scholarly journals The β-glucosidase in the gut contents of the snail Achatina achatina

1976 ◽  
Vol 157 (2) ◽  
pp. 381-387 ◽  
Author(s):  
G M Umezurike
Keyword(s):  
Molecular Weight ◽  
Heat Stability ◽  
Molecular Forms ◽  
Gut Contents ◽  
Ph Optimum ◽  
Optimum Ph ◽  
Michaelis Constants ◽  
Molecular Complexity ◽  
Beta Glucosidase ◽  
Digestive Glands

1. The enzyme beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) from the gut contents of active Achatina achatina exists in two molecular forms, beta-glucosidase C (mol.wt. about 82000) and D (mol.wt. about 41000). 2. Only the lower-molecular-weight species was found in the gut contents of aestivating snails or in extracts from their digestive glands and washed gut walls. 3. On re-activation of some aestivating snails, betion of ATP and Mg2+ to the isolated gut contents or to extracts from washed gut walls led to the formation of higher-molecular-weight forms of the enzyme, beta-glucosidase A (mol.wt. about 329000) and beta-glucosidase B (mol.wt. about 165000). 5. All these forms of the enzyme have similar pH optimum (pH 5.0-5.6). 6. The Michaelis constants (Km) and heat stability of the enzyme increased with increasing molecular complexity.

Multiple molecular forms of avian aldolases. II. Enzymic properties and amino acid composition of chicken (Gattus domesticus) breast muscle aldolase

1969 ◽  
Vol 47 (5) ◽  
pp. 527-534 ◽  
Author(s):  
Ronald R. Marquardt
Keyword(s):  
Amino Acid ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Heat Stability ◽  
Breast Muscle ◽  
Molecular Forms ◽  
Ph Optimum ◽  
Wide Ph Range ◽  
Michaelis Constants ◽  
Ph Range

Several properties of crystalline chicken (Gallus domesticus) breast muscle aldolase (fructose 1,6-diphosphate–D-glyceraldehyde 3-phosphate lyase, EC 4.1.2.13) were determined. The enzyme was found to have a broad pH optimum centered around pH 7.1 and to be remarkably stable over a wide pH range. The temperature coefficient Q10 is 2.6 in the range from 10 to 35 °C. The enzyme is stable at 48 °C for 10 min and almost completely inactivated at 55 °C. The apparent Michaelis constants for fructose 1,6-diphosphate and fructose 1-phosphate were 4.2 × 10−5 M and 1.7 × 10−2 M, respectively. The phosphate inhibitor constant (K1) was 5.5 × 10−3 M.Chicken breast muscle aldolase is similar to the rabbit enzyme in many of the above properties, although there are significant differences in heat stability and amino acid composition.

Glutamate Dehydrogenase from Pea Roots: Purification and Properties of the Enzyme

1971 ◽  
Vol 49 (1) ◽  
pp. 127-138 ◽  
Author(s):  
E. Pahlich ◽  
K. W. Joy
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Glutamate Dehydrogenase ◽  
Constant Ratio ◽  
Ph Optimum ◽  
Purification And Properties ◽  
Activity Ratios ◽  
Single Protein ◽  
Michaelis Constants ◽  
Pea Roots

Glutamate dehydrogenase (L-glutamate: NAD+ oxidoreductase (deaminating), EC 1.4.1.2) has been purified 1250-fold from pea roots. The preparation contains only a single protein, and the molecular weight was estimated to be 208 000 ± 10 000. The enzyme shows NADH (aminating) and NAD+ (deaminating) activities, but the ratio of these activities is not constant and can be changed experimentally. NADPH activity is also present and shows a relatively constant ratio to NAD+ activity. EDTA inhibits NADH activity in intermediate concentrations, but reactivates at higher concentrations. NAD+ (and NADPH) activity is only slightly changed by EDTA. The effects of dioxane and the coenzymes on the enzyme are also reported. Mechanisms which could explain the different activity ratios, in terms of two interconvertible enzyme forms, are discussed.The pH optimum for NADH and NAD+ activities is about pH 8.0. Michaelis constants were found to be: α-ketoglutarate, 3.3 × 10−3 M; ammonium (sulfate), 3.8 × 10−2 M; glutamate, 7.3 × 10−3 M; NADH, 8.6 × 10−4 M; NAD+, 6.5 × 10−4 M. The enzyme is highly specific for the substrates glutamate and α-ketoglutarate, showing no alanine or aspartate dehydrogenase activity, and no deamination with a range of amino acids.

