Characterization of Hexokinase and Fructokinase from Suspension-Cultured Catharanthus roseus Cells

1988 ◽  
Vol 43 (11-12) ◽  
pp. 827-834 ◽  
Author(s):  
Yüko Yamashita ◽  
Hiroshi Ashihara
Keyword(s):  
Catharanthus Roseus ◽  
Divalent Cations ◽  
Ph Optimum ◽  
Optimum Ph ◽  
Phosphate Donor

Abstract Two different hexose-phosphorylating enzymes, hexokinase and fructokinase, were partially purified from suspension-cultured Catharanthus roseus cells. One of the enzymes, hexokinase, catalyzed the phosphorylation of both glucose and fructose. The Km values for glucose and fructose were 0.06 mM and 0.23 mM, respectively. The Vmax of the enzyme with fructose was approximately three times higher than with glucose. This enzyme was specific in its requirement for ATP and its Km value for ATP was 52 μM. The optimum pH was 8.0 and Mg2+ or Mn2+ was required for the activity. The activity was inhibited by considerably higher concentrations of ADP (i.e., 4 mM ADP was required for 50% inhibition). The second enzyme, fructokinase, was specific for fructose, and no activity was detected with glucose as substrate. This enzyme used UTP or CTP as phosphate donor. The Km values of this enzyme for fructose and UTP were 0.13 mM and 0.15 mM, respectively. The pH optimum was 7.2, and Mg2+ or Mn2+ was required for the activity. These divalent cations could be partially replaced by Ca2+. The activity was inhibited noncompetitively by ADP and AMP. 90% inhibition of the activity by 0.5 mM ADP was observed in the presence of 2 mM UTP and 5 mM MgCl2. Fructose-2,6-bisphosphate, glucose-1,6-bisphosphate, glucose-6-phosphate, and fructose-6-phosphate had little or no effect on the activity of both the hexokinase and the fructokinase. Based on these results, a discussion is presented of the role of hexokinase and fructokinase and their involvement in the regulation of the metabolism of sugars in Catharanthus cells.

Characterization of CDPdiacylglycerol hydrolase in mitochondrial and microsomal fractions from rat lung

1988 ◽  
Vol 66 (5) ◽  
pp. 425-435 ◽  
Author(s):  
Amy Mok ◽  
Tanya Wong ◽  
Octavio Filgueiras ◽  
Paul G. Casola ◽  
Don W. Nicholson ◽  
...  
Keyword(s):  
Divalent Cations ◽  
Mitochondrial Fraction ◽  
Liver Mitochondria ◽  
Inhibitory Effect ◽  
Mitochondrial Activity ◽  
Rat Lung ◽  
Ph Optimum ◽  
Low Concentrations ◽  
Mercuric Ions

CDPdiacylglycerol pyrophosphatase (E. C. 3.6.1.26) activity has been examined in rat lung mitochondrial and microsomal fractions. While the mitochondrial hydrolase exhibited a broad pH optimum from pH 6–8, the microsomal activity decreased rapidly above pH 6.5. Apparent Km values of 36.2 and 23.6 μM and Vmax values of 311 and 197 pmol∙min−1∙mg protein−1 were observed for the mitochondrial and microsomal preparations, respectively. Addition of parachloromercuriphenylsulphonic acid led to a marked inhibition of the microsomal fraction but slightly stimulated the mitochondrial activity at low concentrations. Mercuric ions were inhibitory with both fractions. Although biosynthetic reactions utilizing CDPdiacylglycerol require divalent cations, addition of Mg2+, Mn2+, Ca2+, Zn2+, Co2+, and Cu2+ all inhibited the catabolic CDPdiacylglycerol hydrolase activity in both fractions. EDTA and EGTA also produced an inhibitory effect, especially with the mitochondrial fraction. Although addition of either adenine or cytidine nucleotides led to a decrease in activity with both fractions, the marked susceptibility to AMP previously reported for this enzyme in Escherichia coli membranes, guinea pig brain lysosomes, and pig liver mitochondria was not observed. These results indicate that rat lung mitochondria and microsomes contain specific CDPdiacylglycerol hydrolase activities, which could influence the rate of formation of phosphatidylinositol and phosphatidylglycerol for pulmonary surfactant.

