scholarly journals Molecular Characterization of a Thermostable Cyanophycin Synthetase from the Thermophilic Cyanobacterium Synechococcus sp. Strain MA19 and In Vitro Synthesis of Cyanophycin and Related Polyamides

2002 ◽  
Vol 68 (1) ◽  
pp. 93-101 ◽  
Author(s):  
Tran Hai ◽  
Fred Bernd Oppermann-Sanio ◽  
Alexander Steinbüchel
Keyword(s):  
Aspartic Acid ◽  
Thermostable Enzyme ◽  
Exchange Chromatography ◽  
In Vitro Production ◽  
In Vitro Synthesis ◽  
Prolonged Incubation ◽  
Thermophilic Cyanobacterium ◽  
Recombinant Cells ◽  
Cyanophycin Synthetase

ABSTRACT The thermophilic cyanobacterium Synechococcus sp. strain MA19 contained the structural genes for cyanophycin synthetase (cphA) and cyanophycinase (cphB), which were identified, cloned, and sequenced in this study. The translation products of cphA and cphB exhibited high levels of similarity to corresponding proteins of other cyanobacteria, such as Anabaena variabilis and Synechocystis sp. Recombinant cells of Escherichia coli harboring cphA colinear with lacPO accumulated cyanophycin that accounted for up to 25% (wt/wt) of the dry cell matter in the presence of isopropyl-β-d-thiogalactopyranoside (IPTG). The cyanophycin synthetase was enriched 123-fold to electrophoretic homogeneity from the soluble fraction of the recombinant cells by anion-exchange chromatography, affinity chromatography, and gel filtration chromatography. The purified cyanophycin synthetase maintained the parental thermophilic character and was active even after prolonged incubation at 50°C; in the presence of ectoine the enzyme retained 90% of its activity even after 2 h of incubation. The in vitro activity of the enzyme depended on ATP, primers, and both substrates, l-arginine and l-aspartic acid. In addition to native cyanophycin, the purified enzyme accepted a modified cyanophycin containing less arginine, α-arginyl aspartic acid dipeptide, and poly-α,β-dl-aspartic acid as primers and also incorporated β-hydroxyaspartic acid instead of l-aspartic acid or l-canavanine instead of l-arginine at a significant rate. The lack of specificity of this thermostable enzyme with respect to primers and substrates, the thermal stability of the enzyme, and the finding that the enzyme is suitable for in vitro production of cyanophycin make it an interesting candidate for biotechnological processes.

scholarly journals Purification of Synechocystis sp. Strain PCC6308 Cyanophycin Synthetase and Its Characterization with Respect to Substrate and Primer Specificity

2001 ◽  
Vol 67 (5) ◽  
pp. 2176-2182 ◽  
Author(s):  
Elsayed Aboulmagd ◽  
Fred B. Oppermann-Sanio ◽  
Alexander Steinbüchel
Keyword(s):  
Methyl Ester ◽  
Glutamic Acid ◽  
Aspartic Acid ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
In Vitro Synthesis ◽  
Complete Reaction ◽  
Cyanophycin Synthetase

