scholarly journals Purification and characterization of human neuropeptide Y from adrenal-medullary phaeochromocytoma tissue

1984 ◽  
Vol 219 (3) ◽  
pp. 699-706 ◽  
Author(s):  
R Corder ◽  
P C Emson ◽  
P J Lowry
Keyword(s):  
Amino Acid ◽  
Neuropeptide Y ◽  
Amino Acid Analysis ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Carboxypeptidase Y ◽  
Purification And Characterization ◽  
Reverse Phase ◽  
Tryptic Fragment

Human neuropeptide Y was isolated from acid extracts of adrenal-medullary phaeochromocytoma tissue. After (NH4)2SO4 fractionation, the neuropeptide Y-like immunoreactivity was purified from the resolubilized 80%-saturation-(NH4)2SO4 peptide-rich precipitate, by gel filtration, cation-exchange chromatography and reverse-phase high-pressure liquid chromatography. Amino acid analysis of the peptide revealed a composition almost identical with that of the pig peptide, the exception being the loss of one leucine residue and its replacement with methionine. Tryptic digestion of the peptide and subsequent amino acid analysis of the fragments further confirmed the identity of the peptide. Carboxypeptidase Y digestion of the (1-19)-peptide tryptic fragment has shown the methionine to be located at position 17 in human neuropeptide Y.

Preparation and Characterization of NH2-Terminal Fibrinogen Bβ Fragments from N-DSK of Human Fibrinogen

1984 ◽  
Vol 51 (01) ◽  
pp. 016-021 ◽  
Author(s):  
S Birken ◽  
G Agosto ◽  
B Lahiri ◽  
R Canfield
Keyword(s):  
High Performance ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Reverse Phase ◽  
Human Fibrinogen ◽  
Human Volunteers ◽  
Cnbr Cleavage ◽  
Two Peaks

SummaryIn order to investigate the early release of NH2-terminal plasmic fragments from the Bβ chain of fibrinogen, substantial quantities of Bβ 1-42 and Bβ 1-21 are required as immunogens, as radioimmunoassay standards and for infusion into human volunteers to determine the half-lives of these peptides. Towards this end methods that employ selective proteolytic cleavage of these fragments from fibrinogen have been developed. Both the N-DSK fragment, produced by CNBr cleavage of fibrinogen, and Bβ 1-118 were employed as substrates for plasmin with the finding of higher yields from N-DSK. Bβ 1-42 and Bβ 1-21 were purified by gel filtration and ion-exchange chromatography on SP-Sephadex using volatile buffers. When the purified preparation of Bβ 1-42 was chromatographed on reverse-phase high performance liquid chromatography, two peaks of identical amino acid composition were separated, presumably due either to pyroglutamate or to amide differences.

scholarly journals Purification and Characterization of Endoglucanase from local isolate of Aspergillus flavus

2013 ◽  
Vol 10 (3) ◽  
pp. 844-853
Author(s):  
Baghdad Science Journal
Keyword(s):  
Aspergillus Flavus ◽  
Specific Activity ◽  
Gel Filtration ◽  
Temperature Stability ◽  
Exchange Chromatography ◽  
Purification And Characterization ◽  
Original Activity ◽  
A Value ◽  
Optimum Ph

Endoglucanase produced from Aspergillus flavus was purified by several steps including precipitation with 25 % ammonium sulphate followed by Ion –exchange chromatography, the obtained specific activity was 377.35 U/ mg protein, with a yield of 51.32 % .This step was followed by gel filtration chromatography (Sepharose -6B), when a value of specific activity was 400 U/ mg protein, with a yield of 48 %. Certain properties of this purified enzyme were investigated, the optimum pH of activity was 7 and the pH of its stability was 4.5, while the temperature stability was 40 °C for 60 min. The enzyme retained 100% of its original activity after incubation at 40 °C for 60 min; the optimum temperature for enzyme activity was 40 °C.

scholarly journals The purification and characterization of glucokinase from the thermophile Bacillus stearothermophilus