Zebrafish tyrosylprotein sulfotransferase: molecular cloning, expression, and functional characterization

2004 ◽  
Vol 82 (2) ◽  
pp. 295-303 ◽  
Author(s):  
Emi Mishiro ◽  
Ming-Yih Liu ◽  
Yoichi Sakakibara ◽  
Masahito Suiko ◽  
Ming-Cheh Liu
Keyword(s):  
Molecular Cloning ◽  
Divalent Cations ◽  
Functional Characterization ◽  
The Other ◽  
Inhibitory Effects ◽  
Amino Acid Sequence Level ◽  
Ph Optimum ◽  
Rapid Amplification ◽  
High Degree

By employing the reverse transcriptase – polymerase chain reaction technique in conjunction with 3' rapid amplification of cDNA ends, a full-length cDNA encoding a zebrafish (Danio rerio) tyrosylprotein sulfotransferase (TPST) was cloned and sequenced. Sequence analysis revealed that this zebrafish TPST is, at the amino acid sequence level, 66% and 60% identical to the human and mouse TPST-1 and TPST-2, respectively. The recombinant form of the zebrafish TPST, expressed in COS-7 cells, exhibited a pH optimum at 5.75. Manganese appeared to exert a stimulatory effect on the zebrafish TPST. The activity of the enzyme determined in the presence of 20 mM MnCl2 was more than 2.5 times that determined in the absence of MnCl2. Of the other nine divalent metal cations tested at a 10 mM concentration, Co2+ also showed a considerable stimulatory effect, while Ca2+, Pb2+, and Cd2+ exerted some inhibitory effects. The other four divalent cations, Fe2+, Cu2+, Zn2+, and Hg2+, inhibited completely the sulfating activity of the zebrafish TPST. Using the wild-type and mutated P-selectin glycoprotein ligand-1 N-terminal peptides as substrates, the zebrafish TPST was shown to exhibit a high degree of substrate specificity for the tyrosine residue on the C-terminal side of the peptide. These results constitute a first study on the cloning, expression, and characterization of a zebrafish cytosolic TPST.Key words: zebra fish, tyrosylprotein sulfotransferase, molecular cloning.

scholarly journals Identification and Characterization of Bacterial Lipase from Plateu Soil in West Java

2017 ◽  
Vol 18 (02) ◽  
pp. 103-108
Author(s):  
Vivitri Dewi Prasasty ◽  
Vinella Winata ◽  
Muhammad Hanafi
Keyword(s):  
Heat Stability ◽  
Industrial Applications ◽  
West Java ◽  
Ph Optimum ◽  
Good Efficiency ◽  
Optimum Ph ◽  
Glycerol Ester ◽  
Lipase Screening ◽  
Hydrolysis Of

Lipases are known as glycerol ester hydrolases that catalyze the hydrolysis of triglycerides into free fatty acids and glycerol. Lipases are found in human, animal, plant, and microorganisms. The aim of this research is to identify lipase producers and characterize bacterial lipase from West Java plateau soil. Plateau soil bacteria samples were isolated on lipase screening medium containing Rhodamine B. Olive oil was used as a substrate in screening and production medium bacterial lipases. From 16 bacterial isolate of lipase producers, 14 were identified as Bacillus sp. and the others were identified as Pseudomonas alcaligenes. All isolates were taken into production step to determine their lipase activities. Moreover, top 3 lipase activities out of 16 lipase activities were chosen to find the optimum pH and temperature. Both characterizations showed pH optimum and temperature optimum from each lipase. These optimum condition were used in heat stability characterization for each lipase samples. The result showed that lipase from isolate COK 2 in optimum pH 4 and temperature 50oC was the most stable lipase due to this sample has good and stable activity for 1 to 5 hours incubation time. Lipase sample from isolate COK 2 has good efficiency for lipase productivity in acid condition and high temperature. Results of this investigation could encourage utilization of these activity enhancers for various industrial applications.