ABSTRACT Synechocystis sp. strain PCC6308 cyanophycin synthetase was purified 72-fold in three steps by anion exchange chromatography on Q Sepharose, affinity chromatography on the triazine dye matrix Procion Blue HE-RD Sepharose, and gel filtration on Superdex 200 HR from recombinant cells of Escherichia coli. The native enzyme, which catalyzed the incorporation of arginine and aspartic acid into cyanophycin, has an apparent molecular mass of 240 ± 30 kDa and consists of identical subunits of 85 ± 5 kDa. The K m values for arginine (49 μM), aspartic acid (0.45 mM), and ATP (0.20 mM) indicated that the enzyme had a high affinity towards these substrates. During in vitro cyanophycin synthesis, 1.3 ± 0.1 mol of ATP per mol of incorporated amino acid was converted to ADP. The optima for the enzyme-catalyzed reactions were pH 8.2 and 50°C, respectively. Arginine methyl ester (99.5 and 97% inhibition), argininamide (99 and 96%), S-(2-aminoethyl) cysteine (43 and 42%), β-hydroxy aspartic acid (35 and 37%), aspartic acid β-methyl ester (38 and 40%), norvaline (0 and 3%), citrulline (9 and 7%), and asparagine (2 and 0%) exhibited an almost equal inhibitory effect on the incorporation of both arginine and aspartic acid, respectively, when these compounds were added to the complete reaction mixture. In contrast, the incorporation of arginine was diminished to a greater extent than that of aspartic acid, respectively, with canavanine (82 and 53%), lysine (36 and 19%), agmatine (33 and 25%), d-aspartic acid (37 and 30%), l-glutamic acid (13 and 5%), and ornithine (23 and 11%). On the other hand, canavanine (45% of maximum activity) and lysine (13%) stimulated the incorporation of aspartic acid, whereas aspartic acid β-methyl ester (53%) and asparagine (9%) stimulated the incorporation of arginine. [3H]lysine (15% of maximum activity) and [3H]canavanine (13%) were incorporated into the polymer, when they were either used instead of arginine or added to the complete reaction mixture, whereas l-glutamic acid was not incorporated. No effect on arginine incorporation was obtained by the addition of other amino acids (i.e., alanine, histidine, leucine, proline, tryptophan, and glycine). Various samples of chemically synthesized poly-α,β-d,l-aspartic acid served as primers for in vitro synthesis of cyanophycin, whereas poly-α-l-aspartic acid was almost inactive.

A STUDY ON THE "IN VITRO" SYNTHESIS OF CITRULLINE: PART I. EFFECT OF SOME N-ALKYL DERIVATIVES OF ASPARTIG ACID

1964 ◽  
Vol 42 (9) ◽  
pp. 1317-1324 ◽  
Author(s):  
Louis Berlinguet ◽  
René Charbonneau
Keyword(s):  
Aspartic Acid ◽  
Inhibitory Action ◽  
Liver Homogenate ◽  
Chelating Agent ◽  
Magnesium Ion ◽  
Carbamyl Phosphate ◽  
In Vitro Synthesis ◽  
Alkyl Derivatives ◽  
Derivatives Of

The effects of various N-alkyl derivatives of aspartic acid on the synthesis of citrulline in the presence of a particulate fraction obtained from a rat liver homogenate were studied. Even though aspartic acid has no role in this synthesis, both N-methyl and N-isopropyl aspartic acids were found to increase the synthesis of citrulline by 60%, whereas N-cyclohexyl aspartic acid decreased it by 50%. N-Allyl aspartic acid has the strongest effect which is an almost complete inhibition at low concentration of 1.2 × 10−2 M.It seems that N-allyl aspartic acid inhibits directly or indirectly the first step in the synthesis of citrulline leading to the formation of carbamyl phosphate. At various concentrations, none of the intermediates in this synthesis, except magnesium ion, can reverse the inhibition. In order that N-allyl aspartic acid retain its inhibitory action, the ω-carboxyl group has to be free and the double bond in the allyl group must be intact. From these results, it is postulated that N-allyl aspartic acid acts as a chelating agent for magnesium.

Regulation of a metabolic system in vitro: synthesis of threonine from aspartic acid

Biochemistry ◽  
1976 ◽  
Vol 15 (10) ◽  
pp. 2236-2244 ◽  
Author(s):  
Mark Szczesiul ◽  
D. Eugene Wampler
Keyword(s):  
Aspartic Acid ◽  
Metabolic System ◽  
In Vitro Synthesis

Extraction and measurement of circulating angiotensins I and II.