1986 ◽  
Vol 237 (2) ◽  
pp. 415-420 ◽  
Author(s):  
C R Goward ◽  
R Hartwell ◽  
T Atkinson ◽  
M D Scawen
Keyword(s):  
Large Scale ◽  
Bacillus Stearothermophilus ◽  
Specific Activity ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Purification And Characterization ◽  
Km Value ◽  
Extraneous Material ◽  
Sepharose 4B

Homogeneous glucokinase (EC 2.7.1.2) from the thermophile Bacillus stearothermophilus was isolated on the large scale by using four major steps: precipitation of extraneous material at pH 5.5, ion-exchange chromatography on DEAE-Sepharose, pseudo-affinity chromatography on Procion Brown H-3R-Sepharose 4B and gel filtration on Ultrogel AcA 34. The purified enzyme had a specific activity of about 330 units/mg of protein and was shown to exist as a dimer of subunit Mr 33,000. Kinetic parameters for the enzyme were determined with a variety of substrates. The glucokinase was highly specific for alpha-D-glucose, and the only other sugar substrate utilized was N-acetyl-alpha-D-glucosamine. The enzyme shows Michaelis-Menten kinetics, with a Km value of 150 microM for alpha-D-glucose. The glucokinase was maximally active at pH 9.0.

scholarly journals Cadmium-binding proteins of rat testes. Apparent source of the protein of low molecular mass

1984 ◽  
Vol 220 (3) ◽  
pp. 819-824 ◽  
Author(s):  
M P Waalkes ◽  
S B Chernoff ◽  
C D Klaassen
Keyword(s):  
Amino Acid ◽  
Metal Binding ◽  
Amino Acid Analysis ◽  
Binding Proteins ◽  
Binding Protein ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Breakdown Product ◽  
Heat Stable ◽  
Metal Binding Protein

Fractionation of rat testicular cytosolic proteins by gel filtration indicates three major metal-binding proteins, or groups of proteins, termed testicular metal-binding protein (TMBP) 1, 2 and 3 by order of elution. The major heat-stable, metal-binding proteins in testes is TMBP-2, which has an Mr of approx. 25000. In most tissues, metallothionein (MT) is the major heat-stable, metal-binding protein, but it has an Mr of 6000. This testicular protein (TMBP-2) is much larger than MT, and since polymeric forms of MT have been previously reported, further characterization of TMBP-2 was performed. TMBP-2 was separated into two forms by DEAE-Sephadex A-25 anion-exchange chromatography. Amino acid analysis of both forms of TMBP-2 revealed that they differed markedly from MT, having particularly low cysteine contents. However, amino acid analysis showed that TBMP-2 was strikingly similar to TMBP-3, with an approximate stoichiometric relationship of 4:1. Therefore, experiments were conducted to determine if TMBP-3 could be a breakdown product of TMBP-2. Heat treatment of testicular cytosol in room air before gel filtration resulted in a marked increase in TMBP-3 and loss of TMBP-2. Storing intact testes at −20 degrees C for 2 weeks before processing for gel filtration also resulted in an increase in TMBP-3 and a loss of TMBP-2. Addition of a reducing agent (dithiothreitol) or proteinase inhibitor (N-ethylmaleimide) in processing of samples before gel filtration inhibited the appearance of TMBP-3. Results suggest that the low-Mr Cd-binding protein (TMBP-3) of rat testes results from either proteolytic or oxidative breakdown of a higher-Mr species, or from a combination of such factors.

