scholarly journals The action on cellulose and its derivatives of a purified 1,4-β-glucanase from Trichoderma koningii

1981 ◽  
Vol 199 (2) ◽  
pp. 409-417 ◽  
Author(s):  
G Halliwell ◽  
R Vincent
Keyword(s):  
Specific Activity ◽  
High Specificity ◽  
Enzymic Activity ◽  
Transferase Activity ◽  
Beta Glucanase ◽  
Trichoderma Koningii ◽  
Initial Substrate ◽  
Central Bond ◽  
Beta Glucosidase ◽  
Derivatives Of

The specific properties have been examined of the 1,4-beta-glucanase component of Trichoderma koningii that participates in an early and effective stage of random breakdown of native cellulose to short fibres. The enzyme was purified and freed from associated components of the cellulase complex (particularly beta-glucosidase) that interfere with, and complicate interpretation of, the action of such enzymes. Purification increased the specific activity 25-fold over culture filtrates; the enzyme hydrolysed CM-cellulose faster than the purified beta-glucosidase from the same organism hydrolysed any of its substrates (cellobiose or cellodextrins). The specificity of the glucanase was directed towards soluble derivatives of cellulose, CM-cellulose and cellodextrins, and not to insoluble cellulose or alpha-linked polymers. The approximate Km was 2.5 mg of CM-cellulose . ml-1 at 37 degrees C at the optimum pH, 5.5, where enzymic activity was maximal with 6--7 mg of CM-cellulose . ml-1 and inhibited by higher concentrations. The temperature optimum was 60 degrees C. The glucanase attacked larger cellodextrins (cellohexaose to cellotetraose, in that order) much more readily than smaller dextrins (cellobiose and cellotriose) and released a mixture of products, glucose up to cellopentaose, which was quantitatively determined after chromatography on charcoal. Similar examination of hydrolysates of the reduced cellodextrins showed clearly the high specificity of the enzyme for the central bond of its natural substrates (the cellodextrins), whatever their chain length, and indicated the nature of the enzyme as an endoglucanase. Outer bonds shared a weaker, but similar, susceptibility to enzymic cleavage. Transferase activity was absent and no larger dextrins than the initial substrate were formed.

Synthesis and Antimicrobial Activity of Adamantyl Substituted Pyridoxine Derivatives

2019 ◽  
Vol 16 (12) ◽  
pp. 1360-1369 ◽  
Author(s):  
Rail Khaziev ◽  
Nikita Shtyrlin ◽  
Roman Pavelyev ◽  
Raushan Nigmatullin ◽  
Raylya Gabbasova ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Lipid Membranes ◽  
Specific Activity ◽  
Tuberculosis H37rv ◽  
Microbial Pathogens ◽  
Adamantane Derivatives ◽  
Carbon Cage ◽  
H37rv Strain ◽  
Derivatives Of

