Fragmentation of Actin by Thrombin-Like Snake Venom Proteases

1979 ◽  
Author(s):  
M. Hauck ◽  
L. Muszbek
Keyword(s):  
Skeletal Muscle ◽  
Amino Acid ◽  
Snake Venom ◽  
Time Course ◽  
Peptide Bond ◽  
Muscle Actin ◽  
Sds Page ◽  
End Groups ◽  
Bothrops Marajoensis ◽  
Bothrops Moojeni

It has been demonstrated that thrombin can split skeletal muscle actin (Muszbek and Laki, PNAS 1974,71,2208). In the present paper the effect of thrombin-like snake venom proteases (Ancrod and Batroxobins of Bothrops moojeni and Bothrops marajoensis) on actin was studied and compared to the thrombic cleavage of this protein. Only EDTA pretreated G and F actin were split by Ancrod, while, Batroxobins hydrolized native G actin, too. The time course of digestion was followed by SDS PAGE. A split product of 37500 m.w. appeared first which was cleaved further resulting in three lower m.w. fragments. The SDS gel pattern of thrombic fragmentation was well distinguishable from those caused by Ancrod and Batroxobins. The first split products of Batroxobin digestion were isolated and by estimating their N-, and C-terminal end groups and amino acid compositions the peptide bond hydrolyzed first was located in the primary structure of actin. It was established that while thrombin split off two actinopeptides (at Arg ( 28)-Ala(29) and Arg ( 39)-His ( 40) from the N-terminal end of the molecule only Arg ( 39)-His ( 40) was cleaved by Batroxobins.

scholarly journals The mechanism of the reaction between human plasminogen-activator inhibitor 1 and tissue plasminogen activator

1990 ◽  
Vol 265 (1) ◽  
pp. 109-113 ◽  
Author(s):  
T L Lindahl ◽  
P I Ohlsson ◽  
B Wiman
Keyword(s):  
Amino Acid ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Peptide Bond ◽  
Plasminogen Activator Inhibitor 1 ◽  
Sds Page ◽  
Tissue Plasminogen ◽  
Activator Inhibitor ◽  
Pai 1

The structural events taking place during the reaction between PAI-1 (plasminogen-activator inhibitor 1) and the plasminogen activators sc-tPA (single-chain tissue plasminogen activator) and tc-tPA (two-chain tissue plasminogen activator) were studied. Complexes were formed by mixing sc-tPA or tc-tPA with PAI-1 in slight excess (on an activity basis). The complexes were purified from excess PAI-1 by affinity chromatography on fibrin-Sepharose. Examination of the purified complexes by SDS/polyacrylamide-gel electrophoresis (SDS/PAGE) and N-terminal amino acid sequence analysis demonstrated that a stoichiometric 1:1 complex is formed between PAI-1 and both forms of tPA. Data obtained from both complexes revealed the amino acid sequences of the parent molecules and, in addition, a new sequence: Met-Ala-Pro-Glu-Glu-. This sequence is found in the C-terminal portion of the intact PAI-1 molecule and thus locates the reactive centre of PAI-1 to Arg346-Met347. The proteolytic activity of sc-tPA is demonstrated by its capacity to cleave the ‘bait’ peptide bond in PAI-1. The complexes were inactive and dissociated slowly at physiological pH and ionic strength, but rapidly in aq. NH3 (0.1 mol/l). Amidolytic tPA activity was generated on dissociation of the complexes, corresponding to 0.4 mol of tPA/mol of complex. SDS/PAGE of the dissociated complexes indicated a small decrease in the molecular mass of PAI-1, in agreement with proteolytic cleavage of the ‘bait’ peptide bond during complex-formation.

scholarly journals The complete amino acid sequence of chicken skeletal-muscle enolase

1986 ◽  
Vol 236 (1) ◽  
pp. 115-126 ◽  
Author(s):  
G A Russell ◽  
B Dunbar ◽  
L A Fothergill-Gilmore
Keyword(s):  
Skeletal Muscle ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Peptide Bond ◽  
Peptide Bonds ◽  
Complete Amino Acid Sequence ◽  
Arginine Residues ◽  
Cnbr Cleavage ◽  
Yeast Enolase ◽  
Chicken Skeletal Muscle

The complete amino acid sequence of chicken skeletal-muscle enolase, comprising 433 residues, was determined. The sequence was deduced by automated sequencing of hydroxylamine-cleavage, CNBr-cleavage, o-iodosobenzoic acid-cleavage, clostripain-digest and staphylococcal-proteinase-digest fragments. The presence of several acid-labile peptide bonds and the tenacious aggregation of most CNBr-cleavage fragments meant that a commonly used sequencing strategy involving initial CNBr cleavage was unproductive. Cleavage at the single Asn-Gly peptide bond with hydroxylamine proved to be particularly useful. Comparison of the sequence of chicken enolase with the two yeast enolase isoenzyme sequences shows that the enzyme is strongly conserved, with 60% of the residues identical. The histidine and arginine residues implicated as being important for the activity of yeast enolase are conserved in the chicken enzyme. Secondary-structure predictions are analysed in an accompanying paper [Sawyer, Fothergill-Gilmore & Russell (1986) Biochem. J. 236, 127-130].