1977 ◽  
Vol 23 (3) ◽  
pp. 464-468 ◽  
Author(s):  
J D Barrett ◽  
P Eggena ◽  
M P Sambhi
Keyword(s):  
Extraction Procedure ◽  
Isotonic Saline ◽  
Plasma Renin ◽  
Exchange Chromatography ◽  
Angiotensin I ◽  
In Vitro Production ◽  
Angiotensin Peptides ◽  
Before And After ◽  
Vitro Production

Abstract We describe a method for simultaneous extraction of angiotensins I and II from human blood. Blood was diluted with 10 volumes of isotonic saline containing ammonium ethylenediaminetetraacetate, and the angiotensin peptides were extracted by cation-exchange chromatography. The dilution effectively inhibits in vitro production of angiotensin I, conversion of angiotensin I to angiotensin II, and degradation of the peptides. Analytical recovery of exogenous angiotensin I or II added to whole blood in physiological concentrations was essentially complete. The extraction procedure removes blood and plasma components that interfere with radioimmunoassay of angiotensin, thus eliminating the need to correct for nonspecific interference. Circulating angiotensins I and II, measured in patients with essential hypertension before and after a week of diuretic administration, increased in parallel with plasma renin activity.

THE IN VITRO PRODUCTION OF ANDROGENS BY FOLLICULAR TISSUE OF RABBITS

1964 ◽  
Vol 47 (2) ◽  
pp. 306-313 ◽  
Author(s):  
Denis Gospodarowicz
Keyword(s):  
In Vitro Production ◽  
Vitro Production

ABSTRACT Incubation in vitro of rabbit follicles in separate experiments with dehydroepiandrosterone-14C (DHEA-14C), progesterone-14C and pregnenolone-3H in the presence of FSH gave the following results: 39 % of the radioactivity of DHEA-14C is converted to androstenedione and testosterone, while only 3 % of the radioactivity of either progesterone-14C or pregnenolone-3H is found in the androgen fraction. From the ratio of testosterone to androstenedione formed from the three precursors, the results are interpreted to mean that DHEA and pregnenolone, and not progesterone, are precursors of androgens in the follicle.

Co-purification of bone resorbing activity and adenylate cyclase stimulating activity from human tumours associated with the humoral hypercalcaemia of malignancy

1987 ◽  
Vol 114 (1) ◽  
pp. 18-26 ◽  
Author(s):  
Chohei Shigeno ◽  
Itsuo Yamamoto ◽  
Shegiharu Dokoh ◽  
Megumu Hino ◽  
Jun Aoki ◽  
...  
Keyword(s):  
Bone Resorption ◽  
High Performance ◽  
Basic Protein ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Protein Factor ◽  
Human Tumours ◽  
Acid Stable ◽  
Bone Resorbing Activity

Abstract. We have partially purified a tumour factor capable of stimulating both bone resorption in vitro and cAMP accumulation in osteoblastic ROS 17/2 cells from three human tumours associated with humoral hypercalcaemia of malignancy. Purification of tumour factor by sequential acid urea extraction, gel filtration and cation-exchange chromatography, reverse-phase high performance liquid chromatography followed by analytical isoelectric focussing provided a basic protein (pI > 9.3) with a molecular weight of approximately 13 000 as a major component of the final preparation which retained both the two bioactivities. Bone resorbing activity and cAMP-increasing activity in purified factor correlated with each other. cAMP-increasing activity of the factor was heat- and acid-stable, but sensitive to alkaline ambient pH. Treatment with trypsin destroyed cAMP-increasing activity of the factor. Synthetic parathyroid hormone (PTH) antagonist, human PTH-(3– 34) completely inhibited the cAMP-increasing activity of the factor. The results suggest that this protein factor, having its effects on both osteoclastic and osteoblastic functions, may be involved in development of enhanced bone resorption in some patients with humoral hypercalcaemia of malignancy.

