scholarly journals Isolation and Biochemical Characterization of a New Thrombin-Like Serine Protease fromBothrops pirajaiSnake Venom

2014 ◽  
Vol 2014 ◽  
pp. 1-13 ◽  
Author(s):  
Kayena D. Zaqueo ◽  
Anderson M. Kayano ◽  
Rodrigo Simões-Silva ◽  
Leandro S. Moreira-Dill ◽  
Carla F. C. Fernandes ◽  
...  
Keyword(s):  
Serine Protease ◽  
Serine Proteases ◽  
Biochemical Characterization ◽  
Sequence Similarity ◽  
Size Exclusion ◽  
Coagulation Cascade ◽  
Activate Factor ◽  
High Sequence Similarity ◽  
Fibrin Networks ◽  
Bothrops Pirajai

This paper presents a novel serine protease (SP) isolated fromBothrops pirajai, a venomous snake found solely in Brazil that belongs to the Viperidae family. The identified SP, named BpirSP-39, was isolated by three chromatographic steps (size exclusion, bioaffinity, and reverse phase chromatographies). The molecular mass of BpirSP-39 was estimated by SDS-PAGE and confirmed by mass spectrometry (39,408.32 Da). The protein was able to form fibrin networks, which was not observed in the presence of serine protease inhibitors, such as phenylmethylsulfonyl fluoride (PMSF). Furthermore, BpirSP-39 presented considerable thermal stability and was apparently able to activate factor XIII of the blood coagulation cascade, unlike most serine proteases. BpirSP-39 was capable of hydrolyzing different chromogenic substrates tested (S-2222, S-2302, and S-2238) while Cu2+significantly diminished BspirSP-39 activity on the three tested substrates. The enzyme promoted platelet aggregation and also exhibited fibrinogenolytic, fibrinolytic, gelatinolytic, and amidolytic activities. The multiple alignment showed high sequence similarity to other thrombin-like enzymes from snake venoms. These results allow us to conclude that a new SP was isolated fromBothrops pirajaisnake venom.

scholarly journals Purification and Biochemical Characterization of Alkaline Serine Protease from Caesalpinia bonducella

2010 ◽  
Vol 5 (6) ◽  
pp. 1934578X1000500
Author(s):  
Hidayatullah Khan ◽  
Irshad Ali ◽  
Arif-ullah Khan ◽  
Mushtaq Ahmed ◽  
Zamarud Shah ◽  
...  
Keyword(s):  
Serine Protease ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Alkaline Serine Protease ◽  
Protease Enzyme ◽  
Caesalpinia Bonducella

A high molecular weight serine protease has been purified to electrophoretic homogeneity from the seeds of Caesalpinia bonducella Flem. (Caesalpiniaceae) by the combination of size exclusion and ion exchange chromatography. About 524 fold purification was achieved with an overall recovery of 6.8%. The specific activity was found to be 86 U/mg/min at pH 8.0. The calculated Km and Vmax were 1.66 mg/mL and 496.68 units/min per mg of protein, respectively. The molecular mass was estimated to be about 63 kDa by sodium dodecyl sulfate PAGE. The enzyme showed optimum activity at pH 8.0 and exhibited its highest activity at 40°C. The enzyme was strongly inhibited by 2mM phenylmethylsulfonyl fluoride (PMSF), suggesting the presence of a serine residue at the active site. PMSF showed a pure competitive type of inhibition with the serine protease enzyme. It was observed that enzyme activity was enhanced in the presence of dications and was active against a variety of modified substrates and natural proteins.

scholarly journals Purification of enzymes of the kallikrein gene family (rK8 and rK9) from the rat prostate

1994 ◽  
Vol 302 (1) ◽  
pp. 229-235 ◽  
Author(s):  
H Schøyen ◽  
I Wassdal ◽  
K Toft ◽  
M Almendingen ◽  
T Berg
Keyword(s):  
Sequence Analysis ◽  
Submandibular Gland ◽  
Serine Proteases ◽  
Biochemical Characterization ◽  
Sequence Similarity ◽  
Complete Sequence ◽  
Polyclonal Antiserum ◽  
Bean Trypsin Inhibitor ◽  
Rat Prostate ◽  
Ph Range