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.

scholarly journals The subunit structure of β-glucosidase from Botryodiplodia theobromae Pat

1975 ◽  
Vol 145 (2) ◽  
pp. 361-368 ◽  
Author(s):  
G M Umezurike
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Substrate Inhibition ◽  
Gel Filtration ◽  
Heat Stability ◽  
Reduced Form ◽  
Subunit Structure ◽  
Botryodiplodia Theobromae ◽  
Low Concentrations ◽  
Michaelis Constants

1. A homologous series of beta-glcosidase (beta-D-glcoside glcohydrolase, EC 3.2.1.21), which varied in relative amounts in different preparations from cultures of similar and different age, was observed in cultures od Botryodiplodia theobromae Pat grown for 4-8 week on cotton flock (cellulose) as carbon source. 2. Aging of the purified high-molecular-weight species led to some amount of siddociation into a homolous series of lower-molecular-weight speices. 3. Rough molecular-weight estimates, by gel filtration, of the various species derived from the purifeid high-molecular-weight enzyme were 350000-3800000, 170000, 180000, 83000-87000 and 45000-47000. 4. Electron micrographs of the negatively stained 350000-380000-molecular-weight enzyme showed that the molecule is an octamer in which each roughly spherical monomer occupies a corner of a cube with each side about 7.14nm long. 5. Carboxamidomethylation of the reduced form of each molecular-weight species of the enzyme led to irreversible dissociation of the molecules into electrophoretically identical polypeptides with a moleclar weight of 10000-12000. 6. These results suggest a slow association-dissociation of the type (8n)in equilibrium 2 (4n) in equilibrium 4(2n) in equilibrium 8(n), where n is defined as the monomer. The monomer is in turn made up of four polypeptide a subunits whi-ch are non-catalytic. 7. The Michaelis constants (Km) and heat stability of the four wnzymically active molecular species derived from the purified enzyme increased with molecular complexity, whereas all four species were inhibited by glycerol (100nM) at low concentrations of substrate (o-nitrophenyl beta-D-glucopyranoside) but activated at high substrat concentrations. 8. Only the lowest-molecular-weight species (45species (45,000-47000 mol. wt.) showed substrate inhibition.

scholarly journals Partial purification and properties of an acid nucleotidase from the postmicrosomal supernatant of rat spleen

1978 ◽  
Vol 169 (3) ◽  
pp. 597-605 ◽  
Author(s):  
Hans Tjernshaugen
Keyword(s):  
Molecular Weight ◽  
Catalytic Properties ◽  
Partial Purification ◽  
Ph Optimum ◽  
Phosphatase Inhibitors ◽  
Purification And Properties ◽  
Optimum Ph ◽  
Liver Lysosomes ◽  
Rat Liver Lysosomes ◽  
Single Enzyme

1. The dephosphorylation of 3′-AMP, 3′-dAMP, 3′-CMP and 3′-dCMP was studied in the postmicrosomal supernatant of rat spleen and liver. In both organs 3′-AMP and 3′-dAMP were dephosphorylated at an appreciable rate, in both the presence and the absence of Mg2+. The pH optimum for this dephosphorylation was in the range 4.5–5.0. 3′-CMP and 3′-dCMP were very slowly degraded, though the activity towards 3′-dCMP increased somewhat in the presence of Mg2+. The optimum pH for this Mg2+-dependent dephosphorylation was 5.5–6.0. 2. The rate of dephosphorylation of 3′-AMP and 3′-dAMP per mg of protein was about 5 times as high in spleen as in liver. 3. The dephosphorylation of 3′-AMP could be ascribed to a single enzyme with pH optimum about 4.5. The activity towards 3′-dAMP could be resolved into one component coinciding with the 3′-dAMP-degrading enzyme, and one Mg2+-requiring component probably identical with the soluble deoxyinosine-activated nucleotidase. The dephosphorylation of 3′-dCMP seemed to be performed only by the latter enzyme. 4. The enzyme dephosphorylating 3′-AMP was purified 200-fold from the postmicrosomal supernatant and its physical and catalytic properties were compared with those of acid nucleotidase (EC 3.1.3.31) purified from rat liver lysosomes. The two enzymes were identical in all properties tested (substrate specificity, Km, molecular weight, response to phosphatase inhibitors), but some of the data differed from earlier reports on the acid nucleotidase. 5. The subcellular localization of the acid nucleotidase, its relationship to the acid phosphatase(s) and its role in the breakdown of nucleic acid constituents are discussed.

