scholarly journals Poly(adenylic acid) in small amounts, free or covalently linked to substrate, protects RNA from hydrolysis by ribonuclease

1981 ◽  
Vol 193 (1) ◽  
pp. 311-324 ◽  
Author(s):  
T P Karpetsky ◽  
K K Shriver ◽  
C C Levy
Keyword(s):  
Enzyme Activity ◽  
Segment Length ◽  
Human Spleen ◽  
Fixed Amount ◽  
Polyadenylic Acid ◽  
Adenylic Acid ◽  
Covalently Linked ◽  
The Stability ◽  
Bovine Pancreas ◽  
Small Subpopulation

Short lengths (18 residues) of poly(A), covalently linked to the 3′-termini of Escherichia coli 5 S rRNA, induce powerful inhibitions (38-87%) of the activities of RNAases (ribonucleases) from Citrobacter sp., Enterobacter sp., bovine pancreas, human spleen and human plasma. As the polypurine chain length is extended, enzyme activity declines. Furthermore, poly(A) sequences, present only on a small subpopulation of RNA, and accounting for less than 1% of total RNA, serve to protect all RNA, polyadenylated or not, from enzyme-catalysed degradation. The quantity of 3′-terminal adenylic acid residues, relative to the amount of substrate, determines enzyme activity. The exact distribution of a fixed amount of poly(A) residues on the 3′-termini of substrate molecules is unimportant in this respect. Comparison of the efficacies of inhibition of RNAase activity, by using linked poly(A) and similar quantities of free poly(A), revealed that although the free polypurine inhibits RNAase activity, covalent linkage of poly(A) to RNA is more advantageous to the stability of an RNA substrate. However, the ratio of inhibited activities obtained by using linked or free poly(A) may change considerably with alterations in either substrate concentration or polyadenylic acid segment length.

scholarly journals The effect of polyamines on the poly(adenylic acid)-induced inhibition of ribonuclease activity

1981 ◽  
Vol 193 (1) ◽  
pp. 325-337 ◽  
Author(s):  
T P Karpetsky ◽  
K K Shriver ◽  
C C Levy
Keyword(s):  
Human Plasma ◽  
Segment Length ◽  
Side Chain ◽  
Amino Groups ◽  
Human Spleen ◽  
Low Concentrations ◽  
Degradation Rates ◽  
Adenylic Acid ◽  
Bovine Pancreas ◽  
Terminal Poly

Segments of poly(A) at the 3′-termini of 5 S rRNA inhibit the activities of ribonucleases from Citrobacter, Enterobacter, bovine pancreas, human spleen and human plasma. Certain polyamines, or compounds containing polyamine substructures, mediate reversal of this inhibition. Effective compounds contain three amino groups, at least two of which are charged and are separated from the others by no less than three carbon atoms. Spermidine and 9-aminoacridines, which contain substituted propyl- or butylamino moieties at the 9-amino position and which bear two positive charges per molecule, are efficacious at low concentrations (5 microM). A decrease in effectiveness is associated with the removal of one aromatic ring from the 9-aminoacridines. However, the resulting 4-aminoquinolines, unlike the acridines, do not inhibit enzyme activity when present in concentrations above 30 microM. Relocating the diamino side chain from the 4- to the 8-position of the quinoline nucleus causes a decrease in charge density to +1, with the result that such compounds are ineffective. The orders of polyamine efficacy of reversal of inhibition were similar for enzymes from Citrobacter, bovine pancreas, and human plasma, and paralleled the order of binding of polyamines to either poly(A) or 5 S rRNA. This was not the case with Enterobacter and human spleen RNAases, indicating that the identity of the most effective polyamines depends on the RNAase studied. The combination of variable 3′-terminal poly(A) segment length and polyamine identity and concentration constitutes a system by which RNAase activities, and, therefore, substrate-degradation rates, may be easily varied.

scholarly journals EFFECT OF USING L-ASPARAGINASE IN REDUCING ACRYLAMIDE PERCENTAGE IN BISICUT

