scholarly journals Characterization of a new kallikrein-like enzyme (KLP-S3) of the rat submandibular gland

1992 ◽  
Vol 281 (3) ◽  
pp. 819-828 ◽  
Author(s):  
T Berg ◽  
H Schøyen ◽  
I Wassdal ◽  
R Hull ◽  
V P Gerskowitch ◽  
...  
Keyword(s):  
Amino Acid ◽  
Molecular Mass ◽  
Submandibular Gland ◽  
Rat Aorta ◽  
Tissue Kallikrein ◽  
Amino Acid Residues ◽  
Alpha Adrenoceptor ◽  
Adrenoceptor Agonist ◽  
Bean Trypsin Inhibitor ◽  
Rat Submandibular Gland

The submandibular gland of the rat contains several enzymes belonging to the kallikrein family. These include tissue kallikrein, antigen gamma (T-kininogenase), esterase B and tonin. In the present study, a new member of this family, which we have named KLP-S3, was identified and purified from the submandibular gland. KLP-S3 was classified as a kallikrein-like enzyme on the basis of its immunological similarity to other kallikrein-like enzymes and its showing 70% and 73% identity in partial amino acid sequence with tissue kallikrein and tonin respectively. Furthermore, the 44 sequenced amino acid residues showed complete correspondence to the mRNA S3 of the kallikrein gene family, which was the rationale for the name kallikrein-like protein (KLP) S3. KLP-S3 consisted of three isoenzymes with pI 6.75, 6.90 and 6.95, which significantly differed from those of other kallikrein-like enzymes. In conjunction with its immunological relationship to kallikrein, this parameter (pI) was considered robust enough to identify the enzyme during purification, since a specific physiological substrate for KLP-S3 has yet to be identified. In SDS/PAGE the three isoenzymes ran as one band with a molecular mass of 25,800 Da, which after reduction with 2-mercaptoethanol was split into two chains with molecular masses of 16,500 and 13,300 Da. In common with other kallikrein-like enzymes, KLP-S3 was inhibited by phenylmethanesulphonyl fluoride, and was thus classified as a serine protease. It was also inhibited by soya-bean trypsin inhibitor but not by aprotinin. It showed weak reactivity against the chromogenic substrates S2288, S2266, S2366 and S2302 (D-Ile-Pro-Arg 4-nitroanilide, D-Val-Leu-Arg 4-nitroanilide, Glu-Pro-Arg 4-nitroanilide and D-Pro-Phe-Arg 4-nitroanilide respectively) and did not cleave rat T-kininogen or dog high-molecular-mass/low-molecular-mass kininogen. Its specific angiotensin II-generating activity (angiotensin I as substrate) was 0.04% of that of rat tonin. KLP-S3 (1-100 nM) induced a statistically significant angiotensin-independent contraction of isolated rat aorta rings. The maximum contraction was 15% of the response to the alpha-adrenoceptor agonist phenylephrine (1 microM). The concentration of KLP-S3 in the rat submandibular gland was by single radial immunodiffusion estimated to be 47 +/- 3 micrograms/mg of protein.

scholarly journals T-kininogenase activity of the rat submandibular gland is predominantly due to the kallikrein-like serine protease antigen γ

1991 ◽  
Vol 280 (1) ◽  
pp. 19-25 ◽  
Author(s):  
T Berg ◽  
I Wassdal ◽  
T Mindroiu ◽  
K Sletten ◽  
G Scicli ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Molecular Mass ◽  
Submandibular Gland ◽  
Sequence Similarity ◽  
Rat Plasma ◽  
Amino Acid Sequence Similarity ◽  
Tissue Kallikrein ◽  
Reverse Phase ◽  
Rat Submandibular Gland

