scholarly journals Assignment of a single disulphide bridge in human α2-antiplasmin: implications for the structural and functional properties

1997 ◽  
Vol 323 (3) ◽  
pp. 847-852 ◽  
Author(s):  
Søren CHRISTENSEN ◽  
Zuzana VALNICKOVA ◽  
Ida B. THØGERSEN ◽  
Eva H. N. OLSEN ◽  
Jan J. ENGHILD
Keyword(s):  
Functional Analysis ◽  
Acetic Acid ◽  
Blood Coagulation ◽  
Proteinase Inhibitors ◽  
Serine Proteinase ◽  
Thiol Groups ◽  
Disulphide Bridge ◽  
Disulphide Bonds ◽  
Functional Aspects ◽  
Structural And Functional Properties

Human α2-antiplasmin (α2AP) is a serpin involved in the regulation of blood coagulation. Most serpins, unlike smaller serine proteinase inhibitors, do not contain disulphide bridges. α2AP is an exception from this generalization and has previously been shown to contain four Cys residues organized into two disulphide bridges [Lijnen, Holmes, van Hoef, Wiman, Rodriguez and Collen (1987) Eur. J. Biochem. 166, 565–574]. However, we found that α2AP incorporates iodo[14C]acetic acid, suggesting that the protein contains reactive thiol groups. This observation prompted a re-examination of the state of the thiol groups, which revealed (i) a disulphide bridge between Cys43 and Cys116, (ii) that Cys76 is bound to a cysteinyl-glycine dipeptide, and (iii) and Cys125 exists as either a free thiol or in a mixed disulphide with another Cys residue. The disulphide identified between Cys43 and Cys116 appears to be conserved in orthologous proteins since the homologous Cys residues form disulphide bonds in bovine and possibly mouse α2AP. The conservation of this disulphide bridge suggests that it is important for functional aspects of α2AP. However, the structural and functional analysis described in this study does not support this conclusion.

scholarly journals On the disulphide bonds of rhodopsins

1987 ◽  
Vol 246 (1) ◽  
pp. 131-137 ◽  
Author(s):  
S Al-Saleh ◽  
M Gore ◽  
M Akhtar
Keyword(s):  
Acetic Acid ◽  
Indirect Evidence ◽  
Iodoacetic Acid ◽  
The Other ◽  
Cysteine Residues ◽  
Disulphide Bridge ◽  
Disulphide Bonds ◽  
Peptide Purification ◽  
Reducing Conditions ◽  
Bovine Rhodopsin

Carboxymethylation using 14C- or 3H-labelled iodoacetic acid has been used to identify the cysteine residues in bovine rhodopsin involved in the formation of the two intramolecular disulphide bridges. Iodo[2-14C]acetic acid was used to modify 5.8-5.9 residues of cysteine under non-reducing conditions. After dialysis and reduction of disulphide bridges by 2-mercaptoethanol, iodo[2-3H]acetic acid was employed to covalently modify 3.3-3.6 residues of cysteine. Peptide purification and sequencing has unambiguously shown that cysteine residues 322 and 323 are only carboxymethylated after reduction of disulphide bridges. Indirect evidence presented, now coupled with the earlier finding [Findlay & Pappin (1986) Biochem. J. 238, 625-642] suggests that the other disulphide bridge is formed between cysteine residues 110 and 187. A comparison is made of all the sequences of mammalian rhodopsins and colour pigments and attention is drawn to the fact that whereas Cys-322 and Cys-323 are conserved only in three rhodopsins (bovine, ovine and human), the residues corresponding to Cys-110 and Cys-187 are found in all the visual proteins (from rods as well as human cones).

Plasminogen Activator and Plasminogen Activator Inhibitor Associated with Granulomatous Inflammation: A Study with Murine Leprosy

1984 ◽  
Vol 52 (03) ◽  
pp. 243-249 ◽  
Author(s):  
S Izaki ◽  
T Hibino ◽  
Y Isozaki ◽  
P S Hsu ◽  
M Izaki ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Soluble Fraction ◽  
Proteinase Inhibitors ◽  
Granulomatous Inflammation ◽  
Serine Proteinase ◽  
Plasminogen Activator Inhibitor ◽  
Tissue Type ◽  
Weakly Alkaline ◽  
Heat Stable ◽  
Type Plasminogen Activator