2016 ◽  
Vol 47 (4) ◽  
Author(s):  
Jebur & et al.
Keyword(s):  
High Performance Liquid Chromatography ◽  
Enzyme Activity ◽  
Liquid Chromatography ◽  
High Performance ◽  
Specific Activity ◽  
Control Sample ◽  
The Other ◽  
Enzyme Efficiency ◽  
Negative Effect ◽  
The Stability

This study was aimed to know the efficiency of partially purified L- asparaginase produced from local isolate from Erwinia spp. to reduce the percentage of acrylamide formed in Biscuit. Four types of biscuit from wheat flour were prepared (T1, T2, T3, T4),and T1 as control. High performance liquid chromatography technique was used to estimate acrylamide ratio in biscuit , Effect of enzyme addition  on flour chemical and rheological properties was studied, also dough behavior ,gluten percentage, water absorption and amylase enzyme activity was estimated. The results revealed  that  the  addition of  experimental asparaginase ( specific activity 20.5 unite mg-1 ) with 1% of flour weight lead to reduce in acrylamide formation in Biscuit  to 89 %  compared  to  control sample ( in absence of enzyme ) . Moreover, the addition of Asparagine to flour at 0.1 % of its weight, where L- asparaginase was available caused a negative effect on enzyme efficiency in reducing the acrylamide in biscuit. So the level of acrylamide was reduced to 57.7 %. In the other hand , the percentage of acryl amide in biscuit was increased to   233 % when the asparagine was added to mixture in absence of L- asparaginase .Addition of  the enzyme to flour have no effect on the percentage value of gluten but improved the  stability of dough .The  enzyme  addition also led to increase amylases activities.  Addition of experimental enzyme had no effect on quality and sensory evaluation of biscuit.

Morphology and radical reactions of Cu(II) and Co(II) sulfonated phthalocyanines covalently linked to poly(ethyleneamide)

2010 ◽  
Vol 14 (01) ◽  
pp. 69-80 ◽  
Author(s):  
Gustavo T. Ruiz ◽  
Alexander G. Lappin ◽  
Guillermo Ferraudi
Keyword(s):  
Aqueous Solutions ◽  
Redox Reactions ◽  
Radical Reactions ◽  
Stable Bundles ◽  
Carbon Bonds ◽  
Amine Groups ◽  
Covalently Linked ◽  
The Stability ◽  
Sulfonated Phthalocyanines

The tetrasulfonated Cu II( tspc )4-, tspc = 4,4′,4″,4‴-phthalocyaninetetrasulfonate, and the trisulfonated Co II( trspc )3-, trspc = n , n ′, n ″-phthalocyanine-trisulfonate and n, n′ and n″ indicate the sulfonated positions of the various isomers, were covalently linked to a polyethyleneimine, M n ~ 10000 and M w ~ 25000, backbone. A fraction of the amine groups in a strand was converted to Cu II pc (- SO 3)3(- SO 2 N <)\3- or Co II pc (- SO 3)2(- SO 2 N <)\2- pendants and the remaining were acetylated. Strands of the polymers, poly(K3CuIItspc) and poly(K2CoIItrspc) , formed spherical bundles with diameters ~1000 nm for poly(K3CuIItspc) and ~100 nm for poly(K2CoIItrspc) . The stability of the bundles is metal-dependent. Poly(K2CoIItrspc) forms the most stable bundles with respect to hydrolytic and redox reactions. ESR and optical spectroscopies as well as reactions of the pendants with pulse radiolytically generated radicals, namely e - sol , C•H2OH, (CH3)2COHC•H2 , CO 2•-, N 3• and SO 4•-, revealed a distribution of pendants between isolated and aggregated forms in a bundle. The reduction of the Cu(II) to Cu(I) in aqueous solutions of the poly(K3CuIItspc) was associated with pendants present in a mostly hydrophobic environment. Unstable phthalocyanine pendants with CoIII -carbon bonds are formed when CoIIpc(-SO3)2(-SO2N<)\2- reacted with C -centered radicals, ( CH3)2COHC•H2 and C•H2OH . The species with CoIII -carbon bonds were precursors to the formation of CoIpc(-SO3)2(-SO2N<)\3- pendants. The redox reactions of the pendants in these polymers are compared with those of K4CuIItspc and K3CoIItrspc .