Purification and Partial Characterization of Deoxyribonuclease I from Bovine Parotid Gland

1977 ◽  
Vol 56 (3) ◽  
pp. 320-326 ◽  
Author(s):  
Roger L. Lundblad ◽  
Steve Hoffman ◽  
Claudia M. Noyes ◽  
Henry S. Kingdon
Keyword(s):  
Parotid Gland ◽  
Proteolytic Enzymes ◽  
Divalent Cations ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Acid Extraction ◽  
Ph Optimum ◽  
Deoxyribonuclease I ◽  
Dnase A

Deoxyribonuclease I has been purified from bovine parotid gland. The purification procedure utilizes an acid extraction of minced parotid gland, salt fractionation, gel filtration, and ion-exchange chromatography. The last step, chromatography on Sulfopropyl-Sephadex, resolves the enzymatic activity into several fractions. The major fraction, designated DNase A, was subjected to further investigation. This enzyme has, as expected, an alkaline pH optimum and an obligate requirement for divalent cations. The presence of calcium chloride protects DNase A from inactivation by proteolytic enzymes. Despite the previously described immunologic dissimilarity, there appears to be a large amount of homology between the parotid and pancreatic DNase's.

scholarly journals Production and Characterization of a Thermostable Alcohol Dehydrogenase That Belongs to the Aldo-Keto Reductase Superfamily

2006 ◽  
Vol 72 (1) ◽  
pp. 233-238 ◽  
Author(s):  
Ronnie Machielsen ◽  
Agustinus R. Uria ◽  
Servé W. M. Kengen ◽  
John van der Oost
Keyword(s):  
Alcohol Dehydrogenase ◽  
Pyrococcus Furiosus ◽  
Enantiomeric Excess ◽  
Physiological Role ◽  
Secondary Alcohols ◽  
Gene Encoding ◽  
Ph Optimum ◽  
Reduction Of Ketones

ABSTRACT The gene encoding a novel alcohol dehydrogenase that belongs to the aldo-keto reductase superfamily has been identified in the hyperthermophilic archaeon Pyrococcus furiosus. The gene, referred to as adhD, was functionally expressed in Escherichia coli and subsequently purified to homogeneity. The enzyme has a monomeric conformation with a molecular mass of 32 kDa. The catalytic activity of the enzyme increases up to 100°C, and a half-life value of 130 min at this temperature indicates its high thermostability. AdhD exhibits a broad substrate specificity with, in general, a preference for the reduction of ketones (pH optimum, 6.1) and the oxidation of secondary alcohols (pH optimum, 8.8). Maximal specific activities were detected with 2,3-butanediol (108.3 U/mg) and diacetyl-acetoin (22.5 U/mg) in the oxidative and reductive reactions, respectively. Gas chromatrography analysis indicated that AdhD produced mainly (S)-2-pentanol (enantiomeric excess, 89%) when 2-pentanone was used as substrate. The physiological role of AdhD is discussed.

scholarly journals Penentuan pH Optimum Gel Metasilikat Sekam Padi sebagai Media Tumbuh Kristal Tunggal Kalsium Tartrat Tetrahidrat (CaC4H4O6.4H2O)

2019 ◽  
Vol 20 (2) ◽  
pp. 195
Author(s):  
Yunanda FR ◽  
Suriati Eka Putri ◽  
Hasri Hasri ◽  
Ramdani Ramdani
Keyword(s):  
Single Crystal ◽  
Functional Groups ◽  
Sodium Silicate ◽  
Rice Husk ◽  
Rice Husk Ash ◽  
White Crystal ◽  
Ph Optimum ◽  
Calcium Tartrate ◽  
Optimum Ph