The rat kallikrein family consists of multiple closely related proteins. A method for demonstration and identification of kallikrein-like proteins has been developed based on their differences in isoelectric point and their immunological similarity. The method, which involved separation in flat-bed isoelectro-focusing gels (pH range 3-9) and detection by immunoblotting using polyclonal antiserum against one of the family members, has been used in the present study to detect kallikrein-like proteins in the rat prostate. Nine immunoreactive kallikrein-like protein bands were detected with pI ranging from 5.30 to 8.35. Of these, six were completely purified and three were partially purified. Two proteins (pI 5.30 and 6.75-6.90) corresponded to protein bands in gels of rat submandibular-gland extracts, and were identified by partial amino acid sequence analysis as rK8 and rK9 respectively. In addition, sequence analysis revealed complete sequence similarity between rK9 and the immunoreactive prostate proteins with pI 7.15, 7.25, 7.50 and 8.27. On the basis of this finding and immunological and biochemical characterization, we concluded that all the kallikrein-like proteins detected, except for rK8, represented isoenzymes of rK9. The molecular masses of the prostate rK9 isoenzymes (24,600-29,300 Da) were close to that of submandibular-gland rK9 (24,600 Da), although differences were observed after reduction with mercaptoethanol. The prostate rK9 isoenzymes were, like submandibular-gland rK9, inhibited by soya-bean trypsin inhibitor but not by aprotinin, and were classified as serine proteases as they were inhibited by phenylmethanesulphonyl fluoride. rK8 (28,700 Da) showed no activity with any of the substrates tested, and its inhibitory profile could therefore not be studied. No other enzymes of the kallikrein family were found in the rat prostate.

scholarly journals Biochemical and Pharmacological Characterization of TLBbar, a New Serine Protease with Coagulant Activity from Bothrops barnetti Snake Venom

2013 ◽  
Vol 2013 ◽  
pp. 1-11
Author(s):  
Magaly Alejandra Brousett-Minaya ◽  
Paulo Aparecido Baldasso ◽  
Salomón Huancahuire-Vega ◽  
Sérgio Marangoni
Keyword(s):  
Serine Protease ◽  
Snake Venom ◽  
Serine Proteases ◽  
Kinetic Studies ◽  
Size Exclusion ◽  
Reverse Phase Hplc ◽  
Pharmacological Characterization ◽  
Coagulant Activity ◽  
Exclusion Chromatography

A thrombin-like enzyme named TLBbar was isolated from Bothrops barnetti snake venom and its biochemical and pharmacological characteristics were determined. TLBbar was purified using size exclusion chromatography and reverse phase HPLC, showing molecular mass of 28750.7 Da determined by mass spectrometry. TLBbar serine protease is basic (pI 7.4) and its structure shows similarity with other serine proteases of snake venom. Optimal proteolytic activity was at 37°C and pH 8; this activity was strongly inhibited by PMSF and Leupeptin, however; heparin, and soybean trypsin inhibitor (SBT-I) were ineffective. Kinetic studies on BApNA chromogenic substrate have revealed that TLBbar presents a Michaelis-Menten kinetics, with values of Km and Vmax of 0.433 mM and 0.42 nmol/min, respectively. TLBbar showed high clotting activity upon bovine and human plasma, presenting IC of 125 and minimum dose coagulant (MDC) of 2.23 μg/μL. TLBbar cleavages the Aα chain of bovine fibrinogen, with maximal efficiency at 30–40°C in the presence of calcium after two hours incubation; this fibronogenolityc activity was inhibited by PMSF and Leupeptin, confirming its classification in the group of serine proteases. In addition, TLBbar is capable of aggregating platelets in the same way that thrombin in concentrations of 2.5 μg/μL.