scholarly journals Engineering enzyme catalysis: an inverse approach

2019 ◽  
Vol 39 (2) ◽  
Author(s):  
Clare F. Megarity
Keyword(s):  
Enzyme Activity ◽  
Enzyme Catalysis ◽  
Recent Article ◽  
Inverse Approach ◽  
Enzyme Substrate ◽  
Reaction Solution ◽  
Fundamental Research ◽  
Covalently Linked ◽  
Scaffold Molecule ◽  
Polyamine Oxidases

Abstract Enzymes’ inherent chirality confers their exquisite enantiomeric specificity and makes their use as green alternatives to chiral metal complexes or chiral organocatalysts invaluable to the fine chemical industry. The most prevalent way to alter enzyme activity in terms of regioselectivity and stereoselectivity for both industry and fundamental research is to engineer the enzyme. In a recent article by Keinänen et al., published in Bioscience Reports 2018, ‘Controlling the regioselectivity and stereoselectivity of FAD-dependent polyamine oxidases with the use of amine-attached guide molecules as conformational modulators’, an inverse approach was presented that focuses on the manipulation of the enzyme substrate rather than the enzyme. This approach not only uncovered dormant enantioselectivity in related enzymes but allowed for its control by the use of guide molecules simply added to the reaction solution or covalently linked to an achiral scaffold molecule.

scholarly journals The stability, polyadenylic acid content and ribonucleoprotein form of nulcear ribonucleic acid in artichoke

1976 ◽  
Vol 159 (3) ◽  
pp. 585-600 ◽  
Author(s):  
K S Chapman ◽  
J Ingle
Keyword(s):  
Mammalian Cells ◽  
Molecular Size ◽  
Rrna Synthesis ◽  
Kinetic Measurements ◽  
Polyadenylic Acid ◽  
Nuclear Rna ◽  
The Stability ◽  
Time Required ◽  
Total Tissue ◽  
Isopycnic Centrifugation

A nuclear preparation, containing 60-80% of the total tissue DNA and less than 0.5% of the total rRNA, was used to characterize the nuclear RNA species synthesized in cultured artichoke explants. The half-lives of the nuclear RNA species were estimated from first-order-decay analyses to be: hnRNA (heterogeneous nuclear RNA) containing poly(A), 38 min; hnRNA lacking poly(A), 37 min; 2.5 × 10(6)-mol. wt. precursor rRNA, 24 min; 1.4 × 10(6)-mol.wt. precursor rRNA, 58 min; 1.0 × 10(6)-mol.wt. precursor rRNA, 52 min. The shorter half-lives are probably overestimates, owing to the time required for equilibration of the nucleotide-precursor pools. The pathway of rRNA synthesis is considered in terms of these kinetic measurements. The rate of accumulation of cytoplasmic polydisperse RNA suggested that as much as 40% of the hnRNA may be transported to the cytoplasm. The 14-25% of the hnRNA that contained a poly(A) tract had an average molecular size of 0.7 × 10(6) daltons. The poly(A) segment was 40-200 nucleotides long, consisted of at least 95% AMP and accounted for 8-10% of the [32P]orthophosphate incorporated into the poly(A)-containing hnRNA. Ribonucleoprotein particles released from nuclei by sonication, lysis in EDTA or incubation in buffer were analysed by sedimentation through sucrose gradients and by isopycnic centrifugation in gradients of metrizamide and CsCl. More than 50% of the hnRNA remained bound to the chromatin after each treatment. The hnRNA was always associated with protein but the densities of isolated particles suggested that the ratio of protein to RNA was lower than that reported for mammalian cells, The particles separated from chromatin were not enriched for poly(A)-containing hnRNA.

scholarly journals The effects of the site-directed removal of N-glycosylation sites from β-1,4-N-acetylgalactosaminyltransferase on its function