Background: Adamantane derivatives possess multiple pharmacological activities such as antiviral, anticancer, antimycobacterial, antidiabetic, antiparkinsonian and others. The interest of medicinal chemists in adamantane compounds is due to their unique spatial structure, high lipophilicity, and carbon cage rigidity. As a result, these molecules can easily penetrate biological lipid membranes and often have unique target-specific activity profile. Another pharmacophore studied in this work is pyridoxine (vitamin B6). Pyridoxine plays highly important roles in living cells as a key cofactor of many enzymes. On the other hand, its molecular scaffold is a valuable structural platform which has led to the development of several launched drugs (Pyritinol, Pirisudanol, Cycletanine, Mangafodipir) and a wide number of preclinical and clinical drug candidates. Objective: The objective of this study is a synthesis of pyridoxine-adamantane and pyridoxinecyclooctane dipharmacophore molecules. The underlying idea was to assess the antibacterial and antiviral potential of such dipharmacophores, based on multiple examples of promising antiinfective agents which have in their structures adamantane and pyridoxine moieties. Another specific reason was to explore the ability of pyridoxine pharmacophore to suppress the potential of microbial pathogens to develop resistance to drug molecules. Methods: In this study, a series of pyridoxine-adamantane and pyridoxine-cyclooctane dipharmacophore molecules were synthesized based on reactions of three different cycloalkyl amines with the corresponding electrophilic derivatives of pyridoxine aldehydes, chlorides and acetates. All synthesized compounds have been tested for their in vitro activity against M. tuberculosis H37Rv strain and H3N2 (A/Aichi/2/68) influenza virus. Results: Series of pyridoxine-adamantane and pyridoxine-cyclooctane dipharmacophore molecules were synthesized based on reactions of three different cycloalkylamines with the corresponding electrophilic derivatives of pyridoxine aldehydes, chlorides and acetates. Reaction of cycloalkylamines with pyridoxine derivatives, in which meta-hydroxyl and ortho-hydroxymethyl groups are protected by acetyl groups, represents a useful alternative to reductive amination of aldehydes and nucleophilic substitution of alkyl halides. According to a tentative mechanism, it proceeds via paraand ortho-pyridinone methides which readily react with nucleophiles. None of the synthesized dipharmacophore compounds showed activity against M. tuberculosis H37Rv strain. At the same time, three compounds demonstrated some antiviral activity against H3N2 (A/Aichi/2/68) influenza virus (EC50 52-88 µg/mL) that was comparable to the activity of Amantadine, though lower than the activity of Rimantadine. The results of this work can be useful in the design of physiologically active derivatives of pyridoxine and adamantane. Conclusion: The results of this work can be useful in the design of physiologically active derivatives of pyridoxine and adamantane.

Purification of Glycerol Kinase by "Dye-Ligand" Chromatography and Hydrophobic Interaction Chromatography on Bead-Cellulose Derivatives

1993 ◽  
Vol 58 (2) ◽  
pp. 445-451 ◽  
Author(s):  
Vladimír Žúbor ◽  
Albert Breier ◽  
Marta Horváthová ◽  
Dagmar Hagarová ◽  
Peter Gemeiner ◽  
...  
Keyword(s):  
Hydrophobic Interaction ◽  
Specific Activity ◽  
Hydrophobic Interaction Chromatography ◽  
Glycerol Kinase ◽  
Enzymic Activity ◽  
Cellulose Derivatives ◽  
Long Term Storage ◽  
Bead Cellulose ◽  
Term Storage

The crude extract of cytosole enzymes was obtained from homogenized cells of Saccharomyces cerevisiae by partition. The enzyme was then isolated from the lower aqueous phase displaying higher glycerol kinase activity by dye-ligand chromatography on Cibacron Blue (CB) or Remazol Brilliant Blue R (RB)-derivatized bead-cellulose, ATP being the eluent. The specific activity of glycerol kinase rised more than 10 and 7-times after affinity dye-ligand chromatography and hydrophobic interaction chromatography, respectively. Glycerol kinase obtained by the latter method was purified by CB-bead cellulose. The final preparation maintained its enzymic activity without noticeable losses during a long-term storage at 4 °C in dark.

METABOLISM OF PROGESTERONE BY THE RAT OVARY: FORMATION OF 3β-HYDROXY-5α-PREGNAN-20-ONE BY OVARIAN MICROSOMES

1969 ◽  
Vol 61 (4) ◽  
pp. 618-628 ◽  
Author(s):  
A. Zmigrod ◽  
H. R. Lindner
Keyword(s):  
Oxidation Product ◽  
Microsomal Fraction ◽  
Biological Function ◽  
Specific Activity ◽  
Metabolite Identification ◽  
Enzymic Activity ◽  
Chromatographic Behaviour ◽  
Rat Ovary ◽  
Chromatographic Characterization

ABSTRACT A microsomal fraction from rat ovaries incubated with [4-14C] progesterone in the presence of NADPH produced radioactive 3β-hydroxy-5α-pregnan-20-one as the major metabolite. Identification of this compound, not hitherto isolated from mammalian ovaries, was based on (i) paper and gas-chromatographic behaviour of the free compound and its acetate, (ii) gas-chromatographic characterization of the thioketal derivative and the oxidation product, and (iii) recrystallization with authentic carrier to constant specific activity. Ovaries from both pregnant and prooestrous rats showed this enzymic activity. The possible biological function of ovarian steroid reductases is discussed.