Post-proline endopeptidase. Partial purification and characterization of the enzyme from pig kidneys

1982 ◽  
Vol 47 (4) ◽  
pp. 1139-1148 ◽  
Author(s):  
Karel Hauzer ◽  
Linda Servítová ◽  
Tomislav Barth ◽  
Karel Jošt
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Peptide Bond ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Optimum Ph ◽  
Hydrolysis Of ◽  
Prolyl Leucyl Glycinamide

Post-proline endopeptidase was isolated from pig kidneys and partially purified. The procedure consisted of fractionation with ammonium sulphate, ion exchange chromatography on DEAE-Sephadex A-50, gel filtration on Sephadex G-200 and rechromatography on DEAE-Sephadex A-50. The preparation had 55 times higher specific activity than the crude extract and did not contain any contaminating enzymic activities. The enzyme cleaved a number of proline-containing peptides and was strictly specific in catalyzing the hydrolysis of the peptide bond on the carboxyl side of the proline residue. The optimum pH for the hydrolysis of the synthetic peptides benzyl-oxycarbonylglycyl-prolyl-leucyl-glycinamide and benzyloxycarbonyl-glycyl-proline β-naphtylamide was 7.8-8.0 and, in the case of benzyloxycarbonylglycyl-proline p-nitroanilide, 7.2 to 7.5. For the hydrolysis of the tetrapeptide benzyloxycarbonylglycyl-prolyl-leucyl-glycinamide, the Km value of 75 μ mol l-1 was obtained.

Purification and characterization of the extracellular proteinase ofPseudomonas fluorescensNCDO 2085

1986 ◽  
Vol 53 (3) ◽  
pp. 457-466 ◽  
Author(s):  
David J. Fairbairn ◽  
Barry A. Law
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Fold Increase ◽  
Exchange Chromatography ◽  
Purification And Characterization ◽  
Extracellular Proteinase ◽  
Original Activity ◽  
Heat Stable ◽  
D Values

SUMMAEYPseudomonas fluorescensNCDO 2085 produced a single heat-stable extracellular proteinase in Na caseinate medium at 20 °C and pH 7·0. The proteinase was purified to electrophoretic homogeneity using chromatofocusing, gel filtration and ion-exchange chromatography. The purification procedure resulted in a 158-fold increase in the specific activity and a yield of 3·5% of the original activity. The enzyme is a metalloproteinase containing Zn and Ca, with an isoelectric point at 5·40±0·05 and a mol. wt of 40200±2100. It is heat-stable having D-values at 74 and 140 °C of 1·6 and 1·0 min respectively; 40 and 70% of the original activity remained after HTST (74 °C/17 s) and ultra high temperature (140°C/4 s) treatments respectively. The amino acid composition of the proteinase was determined and compared with those from otherPseudomonasspp.

scholarly journals A Boophilus microplus vitellin-degrading cysteine endopeptidase

Parasitology ◽  
2003 ◽  
Vol 126 (2) ◽  
pp. 155-163 ◽  
Author(s):  
A. SEIXAS ◽  
P. C. DOS SANTOS ◽  
F. F. VELLOSO ◽  
I. DA SILVA VAZ ◽  
A. MASUDA ◽  
...  
Keyword(s):  
Ion Exchange ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Boophilus Microplus ◽  
Hard Tick ◽  
Purification And Characterization ◽  
Basic Residue ◽  
Cysteine Endopeptidase ◽  
Sds Page

Here we describe the purification and characterization of a vitellin (VT) degrading cysteine endopeptidase (VTDCE) from eggs of the hard tick Boophilus microplus. A homogeneous enzyme preparation was obtained by chromatographic fractionation on ion-exchange and gel filtration columns and an autolysis step. This step consisted of incubation of a semipurified enzyme (after the first ion-exchange chromatography) at pH 4·0 that dissociated the enzyme from VT, to which VTDCE is naturally tightly associated. The enzyme purity was confirmed by capillary and native gel electrophoresis, and SDS–PAGE suggested the enzyme is a dimer of 17 and 22 kDa. VTDCE was active upon several synthetic substrates, with a preference for a hydrophobic or a basic residue in P1, and a hydrophobic residue in P2. VTDCE also hydrolysed haemoglobin, albumin, gelatin and vitellin. VTDCE is inactive in the absence of DTT and was totally inhibited by E-64, indicating it is a cysteine endopeptidase. Our results suggest that VTDCE is a major enzyme involved in yolk processing during B. microplus embryogenesis.

