scholarly journals Isoelectric-focusing properties and carbohydrate content of pea (Pisum sativum) legumin

1980 ◽  
Vol 185 (2) ◽  
pp. 497-503 ◽  
Author(s):  
John A. Gatehouse ◽  
Ronald R. D. Croy ◽  
Donald Boulter
Keyword(s):  
Pisum Sativum ◽  
Isoelectric Focusing ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Exchange Chromatography ◽  
Preparative Scale ◽  
Labelling Technique ◽  
Basic Subunit ◽  
Possible Cause ◽  
Focusing Properties

Legumin from pea (Pisum sativum) is a molecule made up of six pairs of subunits, each pair consisting of an ‘acidic’ subunit (mol.wt. about 40000) and a ‘basic’ subunit (mol.wt. about 20000) linked by one or more disulphide bonds. The heterogeneity of legumin has been investigated by isoelectric focusing; undissociated legumin could not be focused satisfactorily, but legumin subunits could be analysed under dissociating conditions. 8m-Urea was not found to be a satisfactory medium for isoelectric focusing of legumin, as the ‘basic’ subunits showed a shift in pI with time of incubation in urea. A new dissociating medium for isoelectric focusing, namely 50% (v/v) formamide, was used for analysis of legumin, which gave pI values of 5.0–5.3 for the ‘acidic’ subunits, and 8.3–8.7 for the ‘basic’ subunits. Both types of subunits were shown to be heterogeneous in charge and molecular weight by two-dimensional analysis employing isoelectric focusing in the first dimension and sodium dodecyl sulphate/polyacrylamide gel electrophoresis in the second. The ‘basic’ and ‘acidic’ subunits of legumin were separated on the preparative scale by ion-exchange chromatography in 50% formamide. Carbohydrate attached to the protein was investigated as a possible cause of the heterogeneity of legumin subunits. However, both a fluorescent-labelling technique and a sensitive radioactive-labelling technique failed to show any carbohydrate bound to legumin subunits, and it was concluded that legumin is not a glycoprotein.

scholarly journals Purification and properties of l-3-glycerophosphate dehydrogenase from pig brain mitochondria

1980 ◽  
Vol 192 (1) ◽  
pp. 9-18 ◽  
Author(s):  
I R Cottingham ◽  
C I Ragan
Keyword(s):  
Isoelectric Focusing ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Brain Mitochondria ◽  
Exchange Chromatography ◽  
Glycerophosphate Dehydrogenase ◽  
Pig Brain ◽  
Purification And Properties ◽  
High Concentrations ◽  
Triton X

L-3-Glycerophosphate dehydrogenase (EC 1.1.99.5) was purified from pig brain mitochondria by extraction with deoxycholate, ion-exchange chromatography and (NH4)2SO4 fractionation in cholate, and preparative isoelectric focusing in Triton X-100. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis shows that the purified enzyme consists of a single subunit of mol.wt. 75 000. The enzyme contains non-covalently bound FAD and low concentrations of iron and acid labile sulphide. No substrate reducible e.p.r. signals were detected. The conditions of purification, particularly the isoelectric focusing step, lead to considerable loss of FAD and possibly iron-sulphur centres. It is therefore not possible to decide with certainty whether the enzyme is a flavoprotein or a ferroflavoprotein. The enzyme catalyses the oxidation of L-3-glycerophosphate by a variety of electron acceptors, including ubiquinone analogues. A number if compounds known to inhibit ubiquinone oxidoreduction by other enzymes of the respiratory chain failed to inhibit L-3-glycerophosphate dehydrogenase, except at very high concentrations.

scholarly journals PURIFICATION AND BIOCHEMICAL CHARACTERIZATION OF A T/TN SPECIFIC LECTIN FROM LEPECHINIA BULLATA SEEDS (LAMIACEAE)

2017 ◽  
Vol 9 (10) ◽  
pp. 165
Author(s):  
Wilches Torres A. ◽  
Rojas Caraballo J. ◽  
Sanabria E. ◽  
Reyes MontaÑo E ◽  
FernÁndez Alonso Jl ◽  
...  
Keyword(s):  
Isoelectric Focusing ◽  
Biochemical Characterization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Reducing Conditions ◽  
Sds Page ◽  
Unique Band