scholarly journals The octameric structure of β-glucosidase from Botryodiplodia theobromae Pat

1991 ◽  
Vol 275 (3) ◽  
pp. 721-725 ◽  
Author(s):  
G M Umezurike
Keyword(s):  
Substrate Inhibition ◽  
Hydrolysis Product ◽  
Gel Filtration ◽  
Molecular Forms ◽  
Botryodiplodia Theobromae ◽  
Complete Inactivation ◽  
Alkaline Conditions ◽  
Michaelis Constants ◽  
Beta Glucosidase ◽  
Inactive Protein

1. Whereas only beta-glucosidase A (beta-D-glucoside glucohydrolase, EC 3.2.1.21) was produced by the tropical fungus Botryodiplodia theobromae Pat. (I.M.I. 115626; A.T.C.C. 26123) in young cultures containing D-cellobiose as carbon source, lower-Mr forms (B, C and D) were found in older cultures when the pH had drifted from the initial value of pH 6.2 to pH 7.9. 2. The Michaelis constants (Km) of the various molecular forms of the enzyme were 0.30 +/- 0.03 mM-, 0.26 +/- 0.01 mM-, 0.20 +/- 0.02 mM- and 0.16 +/- 0.01 mM-o-nitrophenyl beta-D-glucopyranoside for beta-glucosidase forms A (Mr 320,000), B (Mr 160,000), C (Mr 80,000) and D (Mr 40,000) respectively. 3. Only beta-glucosidase D showed substrate inhibition. 4. Only L-arginine was found as the N-terminal residue, and beta-glucosidase A contained 31.7 +/- 0.6 mol of N-terminal L-arginine/mol of the enzyme. 5. Storage of purified beta-glucosidase A under mildly alkaline conditions caused its dissociation into the lower-Mr forms, whereas adjustment of the pH of a solution of beta-glucosidase A to pH 12.0 with 1 M-NaOH led to complete inactivation on incubation at 40 degrees C for 1 h and to the release of 25.2 +/- 1.5 mol of inorganic phosphate/mol of the enzyme. 6. O-Phospho-L-serine was isolated from the acid-hydrolysis product of beta-glucosidase A but not from that of beta-glucosidase D. 7. Reduction and carboxamidomethylation of the various forms of beta-glucosidase gave only one enzymically inactive protein with an Mr of 10,000-11,000. 8. After partial succinylation (3-carboxypropionylation) of beta-glucosidase D at pH 5.0 and removal of the precipitated protein formed, the supernatant solution contained beta-glucosidase components similar to the other molecular forms (A, B and C) and an aggregate (beta-glucosidase Xs) that gave a positive result in the alkaline hydroxylamine test, whereas N-succinylated beta-glucosidase D, an aggregate (form Xp) that behaved like beta-glucosidase Xs and traces of forms A, B and C were found by gel filtration of the solution of the precipitate solubilized at neutral pH (7.0-7.7). 9. These observations are discussed in terms of the proposed octameric structure of beta-glucosidase A based on the result of electron microscopy [Umezurike (1975) Biochem. J. 145, 361-368].

Characterization and partial purification of β-hydroxybutyrate dehydrogenase from sporulating cells of Bacillus cereus T

1973 ◽  
Vol 19 (6) ◽  
pp. 673-677 ◽  
Author(s):  
E. D. Thompson ◽  
H. M. Nakata
Keyword(s):  
Molecular Weight ◽  
Bacillus Cereus ◽  
Divalent Cation ◽  
Phosphate Buffer ◽  
Partial Purification ◽  
Ph Optimum ◽  
Michaelis Constants

A soluble NAD+-dependent β-hydroxybutyrate (βHB) dehydrogenase was shown to appear 3 to 4 h after the onset of sporulation of Bacillus cereus T. The enzyme was stable in Tris-chloride buffer when frozen, but required 0.05 to 0.1 M of MgCl2 or other divalent cation such as Mn2+, Ba2+, or Ca2+ for stability at 4C. In the presence of phosphate buffer or EDTA, the enzyme lost all activity within 2 min. βHB dehydrogenase was partially purified and shown to have a molecular weight of about 93 000, pH optimum of 8.0 in 0.1 M Tris-chloride buffer, Michaelis constants, Km, of 2.3 × 10−3 M for β-hydroxybutyrate and 9.5 × 10−4 M for NAD+, and was inhibited 40% by 1 × 10−3 M p-hydroxymercuribenzoate. The enzyme from B. cereus T was compared in these respects with βHB dehydrogenases isolated from several non-sporeforming bacteria.

