Molecular cloning of chicken calcyclin (S100A6) and identification of putative isoforms

1997 ◽  
Vol 75 (6) ◽  
pp. 733-738 ◽  
Author(s):  
Bruce G Allen ◽  
Jacquelyn E Andrea ◽  
Cindy Sutherland ◽  
Brett O Schönekess ◽  
Michael P Walsh
Keyword(s):  
Smooth Muscle ◽  
Amino Acid ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Chain Reaction ◽  
Proteolytic Fragment ◽  
Chicken Gizzard ◽  
Muscle Tissues ◽  
Bona Fide ◽  
Polymerase Chain

A full-length cDNA encoding smooth muscle calcyclin (S100A6) was cloned from chicken gizzard, using reverse transcription - polymerase chain reaction techniques. The deduced amino acid sequence contains 92 residues with 12 substitutions and a 2 amino acid C-terminal extension when compared with human calcyclin. Calcyclin was purified from chicken gizzard by Ca2+-dependent hydrophobic chromatography, heat treatment, and anion-exchange chromatography. N-terminal sequencing of two CNBr peptides confirmed its identity as calcyclin. Two isoforms of calcyclin (A and B), which differ with respect to the presence or absence of a C-terminal lysine, were identified and the native protein was shown to exist as noncovalently associated homodimers (AA and BB) and heterodimers (AB). Incubation of purified calcyclin AA with an extract of chicken gizzard did not result in degradation of calcyclin A or appearance of calcyclin B, suggesting that calcyclin B is a bona fide isoform rather than a proteolytic fragment generated during purification. Western blotting of chicken tissues with anti-(gizzard calcyclin) indicated abundant expression of calcyclin in smooth muscle tissues, including esophagus, large intestine, and trachea, with lower levels in lung, heart, kidney, and brain, and none detectable in liver or skeletal muscle.Key words: Ca2+-binding proteins, calcyclin, smooth muscle, cDNA cloning, isoforms.

scholarly journals Protein kinase C isoenzymes in airway smooth muscle

1997 ◽  
Vol 324 (1) ◽  
pp. 167-175 ◽  
Author(s):  
Benjamin L. J. WEBB ◽  
Mark A. LINDSAY ◽  
Peter J. BARNES ◽  
Mark A. GIEMBYCZ
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Oligonucleotide Primer ◽  
Mrna Levels ◽  
Kinase C ◽  
Gf 109203X ◽  
Bona Fide

The protein kinase C (PKC) isoenzymes expressed by bovine tracheal smooth muscle (BTSM) were identified at the protein and mRNA levels. Western immunoblot analyses reliably identified PKCα, PKCβI and PKCβII. In some experiments immunoreactive bands corresponding to PKCδ, PKCϵ and PKCθ were also labelled, whereas the γ, η and ζ isoforms of PKC were never detected. Reverse transcriptase PCR of RNA extracted from BTSM using oligonucleotide primer pairs designed to recognize unique sequences in the PKC genes for which protein was absent or not reproducibly identified by immunoblotting, amplified cDNA fragments that corresponded to the predicted sizes of PKCδ, PKCϵ and PKCζ, which was confirmed by Southern blotting. Anion-exchange chromatography of the soluble fraction of BTSM following homogenization in Ca2+-free buffer resolved two major peaks of activity. Using ϵ-peptide as the substrate, the first peak of activity was dependent upon Ca2+ and 4β-PDBu (PDBu = phorbol 12,13-dibutyrate), and represented a mixture of PKCs α, βI and βII. In contrast, the second peak of activity, which eluted at much higher ionic strength, also appeared to comprise a combination of conventional PKCs that were arbitrarily denoted PKCα′, PKCβI′ and PKCβII′. However, these novel enzymes were cofactor-independent and did not bind [3H]PDBu, but were equally sensitive to the PKC inhibitor GF 109203X compared with bona fide conventional PKCs, and migrated on SDS/polyacrylamide gels as 81 kDa polypeptides. Taken together, these data suggest that PKCs α′, βI′ and βII′ represent modified, but not proteolysed, forms of their respective native enzymes that retain antibody immunoreactivity and sensitivity to PKC inhibitors, but have lost their sensitivity to Ca2+ and PDBu when ϵ-peptide is used as the substrate.

scholarly journals Chemical identity of tryptensin with angiotensin

1980 ◽  
Vol 187 (3) ◽  
pp. 647-653 ◽  
Author(s):  
K Arakawa ◽  
M Yuki ◽  
M Ikeda
Keyword(s):  
Amino Acid ◽  
Thin Layer ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Cation Exchange Chromatography ◽  
Human Plasma Protein ◽  
Plasma Protein Fraction ◽  
Layer Chromatography