Objective: This study focused on purifying and characterizing a lectin from Lepechinia bullata (L. bullata) seeds, and determining its specificity towards tumour-associated carbohydrate-antigens.Methods: Pigments were removed by washing the seeds with NH4OH 0.1 M pH 9.4 and treating the crude extracts with Pectinex®. The purification procedure consisted of anion exchange chromatography on diethylaminoethyl (DEAE)-Sephadex followed by affinity chromatography. For the characterization, the phase was used polyacrylamide gel electrophoresis-sodium dodecyl sulphate (SDS-PAGE), isoelectric focusing, hemagglutination assays, enzyme-linked lectinosorbent assay (ELLA) and thermal shift assay (TSA).Results: 6.2 mg of lectin were obtained from 100 g of seeds. It was able to agglutinate enzymatically treated erythrocytes with a minimal required lectin concentration of 7 μg. ml-1. Strong binding to asialo bovine submaxillary mucine (aBSM) was determined, corroborating Tn recognition.The isoelectric focusing showed a unique band at pH 8.5. Lectin pure shown bands at 28, 48 and 93 kDa by SDS-PAGE, with an incomplete dissociation of the last species despite trying several reduction conditions. By preparative electrophoresis under different conditions, three species were observed too, in all fractions one band at 28 kDa on Tricine-PAGE in reducing and no reducing conditions were found.Amino acid composition, carbohydrate content, thermal stability and Ca2+and Mn2+requirements were determined. N-acetylgalactosamine (GalNAc) and desialylated mucins inhibited the agglutinant activity on human cells. Fetuin inhibited hemagglutination of rabbit erythrocytes.Conclusion: A new lectin was isolated and characterized from L. bullata seeds, it recognizes T/Tn antigen and shows some similarities with other Lamiaceae lectins.

scholarly journals Hydrophobic-interaction chromatography and anion-exchange chromatography in the presence of acetonitrile. A two-step purification method for human prolactin

1981 ◽  
Vol 199 (3) ◽  
pp. 619-627 ◽  
Author(s):  
Steven C. Hodgkinson ◽  
Philip J. Lowry
Keyword(s):  
Anion Exchange ◽  
Hydrophobic Interaction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Hydrophobic Interaction Chromatography ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Preparative Scale ◽  
Human Prolactin

Described is a two-chromatographic-step preparative-scale technique for the purification of human prolactin from a frozen pituitary homogenate. The method utilizes hydrophobic interaction chromatography on the mildly hydrophobic adsorbent phenyl-Sepharose CL-4B and anion-exchange chromatography on DEAE-cellulose in the presence of acetonitrile. Human prolactin was solubilized at pH10.0 after a prior extraction of pituitaries at pH4.0, the acid pH being ineffective at solubilizing human prolactin but capable of solubilizing large amounts of interfering protein. An 11-fold increase in the potency of the solubilized human prolactin was achieved in this manner. Prolactin could be adsorbed to phenyl-Sepharose at low ionic strengths (I<0.01); few other proteins were adsorbed under these conditions. This is a demonstration of the hydrophobic nature of human prolactin. The amount of phenyl-Sepharose was limited to the minimum (35mg of protein/g of phenyl-Sepharose) necessary to adsorb human prolactin, further reducing the uptake of other pituitary protein. Desorption was achieved by using an acetonitrile gradient (0–30%, v/v), resulting in a purification of human prolactin of 85-fold and recovery of 78%. Acetonitrile (20%, v/v) was also included in all buffers for DEAE-cellulose chromatography, increasing the resolution and recovery of human prolactin, apparently by minimizing non-ionic interactions with the matrix. Prolactin (10mg) was recovered from 63g if pituitaries, an overall recovery of 58%. It was homogeneous by gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, contained less than 0.1% somatotropin (growth hormone), on iodination demonstrated more than 95% binding to excess anti-(human prolactin) serum and could be displaced from anti-(human prolactin) serum in a manner indistinguishable from the serum of a patient with a human prolactin-secreting adenoma.

scholarly journals Purification to homogeneity of pig leucocyte catabolin, a protein that causes cartilage resorption in vitro

1983 ◽  
Vol 215 (2) ◽  
pp. 385-392 ◽  
Author(s):  
J Saklatvala ◽  
V A Curry ◽  
S J Sarsfield
Keyword(s):  
Isoelectric Focusing ◽  
Interleukin 1 ◽  
Polyacrylamide Gel Electrophoresis ◽  
Active Material ◽  
Sodium Dodecyl ◽  
Reversed Phase ◽  
Buffy Coat ◽  
Exchange Chromatography ◽  
Nasal Cartilage