SummaryPlasminogen activator that is associated with the development of hypersensitivity granulomas (gPA) was partially purified from a saline soluble fraction of murine lepromas elicited in “resistant” mice, C57BL/6N. The gPA was shown to consist of two subspecies (23,000 and 48,000 in molecular weight) with essentially identical enzymologic properties. The gPA was found to be a relatively heat stable weakly alkaline serine proteinase with trypsin-like characteristics in the specificity for synthetic substrates and proteinase inhibitors. It showed a high affinity for H- D-Ile-Pro-Arg-pNA (Km = 1.4 × 10-4 M) H-D-Val-Leu-Lys- pNA (Km = 5.2 × 10-4 M), and L-pyroGlu-Gly-Arg-pNA (Km = 9.3 × 10-4 M). The gPA did not demonstrate antigenic cross reaction with urokinase-type or tissue-type plasminogen activator.Two distinct enzymatic regulators of the gPA were also demonstrated in the saline soluble fraction of the hypersensitivity granulomas. The gPA and its regulation are assumed to be correlated with macrophage activation in the hypersensitivity granulomas

scholarly journals Isolation and amino-acid sequence of two inhibitors of serine proteinases, members of the squash inhibitor family, from Echinocystis lobata seeds.

1996 ◽  
Vol 43 (3) ◽  
pp. 507-513 ◽  
Author(s):  
D Stachowiak ◽  
A Polanowski ◽  
G Bieniarz ◽  
T Wilusz
Keyword(s):  
Amino Acid ◽  
Proteinase Inhibitors ◽  
Sequence Similarity ◽  
Serine Proteinase ◽  
Exchange Chromatography ◽  
Association Constants ◽  
Cathepsin G ◽  
Amino Acid Residues ◽  
Mature Seeds ◽  
Echinocystis Lobata

Two serine proteinase inhibitors (ELTI I and ELTI II) have been isolated from mature seeds of Echinocystis lobata by ammonium sulfate fractionation, methanol precipitation, ion exchange chromatography, affinity chromatography on immobilized anhydrotrypsin and HPLC. ELTI I and ELTI II consist of 33 and 29 amino-acid residues, respectively. The primary structures of these inhibitors are as follows: ELTI I KEEQRVCPRILMRCKRDSDCLAQCTCQQSGFCG ELTI II RVCPRILMRCKRDSDCLAQCTCQQSGFCG The inhibitors show sequence similarity with the squash inhibitor family. ELTI I differs from ELTI II only by the presence of the NH2-terminal tetrapeptide Lys-Glu-Glu-Gln. The association constants (Ka) of ELTI I and ELTI II with bovine-trypsin were determined to be 6.6 x 10(10) M-1, and 3.1 x 10(11) M-1, whereas the association constants of these inhibitors with cathepsin G were 1.2 x 10(7) M-1, and 1.1 x 10(7) M-1, respectively.

The First Intrinsic Tenase Complex Inhibitor with Serine Protease Structure Offers a New Perspective in Anticoagulant Therapy

2018 ◽  
Vol 118 (10) ◽  
pp. 1713-1728 ◽  
Author(s):  
Zorica Latinović ◽  
Adrijana Leonardi ◽  
Lidija Kovačič ◽  
Cho Koh ◽  
Jernej Šribar ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Serine Protease ◽  
Blood Coagulation ◽  
Three Dimensional ◽  
Serine Proteinase ◽  
Medical Application ◽  
Structural Features ◽  
Coagulation System ◽  
Three Dimensional Model ◽  
Excessive Bleeding

AbstractComponents of the intrinsic blood coagulation pathway, among them factor VIIIa (FVIIIa), have been recognized as suitable therapeutic targets to treat venous thromboembolism, pathological process behind two very serious cardiovascular diseases, deep vein thrombosis and pulmonary embolism. Here, we describe a unique glycoprotein from the nose-horned viper (Vipera ammodytes ammodytes [Vaa]) venom, Vaa serine proteinase homolog 1 (VaaSPH-1), structurally a serine protease but without an enzymatic activity and expressing potent anticoagulant action in human blood. We demonstrated that one of its targets in the blood coagulation system is FVIIIa of the intrinsic tenase complex, where it antagonizes the binding of FIXa. Anticoagulants with such characteristics are intensively sought, as they would be much safer for medical application as the contemporary drugs, which frequently induce excessive bleeding and other complications. VaaSPH-1 is unlikely to be orally available for chronic usage as it has molecular mass of 35 kDa. However, it represents a very promising template to design low molecular mass FVIIIa-directed anticoagulant substances, based on structural features of the interaction surface between VaaSPH-1 and FVIIIa. To this end, we constructed a three-dimensional model of VaaSPH-1 bound to FVIIIa. The model exposes the 157–loop and the preceding α-helix as the most appropriate structural elements of VaaSPH-1 to be considered as a guideline to synthesize small FVIIIa-binding molecules, potential new generation of anticoagulants.

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