scholarly journals Hagfish slime gland thread cells. II. Isolation and characterization of intermediate filament components associated with the thread.

1984 ◽  
Vol 98 (2) ◽  
pp. 670-677 ◽  
Author(s):  
R H Spitzer ◽  
S W Downing ◽  
E A Koch ◽  
W L Salo ◽  
L J Saidel
Keyword(s):  
Amino Acid ◽  
Present Report ◽  
Amino Acid Content ◽  
Cell Types ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Isolation And Characterization ◽  
Hydroxyamino Acids ◽  
Isoelectric Ph

The slime glands of hagfish have two major cell types, gland thread cells (GTCs) and gland mucous cells (GMCs), both of which upon contact with water contribute to the formation of an abundant quantity of viscous mucus. In previous studies we reported a method for the isolation of GTCs and showed that each ellipsoidal thread cell normally contains a single tapered thread which is uniquely coiled into a space-saving conformation and occupies most of the cell volume. Subsequently, the developing thread was found to consist mainly of intermediate filaments (IFs) aligned in parallel not only to one another but also to a far fewer number of interspersed microtubules (see accompanying paper). In the present report, urea extracts of GTCs were purified and characterized to establish the properties of thread components. One major (alpha) and two minor (beta, gamma) components prepared by anion exchange chromatography were shown to have similar apparent molecular weights of 63,500 +/- 500 daltons but different isoelectric pH values (alpha, 7.56; beta, 5.67; gamma, 5.31). Although the amino acid content of alpha differed significantly from beta and gamma, each of the three was highest in Gly, relatively high in Glx, Ser, Thr, Asx, Ala, Val, and Leu, and relatively low in Cys/2 and Trp. The amino acid compositions of beta and gamma were very similar, and only beta showed evidence of carbohydrate. The threonine content of the alpha component was higher than has been reported for IFs of different origin, and the high content of hydroxyamino acids (18, 19 residues per 100) in alpha, beta, and gamma has been approached only by several IF polypeptides from human or bovine epidermal keratins. Mixtures of the purified components formed 9-11-nm filaments in vitro. The results indicate that the hagfish thread cell is a rich source of IFs, which have a structure that facilitates formation of macrofibrils within the cell.

scholarly journals Isolation and characterization of pig enamelins

1987 ◽  
Vol 243 (2) ◽  
pp. 385-390 ◽  
Author(s):  
H Limeback
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Binding Studies ◽  
Isolation And Characterization ◽  
Sequential Extraction Procedures ◽  
Enamel Proteins

Enamel proteins were extracted from pig developing enamel by sequential extraction procedures. Two proteins identified as enamelins by slab-gel electrophoresis (Mr 67,000 and 63,000) were separated from amelogenins by gel sieving and ion-exchange chromatography. Their enamelin characteristic was confirmed by hydroxyapatite-binding studies and amino acid analysis. Degradation of extracted enamel proteins was also studied in vitro. The larger of the two enamelins appeared to be resistant to degradation by endogenous enamel proteinases. Hydroxyapatite showed strong binding with the enamelins, but did not prevent the degradation of the Mr-63,000 enamelin. These results indicate that at least one high-Mr enamelin in pig developing enamel is a source of enamelin breakdown products.

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.

scholarly journals In vitro and In Silico Approach For Characterization of Antimicrobial Peptide From Probiotics Against Staphylococcus Aureus and Escherichia Coli

2021 ◽  
Author(s):  
Amrutha Bindu ◽  
Lakshmi Devi
Keyword(s):  
Amino Acid ◽  
Antimicrobial Peptide ◽  
Lactobacillus Plantarum ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Proteinase K ◽  
Kinetic Assay ◽  
Wide Range

Abstract The focus of present study was to characterize antimicrobial peptide produced by probiotic cultures, Enterococcus durans DB-1aa (MCC4243), Lactobacillus plantarum Cu2-PM7 (MCC4246) and Lactobacillus fermentum Cu3-PM8 (MCC4233) against Staphylococus aureus and E. coli. The growth kinetic assay revealed 24 h of incubation to be optimum for bacteriocin production. The partially purified compound after ion-exchange chromatography was found to be thermoresistant and stable under wide range of pH. The compound was sensitive to proteinase-K, but resistant to trypsin, a-amylase and lipase. The apparent molecular weight of bacteriocin from MCC4243 and MCC4246 was found to be 3.5 KDa. Translated partial amino acid sequence of plnA gene in MCC4246 displayed 48 amino acid sequences showing 100% similarity with plantaricin A of Lactobacillus plantarum (WP_0036419). The sequence revealed 7 β sheets, 6 α sheets, 6 predicted coils and 9 predicted turns. The functions on cytoplasm show 10.82 isoelectric point and 48.6% hydrophobicity. The molecular approach of using Geneious Prime software and protein prediction data base for characterization of bacteriocin is novel and predicts “KSSAYSLQMGATAIKQVKKLFKKWGW” as peptide responsible for antimicrobial activity. The study provides information about broad spectrum bacteriocin in native probiotic culture and paves a way towards its application in functional foods as biopreservative agents.