Catabolin, a protein that causes proteoglycan resorption in explants of living cartilage, was purified to homogeneity from culture medium conditioned by culturing buffy-coat leucocytes from 60 litres of pig blood in the presence of concanavalin A. The purification steps were (1) gel filtration of concentrated medium, (2) chromatofocusing, (3) hydroxyapatite chromatography, (4) anion-exchange chromatography (Mono Q), (5) reversed-phase high-pressure liquid chromatography (h.p.l.c.) (Zorbax ODS). These achieved approx. 9000-fold purification from the starting material. The purified protein when reduced ran as a single band on sodium dodecyl sulphate (SDS)/polyacrylamide-gel electrophoresis with Mr 21000. On isoelectric focusing its pI was 4.8-5.0, and there was evidence of micro-heterogeneity. The protein co-migrated with active material on h.p.l.c., isoelectric focusing and SDS gels (15 and 12.5% acrylamide) under both reducing and non-reducing conditions. The pure protein caused proteoglycan release from cultured bovine nasal cartilage at 20pM. Its possible identity with interleukin 1 is discussed.

scholarly journals Isolation, partial purification and characterization of phospholipase A2 from Naja Katiensis venom

2021 ◽  
Vol 13 (2) ◽  
pp. 107-112
Author(s):  
C.F. Okechukwu ◽  
P.L. Shamsudeen ◽  
R.K. Bala ◽  
B.G. Kurfi ◽  
A.M. Abdulazeez
Keyword(s):  
Ion Exchange ◽  
Phospholipase A2 ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Crude Venom

The most effective and acceptable therapy for snakebite victims is the immediate administration of antivenin which is limited by problems of hypersensitivity reactions in some individuals and its inability to resolve the local effects of the venom. The aim of this study was to isolate, partially purify and characterize phospholipase A2 from Naja Katiensis venom. Phospholipase A2 was partially purified via a two-step process: gel filtration on Sephadex G-75 and ion exchange chromatography using CM Sephadex, and subjected to SDS-PAGE analysis. From the results, the specific activity of the partially purified PLA2 decreased from 0.67μmol/min/mg in crude venom to 0.29μmol/min/mg after ion exchange chromatography with a yield of 5% and purification fold of 0.43. The optimum temperature of the purified PLA2 was found to be 35ºC and optimum p.H of 7. velocity studies for the determination of kinetic constants using L-a-lecithin as substrate revealed a Km  of 1.47mg/ml and Vmax  of 3.32μ moles/min/mg. The sodium dodecyl sulphate polyacrylamide gel electrophoresis of the purified PLA2 showed a distinct band with molecular weight estimated to be 14KDa. In conclusion, the present study shows that phospholipase A2 was isolated, purified and characterized. This may serve as a promising candidate for future development of a novel anti-venin drug.

scholarly journals Separation and partial characterization of guinea-pig caseins

1978 ◽  
Vol 173 (2) ◽  
pp. 633-641 ◽  
Author(s):  
R K Craig ◽  
D McIlreavy ◽  
R L Hall
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Guinea Pig ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Exchange Chromatography

1. Guinea-pig caseins A, B and C were purified free of each other by a combination of ion-exchange chromatography and gel filtration. 2. Determination of the amino acid composition showed all three caseins to contain a high proportion of proline and glutamic acid, but no cysteine. This apart, the amino acid composition of the three caseins was markedly different, though calculated divergence values suggest that some homology may exist between caseins A and B. Molecular-weight estimates based on amino acid composition were in good agreement with those based on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 3. N-Terminal analysis showed lysine, methionine and lysine to be the N-terminal residues of caseins A, B and C respectively. 4. Two-dimensional separation of tryptic digests revealed a distinctive pattern for each casein. 5. All caseins were shown to be phosphoproteins. The casein C preparation also contained significant amounts of sialic acid, neutral and amino sugars. 6. The results suggest that each casein represents a separate gene product, and that the low-molecular-weight proteins are not the result of a post-translational cleavage of the largest. All were distinctly different from the whey protein alpha-lactalbumin.

Isolation and partial characterization of tammar wallaby luteinizing hormone and development of a radioimmunoassay

1997 ◽  
Vol 9 (4) ◽  
pp. 475 ◽  
Author(s):  
James R. McFarlane ◽  
Carl D. Rudd ◽  
Lynda M. Foulds ◽  
Terry P. Fletcher ◽  
Marilyn B. Renfree
Keyword(s):  
Luteinizing Hormone ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Tammar Wallaby ◽  
Exchange Chromatography ◽  
Significant Rise ◽  
Brushtail Possum ◽  
Response Curves ◽  
Antechinus Stuartii

Tammar wallaby (Macropus eugenii) luteinizing hormone (LH) was purified from pituitaries collected from wild and captive populations by salt sequential precipitation, ion exchange chromatography and gel filtration. Pituitary tissue (5 g) yielded 1·8 mg of purified wallaby luteinizing hormone (ME-14B), as verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A heterologous radioimmunoassay has been developed for measurement of LH in plasma of marsupials using a monoclonal antibody raised against bovine LH (518B7). This assay system was able to measure basal LH concentrations in male and female tammars and detected a significant rise in plasma LH in response to oestradiol benzoate in female tammars and luteinizing hormone-releasing hormone (LHRH) in males. Parallel dose–response curves were also obtained from pituitary extracts from four other species of marsupial (brushtail possum, Trichosurus vulpecula; brown antechinus,Antechinus stuartii; kowari, Dasyuroides byrnei; and Eastern pygmy possum,Cercartetus nanus) in this assay, which suggests its usefulness in the measurement of LH in other marsupial species.

