The Correlation of RNase A Enzymatic Activity with the Changes in the Distance between Nε2-His12 and Nδ1-His119 Upon Addition of Stabilizing and Destabilizing Salts

2006 ◽  
Vol 25 (2) ◽  
pp. 117-125 ◽  
Author(s):  
A. A. Moosavi-Movahedi ◽  
M. Gharanfoli ◽  
S. Jalili ◽  
F. Ahmad ◽  
J. Chamani ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Rnase A

Thermal stability and enzymatic activity of RNase A in the presence of cationic gemini surfactants

2012 ◽  
Vol 50 (4) ◽  
pp. 1151-1157 ◽  
Author(s):  
Razieh Amiri ◽  
Abdol-Khalegh Bordbar ◽  
Douglas V. Laurents ◽  
Ahmad Reza Khosropour ◽  
Iraj Mohammadpoor-Baltork
Keyword(s):  
Thermal Stability ◽  
Enzymatic Activity ◽  
Gemini Surfactants ◽  
Rnase A ◽  
Cationic Gemini Surfactants

Trapping RNase A on MCM41 pores: effects on structure stability, product inhibition and overall enzymatic activity

2014 ◽  
Vol 16 (19) ◽  
pp. 9031-9038 ◽  
Author(s):  
Irina Matlahov ◽  
Yasmin Geiger ◽  
Gil Goobes
Keyword(s):  
Enzymatic Activity ◽  
Product Inhibition ◽  
Rnase A ◽  
Structure Stability

Ribonuclease's activity bound to nano-sized pore openings of MCM41 is severely quenched, but inhibition by the product is reduced.

scholarly journals Switching the enzymatic activity of ribonuclease a based on enzyme/polymer complex formation

2014 ◽  
Vol 20 ◽  
pp. 33-39 ◽  
Author(s):  
Sumon Ganguli
Keyword(s):  
Heat Treatment ◽  
Spectral Analysis ◽  
Complex Formation ◽  
Acrylic Acid ◽  
Enzymatic Activity ◽  
Heat Resistance ◽  
Rnase A ◽  
Polymer Complex ◽  
Poly Ethylene Glycol ◽  
Poly Ethylene

Context: Enzymes are ideal for various applications in both medicine and biotechnology. Switching on/off the enzymatic activity of enzymes by polymeric modification would expand the applications of enzymes in a wide range of research fields. Objectives: On/off switching the enzymatic activity of RNase A and the confirmation of the enzyme/polymer complex formation which leads to improve the heat resistance of RNase A. Materials and Methods: ?-Methoxy-?-methacryloyl poly (ethylene glycol) (PEG-MA) macromonomer and PEAMA-g-PEG were synthesized. Bovine RNase A, cytidine 2?,3?-cyclic monophosphate sodium salt (cCMP), and 3-(N-morpholino)propanesulfonic acid (MOPS) were obtained from Sigma Chemical Co. (St. Louis, USA). PAAc (Mn = 5,000 g/mol) were purchased from Wako (Osaka, Japan). Sodium dihydrogen phosphate dihydrate (NaH2PO4.2H2O) was obtained from Nacalai Tesque Inc. (Kyoto, Japan). The enzymatic activity of RNase A was estimated as follows. The RNase A concentration was determined by measuring the absorbance at 280 nm with an appropritate blank, using an extinction coefficient of 7.10 mL mg-1 cm-1. A total of 1.5 mL of 0.1 mg/mL cCMP solution prepared in 0.1 M MOPS (pH 7.0) was mixed with 10 ?L of the RNase A solution in 50 mM sodium phosphate buffer (pH 7.0). The increase in light scattering intensity of the solution was monitored by measuring the absorbance at 284 nm for 60 s in a UV-vis spectrophotometer at room temperature. After heat treatment, the enzymatic activities were measured. Far-UV and near-UV CD spectra were monitored using a spectropolarimeter (model J-720W; Jasco, Tokyo, Japan). Results: We have found that poly (acrylic acid) (PAAc) suppressed the enzymatic activity of RNase A completely and the recovery of enzymatic activity were observed (94%) by the external addition of PEAMA-g-PEG to the RNase A/PAAc complex. Our present findings suggest that the complexation between PEAMA-g-PEG and RNase A has occurred which improve the heat resistance (64%) of RNase A. Heat treatment has been carried out at 98°C for 10 minutes where the native RNase A lost all of its enzymatic activity. CD spectral analysis also indicates that the conformation of the enzyme was not altered due to the complexation. Conclusion: Poly (N,N-diethylaminoethyl methacrylate)-graft-poly (ethylene glycol) (PEAMA-g-PEG) restored the enzymatic activity of RNase A completely which was inactivated upon the addition of poly(acrylic acid) (PAAc) to RNase A. Complexation of RNase A with PEAMA-g-PEG induced the improvement of heat resistance of RNase A . Circular dichroism (CD) spectral analysis clearly indicated that the complexation of enzyme with polymer has almost no influence on the conformation of enzyme. DOI: http://dx.doi.org/10.3329/jbs.v20i0.17650 J. bio-sci. 20: 33-39, 2012

