scholarly journals Subtilisin modification of monodeamidated ribonuclease-A

1977 ◽  
Vol 165 (2) ◽  
pp. 337-345 ◽  
Author(s):  
B. N. Manjula ◽  
A. Seetharama Acharya ◽  
Paul J. Vithayathil
Keyword(s):  
Acid Treatment ◽  
Structural Changes ◽  
Limited Proteolysis ◽  
Ribonuclease A ◽  
Enzymic Activity ◽  
Initial Reaction ◽  
S Protein ◽  
Acidic Solutions ◽  
Peptide Region ◽  
Protein Part

Limited proteolysis of RNAase-Aa1 (monodeamidated ribonuclease-A) by subtilisin results in the formation of an active RNAase-S type of derivative, namely RNAase-Aa1S. RNAase-Aa1S was chromatographically distinct from RNAase-S, but exhibited very nearly the same enzymic activity, antigenic conformation and susceptibility to trypsin as did RNAase-S. Fractionation of RNAase-Aa1S by trichloroacetic acid yielded RNAase-Aa1S-protein and RNAase-Aa1S-peptide, both of which are inactive by themselves, but regenerate active RNAase-Aa1S′ when mixed together. RNAase-Aa1S-peptide was identical with RNAase-S-peptide, whereas the protein part was distinct from that of RNAase-S-protein. Titration of RNAase-Aa1S-protein with S-peptide exhibited slight but noticeably weaker binding of the peptide to the deamidated S-protein as compared with that of native protein. Unlike the subtilisin digestion of RNAase-A, which gives nearly 100% conversion into RNAase-S, the digestion of RNAase-Aa1 gives only a 50% conversion. The resistance of RNAase-Aa1 to further subtilisin modification after 50% conversion is apparently due to the interaction of RNAase-Aa1 with its subtilisin-modified product. RNAase-S was also found to undergo activity and structural changes in acidic solutions, similar to those of RNAase-A. The initial reaction product (RNAase-Sa1) isolated by chromatography was not homogeneous. Unlike the acid treatment of RNAase-A, which affected only the S-protein part, the acid treatment of RNAase-S affected both the S-protein and the S-peptide region of the molecule.

scholarly journals Structure and enzymic activity of ribonuclease-A esterified at glutamic acid-49 and aspartic acid-53

1978 ◽  
Vol 173 (3) ◽  
pp. 821-830 ◽  
Author(s):  
A. Seetharama Acharya ◽  
Belur N. Manjula ◽  
Paul J. Vithayathil
Keyword(s):  
Methyl Ester ◽  
Glutamic Acid ◽  
Aspartic Acid ◽  
Spectral Characteristics ◽  
Dimethyl Ester ◽  
Limited Proteolysis ◽  
Ribonuclease A ◽  
Enzymic Activity ◽  
S Protein ◽  
Hydrolysis Of

The dimethyl ester of bovine pancreatic ribonuclease-A (dimethyl RNAase-A), the initial product of esterification of RNAase-A in anhydrous methanolic HCl, was isolated in a homogeneous form. The two carboxy functions esterified in this derivative are those of glutamic acid-49 and aspartic acid-53. There were no changes in the u.v.-absorption spectral characteristics, the accessibility of the methionine residues, the resistance of the protein to proteolysis by trypsin and the antigenic behaviour of RNAase-A as a result of the esterification of these two carboxy groups. Dimethyl RNAase-A exhibited only 65% of the specific activity of RNAase-A, but still had the same Km value for both RNA and 2′:3′-cyclic CMP. However, the Vmax. was decreased by about 35%. On careful hydrolysis of the methyl ester groups at pH9.5, dimethyl RNAase-A was converted back into RNAase-A. Limited proteolysis of dimethyl RNAase-A by subtilisin resulted in the formation of an active RNAase-S-type derivative, namely dimethyl RNAase-S, which was chromatographically distinct from dimethyl RNAase-A and had very nearly the same enzymic activity as dimethyl RNAase-A. Fractionation of dimethyl RNAase-S by trichloroacetic acid yielded dimethyl RNAase-S-protein and dimethyl RNAase-S-peptide, both of which were inactive by themselves but regenerated dimethyl RNAase-S when mixed together. Dimethyl RNAase-A-peptide was identical with RNAase-S-peptide. RNAase-S-protein could be generated from dimethyl RNAase-S-protein by careful hydrolysis of the methyl ester groups at pH9.5. The interaction of dimethyl RNAase-S-protein with RNAase-S-peptide appears to be about 4-fold weaker than that between the RNAase-S-protein and RNAase-S-peptide. Conceivably, the binding of the S-peptide ‘tail’ of dimethyl RNAase-A with the remainder of the molecule is similarly weaker than that in RNAase-A, and this brings about subtle changes in the geometrical orientation of the active-site amino acid residues of these modified methyl ester derivatives. It is suggested that these changes could be responsible for the generation of the catalytically less-efficient RNAase-A and RNAase-S molecules (dimethyl RNAase-A and dimethyl RNAase-S respectively).