1995 ◽  
Vol 312 (1) ◽  
pp. 273-280 ◽  
Author(s):  
M Haraguchi ◽  
S Yamashiro ◽  
K Furukawa ◽  
K Takamiya ◽  
H Shiku ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Intracellular Localization ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Kinetics Analysis ◽  
Glycosylation Sites ◽  
In The Wild ◽  
Polymerase Chain ◽  
The Stability

The amino acid sequence deduced from the cloned human cDNA of beta-1,4-N-acetylgalactosaminyltransferase (GalNAc-T; EC 2.4.1.92) gene predicted three potential sites for N-linked glycosylation. Although many glycosyltransferases isolated contain from 2 to 6 N-glycosylation sites, their significance has not been adequately demonstrated. To clarify the roles of N-glycosylation in GalNAc-T function, we generated a series of mutant cDNAs, in which some or all of the glycosylation recognition sites were eliminated by polymerase chain reaction (PCR)-mediated site-directed mutagenesis. Using transcription/translation in vitro, we confirmed that all potential N-glycosylation sites could be used. Although cell lines transfected with mutant cDNAs showed equivalent levels of GalNAc beta 1-->4(NeuAc alpha 2-->3)Gal beta 1-->4Glc-Cer (GM2) to that of the wild-type, the extracts from mutant cDNA transfectants demonstrated lower enzyme activity than in the wild-type. The decrease in enzyme activity was more evident as the number of deglycosylated sites increased, with about 90% decrease in a totally deglycosylated mutant. The enzyme kinetics analysis revealed no significant change of Km among wild-type and mutant cDNA products. The intracellular localization of GalNAc-T expressed in transfectants with wild-type or mutant cDNAs also showed a similar perinuclear pattern (Golgi pattern). These results suggest that N-linked carbohydrates on GalNAc-T are required for regulating the stability of the enzyme structure.

scholarly journals THE CYTOLOGICAL DISTRIBUTION IN PIGEON SKELETAL MUSCLE OF ENZYMES ACTING ON PHOSPHORYLATED NUCLEOTIDES

1953 ◽  
Vol 97 (4) ◽  
pp. 553-572 ◽  
Author(s):  
Amara Kitiyakara ◽  
John W. Harman
Keyword(s):  
Skeletal Muscle ◽  
Enzyme Activity ◽  
Breast Muscle ◽  
Adenylic Acid

A procedure has been described for the centrifugal fractionation of the cytological components of pigeon breast muscle. An analysis of the distribution of enzyme activity among the different particles reveals a predominant location of magnesium-activated ATPase and myokinase in the cytochondria. The myofibrillar nuclear components are the site of calcium-activated ATPase and adenylic acid deaminase.

Steroid Δ4-Reductases of Pig Liver: Partial Purification of Testosterone 5β-Reductase

1971 ◽  
Vol 49 (3) ◽  
pp. 385-392 ◽  
Author(s):  
J. C. Nduaguba ◽  
A. F. Clark
Keyword(s):  
Enzyme Activity ◽  
Soluble Fraction ◽  
Reductase Activity ◽  
Maximum Velocity ◽  
Supernatant Fraction ◽  
Partial Purification ◽  
Pig Liver ◽  
Total Enzyme Activity ◽  
Distribution Studies ◽  
The Stability

Intracellular distribution studies of steroid Δ4-reductase activity in female pig liver were done using testosterone as substrate. About 60% of the total enzyme activity was found in the 100 000 × g soluble fraction. Labelled 17β-hydroxy-5β-androstan-3-one but not 17β-hydroxy-5α-androstan-3-one was isolated from the incubation of 1,2-3H-testosterone with the 100 000 × g supernatant fraction, indicating the presence of 5β-reductase activity. 5β-Reduction may play an important role in the inactivation of some Δ4-3-ketosteroids in the pig liver.Evidence that 5β-reductases differing in substrate specificity are present in the soluble fraction includes (a) variation in the ratios of enzyme activities for several Δ4-3-ketosteroids in different (NH4)2SO4 fractions obtained from the 100 000 × g soluble fraction, (b) kinetic data showing that the maximum velocity for an equimolar mixture of testosterone and hydrocortisone is the sum of the maximum velocities for the substrates when used singly, and (c) separation of the enzyme activity specific for testosterone from that specific for hydrocortisone by use of Sephadex G-100 and hydroxylapatite chromatography.Utilizing (NH4)2SO4 precipitation and chromatography on Sephadex G-100 and hydroxylapatite, a 105-fold purification of testosterone Δ4-5β-reductase from the 100 000 × g supernatant fraction has been attained. The presence of 5 mM β-mercaptoethanolamine increased the stability of the enzyme.