Antibodies to a fragment of the Bothrops moojeni l-amino acid oxidase cross-react with snake venom components unrelated to the parent protein

Toxicon ◽  
2005 ◽  
Vol 46 (3) ◽  
pp. 308-317 ◽  
Author(s):  
Rodrigo Guerino Stábeli ◽  
Lívia Maria Pimenta Magalhães ◽  
Heloísa S. Selistre-de-Araujo ◽  
Eduardo Brandt Oliveira
Keyword(s):  
Amino Acid ◽  
Snake Venom ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Parent Protein ◽  
Bothrops Moojeni

scholarly journals Bmoo FIBMP-I: A New Fibrinogenolytic Metalloproteinase from Bothrops moojeni Snake Venom

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
F. S. Torres ◽  
B. Rates ◽  
M. T. R. Gomes ◽  
C. E. Salas ◽  
A. M. C. Pimenta ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Molecular Mass ◽  
Proteolytic Activity ◽  
Snake Venom ◽  
Enzymatic Digestion ◽  
Single Chain ◽  
Average Molecular Mass ◽  
Sds Page ◽  
Enzyme Isoforms ◽  
Bothrops Moojeni

A new fibrinogenolytic metalloproteinase (Bmoo FIBMP-I) was purified from Bothrops moojeni snake venom. This enzyme was isolated through a combination of three chromatographic steps (ion-exchange, molecular exclusion, and affinity chromatography). Analyses by reverse phase chromatography, followed by mass spectrometry, showed the presence of enzyme isoforms with average molecular mass of 22.8 kDa. The SDS-PAGE analyses showed a single chain of 27.6 kDa, in the presence and absence of reducing agent. The protein has a blocked N-terminal. One of the peptides obtained by enzymatic digestion of a reduced and S-alkylated isoform was completely sequenced by mass spectrometry (MS/MS). Bmoo FIBMP-I showed similarity with hemorrhagic factor and several metalloproteinases (MP). This enzyme degraded Aα-chain faster than the Bβ-chain and did not affect the γ-chain of bovine fibrinogen. The absence of proteolytic activity after treatment with EDTA, together with the observed molecular mass, led us to suggest that Bmoo FIBMP-I is a member of the P-I class of the snake venom MP family. Bmoo FIBMP-I showed pH-dependent proteolytic activity on azocasein, but was devoid of coagulant, defibrinating, or hemorrhagic activities. The kinetic parameters of proteolytic activity in azocasein were determined (Vmax=0.4596 Uh−1nmol−1±0.1031 and Km=14.59 mg/mL±4.610).

scholarly journals Monoclonal antibodies against muscle actin isoforms: epitope identification and analysis of isoform expression by immunoblot and immunostaining in normal and regenerating skeletal muscle

F1000Research ◽  
2016 ◽  
Vol 5 ◽  
pp. 416 ◽  
Author(s):  
Christine Chaponnier ◽  
Giulio Gabbiani
Keyword(s):  
Skeletal Muscle ◽  
Time Course ◽  
High Capacity ◽  
Repair Process ◽  
Muscle Actin ◽  
Muscle Repair ◽  
Self Renewal ◽  
Actin Isoforms ◽  
Actin Isoform

Higher vertebrates express six different highly conserved actin isoforms that can be classified in three subgroups: 1) sarcomeric actins, α-skeletal (α-SKA) and α-cardiac (α-CAA), 2) smooth muscle actins (SMAs), α-SMA and γ-SMA, and 3) cytoplasmic actins (CYAs), β-CYA and γ-CYA. The variations among isoactins, in each subgroup, are due to 3-4 amino acid differences located in their acetylated N-decapeptide sequence. The first monoclonal antibody (mAb) against an actin isoform (α-SMA) was produced and characterized in our laboratory in 1986 (Skalli et al., 1986). We have further obtained mAbs against the 5 other isoforms. In this report, we focus on the mAb anti-α-SKA and anti-α-CAA obtained after immunization of mice with the respective acetylated N-terminal decapeptides using the Repetitive Immunizations at Multiple Sites Strategy (RIMMS). In addition to the identification of their epitope by immunoblotting, we describe the expression of the 2 sarcomeric actins in mature skeletal muscle and during muscle repair after micro-lesions. In particular, we analyze the expression of α-CAA, α-SKA and α-SMA by co-immunostaining in a time course frame during the muscle repair process. Our results indicate that a restricted myocyte population expresses α-CAA and suggest a high capacity of self-renewal in muscle cells. These antibodies may represent a helpful tool for the follow-up of muscle regeneration and pathological changes.

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