scholarly journals The effects of autolysis on the structure of chicken calpain II

1987 ◽  
Vol 248 (2) ◽  
pp. 579-588 ◽  
Author(s):  
C Crawford ◽  
A C Willis ◽  
J Gagnon
Keyword(s):  
Structural Changes ◽  
Structural Integrity ◽  
Large Subunit ◽  
Limited Proteolysis ◽  
Gel Permeation Chromatography ◽  
Enzymic Activity ◽  
Amino Acid Sequences ◽  
Cleavage Sites ◽  
Terminal Amino ◽  
Calpain Ii

When chicken calpain II autolysed in the presence of Ca2+, it underwent limited proteolysis to give peptides of Mr 54,000 and 37,000, and several of Mr approx. 30,000 and 18,000. The autolytic peptides were purified and their N-terminal amino acid sequences determined. By comparison of these sequences with the known sequence of the complete calpain molecule, the autolytic cleavage sites were identified. The structural integrity of the molecule during autolysis was investigated by gel-permeation chromatography. Experiments were also done to test the reversibility of adding EDTA to calpain during autolysis, measured as recoverable enzyme activity assayed in the presence of Ca2+. The results are presented in terms of a model for the structural changes occurring in calpain during autolysis. It was concluded that the loss of enzymic activity, which is a consequence of autolysis, was due to dissociation of the autolytic peptides after cleavage of the calpain large subunit within the third domain.

scholarly journals Effect of Structure and Chemical Activation on the Adsorption Properties of Green Clay Minerals for the Removal of Cationic Dye

2018 ◽  
Vol 8 (11) ◽  
pp. 2302 ◽  
Author(s):  
Abdelfattah Amari ◽  
Hatem Gannouni ◽  
Mohammad Khan ◽  
Mohammed Almesfer ◽  
Abubakr Elkhaleefa ◽  
...  
Keyword(s):  
Clay Minerals ◽  
Pore Volume ◽  
Acid Treatment ◽  
Structural Changes ◽  
Chemical Activation ◽  
Total Pore Volume ◽  
Batch Adsorption ◽  
Acid Activation ◽  
Cationic Dye ◽  
Average Pore

In this study, natural clay minerals with green appearance were treated with sulfuric acid. Mass percentage of acid (wt%), temperature (T), contact time (t) and liquid-to-solid mass ratio (R) are used as the prevailing factors that determine the extent of acid-activation. The values of these factors range from 15–50%, 60–90 °C, 1.5–6 h and 4–7, respectively. The study has focused on the structural changes as well as textural characteristics of the clay. Three activated clay samples were prepared under different treatment conditions. The samples were characterized using X-ray powder diffraction (XRD), fourier transform infrared (FTIR), scanning electron microscope (SEM), chemical analysis and N2 adsorption techniques. Characterization of the treated clay minerals exhibited significant structural changes to a greater extent of acid-activation, from being partially crystalline to being amorphous silica. The surface area and total pore volume of clay increased proportionally with the level of acid treatment. The average pore diameter behaved differently. During the strong acid treatment, a large increase in pore volume and the enlargement of the pore size distribution were observed. This suggests that considerable structural changes and partial destruction may have occurred in this condition. The removal of methylene blue, used as cationic dye, from aqueous solution by the batch adsorption technique on three prepared acid-activated clay samples was studied. The Langmuir model was found to agree well with the experimental data.

scholarly journals SARS CoV-2 Variants and Spike Mutations Involved in Second Wave of COVID-19 Pandemic in India

2021 ◽  
Author(s):  
Muttineni Radhakrishna ◽  
Binitha R ◽  
Kalyani Putty ◽  
Kavitha Marpakala ◽  
Panyam Jaslin ◽  
...  
Keyword(s):  
Biological Significance ◽  
Transmembrane Region ◽  
Genome Sequences ◽  
Neutralising Antibodies ◽  
S Protein ◽  
Terminal Domain ◽  
Alpha Variant ◽  
National Trends ◽  
Second Wave ◽  
Peptide Region