scholarly journals Crystal structure of the M14R mutant of oryctin in complex with trypsin

2014 ◽  
Vol 70 (a1) ◽  
pp. C465-C465
Author(s):  
Desheng Liu ◽  
Tatsuya Suzuki ◽  
Shoichiro Horita ◽  
Takeshi Kawai ◽  
Jun Ishibashi ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Serine Protease ◽  
Serine Proteases ◽  
Sequence Similarity ◽  
Point Mutations ◽  
Structural Similarity ◽  
Leukocyte Elastase ◽  
Subtilisin Carlsberg ◽  
Domain 3 ◽  
Α Helix

Oryctin is a 66-amino-acid protein purified from the larval haemolymph of the coconut rhinoceros beetle Oryctes rhinoceros, which shows no sequence similarity to any other protein known. We determined the solution NMR structure of oryctin, and found that oryctin had a similar backbone fold to the turkey ovomucoid domain 3, OMTKY3, a Kazal-type serine protease inhibitor [1]. Based on the structural similarity, we tested the serine protease inhibitory activity of oryctin, and found that oryctin does inhibit some serine proteases, such as α-chymotrypsin, endopeptidase K, subtilisin Carlsberg, and leukocyte elastase [1]. However, oryctin cannot inhibit trypsin at all. In this study, we have introduced point mutations to the putative inhibition loop of oryctin to obtain oryctin mutants that can inhibit trypsin. Then, we have solved the crystal structure of such an oryctin mutant, M14R-oryctin with a Ki value of 3.410.8 nM, in complex with trypsin to reveal how it binds to and inhibits trypsin. As predicted, the putative inhibition loop lay on the substrate binding cleft of trypsin. Particularly, the side chain of R14 fit into the S1 pocket of trypsin by forming hydrogen/ionic bonds with D191, S192 and G216 at the bottom of the S1 pocket and G195, D196, S197 and S212 at its entrance. In addition, R65 located in the C-terminal α-helix of M14R-oryctin formed hydrogen bonds with S40 and F44 of trypsin. The latter interaction, which is unique to oryctin, enhances its binding affinity to trypsin.

scholarly journals A Serine Protease-Encoding Gene (aprII) of Alteromonas sp. Strain O-7 Is Regulated by the Iron Uptake Regulator (Fur) Protein

2000 ◽  
Vol 66 (9) ◽  
pp. 3778-3783 ◽  
Author(s):  
Hiroshi Tsujibo ◽  
Katsushiro Miyamoto ◽  
Takashi Okamoto ◽  
Hideyuki Orikoshi ◽  
Yoshihiko Inamori
Keyword(s):  
Serine Protease ◽  
Marine Bacterium ◽  
Sequence Similarity ◽  
Iron Acquisition ◽  
Northern Hybridization ◽  
Ferric Uptake Regulator ◽  
Gene Encoding ◽  
High Sequence Similarity ◽  
Fur Protein ◽  
Encoding Gene

ABSTRACT The ferric uptake regulator (Fur) box-like sequence was located upstream of the serine protease-encoding gene (aprII) from a marine bacterium, Alteromonas sp. strain O-7. To clarify whether the production of AprII (the gene product of aprII) is regulated by the environmental iron concentrations, this strain was cultured under iron-depleted or iron-rich conditions and the level of AprII in the culture supernatant was analyzed by Western blotting. The production of AprII was significantly repressed under iron-rich conditions. Northern hybridization analysis demonstrated that AprII biosynthesis was regulated by iron through the control of transcription. These results indicate that aprII is a new member of the iron regulon and plays an important role in the iron acquisition system of the strain. Furthermore, the gene encoding Fur was cloned and sequenced. The deduced amino acid sequence of the cloned Fur showed high sequence similarity with that from gram-negative bacteria.

scholarly journals Identification of novel glycosylation events on human serum-derived Factor IX