T-kininogen, the major kininogen in rat plasma, releases Ile-Ser-bradykinin (T-kinin) when incubated with trypsin, but is not a substrate for tissue kallikrein. Enzymes able to release T-kinins from T-kininogen have been found in the rat submandibular gland, but precise identification of these enzymes and their possible relationship to kallikrein-like enzymes has not been established. We studied T-kininogenase activity in fractionated submandibular gland homogenate. The main T-kininogen catalytic enzyme was purified and characterized, and found to be identical to antigen gamma, a kallikrein-like enzyme which we have previously characterized. Of other identified kallikrein-like enzymes only tonin showed weak T-kininogenase activity, which was about 0.25% of that of antigen gamma. No other T-kininogen catalytic enzymes were observed. Antigen gamma released a kinin which was identified as T-kinin by reverse-phase h.p.l.c. The T-kininogenase activity of antigen gamma had a Km of 29 +/- 4 microM and a kcat/Km of 140 M-1.s-1, and was comparable with its high and low molecular mass-kininogenase activity (7.4 and 10 micrograms of kinin/h per mg respectively). In contrast, tissue kallikrein released 0.2 and 42,200 micrograms of kinin/h per mg respectively. Thus antigen gamma is a weak kininogenase. The isoelectric point of antigen gamma, but not its molecular mass, differed from that of other kallikrein-like enzymes. Isoelectrofocusing in flat-bed gels combined with immunostaining was therefore a convenient method for identification. The kallikrein-like nature of antigen gamma was demonstrated by its immunological similarity to tissue kallikrein and tonin and by 91% and 87% amino acid sequence similarity with tonin and kallikrein respectively (67 amino acids sequenced). Complete identity was also not observed with other sequenced kallikrein genes, mRNAs or proteins.

scholarly journals A Strain of Bacillus thuringiensis Containing a Novel cry7Aa2 Gene that Is Toxic to Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae)

Insects ◽  
2019 ◽  
Vol 10 (9) ◽  
pp. 259 ◽  
Author(s):  
Mikel Domínguez-Arrizabalaga ◽  
Maite Villanueva ◽  
Ana Beatriz Fernandez ◽  
Primitivo Caballero
Keyword(s):  
Amino Acid ◽  
Bacillus Thuringiensis ◽  
Molecular Mass ◽  
Leptinotarsa Decemlineata ◽  
Insect Pests ◽  
Amino Acid Residues ◽  
Cry Protein ◽  
Lc50 Value ◽  
Leptinotarsa Decemlineata Say

The genome of the Bacillus thuringiensis BM311.1 strain was sequenced and assembled in 359 contigs containing a total of 6,390,221 bp. The plasmidic ORF of a putative cry gene from this strain was identified as a potential novel Cry protein of 1138 amino acid residues with a 98% identity compared to Cry7Aa1 and a predicted molecular mass of 129.4 kDa. The primary structure of Cry7Aa2, which had eight conserved blocks and the classical structure of three domains, differed in 28 amino acid residues from that of Cry7Aa1. The cry7Aa2 gene was amplified by PCR and then expressed in the acrystalliferous strain BMB171. SDS-PAGE analysis confirmed the predicted molecular mass for the Cry7Aa2 protein and revealed that after in vitro trypsin incubation, the protein was degraded to a toxin of 62 kDa. However, when treated with digestive fluids from Leptinotarsa decemlineata larvae, one major proteinase-resistant fragment of slightly smaller size was produced. The spore and crystal mixture produced by the wild-type BM311.1 strain against L. decemlineata neonate larvae resulted in a LC50 value of 18.8 μg/mL, which was statistically similar to the estimated LC50 of 20.8 μg/mL for the recombinant BMB17-Cry7Aa2 strain. In addition, when this novel toxin was activated in vitro with commercial trypsin, the LC50 value was reduced 3.8-fold to LC50 = 4.9 μg/mL. The potential advantages of Cry7Aa2 protoxin compared to Cry7Aa1 protoxin when used in the control of insect pests are discussed.

Modèle topologique de la structure d’un antiport vacuolaire de type NHX chez la vigne cultivée (Vitis vinifera)

Botany ◽  
2009 ◽  
Vol 87 (3) ◽  
pp. 339-347 ◽  
Author(s):  
Mohsen Hanana ◽  
Olivier Cagnac ◽  
Ahmed Mliki ◽  
Eduardo Blumwald
Keyword(s):  
Amino Acid ◽  
Vitis Vinifera ◽  
Molecular Mass ◽  
Electrostatic Interactions ◽  
Functional Characterization ◽  
Terminal Region ◽  
Vitis Vinifera L ◽  
Amino Acid Residues ◽  
Transmembrane Segments ◽  
Α Helix