ABSTRAK Penelitian ini bertujuan untuk mengetahui pH optimum sintesis gel metasilikat sekam padi sebagai media tumbuh kristal tunggal kalsium tartrat tetrahidrat (CaTT). Tahapan penelitian ini meliputi preparasi sampel, pembentukan gel metasilikat, sintesis kristal CaTT dan karakterisasi gel metasilikat. Abu sekam padi yang digunakan mengandung SiO2 sebesar 98,45% berpotensi untuk membentuk filtrat natrium silikat (Na2SiO3). Filtrat natrium silikat direaksikan dengan asam tartrat (C4H6O6) menghasilkan gel metasilikat pada pH 5,00; 5,25; 5,50; 5,75 dan 6,00. Supernatan kalsium klorida (CaCl¬2) berdifusi ke dalam gel membentuk kristal CaTT berwarna putih jernih sebanyak 0,2649 g pada pH optimum 5,25. Analisis gugus fungsi gel metasilikat menggunakan spektroskopi FTIR memberikan serapan yang khas untuk gugus fungsi –OH pada 3400,05 cm-1 dan 920,05 cm-1, gugus fungsi C=O pada 1622,13 cm-1 dan C-O pada 1346,31 cm-1, serta gugus Si-O-Si pada 1064,71 cm-1. Karakterisasi gel metasilikat menggunakan XRD mengindikasikan bahwa struktur gel metasilikat hasil sintesis tersusun atas garam Na2C4H4O6.2H2O, senyawa SiO2 dan C-grafit. Berdasarkan analisis gugus fungsi menggunakan FTIR dan karakterisasi menggunakan XRD, dapat disimpulkan bahwa sekam padi berpotensi untuk dijadikan gel metasilikat sebagai media tumbuh kristal tunggal CaTT. Kata kunci: Sekam padi, Natrium Silikat, Gel metasilikat, Kalsium tartrat tetrahidrat ABSTRACT The aims of this study was to determine the optimum pH of metasilicate gel rice husk as medium to grow single crystal of calcium tartrate tetrahydrate (CaC4H4O6.4H2O). This research was carried out inseveral stage namely sample preparation, metasilicate gel synthesis, crystal CaTT synthesis, and metasilicate gel characterization. Rice husk ash that used content SiO2 of 98.45 %, it was potential to be a sodium silicate filtrate. Filtrate of sodium silicate was reacted with tartrat acid (C4H6O6) and produce metasilicate gel with pH 5,00; 5,25; 5,50; 5,75 and 6,00. The supernatant of calcium chloride (CaCl¬2) diffuse into the gel and formed the clear white crystal CaTT as much as 0,2649 g at the optimum pH 5,25. Analysis of functional groups of metasilicate gel by FTIR provides the specific absorption of –OH group at 3400.05 cm-1 and 920.05 cm-1, C=O group at 1622.13 cm-1 and C-O group at 1346.31 cm-1, and the Si-O-Si group at 1064.71 cm-1. Characterization of gel metasilicate by XRD indicated that metasilicate gel produced formed on Na2C4H4O6.2H2O, compound of SiO2 and C-Grafit. Based on the analysis of functional groups by FTIR and characterization by XRD, it was concluded that the rice husk was potentially to be a sources of metasilicate gel as medium to grow single crystal of CaTT. Keywords: Rice Husk, Sodium Silicate, Gel metasilicate, Calcium tartrate tetrahydrate

scholarly journals Characterization of Large Conformational Changes and Autoproteolysis in the Maturation of a T=4 Virus Capsid

2008 ◽  
Vol 83 (2) ◽  
pp. 1126-1134 ◽  
Author(s):  
Tsutomu Matsui ◽  
Gabriel Lander ◽  
John E. Johnson
Keyword(s):  
Conformational Changes ◽  
Cleavage Site ◽  
Cleavage Rate ◽  
Virus Particles ◽  
Wild Type Phenotype ◽  
Optimum Ph ◽  
Reaction Proceeds ◽  
Intermediate Size