scholarly journals Structural and Catalytic Characterization of TsBGL, a β-Glucosidase From Thermofilum sp. ex4484_79

2021 ◽  
Vol 12 ◽  
Author(s):  
Anke Chen ◽  
Dan Wang ◽  
Rui Ji ◽  
Jixi Li ◽  
Shaohua Gu ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Specific Activity ◽  
Maximum Activity ◽  
Homologous Proteins ◽  
Glycosidic Bonds ◽  
Catalytic Sites ◽  
Potential Applications ◽  
Beta Glucosidase ◽  
Hydrolysis Of

Beta-glucosidase is an enzyme that catalyzes the hydrolysis of the glycosidic bonds of cellobiose, resulting in the production of glucose, which is an important step for the effective utilization of cellulose. In the present study, a thermostable β-glucosidase was isolated and purified from the Thermoprotei Thermofilum sp. ex4484_79 and subjected to enzymatic and structural characterization. The purified β-glucosidase (TsBGL) exhibited maximum activity at 90°C and pH 5.0 and displayed maximum specific activity of 139.2μmol/min/mgzne against p-nitrophenyl β-D-glucopyranoside (pNPGlc) and 24.3μmol/min/mgzen against cellobiose. Furthermore, TsBGL exhibited a relatively high thermostability, retaining 84 and 47% of its activity after incubation at 85°C for 1.5h and 90°C for 1.5h, respectively. The crystal structure of TsBGL was resolved at a resolution of 2.14Å, which revealed a classical (α/β)8-barrel catalytic domain. A structural comparison of TsBGL with other homologous proteins revealed that its catalytic sites included Glu210 and Glu414. We provide the molecular structure of TsBGL and the possibility of improving its characteristics for potential applications in industries.

scholarly journals Developmentally regulated enzymes and cyclic AMP-binding sites in Dictyostelium discoideum cells blocked during development by α-chymotrypsin

1982 ◽  
Vol 206 (2) ◽  
pp. 185-193
Author(s):  
J A Schmidt ◽  
J L Stirling
Keyword(s):  
Cyclic Amp ◽  
Dictyostelium Discoideum ◽  
Binding Sites ◽  
Glycogen Phosphorylase ◽  
Specific Activity ◽  
Tyrosine Aminotransferase ◽  
Carbohydrate Binding ◽  
Threonine Deaminase ◽  
Developmentally Regulated ◽  
Beta Glucosidase

When cells of the slime mould Dictyostelium discoideum are allowed to starve in the presence of alpha-chymotrypsin, they are blocked in development at the stage where tight aggregates form tips. Analysis of developmentally regulated enzymes has shown that alpha-mannosidase, beta-N-acetylglucosaminidase, threonine deaminase, tyrosine aminotransferase, beta-glucosidase and the carbohydrate-binding protein discoidin are unaffected, but enzymes that show an increase in specific activity during post-aggregative development, namely glycogen phosphorylase, UDP-glucose pyrophosphorylase, UDP-galactose 4-epimerase, UDP-galactose polysaccharide transferase and alkaline phosphatase, did not show the characteristic increase when development was blocked by alpha-chymotrypsin. Recovery of cells from the effects of alpha-chymotrypsin was accompanied by the formation of fruiting bodies and a concomitant increase in the specific activity of UDP-glucose pyrophosphorylase. Uptake or efflux of 45Ca2+ was not altered in the presence of alpha-chymotrypsin. Cells allowed to develop in alpha-chymotrypsin, or treated with the enzyme for 15 min, had a markedly reduced ability to bind cyclic AMP with low affinity; high-affinity binding was unaffected. Pronase had a similar effect on cyclic AMP binding, but trypsin, which does not alter developmental processes, has no effect on cyclic AMP binding to D. discoideum cells.