Against the backdrop of the second wave of COVID-19 pandemic in India that started in March 2021, we have monitored the spike (S) protein mutations in all the reported (GISAID portal) whole genome sequences of SARS CoV-2 circulating in India from 1 January 2021 to 31 August 2021. In the 43,102 SARS-CoV-2 genomic sequences analysed, we have identified 24, 260 mutations in the S protein, based on which 265 pango lineages could be categorised. The dominant lineage in most of the 28 states of India and its 8 union territories was B.1.617.2 (the delta variant). However, the states Madhya Pradesh, Jammu & Kashmir, and Punjab had B.1.1.7 (alpha variant) as the major lineage, while the Himachal Pradesh state reported B.1.36 as the dominating lineage. A detailed analysis of various domains of S protein was carried out for detecting mutations having a prevalence of >1%; 70, 18, 7, 3, 9, 4, and 1 (N=112) such mutations were observed in the N -terminal domain, receptor binding domain, C -terminal domain, fusion peptide region, heptapeptide repeat (HR)-1 domains, signal peptide domain, and transmembrane region, respectively. However, no mutations were recorded in the HR-2, and cytoplasmic domains of the S protein. Interestingly, 13.39% (N=15) of these mutations were reported to increase the infectivity and pathogenicity of the virus; 2%(N=3) were known to be vaccine breakthrough mutations; and 0.89%(N=1) were known to escape neutralising antibodies. Biological significance of 82% (N=92) of the reported mutations is yet unknown. As SARS-CoV-2 variants are emerging rapidly, it is critical to continuously monitor local viral mutations to understand national trends of virus circulation. This can tremendously help in designing better preventive regimens in the country, and avoid vaccine breakthrough infections.

scholarly journals Ligand binding on to maize (Zea mays) malate synthase: a structural study

1994 ◽  
Vol 303 (2) ◽  
pp. 413-421 ◽  
Author(s):  
S Beeckmans ◽  
A S Khan ◽  
L Kanarek ◽  
E Van Driessche
Keyword(s):  
Zea Mays ◽  
Ligand Binding ◽  
Conformational Change ◽  
Limited Proteolysis ◽  
Enzymic Activity ◽  
Malate Synthase ◽  
Acetyl Coa ◽  
Original Activity ◽  
Kinetic Measurements ◽  
Michaelis Constants

A kinetic and ligand binding study on maize (Zea mays) malate synthase is presented. It is concluded from kinetic measurements that the enzyme proceeds through a ternary-complex mechanism. Michaelis constants (Km,glyoxylate and Km,acetyl-CoA) were determined to be 104 microM and 20 microM respectively. C.d. measurements in the near u.v.-region indicate that a conformational change is induced in the enzyme by its substrate, glyoxylate. From these studies we are able to calculate the affinity for the substrate (Kd,glyoxylate) as 100 microM. A number of inhibitors apparently trigger the same conformational change in the enzyme, i.e. pyruvate, glycollate and fluoroacetate. Another series of inhibitors bearing more bulky groups and/or an extra carboxylic acid also induce a conformational change, which is, however, clearly different from the former one. Limited proteolysis with trypsin results in cleavage of malate synthase into two fragments of respectively 45 and 19 kDa. Even when no more intact malate synthase chains are present, the final enzymic activity still amounts to 30% of the original activity. If trypsinolysis is performed in the presence of acetyl-CoA, the cleavage reaction is appreciably slowed down. The dissociation constant for acetyl-CoA (Kd,acetyl-CoA) was calculated to be 14.8 microM when the glyoxylate subsite is fully occupied by pyruvate and 950 microM (= 50 x Km) when the second subsite is empty. It is concluded that malate synthase follows a compulsory-order mechanism, glyoxylate being the first-binding substrate. Glyoxylate triggers a conformational change in the enzyme and, as a consequence, the correctly shaped binding site for acetyl-CoA is created. Demetallization of malate synthase has no effect on the c.d. spectrum in the near u.v.-region. Moreover, glyoxylate induces the same spectral change in the absence of Mg2+ as in its presence. Nevertheless, malate synthase shows no activity in the absence of the cation. We conclude that Mg2+ is essential for catalysis, rather than for the structure of the enzyme's catalytic site.