Ca2+-binding proteins of cilia and infraciliary lattice ofParamecium tetraurelia: their phosphorylation by purified endogenous Ca2+-dependent protein kinases

2002 ◽  
Vol 115 (9) ◽  
pp. 1973-1984
Author(s):  
Kwanghee Kim ◽  
Min Son ◽  
Joan B. Peterson ◽  
David L. Nelson
Keyword(s):  
Protein Kinases ◽  
Calcium Binding ◽  
Binding Proteins ◽  
Soluble Fraction ◽  
Biological Activities ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Paramecium Tetraurelia ◽  
Dependent Protein

We purified two small, acidic calcium-binding proteins(ParameciumCa2+-binding proteins, PCBP-25α and PCBP-25β) from Paramecium tetraurelia by Ca2+-dependent chromatography on phenyl-Sepharose and by anion-exchange chromatography. The proteins were immunologically distinct. Monoclonal antibodies against PCBP-25β did not react with PCBP-25α, and antibodies against centrin from Chlamydomonas reacted with PCBP-25α but not with PCBP-25β. Like the centrins described previously, both PCBPs were associated with the infraciliary lattice (ICL), a fibrillar cytoskeletal element in Paramecium. Both were also present in isolated cilia, from which they could be released (with dynein) by a high-salt wash, and both PCBPs cosedimented with dynein in a sucrose gradient. PCBP-25β was especially prominent in cilia and in the deciliation supernatant, a soluble fraction released during the process of deciliation. The results of immunoreactivity and localization experiments suggest that PCBP-25α is a Paramecium centrin and that PCBP-25β is a distinct Ca2+-binding protein that confers Ca2+ sensitivity on some component of the cilium, ciliary basal body or ICL.We characterized these proteins and Paramecium calmodulin as substrates for two Ca2+-dependent protein kinases purified from Paramecium. PCBP-25α and calmodulin were in vitro substrates for one of the two Ca2+-dependent protein kinases (CaPK-2), but only PCBP-25α was phosphorylated by CaPK-1. These results raise the possibility that the biological activities of PCBP-25α and calmodulin are regulated by phosphorylation.

Amino Acid Content and In Vitro Protein Quality of Different Corn Varieties

1985 ◽  
pp. 421-437
Author(s):  
Samy Fangus Sharobeem ◽  
Radomir Lásztity ◽  
Máté Hidvégi ◽  
András Salgó ◽  
Livia Simon-Sarkadi
Keyword(s):  
Amino Acid ◽  
Acid Content ◽  
Protein Quality ◽  
Amino Acid Content ◽  
Vitro Protein

scholarly journals Role of individual gamma-carboxyglutamic acid residues of activated human protein C in defining its in vitro anticoagulant activity

Blood ◽  
1992 ◽  
Vol 80 (4) ◽  
pp. 942-952 ◽  
Author(s):  
L Zhang ◽  
A Jhingan ◽  
FJ Castellino
Keyword(s):  
Protein C ◽  
Anticoagulant Activity ◽  
Coagulation Factor ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Factor Ix ◽  
Human Protein ◽  
Complete Disappearance ◽  
Carboxyglutamic Acid

