scholarly journals Ecdysone 20-mono-oxygenase in the desert locust, Schistocerca gregaria

1984 ◽  
Vol 223 (3) ◽  
pp. 837-847 ◽  
Author(s):  
D R Greenwood ◽  
H H Rees
Keyword(s):  
Enzyme Activity ◽  
Schistocerca Gregaria ◽  
Specific Activity ◽  
Monochromatic Light ◽  
Malpighian Tubule ◽  
Malpighian Tubules ◽  
Lower Sensitivity ◽  
Marker Enzyme ◽  
Energy Dependent ◽  
Cytochrome P 450

The enzyme catalysing the hydroxylation of ecdysone to 20-hydroxyecdysone, ecdysone 20-mono-oxygenase (EC 1.14.99.22), was investigated in the Malpighian tubules of fifth-instar locusts, Schistocerca gregaria. Enzyme activity was optimal at 35 degrees C and pH 6.8-8.0. Under these conditions the mono-oxygenase exhibited an apparent Km for ecdysone of 7.1 × 10(-7) M, a maximal specific activity of 1.1 nmol/h per mg of protein and was competitively inhibited by 20-hydroxyecdysone with an apparent Ki of 6.3×10(-7) M. Enzyme activity was decreased in the presence of Ca2+, Mg2+, EDTA and non-ionic detergents. The Malpighian tubule ecdysone 20-mono-oxygenase was localized primarily in the subcellular fraction sedimenting at 7500 g and, on the basis of marker enzyme profiles, was assigned mainly to the mitochondria. NADPH was required for activity, although addition of NADH together with NADPH had a synergistic effect. NADP+-dependent isocitrate dehydrogenase (EC 1.1.1.42) and an energy-dependent NAD(P) transhydrogenase (EC 1.6.1.1.) appeared to be the major sources of reducing equivalents, with the contribution from the ‘malic enzyme’ (EC 1.1.1.40) being less important. The monooxygenase was characterized as a cytochrome P-450-containing mixed-function oxidase from the inhibition patterns with metyrapone, CO and cyanide; CO inhibition was reversible with monochromatic light at 450 nm. However, the ecdysone 20-mono-oxygenase shows much lower sensitivity to CO inhibition and to photodissociation of the CO-inhibited complex than do vertebrate cytochrome P-450-dependent hydroxylation systems. The concentration of cytochrome P-450 in the Malpighian tubule mitochondria was 30 pmol/mg of protein. The properties of the mono-oxygenase are discussed in relation to hydroxylation enzymes from other sources.

Amino-Acid Metabolism in Locust Tissues

1957 ◽  
Vol 34 (2) ◽  
pp. 276-289
Author(s):  
B. A. KILBY ◽  
ELISABETH NEVILLE
Keyword(s):  
Amino Acid ◽  
Amino Acid Metabolism ◽  
Acid Metabolism ◽  
Schistocerca Gregaria ◽  
Fat Body ◽  
Malpighian Tubule ◽  
Malpighian Tubules ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Mitochondrial Fractions

1. Homogenates of fat-body of Schistocerca gregaria Forsk. were shown to catalyse transamination reactions between α-ketoglutarate and numerous α-amino acids. The aspartate/glutamate and alanine/glutamate transaminases were the most active. They were present in both the ‘soluble’ and the mitochondrial fractions of fat-body cells and also in Malpighian tubules and mid-gut wall. The other transaminases in the fat-body were confined to the mitochondrial fraction. 2. Fat-body, Malpighian tubule and mid-gut wall homogenates were able to convert glutamic acid into glutamine, a compound which could also act as an amino-group donor in some transamination reactions. 3. A glutamate-cytochrome c reductase system which involved diphosphopyridine nucleotide was present in fat-body. 4. Fat-body contained an active arginase, but urease could not be detected. A D-amino-acid oxidase was present, together with a less active L-amino-acid oxidase. 5. In general, it appears that amino-acid metabolism in the locust resembles that in higher animals.

scholarly journals The mechanism of C-20 hydroxylation of α-ecdysone in the desert locust, Schistocerca gregaria

1977 ◽  
Vol 168 (3) ◽  
pp. 513-520 ◽  
Author(s):  
P Johnson ◽  
H H Rees
Keyword(s):  
Schistocerca Gregaria ◽  
Fat Body ◽  
Difference Spectrum ◽  
Maximum Activity ◽  
Malpighian Tubules ◽  
Desert Locust ◽  
Ph Optimum ◽  
Developmental Variation ◽  
Moulting Hormone ◽  
Cytochrome P 450

