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 Does Covera-19 know ‘when to hold ‘em or ‘when to fold ‘em? A translational thought experiment

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Gerald Dieter Griffin
Keyword(s):  
Conformational Changes ◽  
Structural Changes ◽  
Structural Integrity ◽  
Thought Experiment ◽  
Amino Acid Sequences ◽  
Acid And Base ◽  
Effects Of Ph ◽  
Base Balance ◽  
Environmental Milieu ◽  
And Function

AbstractThe function of proteins depends on their structure. The structural integrity of proteins is dynamic and depends on interacting nearby neighboring moieties that influence their properties and induce folding and structural changes. The conformational changes induced by these nearby neighbors in the micro-environmental milieu at that moment are guided by chemical or electrical bonding attractions.There are few literature references that describe the potential for environmental milieu changes to disfavor SARS-CoV-2 attachment to a receptor for survival outside of a host. There are many studies on the effects of pH (acid and base balance) supporting its importance for protein structure and function, but few focus on pH role in extracellular or intracellular protein or actionable requirements of Covera-19.‘Fold ‘em or Hold ‘em’ is seen by the various functions and effects of furin as it seeks an acidic milieu for action or compatible amino acid sequences which is currently aided by its histidine component and the structural changes of proteins as they enter or exit the host. Questions throughout the text are posed to focus on current thoughts as reviewing applicable COVID-19 translational research science in order to understand the complexities of Covid-19.The pH needs of COVID-19 players and its journey through the human host and environment as well as some efficacious readily available repurposed drugs and out-of-the box and easily available treatments are reviewed.

Production of multiple δ-endotoxins by Bacillus thuringiensis: δ-endotoxins produced by strains of the subspecies galleriae and wuhanensis

1994 ◽  
Vol 40 (12) ◽  
pp. 1026-1034 ◽  
Author(s):  
Galina G. Chestukhina ◽  
Lyubov I. Kostina ◽  
Igor A. Zalunin ◽  
Lyudmila P. Revina ◽  
Alla L. Mikhailova ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Limited Proteolysis ◽  
Protein Composition ◽  
Amino Acid Sequences ◽  
Sequence Determination ◽  
Crystal Proteins ◽  
Sds Page ◽  
Functional Consequences ◽  
Terminal Amino ◽  
Fast Performance Liquid Chromatography

A method was developed to assess the number of δ-endotoxins contained in Bacillus thuringiensis entomocidal crystals. It utilized proteolytic conversion of 130-kDa protoxin into 60- to 65-kDa "true" toxin via limited proteolysis with trypsin and separation of stable N-terminal domains by fast-performance liquid chromatography. Immunodiffusion experiments and N-terminal sequence determination (applied to the major component isolated by SDS-PAGE) completed the analysis of the crystal protein composition. The application of this approach to crystals produced by cells of B. thuringiensis subsp. galleriae and wuhanensis allowed us to identify at least seven and eight different δ-endotoxins, respectively. Among those δ-endotoxins assigned to previously described families, CryIA, CryID, Cry IF, and CryIG were found, as well as crystal proteins, which possess N-terminal amino acid sequences very different from those of all known δ-endotoxins. Possible functional consequences of δ-endotoxin multiplicity are discussed.Key words: Bacillus thuringiensis, δ-endotoxins, multiplicity of crystal proteins, separation of true toxins.

Isolation of inhibin from ovine follicular fluid

1987 ◽  
Vol 113 (2) ◽  
pp. 213-221 ◽  
Author(s):  
L. J. Leversha ◽  
D. M. Robertson ◽  
F. L. de Vos ◽  
F. J. Morgan ◽  
M. T. W. Hearn ◽  
...  
Keyword(s):  
Amino Acid ◽  
Follicular Fluid ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Permeation Chromatography ◽  
Reversed Phase ◽  
Amino Acid Sequences ◽  
Molecular Weights ◽  
Terminal Amino ◽  
Inhibin Subunits

ABSTRACT Two forms of inhibin with molecular weights of 65 000 and 30 000 (65 and 30 kD) were isolated from ovine follicular fluid using a combination of gel permeation chromatography, reversed-phase high-performance liquid chromatography and preparative polyacrylamide gel electrophoresis. The 65 kD form was partially purified approximately 315-fold whilst the 30 kD form was isolated as two isoforms (29 and 30 kD) of similar biological activity and in >95% purity (1210-fold purification and 4·2% recoveries). On reduction the 30 kD form resolved into four components of 36, 31, 20–21 and 16 kD of which the 20–21 and 16 kD components were similar to the corresponding inhibin subunits isolated from porcine and bovine follicular fluid. The 36 kD component was established as a non-reducible inhibin-like material, based on its binding to antiserum raised against bovine 58 kD inhibin. The nature of the remaining non-reducible 31 kD component is unknown. Two NH2-terminal amino acid sequences (first 13 amino acids) identified in purified 30 kD inhibin were identical to the corresponding subunit amino acid sequences of bovine 31 kD inhibin. J. Endocr. (1987) 113, 213–221

scholarly journals Calpain inhibition by peptide epoxides

1985 ◽  
Vol 230 (2) ◽  
pp. 509-516 ◽  
Author(s):  
C Parkes ◽  
A A Kembhavi ◽  
A J Barrett
Keyword(s):  
Smooth Muscle ◽  
Active Site ◽  
Rate Constants ◽  
Cysteine Proteinase ◽  
Limited Proteolysis ◽  
Enzymic Activity ◽  
Isoelectric Precipitation ◽  
Structural Analogues ◽  
Calpain Inhibition ◽  
Calpain Ii