Enzymic activity and .gamma.-phosphorus-32 labeling of fluorescent derivatives of cytidine triphosphate and adenosine triphosphate

1973 ◽  
Vol 95 (3) ◽  
pp. 961-962 ◽  
Author(s):  
Jorge R. Barrio ◽  
Laurence G. Dammann ◽  
Leslie H. Kirkegaard ◽  
Robert L. Switzer ◽  
Nelson J. Leonard
Keyword(s):  
Adenosine Triphosphate ◽  
Enzymic Activity ◽  
Derivatives Of ◽  
Fluorescent Derivatives

Acridinium esters as high-specific-activity labels in immunoassay.

1983 ◽  
Vol 29 (8) ◽  
pp. 1474-1479 ◽  
Author(s):  
I Weeks ◽  
I Beheshti ◽  
F McCapra ◽  
A K Campbell ◽  
J S Woodhead
Keyword(s):  
Monoclonal Antibodies ◽  
Detection Limit ◽  
Specific Activity ◽  
High Specific Activity ◽  
Derivatives Of ◽  
Acridinium Ester

Abstract A chemiluminescent acridinium ester has been synthesized that reacts spontaneously with proteins to yield stable, immunoreactive derivatives of high specific activity. The compound has been used to prepare chemiluminescent monoclonal antibodies to human alpha 1-fetoprotein having average incorporation ratios as great as 2.8 mol of label per mole of antibody, which corresponds to a detection limit of approximately 8 X 10(-19) mol. These antibodies have been used in the preliminary development of a two-site immunochemiluminometric assay for human alpha 1-fetoprotein, which requires only a 30-min incubation and a quantification time of 5 s per sample.

scholarly journals Partial Chemical Characterization of S-Carboxymethylated Chains of Rabbit Fibrin(OGEN): Evidence for Separate Chain Biosynthesis

1977 ◽  
Author(s):  
B. Alving ◽  
G. Murano ◽  
D. Walz
Keyword(s):  
Amino Acid ◽  
Specific Activity ◽  
Chemical Characterization ◽  
Molecular Size ◽  
Exchange Chromatography ◽  
12 Steps ◽  
Polypeptide Chains ◽  
Sds Electrophoresis ◽  
Derivatives Of

The purpose of this study was twofold: 1) chemically characterize the isolated polypeptide chains of rabbit fibrin(ogen), and 2) explore their mode of biosynthesis. The three S-carboxy-methyl polypeptide chain derivatives of rabbit fibrin (α, β and γ) were isolated by cation exchange chromatography. Their amino acid composition was similar to the human with a methionine distribution (mole/mole) as follows: γ = 9; β = 14, α = 14. Their molecular size, (SDS electrophoresis) was estimated as follows: γ = 46,000; β = 54,000; α = 63,500. The N-terminal amino acid sequence (12 steps) of the β derivative was:Gly-His-Arg-Pro-Ile-Asp-Arg-Arg-Arg-Glu-Glu-Leu-. To determine whether the three chains are synthesized sequentially (one continuous chain, later split into three) or in parallel, turpentine-stimulated male New Zealand rabbits were given ~40 μCi of [75Se] selenomethionine (SeM) and its incorporation into fibrinogen (F) was followed. F was clotted from plasma samples, washed, reduced, and constituent chains separated by gel electrophoresis in the presence of SDS-urea. The radioactivity of each chain (expressed as percent of total F radioactivity) was determined, and the specific methionine radioactivity calculated for each chain isolated at 20, 25, and 30 min after SeM injection. During this interval the specific activity of the α and the γ chains was essentially the same (within 3%) while that of the β chain was 42 to 97% greater than that of the α chain. The similar activity of the α and γ chains during the early phase of SeM incorporation suggests that these two chains are not synthesized sequentially, rather they are synthesized in parallel.