1. The C-20 hydroxylation of alpha-ecdysone to produce beta-ecdysone was investigated in the desert locust, Schistocerca gregaria. 2. alpha-Ecdysone C-20 hydroxylase activity was located primarily in the fat-body and Malpighian tubules. The properties of the hydroxylation system from Malpighian tubules investigated further. 3. The enzyme system was mitochondrial, had a pH optimum of 6.5, an apparent Km of 12.5 micron and required O2 and NADPH. 4. The activity of the hydroxylation system showed developmental variation within the fifth instar, the maximum activity corresponding to the maximum tire of endogenous moulting hormone. The significance of these results is assessed in relation to the control of the endogenous titre of beta-ecdysone. 5. The mechanism of the hydroxylation system was investigated by using known inhibitors of hydroxylation reactions such as CO, metyrapone and cyanide. 6. The CO difference spectrum of the reduced mitochondrial preparation indicated the presence of cytochrome P-450 in the preparation. 7. It concluded that the alpha-ecdysone C-20 hydroxylase system is a cytochrome P-450-deendent mono-oxygenase.

Acid-Base Variables in Malpighian Tubule Secretion and Response to Acidosis

1991 ◽  
Vol 159 (1) ◽  
pp. 433-447 ◽  
Author(s):  
ANDREW P. STAGG ◽  
JON F. HARRISON ◽  
JOHN E. PHILLIPS
Keyword(s):  
Schistocerca Gregaria ◽  
Malpighian Tubule ◽  
Malpighian Tubules ◽  
Acid Base ◽  
Acid Injection ◽  
Before And After ◽  
Acid Load ◽  
Acid Base Status ◽  
Tubule Fluid

1. Malpighian tubule fluid from Schistocerca gregaria adults, starved for 1 day, was collected in situ by cannulation of the gut, both before and after injecting 10 μmol of HCl or NaCl into the haemocoel. 2. Haemolymph pH at the neck remained depressed by 0.3 units for at least 6 h in HCl- as compared to NaCl-injected locusts. A lower haemolymph pH persisted near the acid injection site for several hours. 3. The pH of tubule fluid remained about 0.5 units more acid than haemolymph under all conditions. Thus, net tubular acid secretion was proportional to haemolymph acid-base status. 4. The greater acidity of tubular fluid after acid injection was associated with lower estimated bicarbonate concentrations and higher Pcoco2 without any change in total CO2 when compared to controls. 5. The combined contribution of bicarbonate, phosphate and urate to total buffering capacity of tubular fluid was estimated to be 75°, with bicarbonate responsible for 55° of the total. 6. The maximum rate of acid removal by all Malpighian tubules of starved locusts, including H+ trapped in ammonium ions, was calculated to be very small in relation to the acid load injected into the haemocoel Note: To whom reprint requests should be directed.

scholarly journals Isolation of rat liver lysosomes by isopycnic centrifugation in a metrizamide gradient.

1978 ◽  
Vol 78 (2) ◽  
pp. 349-368 ◽  
Author(s):  
R Wattiaux ◽  
S Wattiaux-De Coninck ◽  
M F Ronveaux-dupal ◽  
F Dubois
Keyword(s):  
Enzyme Activity ◽  
Plasma Membrane ◽  
Rat Liver ◽  
Specific Activity ◽  
Mitochondrial Fraction ◽  
Marker Enzyme ◽  
Marker Enzymes ◽  
Liver Lysosomes ◽  
Rat Liver Lysosomes ◽  
Isopycnic Centrifugation

A preparation, similar to the light mitochondrial fraction of rat liver (L fraction of de Duve et al, (1955, Biochem. J. 60: 604-617), was subfractionated by isopycnic centrifugation in a metrizamide gradient and the distribution of several marker enzymes was established. The granules were layered at the top or bottom of the gradient. In both cases, as ascertained by the enzyme distributions, the lysosomes are well separated from the peroxisomes. A good separation from mitochondria is obtained only when the L fraction if set down underneath the gradient. Taking into account the analytical centrifugation results, a procedure was devised to purify lysosomes from several grams of liver by centrifugation of an L fraction in a discontinuous metrizamide gradient. By this method, a fraction containing 10--12% of the whole liver lysosomes can be prepared. As inferred from the relative specific activity of marker enzymes, it can be estimated that lysosomes are purified between 66 and 80 times in this fraction. As ascertained by plasma membrane marker enzyme activity, the main contaminant could be the plasma membrane components. However, cytochemical tests for 5'AMPase and for acid phosphatase suggest that a large part of the plasma membrane marker enzyme activity present in the purified lysosome preparation could be associated with the lysosomal membrane. The procedure for the isolation of rat liver lysosomes described in this paper is compared with the already existing methods.