Oxygenation of 18-hydroxycorticosterone as the final reaction for aldosterone biosynthesis

1984 ◽  
Vol 107 (3) ◽  
pp. 395-400 ◽  
Author(s):  
Itaru Kojima ◽  
Etsuro Ogata ◽  
Hiroshi Inano ◽  
Bun-ichi Tamaoki
Keyword(s):  
Carbon Monoxide ◽  
Hydroxysteroid Dehydrogenase ◽  
Dependent Manner ◽  
In Vitro Production ◽  
Mitochondrial Preparation ◽  
Final Reaction ◽  
Vitro Production ◽  
Dose Dependent ◽  
Cytochrome P 450

Abstract. Incubation of 18-hydroxycorticosterone with the sonicated mitochondrial preparation of bovine adrenal glomerulosa tissue leads to the production of aldosterone, as measured by radioimmunoassay. The in vitro production of aldosterone from 18-hydroxycorticosterone requires both molecular oxygen and NADPH, and is inhibited by carbon monoxide. Cytochrome P-450 inhibitors such as metyrapone, SU 8000. SU 10603, SKF 525A, amphenone B and spironolactone decrease the biosynthesis of aldosterone from 18-hydroxycorticosterone. These results support the conclusion that the final reaction in aldosterone synthesis from 18-hydroxycorticosterone is catalyzed by an oxygenase, but not by 18-hydroxysteroid dehydrogenase. By the same preparation, the production of [3H]aldosterone but not [3H]18-hydroxycorticosterone from [1,2-3H ]corticosterone is decreased in a dose-dependent manner by addition of non-radioactive 18-hydroxycorticosterone.

Chickpea (Cicer arietinum L.) Lectin Exhibit Inhibition of ACE-I, α-amylase and α-glucosidase Activity

2019 ◽  
Vol 26 (7) ◽  
pp. 494-501 ◽  
Author(s):  
Sameer Suresh Bhagyawant ◽  
Dakshita Tanaji Narvekar ◽  
Neha Gupta ◽  
Amita Bhadkaria ◽  
Ajay Kumar Gautam ◽  
...  
Keyword(s):  
Biological Effects ◽  
Exchange Chromatography ◽  
Health Concern ◽  
Carbohydrate Binding ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Ic50 Values ◽  
Chickpea Lectin

Background: Diabetes and hypertension are the major health concern and alleged to be of epidemic proportions. This has made it a numero uno subject at various levels of investigation. Glucosidase inhibitor provides the reasonable option in treatment of Diabetes Mellitus (DM) as it specifically targets post prandial hyperglycemia. The Angiotensin Converting Enzyme (ACE) plays an important role in hypertension. Therefore, inhibition of ACE in treatment of elevated blood pressure attracts special interest of the scientific community. Chickpea is a food legume and seeds contain carbohydrate binding protein- a lectin. Some of the biological properties of this lectin hitherto been elucidated. Methods: Purified by ion exchange chromatography, chickpea lectin was tested for its in vitro antioxidant, ACE-I inhibitory and anti-diabetic characteristic. Results: Lectin shows a characteristic improvement over the synthetic drugs like acarbose (oral anti-diabetic drug) and captopril (standard antihypertensive drug) when, their IC50 values are compared. Lectin significantly inhibited α-glucosidase and α-amylase in a concentration dependent manner with IC50 values of 85.41 ± 1.21 ҝg/ml and 65.05 ± 1.2 µg/ml compared to acarbose having IC50 70.20 ± 0.47 value of µg/ml and 50.52 ± 1.01 µg/ml respectively. β-Carotene bleaching assay showed antioxidant activity of lectin (72.3%) to be as active as Butylated Hydroxylanisole (BHA). In addition, lectin demonstrated inhibition against ACE-I with IC50 value of 57.43 ± 1.20 µg/ml compared to captopril. Conclusion: Lectin demonstrated its antioxidant character, ACE-I inhibition and significantly inhibitory for α-glucosidase and α-amylase seems to qualify as an anti-hyperglycemic therapeutic molecule. The biological effects of chickpea lectin display potential for reducing the parameters of medically debilitating conditions. These characteristics however needs to be established under in vivo systems too viz. animals through to humans.

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