Synthesis of phosphatidylcholine by rat lung during choline deficiency

1986 ◽  
Vol 61 (6) ◽  
pp. 2040-2044 ◽  
Author(s):  
R. W. Yost ◽  
A. Chander ◽  
C. Dodia ◽  
A. B. Fisher
Keyword(s):  
Fatty Acid ◽  
Soy Protein ◽  
De Novo ◽  
Specific Activity ◽  
Choline Chloride ◽  
Transferase Activity ◽  
Rat Lung ◽  
Choline Deficiency ◽  
Glucose Incorporation ◽  
Choline Phosphate

The effect of choline deficiency on the de novo pathway for phosphatidylcholine (PC) synthesis in the lung was investigated in rats fed a washed soy protein (lipotrophic) diet deficient in choline and methionine for 2–3 wk. Lungs from lipotrophic rats showed a decreased content of choline and choline-phosphate (P less than 0.05) compared with control but no change in content of cytidine 5′-diphosphocholine or PC. Isolated perfused lungs from lipotrophic rats were evaluated for choline and fatty acid utilization for PC synthesis. Lipotrophic lungs perfused with 5 microM [14C-methyl]-choline chloride showed increased incorporation into PC while there was no significant effect at saturating levels of choline (100 microM). There was increased incorporation of [1-14C]-palmitic acid into PC and diglyceride and increased incorporation of D-[U-14C]glucose into fatty acids of PC. Increased choline and glucose incorporation was not due to alteration of intracellular specific activity of these substrates. This study indicates the utilization of choline and fatty acid for PC synthesis is stimulated as a result of choline deficiency while lung CDP-choline concentration is maintained, possibly through regulation of choline phosphate cytidyl transferase activity. These mechanisms compensate for decreased choline availability to maintain the PC content of lungs.

scholarly journals DISTRIBUTION OF NEWLY SYNTHESIZED AMYLASE IN MICROSOMAL SUBFRACTIONS OF GUINEA PIG PANCREAS

1966 ◽  
Vol 30 (3) ◽  
pp. 519-530 ◽  
Author(s):  
P. Siekevitz ◽  
G. E. Palade
Keyword(s):  
Guinea Pig ◽  
High Speed ◽  
Specific Activity ◽  
Specific Radioactivity ◽  
Enzymic Activity ◽  
Pellet Fraction ◽  
Light Region ◽  
Centrifuge Tube ◽  
In Vivo Labeling

Amylase distribution was studied in guinea pig pancreas microsomes fractionated by centrifuging, for 2 hr at 57,000 g in a linear 10 to 30% sucrose gradient, a resuspended high speed pellet obtained after treating microsomes with 0.04% deoxycholate (DOC).1 Amylase appeared in the following positions in the gradient: (a) a light region which contained ∼35% of total enzymic activity and which coincided with a monomeric ribosome peak; (b) a heavy region which contained ∼10% of enzymic activity in a sharp peak but which had very little accompanying OD260 absorption; (c) a pellet at the bottom of the centrifuge tube which contained ∼20% of the enzymic activity. After 5 to 20 min' in vivo labeling with leucine-1-C14, radioactive amylase was solubilized from these three fractions by a combined DOC-spermine treatment and purified by precipitation with glycogen, according to Loyter and Schramm. In all cases, the amylase found in the pellet had five to ten times the specific activity (CPM/enzymic activity) of the amylase found in the light or heavy regions of the gradient. The specific radioactivity (CPM/mg protein) of the proteins or peptides not extracted by DOC-spermine was similar for all three fractions. Hypotonic treatment of the fractions solubilized ∼80% of the total amylase in the fraction from the heavy region of the gradient, but only ∼20% of the amylase in the monomer or pellet fraction. Electron microscope observation indicates that the monomer region of the gradient contained only ribosomes, that the heavy region of the gradient contained small vesicles with relatively few attached ribosomes, and that the pellet was composed mostly of intact or ruptured microsomes with ribosomes still attached to their membranes. It is concluded from the above, and from other evidence, that most of the amylase activity in the monomer region is due to old, adsorbed enzyme; in the heavy region mostly to enzyme already inside microsomal vesicles; and in the pellet to a mixture of newly synthesized and old amylase still attached to ribosomes. Furthermore, the ribosomes with nascent, finished protein still bound to them are more firmly attached to the membranes than are ribosomes devoid of nascent protein.

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