Isolation and Characterization of Plasminogen Activator from Pig Leucocytes

1974 ◽  
Vol 31 (01) ◽  
pp. 072-085 ◽  
Author(s):  
M Kopitar ◽  
M Stegnar ◽  
B Accetto ◽  
D Lebez
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Isolation And Characterization ◽  
Ph Range ◽  
Inhibition Studies ◽  
Final Purification

SummaryPlasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

Purine nucleoside phosphorylase: Isolation and characterization

1990 ◽  
Vol 55 (12) ◽  
pp. 2987-2999 ◽  
Author(s):  
Katarina Šedivá ◽  
Ivan Votruba ◽  
Antonín Holý ◽  
Ivan Rosenberg
Keyword(s):  
Molecular Weight ◽  
Purine Nucleoside Phosphorylase ◽  
Purine Nucleoside ◽  
Leukemia Cells ◽  
Ph Optimum ◽  
Nucleoside Phosphorylase ◽  
Isolation And Characterization ◽  
Reaction Proceeds ◽  
The Matrix ◽  
Sepharose 4B

Purine nucleoside phosphorylase (PNP) from mouse leukemia cells L1210 was purified to homogeneity by a combination of ion exchange and affinity chromatography using AE-Sepharose 4B and 9-(p-succinylaminobenzyl)hypoxanthine as the matrix and the ligand, respectively. The native enzyme has a molecular weight of 104 000 and consists of three subunits of equal molecular weight of 34 000. The results of isoelectric focusing showed that the enzyme is considerably microheterogeneous over the pI-range 4.0-5.8 and most likely consists of eight isozymes. The temperature and pH-optimum of phosphorolysis, purine nucleoside synthesis and also of transribosylation is identical, namely 55 °C and pH 7.4. The transribosylation reaction proceeds in the presence of phosphate only. The following Km-values (μmol l-1) were determined for phosphorolysis: inosine 40, 2'-deoxyinosine 47, guanosine 27, 2'-deoxyguanosine 32. The Km-values (μmol l-1) of purine riboside and deoxyriboside synthesis are lower than the values for phosphorolysis (hypoxanthine 18 and 34, resp., guanine 8 and 11, resp.). An affinity lower by one order shows PNP for (-D-ribose-1-phosphate, (-D-2-deoxyribose-1-phosphate (Km = 200 μmol l-1 in both cases) and phosphate (Km = 805 μmol l-1). The substrate specificity of the enzyme was also studied: positions N(1), C(2) and C(8) are decisive for the binding of the substrate (purine nucleoside).

Investigating the Rheological Properties and Heat Stability of Polyarylsulphone Sulphides of Domestic Grades

2017 ◽  
Vol 44 (1) ◽  
pp. 23-26
Author(s):  
A.B. Baranov ◽  
I.D. Simonov-Emel'yanov ◽  
T.I. Andreeva ◽  
T.N. Prudskova ◽  
V.I. Sazikov
Keyword(s):  
Molecular Weight ◽  
Heat Stability ◽  
Chain Structure ◽  
Low Flow ◽  
Processing Temperature ◽  
Rheological Characteristics ◽  
Melt Viscosity ◽  
Full Curve ◽  
Temperature Range ◽  
Service Characteristics

The rheological characteristics of melts of polyarylsulphone sulphides of domestic grades were investigated in the temperature range 240–360°C. Full curves of heat stability were obtained, and formulae were proposed for their description. Polyarylsulphone sulphides (PSPSs) are heat-resistant polymers of structural designation of the polysulphone (PSP) class, the domestic production technology of which was developed at OAO “Institut plastmass”, which makes it possible to carry out the production of a wide grade range of PSP of different polymer chain structure and molecular weight. The main problem in the processing of PSPSs is their high melt viscosity and fairly low flow. Increase in the processing temperature lowers their melt viscosity, but there is then the danger of thermooxidative degradation of the polymer and consequently of deterioration in the service characteristics and appearance of articles. The aim of this work was to investigate the rheological characteristics and to obtain the full curve of heat stability for domestic grades of PSPSs and PSP of different molecular weight in a wide processing temperature range.

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