After identifying and isolating a grapevine ( Vitis vinifera L.) NHX vacuolar antiporter and before initializing functional genomic studies, we juged necessary to acquire a minimum of knowledge about the VvNHX1 protein. Thus, we realized a bioinformatic analysis to determine its basic characteristics and to get structural informations that could guide us through the functional characterization. We have determined important physico-chemical parameters (molecular mass, isoelectric point, hydrophobic regions, etc.) and obtained interesting structural data (primary, secondary, and tertiary structures; conserved domains and interaction motives; etc.). The VvNHX1 gene, which encodes this 541 amino-acid protein with a predicted molecular mass of 60 kDa, is made of 14 exons and measures 6.5 kb. The amino-acidic composition of this protein is very important, in particular, for the establishment of the α-helix structure, which represents more than 50% of the protein, but also for charge distribution, which generates critical electrostatic interactions for the ionic flux. The secondary structure of VvNHX1 contains multiple transmembrane α-helix segments that are made of hydrophobic amino-acid residues, thus facilitating its insertion in the membrane. Globally, VvNHX1 has one hydrophobic N-terminal region, made of 10 transmembrane segments with 440 amino-acid residues, and one hydrophilic C-terminal region, made of 100 residues. The region located between the fourth and fifth transmembrane segments represents, with its structure mainly helicoidal and the presence of a favourable electrostatic environment, the pore where cation flux is performed across the membrane. VvNHX1 contains various interaction domains as well as several putative posttranslational modification sites, mainly at the C-terminus but also at the N-terminus, that play an important part in regulating protein activities, influence protein structural stability, or interact with other proteins or signalling molecules.

scholarly journals A Strain of Bacillus thuringiensis Containing a Novel cry7Aa2 Gene That Is Highly Toxic to Leptinotarsa decemlineata (Say) (Coleoptera; Chrysomelidae)

2019 ◽  
Author(s):  
Mikel Dominguez- ◽  
Maite Villanueva ◽  
Ana Beatriz Fernandez ◽  
Primitivo Caballero
Keyword(s):  
Amino Acid ◽  
Bacillus Thuringiensis ◽  
Molecular Mass ◽  
Leptinotarsa Decemlineata ◽  
Insect Pests ◽  
Amino Acid Residues ◽  
Cry Protein ◽  
Sds Page ◽  
Neonate Larvae

The genome of the Bacillus thuringiensis BM311.1 strain was sequenced and assembled in 359 contigs containing a total of 6,390,221 bp. The plasmidic ORF of a putative cry gene from this strain was identified as a potential novel Cry protein of 1138 amino acid residues with a 98% identity respect to Cry7Aa1 protein and a predicted molecular mass of 129.4 kDa. The primary structure of this Cry7Aa2 protein, which revealed the presence of eight conserved blocks and the classical structure of three domains, differed in 28 amino acid residues from that of Cry7Aa1. The cry7Aa2 gene was amplified by PCR and then expressed in the acrystalliferous strain BMB171. SDS-PAGE analysis confirmed the predicted molecular mass for the Cry7Aa2 protein and revealed that, after in vitro trypsin incubation, it was degraded to a toxin of 62 kDa. However, when treated with digestive fluids from Leptinotarsa decemlineata larvae two proteinase-resistant fragments of 60 and 65 kDa were produced. Spore and crystal mixture produced by the wild-type BM311.1 strain against L. decemlineata neonate larvae resulted in a LC50 (18.8 μg/ml), which was statistically equal to the estimated LC50 (20.8 μg/mL) for the recombinant BMB17-Cry7Aa2 strain. In addition, when this novel toxin was activated in vitro with commercial trypsin, the LC50 value was reduced 4 times approximately (LC50 = 4.9 μg/mL). The advantages of Cry7Aa2 protoxin compared to Cry7Aa1 protoxin when used in the control of insect pests are discussed.

scholarly journals Binding of high-molecular-mass kininogen to the Apple 1 domain of factor XI is mediated in part by Val64 and Ile77

1994 ◽  
Vol 304 (3) ◽  
pp. 715-721 ◽  
Author(s):  
F S Seaman ◽  
F A Baglia ◽  
J A Gurr ◽  
B A Jameson ◽  
P N Walsh
Keyword(s):  
Amino Acid ◽  
Molecular Mass ◽  
Binding Site ◽  
Expression System ◽  
High Molecular Mass ◽  
Amino Acid Residues ◽  
Factor Xi ◽  
Specific Amino Acid ◽  
Conformationally Constrained ◽  
A1 Domain