Fine Structure of the Malpighian Tubules of the Mosquito, Culex pipiens

1971 ◽  
Vol 29 ◽  
pp. 402-403
Author(s):  
Brendan Clifford
Keyword(s):  
Fine Structure ◽  
Culex Pipiens ◽  
Stellate Cell ◽  
Malpighian Tubule ◽  
Cell Types ◽  
Instar Larva ◽  
Malpighian Tubules ◽  
Calliphora Erythrocephala ◽  
Distinct Cell ◽  
Basal Plasma

An ultrastructural investigation of the Malpighian tubules of the fourth instar larva of Culex pipiens was undertaken as part of a continuing study of the fine structure of transport epithelia.Each of the five Malpighian tubules was found to be morphologically identical and regionally undifferentiated. Two distinct cell types, the primary and stellate, were found intermingled along the length of each tubule. The ultrastructure of the stellate cell was previously described in the Malpighian tubule of the blowfly, Calliphora erythrocephala by Berridge and Oschman.The basal plasma membrane of the primary cell is extremely irregular, giving rise to a complex interconnecting network of basal channels. The compartments of cytoplasm entrapped within this system of basal infoldings contain mitochondria, free ribosomes, and small amounts of rough endoplasmic reticulum. The mitochondria are distinctive in that the cristae run parallel to the long axis of the organelle.

scholarly journals Study of Extraction and Enzymatic Properties of Cell-Envelope Proteinases from a Novel Wild Lactobacillus plantarum LP69

Catalysts ◽  
2018 ◽  
Vol 8 (8) ◽  
pp. 325 ◽  
Author(s):  
He Chen ◽  
Jie Huang ◽  
Binyun Cao ◽  
Li Chen ◽  
Na Song ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Lactobacillus Plantarum ◽  
Industrial Development ◽  
Extraction Efficiency ◽  
Specific Activity ◽  
Cell Envelope ◽  
Serine Proteinase ◽  
Kinetic Studies ◽  
Predicted Values ◽  
Hydrolyze Whey Protein

Lactobacilli cell-envelope proteinases (CEPs) have been widely used in the development of new streams of blockbuster nutraceuticals because of numerous biopharmaceutical potentials; thus, the development of viable methods for CEP extraction and the improvement of extraction efficiency will promote their full-scale application. In this study, CEP from a novel wild Lactobacillus plantarum LP69 was released from cells by incubating in calcium-free buffer. The extraction conditions of CEP were optimized by response surface methodology with the enzyme activity and specific activity as the detective marker. The optimal extraction conditions were: time of 80 min, temperature of 39 °C and buffer pH of 6.5. Under these conditions, enzyme activity and specific activity were (23.94 ± 0.86) U/mL and (1.37 ± 0.03) U/mg, respectively, which were well matched with the predicted values (22.12 U/mL and 1.36 U/mg). Optimal activity of the crude CEP occurred at pH 8.0 and 40 °C. It is a metallopeptidase, activated by Ca2+, inhibited by Zn2+ and ethylene-diamine-tetra-acetic acid, and a serine proteinase which is inhibited by phenylmethylsulfonyl fluoride. Kinetic studies showed that CEP from LP69 could hydrolyze whey protein, lactoglobulin and casein. Our study improves the extraction efficiency of CEPs from LP69, providing the reference for their industrial development.

Binding of 3-phosphoglycerate leads to both activation and stabilisation of ADP-glucose pyrophosphorylase from apple leaves

2005 ◽  
Vol 32 (9) ◽  
pp. 839
Author(s):  
Rui Zhou ◽  
Lailiang Cheng
Keyword(s):  
Heat Treatment ◽  
Thermal Stability ◽  
Enzyme Activity ◽  
Inorganic Phosphate ◽  
Thermal Inactivation ◽  
Specific Activity ◽  
Apple Leaves ◽  
Apparent Homogeneity ◽  
Adp Glucose Pyrophosphorylase ◽  
High Concentrations

Apple leaf ADP-glucose pyrophosphorylase was purified 1436-fold to apparent homogeneity with a specific activity of 58.9 units mg–1. The enzyme was activated by 3-phosphoglycerate (PGA) and inhibited by inorganic phosphate (Pi) in the ADPG synthesis direction. In the pyrophosphorolytic direction, however, high concentrations of PGA (> 2.5 mm) inhibited the enzyme activity. The enzyme was resistant to thermal inactivation with a T0.5 (temperature at which 50% of the enzyme activity is lost after 5 min incubation) of 52°C. Incubation with 2 mm PGA or 2 mm Pi increased T0.5 to 68°C. Incubation with 2 mm dithiothreitol (DTT) decreased T0.5 to 42°C, whereas inclusion of 2 mm PGA in the DTT incubation maintained T0.5 at 52°C. DTT-induced decrease in thermal stability was accompanied by monomerisation of the small subunits. Presence of PGA in the DTT incubation did not alter the monomerisation of the small subunits of the enzyme induced by DTT. These findings indicate that binding of PGA renders apple leaf AGPase with a conformation that is not only more efficient in catalysis but also more stable to heat treatment. The physiological significance of the protective effect of PGA on thermal inactivation of AGPase is discussed.