Plant NAD-Dependent Glutamate Dehydrogenase. Purification, Molecular Properties and Metal Ion Activation of the Enzymes from Lemna minor and Pisum sativum

1980 ◽  
Vol 35 (3-4) ◽  
pp. 213-221 ◽  
Author(s):  
Heinz-Walter Scheid ◽  
Adelheid Ehmke ◽  
Thomas Hartmann
Keyword(s):  
Amino Acid ◽  
Pisum Sativum ◽  
Glutamate Dehydrogenase ◽  
Gel Electrophoresis ◽  
Metal Ion ◽  
Lemna Minor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Equilibrium ◽  
Electron Microscopic

Abstract Glutamate dehydrogenase (ʟ-glutamate: NAD+ oxidoreductase (deaminating) EC 1.4.1.2) has been purified to homogeneity from Lemna minor and seeds of Pisum sativum. As established by polyacrylamide gel electrophoresis the Pisum-enzyme constitutes a multiple pattern of seven char­ge isoenzymes whereas the Lemna enzyme shows one single protein band. Molecular weights of 230 000 were calculated for both enzymes by sedimentation equilibrium measurements (Pisum-enzyme) and comparative gel filtration (Lemna-enzyme). Sodium dodecyl sulfate gel electrophoresis and electron microscopic observations revealed that both enzymes are composed of four identical subunits (molecular weight 58 500) arranged in a tetraedric structure. The amino acid compositions of both enzymes are similar to those of various hexameric glutamate dehydrogenases. The N-terminal amino acid of the Pisum-enzyme is alanine. Both enzymes require Ca2+ for maximal catalytic activity. For the Lemna-enzyme the K0.5 values for Ca2+ are 22 µᴍ (NADH-dependent reaction) and 4 µᴍ (NAD+ -dependent reaction), respectively. Ca2+ which to some extent can be replaced by Zn2+ does not affect the enzyme aggregation but seems to govern a reversible equilibrium between catalytically active and inactive enzyme forms.

scholarly journals The 47-kD fragment of talin is a substrate for protein kinase P

Blood ◽  
1993 ◽  
Vol 82 (11) ◽  
pp. 3343-3349 ◽  
Author(s):  
PC Simons ◽  
L Elias
Keyword(s):  
Protein Kinase ◽  
Ion Exchange ◽  
Sequence Data ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Exchange Chromatography ◽  
Myelogenous Leukemia ◽  
Blue Staining ◽  
Major Protein

Abstract This laboratory has been characterizing protein serine/threonine kinase reactions of hematopoietic tissues, whose most distinguishing characteristics in vitro are stimulation with vesicular phosphatidyl glycerol, and the ability to function using Mn2+ as the sole divalent cation. The major protein substrates are a 73-kD protein and a protein migrating near ovalbumin on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 47-kD protein was partially purified from cells harvested by leukapheresis from a patient with acute myelogenous leukemia, using ammonium sulfate precipitation and ion exchange chromatography. This partially purified ion-exchange fraction contained an endogenous kinase activity with characteristics similar to those we previously described of protein kinase P (protein kinase, phospholipid- stimulable: PK-P), but not typical of any form of protein kinase C (PK- C). With longer phosphorylation, the 47-kD band showed increasingly lower mobility demonstrable both by Coomassie blue staining and autoradiography, suggesting both that it was multiply phosphorylated, and that the excisable band was pure. The protein was thus eluted from preparative gel slices and digested with endoproteinase lys C. Sequence data from the fragments identified the protein as the 47-kD calpain fragment of talin, a protein found in focal adhesion plaques and some cell-cell contacts. PK-C phosphorylated the 47-kD protein, as has been reported previously, and phosphopeptide mapping disclosed a similar pattern of phosphorylation using either PK-C or the endogenous activity. The 47-kD protein labeled with the endogenous kinase contained predominantly phosphoserine, with some phosphothreonine and a trace of phosphotyrosine. Intact, purified talin was also phosphorylated by PK-P in a phospholipid-stimulable manner, but at 1/20 the rate of the 47-kD fragment.

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