scholarly journals Effect of metal ions and chemical agents on the activity of endoglucanase GH5 exploited from goats-rumen bacterial metagenomic DNA data

2021 ◽  
Vol 19 (3) ◽  
pp. 509-517
Author(s):  
Nguyen Khanh Hoang Viet ◽  
Ha Thi Thuy Hoa ◽  
Truong Nam Hai ◽  
Do Thi Huyen
Keyword(s):  
Enzyme Activity ◽  
Metal Ions ◽  
Specific Binding ◽  
Tween 80 ◽  
Conserved Residues ◽  
E Coli ◽  
Chemical Agents ◽  
Gene Coding ◽  
The Stability ◽  
Triton X

A gene coding for GH5 endoglucanase exploited from metagnomic DNA data of bacteria in Vietnamese goats’ rumen was modularity structure including a catalytic module, a fibronectin-3 like module and an X module. The recombinant enzyme was sucessfully expressed in E. coli and purified.  To study the effect of some metal ions and chemicals on enzyme activity, in this study, we used some tools including Swiss-Prot, ProFunc, COFACTOR for prediction of enzyme structure and ligands interaction. The obtained results indicated that the most similar structure with enzyme had two conserved residues (Asp-190 và Asp-192) linked with Mn2+ within a radius of ~ 3.5 Å from the center of ion Mn2+ and enzyme molecule contained a disulphide bond. Experimental results for essessment of the effect of some metal ions (Ca2 +, Mn2 +, Mg2+, Ni2+, K+, Co2+, Cu2+, Zn2+, Fe3+) at the final concentration of 10 mM and of six common chemicals including SDS (1%), urea (1 µM), 2-mercaptoethanol (1 µM), EDTA (1 µM), tween 80 (1mM), triton X-100 (1 µM) showed that only Mn2+ increased enzyme activity slightly at concentration of 10 mM and two times at the concentration of 40 mM Mn2+. The Mn2+ has been identified as a specific binding agent may increase the stability and activity of endoglucanase GH5.

scholarly journals Directed Evolution and Immobilization of a Novel Lipase LIP 906

2020 ◽  
Author(s):  
Shuang Dai ◽  
He Li
Keyword(s):  
Enzyme Activity ◽  
Directed Evolution ◽  
Early Stage ◽  
Temperature Stability ◽  
Mutant Enzyme ◽  
Random Mutation ◽  
Optimum Reaction ◽  
Pcr Technology ◽  
Solid Foundation ◽  
The Stability

Abstract Background: The object of this experimental study is a new lipase screened from metagenomic libraries in the early stage of the laboratory and named it LIP 906. In order to improve the stability of the enzyme and develop and apply it as soon as possible, experiments use directed evolution and immobilization.Results: A random mutation library was constructed by error-prone PCR technology, and finally a mutant lipase LIP 5-D with improved enzyme activity was screened out and then immobilized. Compared with the wild-type lipase LIP 906, the enzyme activity of the mutant enzyme LIP 5-D increased 4 times; the optimum reaction temperature was increased by 4 °C by mutation and 3 ℃ by immobilization; and the optimum reaction pH is changed from 7.8 to 7.5; temperature stability and pH stability has been improved. The mutant enzyme LIP 5-D can maintain a relative enzyme activity of about 70% at a temperature below 65 °C for 2 hours, and can also maintain a relative enzyme activity of about 60% at different pH 3 -10.Conclusions: Error-prone PCR and immobilization improved the catalytic activity and stability of the enzyme, and promoted its development and application in many industries. The research on the properties and modification of the new lipase LIP906 provides a solid foundation for my next innovative research in application and environmental protection.

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