2019 ◽  
Author(s):  
Cassandra L. Pegg ◽  
Lucia F. Zacchi ◽  
Dinora Roche Recinos ◽  
Christopher B. Howard ◽  
Benjamin L. Schulz
Keyword(s):  
Human Serum ◽  
Serine Protease ◽  
Factor Ix ◽  
Coagulation Cascade ◽  
Clotting Factor ◽  
Activate Factor ◽  
Protease Domain ◽  
Structural Characterisation ◽  
Serine Protease Domain ◽  
Human Factor Ix

ABSTRACTHuman Factor IX is a highly post-translationally modified protein that is an important clotting factor in the blood coagulation cascade. Functional deficiencies in Factor IX result in the bleeding disorder haemophilia B, which is treated with plasma-derived or recombinant Factor IX concentrates. Here, we investigated the post-translational modifications of human serum-derived Factor IX and report previously undescribed O-linked monosaccharide compositions at serine 141 and a novel site of glycosylation. At serine 141 we observed two monosaccharide compositions, with HexNAc1Hex1NeuAc2 dominant and a low level of HexNAc1Hex1NeuAc1. This O-linked site lies N-terminal to the first cleavage site for the activation peptide, an important region of the protein that is removed to activate Factor IX. The novel site is an N-linked site in the serine protease domain with low occupancy in a non-canonical consensus motif at asparagine 258, observed with a HexNAc4Hex5NeuAc2 monosaccharide composition attached. This is the first reported instance of a site of modification in the serine protease domain. The description of these glycosylation events provides a basis for future functional studies and contributes to structural characterisation of native Factor IX for the production of effective therapeutic biosimilars and biobetters.

scholarly journals Biochemical characterization and comparative analysis of two distinct serine proteases from Bothrops pirajai snake venom

Biochimie ◽  
2012 ◽  
Vol 94 (12) ◽  
pp. 2545-2558 ◽  
Author(s):  
Danilo Luccas Menaldo ◽  
Carolina Petri Bernardes ◽  
Norival Alves Santos-Filho ◽  
Laura de Andrade Moura ◽  
André Lopes Fuly ◽  
...  
Keyword(s):  
Comparative Analysis ◽  
Snake Venom ◽  
Serine Proteases ◽  
Biochemical Characterization ◽  
Bothrops Pirajai

scholarly journals Serine Protease Variants Encoded byEchis ocellatusVenom Gland cDNA: Cloning and Sequencing Analysis

2010 ◽  
Vol 2010 ◽  
pp. 1-12 ◽  
Author(s):  
S. S. Hasson ◽  
R. A. Mothana ◽  
T. A. Sallam ◽  
M. S. Al-balushi ◽  
M. T. Rahman ◽  
...  
Keyword(s):  
Serine Protease ◽  
Serine Proteases ◽  
Tertiary Structure ◽  
Indian Subcontinent ◽  
Sequence Similarity ◽  
Sequence Conservation ◽  
Sequencing Analysis ◽  
Cdna Sequences ◽  
Venom Glands ◽  
High Degree

Envenoming byEchissaw-scaled viper is the leading cause of death and morbidity in Africa due to snake bite. Despite its medical importance, there have been few investigations into the toxin composition of the venom of this viper. Here, we report the cloning of cDNA sequences encoding four groups or isoforms of the haemostasis-disruptive Serine protease proteins (SPs) from the venom glands ofEchis ocellatus. All these SP sequences encoded the cysteine residues scaffold that form the 6-disulphide bonds responsible for the characteristic tertiary structure of venom serine proteases. All theEchis ocellatus EoSPgroups showed varying degrees of sequence similarity to published viper venom SPs. However, these groups also showed marked intercluster sequence conservation across them which were significantly different from that of previously published viper SPs. Because viper venom SPs exhibit a high degree of sequence similarity and yet exert profoundly different effects on the mammalian haemostatic system, no attempt was made to assign functionality to the newEchis ocellatus EoSPson the basis of sequence alone. The extraordinary level of interspecific and intergeneric sequence conservation exhibited by theEchis ocellatus EoSPsand analogous serine proteases from other viper species leads us to speculate that antibodies to representative molecules should neutralise (that we will exploit, by epidermal DNA immunization) the biological function of this important group of venom toxins in vipers that are distributed throughout Africa, the Middle East, and the Indian subcontinent.