We have previously demonstrated the presence of a binding site for high-molecular-mass kininogen (HK), spanning residues Val59-Lys83, in the first Apple (A1) domain in the heavy-chain region of factor XI. We have now prepared conformationally constrained synthetic peptides and recombinant A1 domain (rA1) constructs to identify the specific amino acid residues that constitute the HK-binding site. Expression of the A1 domain (Glu1-Ser90) was achieved in a bacterial expression system following PCR amplification of the A1 domain from factor XI cDNA and ligation into an expression plasmid. The rA1 inhibited factor XI binding to HK [Ki approximately (2-3) x 10(-7) M] in a manner indistinguishable from purified factor XI, indicating that all the information necessary for binding HK is contained within the A1 domain. To identify specific amino acid residues involved in binding HK, conformationally constrained peptides were synthesized containing conservative amino acid substitutions at residues suspected to contain side chains involved in binding, including Val64-->Ala, Glu66-->Ala, Arg73-->Ala and Ile77-->Ala. Because normal results were obtained with all peptides with the exception of Val64-->Ala and Ile77-->Ala, which failed to compete normally with factor XI for binding to HK, we prepared two mutant rA1 domains (Val64-->Ala and Ile77-->Ala) by PCR-based site-directed mutagenesis, both of which exhibited diminished capacity to inhibit factor XI binding to HK. Competition studies with prekallikrein (PK) and a PK-dependent synthetic peptide suggested that PK and factor XI have a common surface in the A1 domain for binding HK of which Val64 is a part. We conclude that the binding of factor XI to HK is mediated at least in part by Val64 and Ile77 in the A1 domain of factor XI.

Antibody-hapten interactions in solution

1975 ◽  
Vol 272 (915) ◽  
pp. 53-74 ◽  
Keyword(s):  
Amino Acid ◽  
Molecular Mass ◽  
Conformational Changes ◽  
Difference Spectrum ◽  
Spin Labels ◽  
Amino Acid Residues ◽  
Difference Spectroscopy ◽  
Relaxation Rates ◽  
Reciprocal Effect ◽  
Myeloma Protein

This paper reports the initial progress in a research programme to identify and obtain the relative orientations, in solution, of the amino acid residues that constitute the combining site of the myeloma protein MOPC 315. This protein has a molecular mass of 150000, but enzymic digestion yields the Fv fragment of molecular mass 25000 which still has the combining site intact, as judged by the affinity for dinitrophenyl haptens. Analysis of the e.s.r. spectra of a series of dinitrophenyl spin labelled haptens has allowed the dimensions, rigidity and polarity profile of the combining site to be determined. The combining site is a cleft of overall dimensions 1.1 nm x 0.9 nm x 0.6 nm which has considerable structural rigidity. One of these spin labels has also been used to perturb the n.m.r. spectrum of the Fv and using difference spectroscopy the 270 MHz proton n.m.r. spectrum of the amino acid residues in and around the combining site has been obtained. This spectrum contains only the equivalent of about 30 aromatic and 21 aliphatic protons. Comparison of this difference spectrum with that obtained using a diamagnetic analogue suggests that any conformational changes on hapten binding are mainly localized to the combining site. By the use of (n.m.r.) difference spectroscopy the protons of the three histidine residues in the Fv are observed to titrate with pH and have pK a values of about 8.1, 6.9 and 6.1. The histidine resonances with pK a values 6.9 and 6.1 alter slightly in the presence of haptens and also appear in the spin label difference spectrum, and must therefore be in or near to the combining site. These are assigned to His 102 H and His 97 L . The existence of lanthanide binding sites on the Fv, necessary for the mapping studies, has been demonstrated by measurements of Gd hi water relaxation rates in Fv solutions and also by the changes in the Fv tryptophan fluorescence on addition of Gd hi. At pH 5.5 there is one tight binding site for the lanthanides (K D ≈ 80 μm) but in the presence of hapten this is weakened 10-20 fold with a reciprocal effect on the hapten binding. Measurements of the Gd hi quenching of the e.s.r. spectrum of a spin labelled hapten bound to Fv indicate that the lanthanide site is ca. 1.5 nm from the nitroxide moiety.