Tryptensin, a vasopressor substance generated from human plasma protein fraction IV-4 by trypsin, has been isolated and the amino acid composition analysed. The procedures used for the isolation were: (a) adsorption of the formed tryptensin on Dowex 50W (X2; NH4+ form); (b) gel filtration through Sephadex G-25; (c) cation-exchange chromatography on CM-cellulose; (d) anion-exchange chromatography on DEAE-cellulose; (e) re-chromatography on CM-cellulose; (f) gel filtration on Bio-Gel P-2; (g) partition chromatography on high-pressure liquid chromatography. The homogeneity of the isolated tryptensin was confirmed by thin-layer chromatography and thin-layer electrophoresis. The amino acid analysis of the hydrolysate suggested the following proportional composition: Asp, 1; Val, 1; Ile, 1; Tyr, 1; Phe, 1; His, 1; Arg, 1; Pro, 1. This composition is identical with that of human angiotensin.

Separation of bovine κ-casein glycomacropeptide from sweet whey protein products with undetectable level of phenylalanine by protein precipitation followed by anion exchange chromatography

2018 ◽  
Vol 85 (1) ◽  
pp. 110-113 ◽  
Author(s):  
Takuo Nakano ◽  
Lech Ozimek ◽  
Mirko Betti
Keyword(s):  
Heat Treatment ◽  
Amino Acid ◽  
Whey Protein ◽  
Anion Exchange ◽  
Large Scale ◽  
Dry Weight ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Ph Shift ◽  
Sweet Whey

Bovine κ-casein glycomacropeptide (GMP) found in sweet whey is a 64 amino acid residue glycopeptide, which does not contain phenylalanine or other aromatic amino acids. There is, however, little information available concerning isolation of phenylalanine free GMP from sweet whey. In the study reported in this Research Communication, GMP was purified from three samples of sweet whey protein products (SWPP) by a procedure involving: (1) precipitation of protein by heat treatment; (2) precipitation of protein by pH shift to 4·6; and (3) diethylaminoethyl (DEAE)-Sephacel anion exchange chromatography of soluble portion of each sample obtained after removal of protein precipitates. The total protein precipitated with both heat treatment and pH shift accounted for average 61% of dry weight of SWPP. The GMP fraction obtained by DEAE-Sephacel chromatography accounted for average 7·5% of dry weight of SWPP. Amino acid analysis showed that there was no detectable level of phenylalanine in GMP fractions from all samples examined. The present method may help develop large scale methods of production of GMP.

Antithrombin Milano, Single Amino Acid Substitution at the Reactive Site, Arg393 to Cys

1988 ◽  
Vol 60 (03) ◽  
pp. 471-475 ◽  
Author(s):  
H Erdjument ◽  
D A Lane ◽  
H Ireland ◽  
V Di Marzo ◽  
M Panico ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Fast Atom Bombardment ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid ◽  
Reducing Conditions ◽  
Sds Page ◽  
Two Peaks

SummaryAntithrombin Milano is an unusual antithrombin variant, exhibiting an abnormal, fast moving component on crossed immunoelectrophoresis (in the absence of heparin). Antithrombin isolated from the propositus could be resolved into two peaks on anion-exchange chromatography; anti thrombin Milano peak 1 of Mr ∼60,000 which could inhibit thrombin, and antithrombin Milano peak 2 of Mr ∼120,000 which was inactive. The latter component also reacted with antisera to both antithrombin and albumin on immunoblotting. Under reducing conditions, the ∼120,000 Mr component migrated on SDS-PAGE as two distinct bands with Mr ∼60,000, one of which reacted with antiserum to antithrombin and the other (of slower mobility) of which reacted with antiserum to albumin only. These and other results established the ∼120,000 Mr component to be an inactive, disulphide-linked variant antithrombin and albumin complex. The variant antithrombin was isolated, following reduction and S-carboxy-methylation, by reverse-phase HPLC and then it was fragmented with CNBr. A major CNBr pool containing the sequence Gly339-Met423 was treated with trypsin, followed by V8 protease. The resulting peptides were analysed by fast atom bombardment mass spectrometry (Fab-MS) mapping. A peptide of molecular mass 1086, corresponding to the normal sequence Ala382-Arg393, was almost absent from the mass spectrum, but an additional peptide of mass number 1772 was present. These results are almost identical to those found in another variant antithrombin, North-wick Park (Erdjument et al., J Biol Chem, 262: 13381, 1987; Erdjument et al., J Biol Chem, 263: 5589-5593, 1988), indicating the same single amino acid substitution of Arg393 to Cys.