5α-Reductase activity in stroma and epithelium of rat prostate and epididymis. A contribution to elucidation of the mechanism for development of hyperplastic growth of prostatic tissue

1983 ◽  
Vol 103 (2) ◽  
pp. 273-281 ◽  
Author(s):  
Ole Djøseland ◽  
Nicholas Bruchovsky ◽  
Paul S. Rennie ◽  
Navdeep Otal ◽  
Sian Høglo
Keyword(s):  
Enzyme Activity ◽  
Specific Activity ◽  
Reductase Activity ◽  
Major Change ◽  
Growth Stimulation ◽  
Total Activity ◽  
Prostatic Tissue ◽  
Ventral Prostate ◽  
Total Enzyme Activity ◽  
Abnormal Growth

Abstract. The 5α-reductase activity was assayed in homogenates of stroma and epithelium in the rat ventral prostate and epididymis. Samples consisting of 0.3 mg/ml tissue protein in TES buffer, pH 7.0 were incubated at 37°C for 30 min in the presence of 50 nm [1,2-3H]testosterone and a NADPH-generating system started with 5 × 10−4 m NADP. The yield of 5α-reduced metabolites, as established by using thin-layer chromatography, gave an estimate of enzyme activity. Whereas the specific activity of 5α-reductase was highest in prostatic stroma and epididymal epithelium, most of the total enzyme activity was associated with the epithelium in both the prostate and epididymis. The effect of dihydrotestosterone on specific activity of 5α-reductase was studied by administering the hormone to 7-day castrated rats. In prostate, the specific activity of both stromal and epithelium forms of the enzyme reached a maximum after 4 days of treatment. In epididymis only the epithelial form of 5α-reductase underwent a major change in specific activity, the latter peaking after 8–12 days of treatment. Furthermore, while the total activity of 5α-reductase in the prostatic tissue fractions could be induced by as much as 4-fold the normal control values, the epididymal enzyme could not be induced above the normal level either in the stroma or the epithelium. This may explain the relative resistance of epididymis to abnormal growth stimulation under the influence of hormones.

Inhibitory Effects of Vitamin A on TCDD-induced Cytochrome P-450 1A1 Enzyme Activity and Expression

2005 ◽  
Vol 85 (1) ◽  
pp. 727-734 ◽  
Author(s):  
Yan-Mei Yang ◽  
Dong-Yang Huang ◽  
Ge-Fei Liu ◽  
Jiu-Chang Zhong ◽  
Kun Du ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Vitamin A ◽  
Inhibitory Effects ◽  
Cytochrome P 450

Anti-diuresis in the blood-feeding insect Rhodnius prolixus Stål: the peptide CAP2b and cyclic GMP inhibit Malpighian tubule fluid secretion.

1997 ◽  
Vol 200 (17) ◽  
pp. 2363-2367 ◽  
Author(s):  
M C Quinlan ◽  
N J Tublitz ◽  
M J O'Donnell
Keyword(s):  
Cyclic Gmp ◽  
Physiological Role ◽  
Malpighian Tubule ◽  
Fluid Secretion ◽  
Blood Feeding ◽  
Rhodnius Prolixus ◽  
Malpighian Tubules ◽  
Tubule Fluid ◽  
Secretion Rates

Rhodnius prolixus eliminates NaCl-rich urine at high rates following its infrequent but massive blood meals. This diuresis involves stimulation of Malpighian tubule fluid secretion by diuretic hormones released in response to distention of the abdomen during feeding. The precipitous decline in urine flow that occurs several hours after feeding has been thought until now to result from a decline in diuretic hormone release. We suggest here that insect cardioacceleratory peptide 2b (CAP2b) and cyclic GMP are part of a novel mechanism of anti-diuresis. Secretion rates of 5-hydroxytryptamine-stimulated Malpighian tubules are reduced by low doses of CAP2b or cyclic GMP. Maximal secretion rates are restored by exposing tubules to 1 mmol l-1 cyclic AMP. Levels of cyclic GMP in isolated tubules increase in response to CAP2b, consistent with a role for cyclic GMP as an intracellular second messenger. Levels of cyclic GMP in tubules also increase as urine output rates decline in vivo, suggesting a physiological role for this nucleotide in the termination of diuresis.

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