Purification and Characterization of Superoxide Dismutase from Martianus Dermestoides Chevrola

2013 ◽  
Vol 773 ◽  
pp. 336-341 ◽  
Author(s):  
Wen Ge Yu ◽  
Bei Bei Zhang ◽  
Yong Jian Shen ◽  
Ying Li ◽  
Yong Bin Tian ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Ph Value ◽  
Gel Filtration ◽  
Inhibitory Effect ◽  
Exchange Chromatography ◽  
Enzyme Activation ◽  
Purification And Characterization ◽  
Sds Page ◽  
Optimum Reaction

In this paper, the superoxide dismutase fromMartianus dermestoidesis purified by the following methods: heat treatment, polyethylene concentration, Sephadex G-75 gel filtration, and DEAE-Sepharose FF ion exchange chromatography. The result shows that the purification multiple is 3.86, the activation yield is 21.89% and the specific activation of the enzyme is 447.6 U/mg. The purified SOD appears to be a sole protein on SDS-PAGE and the molecular weight is estimated to be 40.58 kDa. H2O2can obviously inhibit the enzyme activation and CHCl3-CH3CH2OH only demonstrates basically no inhibitory effect. The type of the dermestoides SOD might be Cu/Zn-SOD. After purification, some enzymatic characterizations of the SOD are studied. The optimum reaction temperature of purified SOD is 50°C. The optimum reaction pH value of purification is 6. The dermestoide SOD has a preferable stability below 50°C and at pH values between 5-8.

Preliminary Characterization of Resilin Isolated from the Cockroach, Periplaneta Americana

1992 ◽  
Vol 292 ◽  
Author(s):  
Elizabeth Craig Lombardi ◽  
David L. Kaplan
Keyword(s):  
Mechanical Properties ◽  
Amino Acid ◽  
Amino Acid Analysis ◽  
Periplaneta Americana ◽  
Insect Cuticle ◽  
Oligonucleotide Probes ◽  
Protein Matrix ◽  
Tryptic Fragment ◽  
Good Agreement

AbstractWe would like to mimic the mechanical properties of animal systems for the development of novel materials. Insect cuticle serves as one source of inspiration for the design of these materials. Cuticle is composed of chitin embedded in a protein matrix which may also contain plasticizers, fillers, crosslinkers, and minerals. The specific properties of the cuticle depend on the type, amount and interactions between each component. We are renewing the investigation of the elastic cuticle, resilin. Resilin, a protein-based elastomer first described in the early 1960s, has properties which have been reported to be most like those of ideal rubbers. We have examined resilin isolated from the prealar arms of the cockroach, Periplaneta americana. The results of amino acid analysis are in good agreement with earlier data reported for resilin. A series of tryptic fragments have been isolated and sequenced. These peptides have been used for the design of oligonucleotide probes for the identification of the gene(s) from a teneral cockroach cDNA library. A biopolymer, based on one tryptic fragment, has been designed and synthesized. We are continuing to treat resilin with residue specific proteases in order to map the resilin protein.

Partial Amino Acid Sequences of Two Proteins in Developing Porcine Enamel

1979 ◽  
Vol 58 (2_suppl) ◽  
pp. 1000-1001 ◽  
Author(s):  
M. Fukae ◽  
H. Ijiri ◽  
T. Tanabe ◽  
M. Shimizu
Keyword(s):  
Amino Acid ◽  
Early Stage ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Carboxypeptidase Y ◽  
Sequence Analyses ◽  
Stage Of Development ◽  
Enamel Proteins ◽  
Main Components

The enamel proteins prepared from porcine enamel samples at an early stage of development are separated at least 4 fractions by Sephadex G-100 gel filtration at pH 10.8. The main components of secondary and fourthly-eluted fraction were further purified by ion exchange chromatography. The results of sequence analyses of these proteins by sequential Edman degradation and by hydrolysis with carboxypeptidase Y showed that the partial amino acid sequences of these proteins were iden tical.

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