scholarly journals Biological and Biochemical Characterization of Coronado Island Rattlesnake (Crotalus helleri caliginis) Venom and Antivenom Neutralization

Toxins ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 582
Author(s):  
Cristian Franco-Servín ◽  
Edgar Neri-Castro ◽  
Melisa Bénard-Valle ◽  
Alejandro Alagón ◽  
Ramsés Alejandro Rosales-García ◽  
...  
Keyword(s):  
Serine Proteases ◽  
Biochemical Characterization ◽  
Two Dimensions ◽  
Size Exclusion ◽  
Muscle Twitch ◽  
Amino Terminal ◽  
Sds Page ◽  
Exclusion Chromatography ◽  
Main Components

The Baja California Peninsula has over 250 islands and islets with many endemic species. Among them, rattlesnakes are the most numerous but also one of the least studied groups. The study of island rattlesnake venom could guide us to a better understanding of evolutionary processes and the description of novel toxins. Crotalus helleri caliginis venom samples were analyzed to determine possible ontogenetic variation with SDS-PAGE in one and two dimensions and with RP-HPLC. Western Blot, ELISA, and amino-terminal sequencing were used to determine the main components of the venom. The biological and biochemical activities demonstrate the similarity of C. helleri caliginis venom to the continental species C. helleri helleri, with both having low proteolytic and phospholipase A2 (PLA2) activity but differing due to the absence of neurotoxin (crotoxin-like) in the insular species. The main components of the snake venom were metalloproteases, serine proteases, and crotamine, which was the most abundant toxin group (30–35% of full venom). The crotamine was isolated using size-exclusion chromatography where its functional effects were tested on mouse phrenic nerve–hemidiaphragm preparations in which a significant reduction in muscle twitch contractions were observed. The two Mexican antivenoms could neutralize the lethality of C. helleri caliginis venom but not the crotamine effects.

scholarly journals Phenotypic Analysis of Mice Lacking the Tmprss2-Encoded Protease

2006 ◽  
Vol 26 (3) ◽  
pp. 965-975 ◽  
Author(s):  
Tom S. Kim ◽  
Cynthia Heinlein ◽  
Robert C. Hackman ◽  
Peter S. Nelson
Keyword(s):  
Serine Protease ◽  
Serine Proteases ◽  
Biological Activities ◽  
Type Ii ◽  
Phenotypic Analysis ◽  
Normal Prostate ◽  
Prostate Hyperplasia ◽  
Transmembrane Serine Protease

ABSTRACT Tmprss2 encodes an androgen-regulated type II transmembrane serine protease (TTSP) expressed highly in normal prostate epithelium and has been implicated in prostate carcinogenesis. Although in vitro studies suggest protease-activated receptor 2 may be a substrate for TMPRSS2, the in vivo biological activities of TMPRSS2 remain unknown. We generated Tmprss2 −/− mice by disrupting the serine protease domain through homologous recombination. Compared to wild-type littermates, Tmprss2 −/− mice developed normally, survived to adulthood with no differences in protein levels of prostatic secretions, and exhibited no discernible abnormalities in organ histology or function. Loss of TMPRSS2 serine protease activity did not influence fertility, reduce survival, result in prostate hyperplasia or carcinoma, or alter prostatic luminal epithelial cell regrowth following castration and androgen replacement. Lack of an observable phenotype in Tmprss2 −/− mice was not due to transcriptional compensation by closely related Tmprss2 homologs. We conclude that the lack of a discernible phenotype in Tmprss2 −/− mice suggests functional redundancy involving one or more of the type II transmembrane serine protease family members or other serine proteases. Alternatively, TMPRSS2 may contribute a specialized but nonvital function that is apparent only in the context of stress, disease, or other systemic perturbation.

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