Substrate-leash amplification with ribonuclease S-peptide and S-protein.

1986 ◽  
Vol 32 (9) ◽  
pp. 1622-1630 ◽  
Author(s):  
M Ehrat ◽  
D J Cecchini ◽  
R W Giese
Keyword(s):  
Enzymatic Activity ◽  
Rnase A ◽  
S Protein ◽  
Protein Fragments ◽  
Polycytidylic Acid ◽  
Amplification System ◽  
Protein Gels ◽  
Acid Substrate

Abstract The S-peptide and S-protein fragments of ribonuclease S (RNase S, no EC no. assigned) have been immobilized onto separate Sepharose gels via a "leash" of polycytidylic acid substrate. Each of these gels releases its RNase fragment when treated with the complementary enzyme fragment or with RNase A (EC 3.1.27.5), and the released fragments recombine to give RNase S activity. Thus this system provides substrate-leash amplification (SLA), such that more enzymatic activity is eluted from the system than is applied. For example, 100 pg of RNase applied to the S-peptide gel is amplified by 1.9 X 10(4) to the equivalent of 1.9 micrograms of activity in 20 h, when followed by combination of the released S-peptide with excess S-protein. We also tested a three-stage amplification system, with a pair of S-peptide and S-protein gels at each stage. In this system the cumulative amplification of the initial 1-ng dose of RNase A is 4.9, 52, and 25-fold after each stage, respectively. Only 2 mg of each SLA gel is used per stage in these experiments, reflecting the magnitude of their production of RNase S activity.

Portein-gold labelling of ultrathin sections of wheat spot mosaic-affected wheat plants

1990 ◽  
Vol 48 (3) ◽  
pp. 680-681
Author(s):  
K. S. Zaychuk ◽  
M. H. Chen ◽  
C. Hiruki
Keyword(s):  
Osmium Tetroxide ◽  
Rnase A ◽  
Leaf Ultrastructure ◽  
Double Membrane ◽  
Young Root ◽  
Leaf Tissues ◽  
Membrane Bound ◽  
Ultrathin Sections ◽  
Gold Labelling ◽  
Electron Dense Core

Wheat spot mosaic (WSpM), which frequently occurs with wheat streak mosaic virus was first reported in 1956 from Alberta. Singly isolated, WSpM causes chlorotic spots, chlorosis, stunting, and sometimes death of the wheat plants. The vector responsible for transmission is the eriophyid mite, Eriophyes tulipae Kiefer. The examination of leaf ultrastructure by electron microscopy has revealed double membrane bound bodies (DMBB’s) 0.1-0.2 μm in diameter. Dispersed fibrils within these bodies suggested the presence of nucleic acid. However, neither ribosomes characteristic of bacteria, mycoplasma and the psittacosis group of organisms nor an electron dense core characteristic of many viruses was commonly evident.In an attempt to determine if the DMBB’s contain nucleic acids, RNase A, DNase I, and lactoferrin protein were conjugated with 10 nm colloidal gold as previously described. Young root and leaf tissues from WSpM-affected wheat plants were fixed in glutaraldehyde, postfixed in osmium tetroxide,and embedded in Spurr’s resin.

