrimp

Characterization of α-Farnesene Synthase from `Delicious' Apples

HortScience ◽  
1998 ◽  
Vol 33 (3) ◽  
pp. 532d-532
Author(s):  
H.P.V. Rupasinghe ◽  
G. Paliyath ◽  
D.P. Murr
Keyword(s):  
Enzyme Activity ◽  
Metal Ion ◽  
Divalent Metal ◽  
Farnesyl Pyrophosphate ◽  
Anaerobic Conditions ◽  
Ph Optimum ◽  
Divalent Metal Ion ◽  
Apple Cultivars ◽  
Inherent Nature

α-Farnesene metabolism is associated with the occurrence of superficial scald in pome fruits. Trans,trans-α-farnesene synthase, which catalyzes the terminal step of α-farnesene biosynthesis viz. conversion of farnesyl pyrophosphate to α-farnesene, has been characterized in the extract from skin tissues of `Delicious' apples (Malus domestica Borkh.). The total and specific activities of the enzyme were the highest in the cytosolic fraction when compared to that in membrane fractions. The enzyme possessed a pH optimum of 5.6 and required a divalent metal ion (Mg+2 and Mn+2 were preferred). The activity was highest between 10 and 20 °C, although 50 % of the activity was still retained at 0 °C. The presence of thiol reagents, pyridine dinucleotide effectors, anaerobic conditions or antioxidants did not significantly affect enzyme activity. α-Farnesene synthase activity was similar in the extract of skin tissue from scald-developing and non-scald-developing apples. The enzyme activity was not correlated to the inherent nature of scald-susceptibility or resistance in eight different apple cultivars tested.

Sinapine Esterase I. Characterization of Sinapine Esterase from Cotyledons of Raphanus sativus

1979 ◽  
Vol 34 (9-10) ◽  
pp. 715-720 ◽  
Author(s):  
Gerhild Nurmann ◽  
Dieter Strack
Keyword(s):  
Enzyme Activity ◽  
Esterase Activity ◽  
Raphanus Sativus ◽  
Sinapic Acid ◽  
Inhibitory Effect ◽  
Ph Optimum ◽  
Diisopropyl Fluorophosphate ◽  
E 600 ◽  
Red Radish

Abstract From cotyledons of Raphanus sativus (red radish) an esterase activity which catalyzes the hy­drolysis of sinapine into sinapic acid and choline has been isolated. The enzyme, which has a near absolute specificity, is not analogous with any esterase described in the literature. The reaction has a pH optimum of 8.5 and the apparent Km is 1.95 × 10-5 m. The enzyme is relatively insensi­tive to both physostigmine (eserine) {Ki = 1.73 × 10-4 m) and neostigmine (Ki = 2 .1 3 × 10-4 ᴍ). Diisopropyl fluorophosphate (DFP) showed no inhibition and diethyl p-nitrophenylphosphate (E 600) only a slight inhibitory effect at 10-5 ᴍ, respectively. Choline (10-2 ᴍ) was inhibitory but acetylcholine (10-2 ᴍ) stimulated the enzyme activity.

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

1978 ◽  
Vol 175 (1) ◽  
pp. 171-180 ◽  
Author(s):  
Prakash V. Sulakhe ◽  
Njanoor Narayanan
Keyword(s):  
Enzyme Activity ◽  
Sarcoplasmic Reticulum ◽  
Adenylate Cyclase ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Protein Ratio ◽  
Guinea Pig Heart ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Ionic Detergent ◽  
Triton X

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. Acta451, 48–61]. Further, the results of this study show that cardiac sarcoplasmic-reticulum membranes possess this enzyme.

Effects of benzyl alcohol on PTH receptor-adenylate cyclase system of canine kidney

1985 ◽  
Vol 248 (1) ◽  
pp. E31-E35
Author(s):  
K. J. Martin ◽  
C. L. McConkey ◽  
T. J. Stokes
Keyword(s):  
Enzyme Activity ◽  
Adenylate Cyclase ◽  
Benzyl Alcohol ◽  
Membrane Fluidity ◽  
Phospholipid Composition ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Adenylate Cyclase System ◽  
Hormone Sensitive ◽  
Canine Kidney

In many systems, perturbations of membrane architecture by changes of lipid and phospholipid composition have been shown to alter the activity of membrane-bound enzymes. The present studies examined the effect of benzyl alcohol, an agent that has been shown to increase membrane fluidity, on the parathyroid hormone (PTH)-sensitive adenylate cyclase system of canine kidney. Benzyl alcohol progressively increased basal adenylate cyclase activity up to fourfold and maximal enzyme activity in the presence of PTH, GTP, guanylimidodiphosphate, and sodium fluoride by four- to sixfold. In the presence of 20 mM Mn2+ (no Mg2+), conditions under which enzyme activity is devoid of influence of guanine nucleotides or hormones, benzyl alcohol was without effect. PTH binding was increased by 25% in the presence of benzyl alcohol without a change in binding affinity. Fluorescent polarization studies using diphenylhexatriene showed a decrease in fluorescence anisotropy in the presence of benzyl alcohol. The results suggest that benzyl alcohol facilitates the interaction of the components of the adenylate cyclase system, presumably by increasing membrane fluidity. Alterations of membrane fluidity may be a potent means of regulating hormone sensitive adenylate cyclase activity.

scholarly journals Catecholamine-sensitive adenylate cyclase of caudate nucleus and cerebral cortex. Effects of guanine nucleotides

1977 ◽  
Vol 164 (1) ◽  
pp. 67-74 ◽  
Author(s):  
P V Sulakhe ◽  
N L Leung ◽  
A T Arbus ◽  
S J Sulakhe ◽  
S H Jan ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Cerebral Cortex ◽  
Adenylate Cyclase ◽  
Caudate Nucleus ◽  
Protein S ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Biphasic Effect ◽  
Site Binding ◽  
Stimulation Of

1. GTP and GMP-P(NH)P (guanyl-5'-yl imidodiphosphate) were observed to increase the stimulation of neural adenylate cyclase by dopamine (3,4-dihydroxyphenethylamine) and noradrenaline. 2. GMP-P(NH)P had a biphasic effect on the enzyme activity. 3. Preincubation of membranes with GMP-P(NH)P activated the enzyme by a process dependent on time and temperature. Catecholamines increased the speed and the extent of this activation. 4. Membrane fractions contained high- and low-affinity sites for GMP-P(NH)P binding: this binding was due to protein(s) of the membrane preparations. 5. Low-affinity-site binding of GMP-P(NH)P appeared to be related to the stimulatory effect on the adenylate cyclase activity.

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