Abstract To evaluate the contributions of individual gamma-carboxyglutamic acid (gla) residues to the overall Ca(2+)-dependent anticoagulant activity of activated human protein C (APC), we used recombinant (r) DNA technology to generate protein C (PC) variants in which each of the gla precursor glutamic acid (E) residues (positions 6, 7, 14, 16, 19, 20, 25, 26, and 29) was separately altered to aspartic acid (D). In one case, a gla26V mutation ([gla26V]r-PC) was constructed because a patient with this particular substitution in coagulation factor IX had been previously identified. Two additional r-PC mutants were generated, viz, an r-PC variant containing a substitution at arginine (R) 15 ([R15]r-PC), because this particular R residue is conserved in all gla- containing blood coagulation proteins, as well as a variant r-PC with substitution of an E at position 32 ([F31L, Q32E]r-PC), because gla residues are found in other proteins at this sequence location. This latter protein did undergo gamma-carboxylation at the newly inserted E32 position. For each of the 11 recombinant variants, a subpopulation of PC molecules that were gamma-carboxylated at all nonmutated gla- precursor E residues has been purified by anion exchange chromatography and, where necessary, affinity chromatography on an antihuman PC column. The r-PC muteins were converted to their respective r-APC forms and assayed for their amidolytic activities and Ca(2+)-dependent anticoagulant properties. While no significant differences were found between wild-type (wt) r-APC and r-APC mutants in the amidolytic assays, lack of a single gla residue at any of the following locations, viz, 7, 16, 20, or 26, led to virtual complete disappearance of the Ca(2+)-dependent anticoagulant activity of the relevant r-APC mutant, as compared with its wt counterpart. On the other hand, single eliminations of any of the gla residues located at positions 6, 14, or 19 of r-APC resulted in variant recombinant molecules with substantial anticoagulant activity (80% to 92%), relative to wtr-APC. Mutation of gla residues at positions 25 and 29 resulted in r-APC variants with significant but low (24% and 9% of wtr-APC, respectively) levels of anticoagulant activity. The variant, [R15L]r-APC, possessed only 19% of the anticoagulant activity of wrt-APC, while inclusion of gla at position 32 in the variant, [F31L, Q32gla]r-APC, resulted in a recombinant enzyme with an anticoagulant activity equivalent to that of wtr-APC.

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.

scholarly journals Isolation and Characterization of a Lectin-like Protein (SBLP) from the Dried Roots of Scutellaria baicalensis (Lamiaceae)

2018 ◽  
Vol 13 (12) ◽  
pp. 1934578X1801301
Author(s):  
Huiqin Wang ◽  
Guanzhen Gao ◽  
Lijing Ke ◽  
Jianwu Zhou ◽  
Pingfan Rao
Keyword(s):  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Scutellaria Baicalensis ◽  
Dependent Manner ◽  
Scutellaria Baicalensis Georgi ◽  
Isolation And Characterization ◽  
Pas Staining ◽  
Ph Range ◽  
Dose Dependent

A novel lectin-like protein with MW 63.2 kDa, designated as SBLP, has been isolated and characterized from the dried roots of Scutellaria baicalensis Georgi (Lamiaceae). SBLP was purified by ammonium sulfate precipitation and anion exchange chromatography. It is a glycoprotein according to a PAS staining assay and consisting of protein (86.0%) and sugar (14.0%). Its N-terminal amino acid sequence was determined as GSAVGFLY by Edman degradation. SBLP showed hemagglutinating activity against human and rooster erythrocytes, which were stable below 60°C and in the pH range of 4 −10. Furthermore, SBLP was found to be stimulated by Ca2+, Na+, Ba2+, Zn2+ ions, which suggested it was a metal-dependent lectin. SBLP inhibited the growth of Fusarium oxysporum f.sp. lycopersici and Alternaria eichhorniae in the a dose-dependent manner, and suppressed the proliferation of HepG2 tumor cells with an IC50 of 1.00 μM. This is the first report of a lectin from Radix Scutellariae.

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.

SUDAY ON A SUBSTANCE FUNCTIONALLY LIKE PROTEIN C IN PLASMA

1987 ◽  
Author(s):  
I TSUNEIZUMI ◽  
T MEGURO ◽  
K YAMADA
Keyword(s):  
Molecular Weight ◽  
Protein C ◽  
Present Report ◽  
Newborn Infants ◽  
Exchange Chromatography ◽  
Isolation And Characterization ◽  
K Deficiency ◽  
Clinically Significant ◽  
Aluminium Hydroxide Gel

The present report first deals with the isolation and characterization of a substance found to demonstrate protein C like activity(PCLA). When it was activated by thrombin, the PCLA substance prolonged the activated partial thromboplastin time(aPTT). The activated PCLA substance also hydrolyzed synthetic substrates such as S�2238, S�2366 and S�2266,while the PCLA substance was not cross reacted with anti-protein C serum(Behring Manheim). The PCLA substance was adsorbed by both aluminium hydroxide gel and barium sulfate. The level of Km, optimum pH and optimum I.S. in the amido-lytic reaction with synthetic substrate is shown in the table.In the chromatography of Sepharose CL-6B gel, the PCLA substance was eluted in the fraction of higher molecular weight, while protein C was eluted with a lower fraction.This prompts an estimate that the molecular weight of the PCLA substance is approximately 200,000. Through DEAE-Sepharose CL-6B gel ion exchange chromatography, the PCLA substance was eluted by a lower ionic strength than was protein C.The levels of a PCLA substance were low in the patients with a vitamin K deficiency and in newborn infants. It is expected that our findings regarding the PCLA substance will be as clinically significant as that which we know about protein C at present.

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