Conformational Alterations of α2-Antiplasmin Induced by Complex Formation with Plasmin

1979 ◽  
Author(s):  
E.F. Plow ◽  
B. Wiman ◽  
D. Collen
Keyword(s):  
Complex Formation ◽  
Structural Changes ◽  
Limited Proteolysis ◽  
Terminal Region ◽  
The Other ◽  
Antigenic Determinants ◽  
Chemical Analyses ◽  
Large Fragment ◽  
Reactive Site ◽  
Immunodiffusion Analysis

The conformational and structural changes induced in the α2-antiplasmin (AP) molecule by complex formation with plasmin have been analyzed utilizing quantitative radioimmuno-chemical analyses. Complexes prepared in plasmin excess -(PAP-P) and therefore subjected to limited proteolysis and complexes prepared in AP excess (PAP-A) have been compared with free AP. With AP antiserum, PAP-A, PAP-P and AP yielded reactions of complete identity by immunodiffusion analysis. In radioimmunoassay, however, these were clearly distinguished, and four distinct sets of antigenic determinants were delineated. Set I determinants were expressed equivalently by PAP-P, PAP-A and AP and were, therefore, not altered by complex formation. This set was recognized by 90% of the antibodies, and the determinants were all included within a large fragment of Mr 60,000 derived from the NH2-terminal region of AP. The other three sets of determinants were modulated by complex formation. Set II was expressed by PAP-A and AP but not by PAP-P, and these were sensitive to proteolysis by plasmin. Set III determinants were expressed only by AP and were localized to a peptide of Mr 8,000 derived from the COOH-terminal region of AP. Set IV determinants were also present only on AP but were not present in the peptide and required an intact reactive site in AP for expression. Thus, there is evidence for multiple conformational modulations in AP induced by complex formation, and these modulations can be pinpointed to specific loci within the AP molecule.

scholarly journals Functional domain organization of the potato α-glucan, water dikinase (GWD): evidence for separate site catalysis as revealed by limited proteolysis and deletion mutants

2005 ◽  
Vol 385 (2) ◽  
pp. 355-361 ◽  
Author(s):  
René MIKKELSEN ◽  
Andreas BLENNOW
Keyword(s):  
Structural Changes ◽  
Sequence Similarity ◽  
Limited Proteolysis ◽  
Phosphoryl Group ◽  
Deletion Mutants ◽  
Functional Domain ◽  
Partial Reaction ◽  
Atp Binding Site ◽  
Binding Domains ◽  
Terminal Domains

The potato tuber (Solanum tuberosum) GWD (α-glucan, water dikinase) catalyses the phosphorylation of starch by a dikinase-type reaction mechanism in which the β-phosphate of ATP is transferred to the glucosyl residue of amylopectin. GWD shows sequence similarity to bacterial pyruvate, water dikinase and PPDK (pyruvate, phosphate dikinase). In the present study, we examine the structure–function relationship of GWD. Analysis of proteolytic fragments of GWD, in conjunction with peptide microsequencing and the generation of deletion mutants, indicates that GWD is comprised of five discrete domains of 37, 24, 21, 36 and 38 kDa. The catalytic histidine, which mediates the phosphoryl group transfer from ATP to starch, is located on the 36 kDa fragment, whereas the 38 kDa C-terminal fragment contains the ATP-binding site. Binding of the glucan molecule appears to be confined to regions containing the three N-terminal domains. Deletion mutants were generated to investigate the functional interdependency of the putative ATP- and glucan-binding domains. A truncated form of GWD expressing the 36 and 38 kDa C-terminal domains was found to catalyse the E+ATP→E-P+AMP+Pi (where Pi stands for orthophosphate) partial reaction, but not the E-P+glucan→E+glucan-P partial reaction. CD experiments provided evidence for large structural changes on autophosphorylation of GWD, indicating that GWD employs a swivelling-domain mechanism for enzymic phosphotransfer similar to that seen for PPDK.

scholarly journals The effect of surfactant structure on ribonuclease A–surfactant interactions

1973 ◽  
Vol 135 (3) ◽  
pp. 547-549 ◽  
Author(s):  
Cecilia Blinkhorn ◽  
Malcolm N. Jones
Keyword(s):  
Anionic Surfactant ◽  
Cationic Surfactants ◽  
Ribonuclease A ◽  
Enzymic Activity ◽  
Head Groups ◽  
Surfactant Interactions ◽  
Dodecylamine Hydrochloride

The enzymic activity of ribonuclease A was measured in the presence of several surfactants at pH7.2. Cationic surfactants with trimethylammonium and pyridinium head groups do not deactivate or denature the enzyme, whereas n-dodecylamine hydrochloride, like the anionic surfactant sodium n-dodecyl sulphate, deactivates and denatures ribonuclease A.

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