ABSTRACT Nudaurelia capensis ω virus-like particles have been characterized as a 480-Å procapsid and a 410-Å capsid, both with T=4 quasisymmetry. Procapsids transition to capsids when pH is lowered from 7.6 to 5.0. Capsids undergo autoproteolysis at residue 570, generating the 74-residue C-terminal polypeptide that remains with the particle. Here we show that the particle size becomes smaller under conditions between pH 6.8 and 6.0 without activating cleavage and that the particle remains at an intermediate size when the pH is carefully maintained. At pH 5.8, cleavage is very slow, becoming detectable only after 9 h. The optimum pH for cleavage is 5.0 (half-life, ∼30 min), with a significant reduction in the cleavage rate at pH values below 5. We also show that lowering the pH is required only to make the virus particles compact and to presumably form the active site for autoproteolysis but not for the chemistry of cleavage. The cleavage reaction proceeds at pH 7.0 after ∼10% of the subunits cleave at pH 5.0. Employing the virion crystal structure for reference, we investigated the role of electrostatic repulsion of acidic residues in the pH-dependent large conformational changes. Three mutations of Glu to Gln that formed procapsids showed three different phenotypes on maturation. One, close to the threefold and quasithreefold symmetry axes and far from the cleavage site, did not mature at pH 5, and electron cryomicroscopy reconstruction showed that it was intermediate in size between those of the procapsid and capsid; one near the cleavage site exhibited a wild-type phenotype; and a third, far from the cleavage site, resulted in cleavage of 50% of the subunits after 4 h, suggesting quasiequivalent specificity of the mutation.

scholarly journals Synthesis & Characterization of Magnetit (Fe3O4) and Its Applications As Adsorbent Methylene Blue

2017 ◽  
Vol 11 (2) ◽  
pp. 61
Author(s):  
Retno Agnestisia
Keyword(s):  
Methylene Blue ◽  
Contact Time ◽  
Coprecipitation Method ◽  
X Ray Diffraction ◽  
Ph Optimum ◽  
Adsorption Study ◽  
Batch System ◽  
Optimum Ph ◽  
Blue Solution

Synthesis, characterization and adsorption study of magnetite have beenconducted. Magnetite was synthesized by coprecipitation method. The characterizations of magnetite were carried out with spectroscopy FTIR (Fourier Transform Infrared) and XRD (X-ray diffraction). The adsorption study was conducted using a batch system with the studied adsorption study including optimum pH, optimum contact time and adsorption equilibrium. The results showed that coprecipitation method has succeeded to form magnetite that has magnetism properties. Magnetite can adsorbed methylene blue from aqueous phase, with the maximum adsorption at pH 5 and contact time of 90 minutes.Adsorption of methylene blue by magnetite follows the adsorption pattern of the Langmuir isotherm with the adsorption energy of 25.59 kJ/mol and adsorption capacity of 43.86 mg/g. The results of magnetite synthesis can accelerate the process of separating the adsorbent particles in a methylene blue solution using an external magnetic field.Keywords : magnetite, coprecipitation, adsorption, and methylene blue.

scholarly journals SCREENING DAN KARAKTERISASI PEKTINESTERASE SEBAGAI ENZIM POTENSIAL DALAM KLARIFIKASI SARI BUAH JERUK KEPROK GARUT (Citrus nobilis var.chrysocarpa)

2015 ◽  
Vol 35 (04) ◽  
pp. 422
Author(s):  
Rohula Utami ◽  
Esti Widowati ◽  
Arifah Rahayu
Keyword(s):  
Thermal Stability ◽  
Optimum Temperature ◽  
Citrus Pectin ◽  
Ph Optimum ◽  
Citrus Juice ◽  
Optimum Ph ◽  
Km Value ◽  
Screening Result ◽  
Optimum Ph And Temperature