Serine protease isoforms of Deinagkistrodon acutus venom: cloning, sequencing and phylogenetic analysis

2001 ◽  
Vol 354 (1) ◽  
pp. 161-168 ◽  
Author(s):  
Ying-Ming WANG ◽  
Suei-Rong WANG ◽  
Inn-Ho TSAI
Keyword(s):  
Amino Acid ◽  
Serine Proteases ◽  
Parallel Evolution ◽  
Gel Filtration ◽  
Venom Gland ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Cdna Clones ◽  
Single Chain

The major coagulating fibrinogenase of Deinagkistrdon acutus venom, designated acutobin, was purified by anion-exchange chromatography, gel filtration and reverse-phase HPLC. Approximately 80% of its protein sequence was determined by sequencing the various fragments derived from CNBr cleavage and digestion with endoprotease. Extensive screening of the venom gland cDNA species after amplification by PCR resulted in the isolation of four distinct cDNA clones encoding acutobin and three other serine proteases, designated Dav-PA, Dav-KN and Dav-X. The complete amino acid sequences of these enzymes were deduced from the cDNA sequences. The amino-acid sequence of acutobin contains a single chain of 236 residues including four potential N-glycosylation sites. The purified acutobin (40kDa) contains approx. 30% carbohydrate by weight, which could be partly removed by N-glycanase. The phylogenetic tree of the complete amino acid sequences of 40 serine proteases from 18 species of Crotalinae shows functional clusters reflecting parallel evolution of the three major venom enzyme subtypes: coagulating enzymes, kininogenases and plasminogen activators. The possible structural elements responsible for the functional specificity of each subtype are discussed.

scholarly journals Natural infection of parvovirus in wild fishing cats (Prionailurus viverrinus) reveals extant viral localization in kidneys

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0247266
Author(s):  
Chutchai Piewbang ◽  
Sabrina Wahyu Wardhani ◽  
Jira Chanseanroj ◽  
Jakarwan Yostawonkul ◽  
Suwimon Boonrungsiman ◽  
...  
Keyword(s):  
Amino Acid ◽  
Natural Infection ◽  
Chain Reaction ◽  
Feline Panleukopenia Virus ◽  
Wild Carnivores ◽  
Transmission Electron ◽  
Polymerase Chain ◽  
Amino Acid Mutations ◽  
Atypical Localization

Carnivore protoparvovirus-1 (CPPV-1), a viral species containing feline panleukopenia virus (FPV) and canine parvovirus (CPV) variants, are widely spread among domestic and wild carnivores causing systemic fatal diseases. Wild fishing cats (Prionailurus viverrinus), a globally vulnerable species, have been found dead. Postmortem examination of the carcasses revealed lesions in intestine, spleen and kidney. CPPV-1 antigen identification in these tissues, using polymerase chain reaction (PCR) and immunohistochemistry (IHC), supported the infection by the virus. PCR- and IHC-positivity in kidney tissues revealed atypical localization of the virus while in situ hybridization (ISH) and transmission electron microscopy (TEM) with the pop-off technique confirmed the first description of viral localization in kidneys. Complete genome characterization and deduced amino acid analysis of the obtained CPPV-1 from the fishing cats revealed FPV as a causative agent. The detected FPV sequences showed amino acid mutations at I566M and M569R in the capsid protein. Phylogenetic and evolutionary analyses of complete coding genome sequences revealed that the fishing cat CPPV-1 genomes are genetically clustered to the FPV genomes isolated from domestic cats in Thailand. Since the 1970s, these genomes have also been shown to share a genetic evolution with Chinese FPV strains. This study is the first evidence of CPPV-1 infection in fishing cats and it is the first to show its localization in the kidneys. These findings support the multi-host range of this parvovirus and suggest fatal CPPV-1 infections may result in other vulnerable wild carnivores.

scholarly journals Human FLT3/FLK2 gene: cDNA cloning and expression in hematopoietic cells

Blood ◽  
1993 ◽  
Vol 82 (4) ◽  
pp. 1110-1119 ◽  
Author(s):  
O Rosnet ◽  
C Schiff ◽  
MJ Pebusque ◽  
S Marchetto ◽  
C Tonnelle ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Tyrosine Kinase ◽  
B Cell ◽  
Cloning And Expression ◽  
Colony Stimulating Factor ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Stimulating Factor

The human FLT3 cDNA was cloned from a pre-B cell line and characterized. The deduced amino acid sequence shows that FLT3 codes for a receptor-type tyrosine kinase of 993 residues, presenting a strong similarity with the corresponding mouse FLT3/FLK2 protein as well as with the receptors for colony-stimulating factor 1 (CSF1R/FMS) and steel locus factor (SLFR/KIT). An analysis of the expression of the gene using amplification of reverse transcribed FLT3 mRNA by polymerase chain reaction shows that FLT3 is expressed in various lymphohematopoietic cells and tissues, including a series of immature cell lines and leukemias of lymphocytic origin.

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