A Ca2+-activated cysteine proteinase (calpain II) was purified from chicken gizzard smooth muscle by use of isoelectric precipitation, (NH4)2SO4 fractionation, chromatography on DEAE-Sepharose CL-6B, Reactive-Red 120-agarose and Mono Q. The apparent second-order rate constants for the inactivation of calpain by a series of structural analogues of L-3-carboxy-trans-2, 3-epoxypropionyl-leucylamido-(4-guanidino)butane (E-64) were determined. The fastest rate of inactivation was observed with L-3-carboxy-trans-2, 3-epoxypropionyl-leucylamido-(4-benzyloxy-carbonylamino)buta ne. It was possible to determine the active-site molarity of solutions of calpain by titration with E-64. When incubated with Ca2+, calpain underwent several steps of intermolecular limited proteolysis, via multiple pathways, followed by a slower loss of enzymic activity. The proteolytic steps preceding the loss of activity did not affect the rates of reaction of calpain with E-64 analogues.

scholarly journals Activation of the cellular src gene by transducing retrovirus.

1986 ◽  
Vol 6 (7) ◽  
pp. 2420-2428 ◽  
Author(s):  
S Ikawa ◽  
K Hagino-Yamagishi ◽  
S Kawai ◽  
T Yamamoto ◽  
K Toyoshima
Keyword(s):  
Amino Acid ◽  
Reverse Transcriptase ◽  
Structural Changes ◽  
Nucleotide Sequences ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Src Gene ◽  
Terminal Amino ◽  
Sequence Encoding ◽  
Carboxy Terminal

Newly isolated strains of avian sarcoma virus, S1 and S2, were shown to have the transduced cellular src gene as their viral transforming gene (Yamagishi et al., Virology 137:266-275, 1984). In this work, the S1 and S2 genomes were molecularly cloned, and the junction sequences between the viral genomes and the c-src genes and the complete nucleotide sequences of the v-src genes transduced in these viruses were determined. Data on the junction sequences suggested that 5' recombination had occurred between the 5'-noncoding region of c-src and the 5' region of the gag sequence encoding p19 in both viruses and that 3' recombination had occurred in the last coding exon of c-src with either the middle portion of the env sequence encoding gp85 for S1 or the 3' portion of pol coding for reverse transcriptase for S2. Comparison of the amino acid sequences of the S1 and S2 src products deduced from the nucleotide sequences (pp62S1-src and pp62S2-src with that of c-src protein (pp60c-src) indicated that in pp62S1-src the 8 carboxy-terminal amino acid residues of the total of 533 in pp60c-src are replaced by 43 residues translated from the env sequence at the wrong frame. In pp62S2-src, on the other hand, the 14 carboxy-terminal amino acids of pp60c-src are replaced by the 38 carboxy-terminal residues of reverse transcriptase. The mechanism of c-src transduction and the structural changes necessary for pp60c-src activation are discussed.

scholarly journals Structural insights on the effects of mutation of a charged binding pocket residue on phosphopeptide binding to 14-3-3 ζ Protein

2021 ◽  
Author(s):  
Sreevidya T S ◽  
Somavally Dalvi ◽  
Prasanna Venkatraman ◽  
Satyavani Vemparala
Keyword(s):  
Electrostatic Interactions ◽  
Structural Changes ◽  
Structural Integrity ◽  
Peptide Binding ◽  
Limited Proteolysis ◽  
Binding Pocket ◽  
Mutant Protein ◽  
Amino Acid Residues ◽  
Salt Bridges ◽  
Dynamics Simulations

Mutation of an invariant aspartate residue in the binding pocket of 14-3-3ζ isoform to alanine dramatically reduced phosphopeptide binding and induced opening of the binding pocket. Here we use extensive molecular dynamics simulations to understand the role of D124 residue in ligand binding. The simulations show that in the absence of phosphopeptide, the D124A mutation leads to binding pocket reorganization including widening up of the binding pocket at the major groove and repositioning of N173, a key residue that interacts with the main chain of phosphopeptide. These structural changes would interfere with the efficient binding of the peptide, corroborating the experimental observations. Both gain and loss of electrostatic interactions in the form of salt bridges strongly indicate a rearrangement of the network of interactions within the binding pocket. Limited proteolysis coupled mass spectrometry (lip-MS) of the apo and holo forms of WT and mutant protein shows a peptide binding helix otherwise buried in the WT protein was particularly accessible to trypsin in the apo form of the mutant protein and the region was mapped to 158-186 amino acid residues of 14-3-3ζ. These results further confirm the dynamic nature of D124A mutant. Unlike other basic residues, the invariant D124 facilitates peptide binding by maintaining the geometry of interacting residues and by enforcing the structural integrity of amphipathic pocket.

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.

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