A Method for the Colorimetric Determination of β-Glucuronidase in Urine, Serum, Tissue; Assay of Enzymatic Activity in Health and Disease

1959 ◽  
Vol 36 (2) ◽  
pp. 193-201 ◽  
Author(s):  
Julius A. Goldbarg ◽  
Esteban P. Pineda ◽  
Benjamin M. Banks ◽  
Alexander M. Rutenburg
Keyword(s):  
Enzymatic Activity ◽  
Colorimetric Determination ◽  
Health And Disease ◽  
Urine Serum

Physiological and enzymatic activity of pepper seeds (Capsicum annuum) during priming

1991 ◽  
Vol 82 (3) ◽  
pp. 433-439 ◽  
Author(s):  
Patrick T. Smith ◽  
B. Greg Cobb
Keyword(s):  
Enzymatic Activity ◽  
Capsicum Annuum

Isolation of Antithrombin III from Normal and α1-Antitrypsin-Deficient Human Plasma

1977 ◽  
Vol 38 (02) ◽  
pp. 0475-0485 ◽  
Author(s):  
Anna D. Borsodi ◽  
Ralph A. Bradshaw
Keyword(s):  
Enzymatic Activity ◽  
Isoelectric Focusing ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Antithrombin Activity ◽  
Bovine Trypsin ◽  
Normal Plasma ◽  
Antitrypsin Deficiency ◽  
Simplified Procedure ◽  
Stimulation Of

SummaryThe plasma of individuals, hetero- or homozygous for α1-antitrypsin deficiency, contains greatly decreased amounts of antithrombin activity as assayed against factor Xa. However, heparin stimulation of the residual antithrombin activity is observed, which is comparable to that of normal plasma. Antithrombins isolated from both normal and α1-antitrypsin deficient plasma by a simplified procedure are indistinguishable in both properties and yields. The microheterogeneity observed on isoelectric focusing of both preparations can be eliminated by treatment with neuraminidase. Neither purified human antithrombin nor α1-antitrypsin, when assayed against bovine trypsin, is stimulated by heparin. These results clearly establish the unique natures of antithrombin and α1-antitrypsin and show that about 75% of the antithrombin activity measured in normal plasma is due to α1-antitrypsin. Estimates of anti thrombin III activity in normal plasma by assays dependent on enzymatic activity can probably be obtained only in the presence of heparin.

Mechanism of Inhibitory Action on Platelet Activation of a Phospholipase A2 Isolated from Lachesis muta (Bushmaster) Snake Venom

1997 ◽  
Vol 78 (05) ◽  
pp. 1372-1380 ◽  
Author(s):  
André L Fuly ◽  
Olga L T Machado ◽  
Elias W Alves ◽  
Célia R Carlinis
Keyword(s):  
Platelet Aggregation ◽  
Enzymatic Activity ◽  
Phospholipase A2 ◽  
Inhibitory Activity ◽  
Thermal Inactivation ◽  
Anticoagulant Activity ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Crude Venom ◽  
Lachesis Muta

SummaryCrude venom from Lachesis muta exhibited procoagulant, proteolytic and phospholipase A2 activities. A phospholipase A2, denoted LM-PLA2 was purified from L. muta venom to homogeneity, through a combination of chromatographic steps involving gel-filtration on Sephacryl S-200 HR and reverse phase chromatography on a C2/C18 column. LM-PLA2 presented a single polypeptide chain with an isoelectric point at pH 4.7 and apparent molecular weight of 17 kDa. Partial aminoacid sequence indicated a high degree of homology for LM-PLA2 with other PLA2 from different sources.LM-PLA2 displayed a potent enzymatic activity as measured by indirect hemolysis of red blood cells but it was neither lethal when injected i.p. into mice nor did it present anticoagulant activity. Furthermore, LM-PLA2 displayed a moderate inhibitory activity on the aggregation of rabbit platelets induced by low levels of ADP, thrombin and arachidonate. In contrast, platelet aggregation induced by high doses of collagen was strongly inhibited by LM-PLA2 as well as ATP-release. Treatment of the protein with p-bromophenacyl bromide or 2-mercapto-ethanol, as well as thermal inactivation studies, suggested that the platelet inhibitory effect of LM-PLA2 is dependent on its enzymatic activity. Thus, the platelet inhibitory activity of LM-PLA2 was shown to be dependent on the hydrolysis of plasma phospholipids and/or lipoproteins, most probably those rich in phosphatidylcholine. Surprisingly, lyso-phosphatidylcholine released by LM-PLA2 from plasma was shown to preferentially inhibited collagen-induced platelet aggregation, in contrast to other PLA2s, whose plasma hydrolytic products indistinctly affect platelet’s response to several agonists.

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