Microsomal glutathione S-transferase A1-1 with glutathione peroxidase activity from sheep liver: molecular cloning, expression and characterization

2001 ◽  
Vol 360 (2) ◽  
pp. 345-354 ◽  
Author(s):  
K. Sandeep PRABHU ◽  
Padala V. REDDY ◽  
Eric GUMPRICHT ◽  
George R. HILDENBRANDT ◽  
Richard W. SCHOLZ ◽  
...  
Keyword(s):  
Amino Acid ◽  
Glutathione Peroxidase ◽  
Molecular Mass ◽  
Peroxidase Activity ◽  
Sequence Similarity ◽  
Liver Microsomes ◽  
Amino Acid Residues ◽  
Glutathione Peroxidase Activity ◽  
Glutathione S Transferase ◽  
Sheep Liver

A 25kDa subunit of glutathione S-transferase (GST) from sheep liver microsomes (microsomal GSTA1-1) with a significant selenium-independent glutathione peroxidase activity has been isolated and characterized. Several analytical criteria, including EDTA stripping, protease protection assay and extraction with alkaline Na2CO3, indicate that the microsomal GSTA1-1 is associated with the inner microsomal membrane. The specific cDNA nucleotide sequence reveals that the enzyme is made up of 222 amino acid residues and shares approx. 73–83% sequence similarity to Alpha-class GSTs from different species. The molecular mass, as determined by electrospray mass ionization, is 25611.3Da. The enzyme is distinct from the previously reported rat liver microsomal GST in both amino acid sequence and catalytic properties [Morgenstern, Guthenberg and DePierre (1982) Eur. J. Biochem. 128, 243–248]. The microsomal GSTA1-1 differs from the sheep liver cytosolic GSTs, reported previously from this laboratory, in its substrate specificity profile and molecular mass [Reddy, Burgess, Gong, Massaro and Tu (1983) Arch. Biochem. Biophys. 224, 87–101]. In addition to catalysing the conjugation of 4-hydroxynonenal with GSH, the enzyme also exhibits significant glutathione peroxidase activity towards physiologically relevant fatty acid hydroperoxides, such as linoleic and arachidonic acid hydroperoxides, as well as phosphatidylcholine hydroperoxide, but not with H2O2. Thus the microsomal GSTA1-1 isoenzyme might have an important role in the protection of biological membranes against oxidative damage.

scholarly journals Primary sequence and domain structure of chicken vinculin

1989 ◽  
Vol 259 (2) ◽  
pp. 453-461 ◽  
Author(s):  
G J Price ◽  
P Jones ◽  
M D Davison ◽  
B Patel ◽  
R Bendori ◽  
...  
Keyword(s):  
Amino Acid ◽  
Molecular Mass ◽  
Domain Structure ◽  
Structure Prediction ◽  
Secondary Structure Prediction ◽  
Phosphorylation Site ◽  
Cdna Clones ◽  
Amino Acid Residues ◽  
Untranslated Sequence ◽  
Globular Head

We have determined the complete sequence of chick vinculin from two overlapping cDNA clones. The vinculin mRNA consists of 262 bp of 5' untranslated sequence, an open reading frame of 3195 bp (excluding the initiation codon) and a long 3' untranslated sequence (greater than 2 kb). Chick vinculin contains 1066 amino acid residues, and has a deduced molecular mass of 116,933 Da. Analysis of the domain structure of vinculin shows that the molecule can be cleaved by V8 proteinase into a 90 kDa globular head and a 32 kDa tail region, the latter of which could further be cleaved into a 27 kDa polypeptide. The 90 kDa globular head contains the N-terminus of vinculin, three 112-residue repeats (residues 259-589), and extends to approximately residue 850. Gel overlay experiments show that it also contains a binding site for the cytoskeletal protein talin. The talin-binding domain was further localized to the N-terminal 398 amino acid residues of the protein by expression in vitro of this region from a vinculin cDNA cloned into the Bluescript SK+ vector. The head and tail domains are apparently separated by a proline-rich region that contains V8-proteinase-cleavage sites and a candidate tyrosine (822)-phosphorylation site. Secondary-structure prediction suggests that the head and tail domains contain alpha-helical regions separated by short stretches of turn/coil. Comparison of the chick with a partial human sequence reveals that vinculin is a highly conserved protein. In chickens Southern-blot analysis is consistent with a single vinculin gene, and it is therefore likely that vinculin, and its higher-molecular-mass isoform termed metavinculin, arise through alternative splicing.

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