The objective of this research was screening of pectinesterase (PE) producing bacteria which are potential in clarification of keprok garut citrus juice (Citrus nobilis var microcarpa) and characterization of the resulted pectinesterase (optimum pH and temperature, pH and thermal stability, KM and Vmaks). The screening result showed that enzyme of isolates AR2, AR 4, AR 6, and KK 2 was found to be a potential enzyme for clarification of keprok garut citrus juice. Enzyme pektinesterase of isolates AR 2, AR 4, AR 6, and KK 2 had optimum pH at 8; 7.5; 8.5; and 6.5 and stable at pH 4-9, 4-9, 6-9, and 3-8. The optimum temperature enzyme of isolates AR 2 and AR 6 were 55ºC and that of AR 4 and KK 2 were 60ºC. Enzyme of isolate AR 2 was stable at 30-50ºC and inactive at 80ºC, AR 4 and KK 2 were stable at 30-60ºC and inactive at 90ºC whereas AR6 was stable at 30-60ºC and still wasn’t inactive at 90ºC. KM value of isolates AR 2, AR 4, AR 6, and KK 2 were 0.604; 0.338; 0.971; and 0.392 mg/ml. Vmaks value of isolates AR 2, AR 4, AR 6, and KK 2 were 1.218; 0.826; 0.969; and 1.080 u/ml. Pectinesterase enzyme of isolates KK 2 was found to be the most potential enzyme for clarification of keprok garut citrus juice.Keywords: Clarification, enzyme, keprok garut citrus, pectin, pectinesterase ABSTRAKTujuan dari penelitian ini adalah untuk melakukan screening bakteri penghasil enzim pektinesterase (PE) yang berpotensi dalam proses klarifikasi sari buah jeruk keprok garut (Citrus nobilis var microcarpa) serta mengetahui karakteristik enzim pektinesterase yang dihasilkan (pH optimum, suhu optimum, kestabilan pH dan suhu, serta nilai KMdan Vmaks). Hasil screening didapatkan isolat AR 2, AR 4, AR 6, dan KK 2 sebagai isolat penghasil enzim pektinesterase yang berpotensi dalam proses klarifikasi sari buah jeruk keprok garut. Aktivitas enzim pektinesterase isolat AR 2, AR 4, AR 6 dan KK 2 berturut-turut optimum pada pH 8; pH 7,5; pH 8,5; dan pH 6,5, serta stabil pada pH 4-9, pH4-9, pH 6-9, dan pH 3-8. Suhu optimum enzim pektinesterase isolat AR 2, AR 4, AR 6, dan KK 2 berturut-turut adalah 55ºC, 60ºC, 55ºC, dan 60ºC. Enzim pektinesterase isolat AR 2 stabil pada suhu 30-50ºC dan inaktif pada suhu 80ºC, enzim pektinesterase isolat AR 4 dan KK 2 stabil pada suhu 30-60ºC dan inaktif pada suhu 90ºC, sedangkan enzim pektinesterase isolat AR 6 stabil pada suhu 30-60ºC namun belum inaktif pada suhu 90ºC. Nilai konstanta Michaelis-Menten (KM) enzim pektinesterase isolat AR 2, AR 4, AR 6, dan KK 2 berturut-turut adalah 0,604; 0,338; 0,971; dan 0,392 mg/ml. Sedangkan nilai kecepatan maksimum (Vmaks) enzim pektinesterase isolat AR 2, AR 4, AR6, dan KK 2 berturut-turut adalah 1,218; 0,826; 0,969; dan 1,080 U/ml. Enzim pektinesterase isolat KK 2 memiliki karakteristik yang paling sesuai untuk aplikasi dalam klarifikasi sari buah jeruk keprok garut dibandingkan dengan enzim pektinesterase isolat lainnya.Kata kunci: Enzim, klarifikasi, pektin, pektinesterase, jeruk keprok garut

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