scholarly journals Purification and characterization of human transcortin

1981 ◽  
Vol 197 (3) ◽  
pp. 645-653 ◽  
Author(s):  
U W Mueller ◽  
J M Potter
Keyword(s):  
Isoelectric Focusing ◽  
Equilibrium Dialysis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Binding Activity ◽  
Spontaneous Polymerization ◽  
Apparent Homogeneity ◽  
Step Procedure ◽  
Apparent Association

Human transcortin was purified to apparent homogeneity from plasma by a two-step procedure involving affinity and hydroxyapatite chromatography. The affinity gel incorporated denatured bovine serum albumin as the spacer and cortisol hemisuccinate as the ligand. Although isolated transcortin showed a propensity for spontaneous polymerization according to a geometric progression (1, 3, 9) only one band was observed on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Cortisol-binding activity of the isolated protein gave an apparent association constant of 2.5 × 10(8) M-1 at 4 degree C in equilibrium dialysis. Isoelectric focusing of purified native transcortin showed six discrete bands, five between pH 3.75 and 4.15 and another, possibly desialylated, at pH 6.15. Desialylated transcortin also gave six bands on isoelectric focusing, with pI values ranging from 4.90 to 6.30.

scholarly journals Isolation and characterization of human plasma α 1-proteinase inhibitor and a conformational study of its interaction with proteinases

1976 ◽  
Vol 157 (2) ◽  
pp. 339-351 ◽  
Author(s):  
J Saklatvala ◽  
G C Wood ◽  
D D White
Keyword(s):  
Human Plasma ◽  
Isoelectric Focusing ◽  
Proteinase Inhibitor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Equilibrium ◽  
Isolation And Characterization ◽  
Step Procedure ◽  
Helical Content

1. alpha 1-Proteinase inhibitor was isolated from human plasma by a five-step procedure. Isoelectric focusing showed that six components focused between pH4.85 and 4.95. 2. The mol.wt. of the inhibitor was 52000 by sedimentation equilibrium and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The amino acid and carbohydrate compositions of the inhibitor were also determined. 3. The far-u.v.c.d. (circular-dichroism) spectrum indicated that the inhibitor had about 36% alpha-helical content. 4. The loss of proteinase-inhibitory activity when the inhibitor was exposed to pH values less than 5.0 or greater than 10.5 was accompanied by small changes in the far-u.v.c.d. spectrum and large changes in the near-u.v.c.d. spectrum. The change at alkaline pH was associated with ionization of tyrosine residues. 5. Interaction of inhibitor with chymotrypsin caused perturbation of the c.d. spectrum and this was used to follow the interaction and show a 1:1 stoicheiometry. 6. C.d., electrophoresis and isoelectric focusing showed that the inhibitor-enzyme complex is degraded by free enzyme. 7. Parallel studies with trypsin indicated that it too forms a 1:1 complex with inhibitor and is degraded by excess of enzyme.

Purification and characterization of ferro- and cobalto-chelatases

1988 ◽  
Vol 66 (1) ◽  
pp. 32-39 ◽  
Author(s):  
Eduardo T. Cánepa ◽  
Elena B.C. Llambías
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Purification And Characterization ◽  
Degree Of Unsaturation ◽  
Apparent Homogeneity ◽  
Optimum Ph ◽  
Single Protein ◽  
Metal Insertion

Pig liver ferrochelatase was purified 465-fold with about 30% yield, to apparent homogeneity, by a procedure involving solubilization from mitochondria, ammonium sulfate fractionation, and Sephacryl S-300 chromatography. The fraction of each purification step had cobaltochelatase as well as ferrochelatase activity. A purified protein of molecular weight 40 000 was found by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. A molecular weight of approximately 240 000 was obtained by Sephacryl S-300 chromatography. Both activities of the purified fraction increased linearly with time until 2 h. but nonlinear plots were obtained with increasing concentrations of protein. Their optimum pH values were similar. Km values were, for ferrochelatase activity, 23.3 μM for the metal and 30.3 μM for mesoporphyrin. and for cobaltochelatase activity. 27 and 45.5 μM, respectively. Fe2+ and Co2+ each protected against inactivation by heat. Pb2+, Zn2+, Cu2+, or Hg2+ inhibited both activities, while Mn2+ slightly activated; Mg2+ had no effect, at the concentrations tested. There appeared to be an involvement of sulfhydryl groups in metal insertion. Lipids, in correlation with their degree of unsaturation, activated both purified activities; phospholipids also had activation effects. We conclude that a single protein catalyzes the insertion of Fe2+ or Co2+ into mesoporphyrin.

scholarly journals Purification and photoaffinity labelling of a rat cytosolic binding protein specific for 3-methylcholanthrene

1987 ◽  
Vol 242 (2) ◽  
pp. 375-381 ◽  
Author(s):  
P S Arnold ◽  
R C Garner ◽  
B Tierney
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Binding Activity ◽  
Liver Protein ◽  
Photoaffinity Labelling ◽  
High Affinity ◽  
Apparent Homogeneity ◽  
Stokes Radius

Rat hepatic cytosolic proteins which sediment at 4-5 S on sucrose gradients exhibit high-affinity saturable binding for the carcinogen 3-methylcholanthrene. A rat liver protein of Stokes' radius 3 nm, Mr by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of 39,000 and with specific 3-methylcholanthrene-binding activity sedimenting at 4.5 S, has been purified 315-fold to apparent homogeneity by using affinity chromatography on a column of 1-hydroxy-3-methylcholanthrene coupled to epoxy-activated Sepharose 6B, in conjunction with two gel-filtration steps. The protein purified by this technique was shown to be associated with the observed specific 3-methylcholanthrene-binding activity by photoaffinity labelling with 1-oxo-3-methylcholanthrene.

scholarly journals Purification and characterization of a saliva-interacting cell-wall protein from Streptococcus mutans serotype f by using monoclonal-antibody immunoaffinity chromatography

1985 ◽  
Vol 228 (1) ◽  
pp. 211-217 ◽  
Author(s):  
F Ackermans ◽  
J P Klein ◽  
J Ogier ◽  
H Bazin ◽  
F Cormont ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Wall ◽  
Streptococcus Mutans ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Immunoaffinity Chromatography ◽  
Binding Activity ◽  
Immunoglobulin M ◽  
Single Polypeptide Chain

A rat monoclonal antibody, LO SM2, of the immunoglobulin M class, specific for a saliva receptor (SR) from Streptococcus mutans serotype f, was able to precipitate the SR from crude cell-wall-associated antigens (WEA) of this bacteria in presence of a detergent mixture. We have then used the technique of monoclonal-antibody immunoaffinity chromatography to purify the S. mutans SR. Pure SR was obtained from a crude WEA fraction with a single chromatographic step. The active SR could be eluted from the column in a highly purified form with 0.2 M-glycine/HC1, pH 2.8. The final yield was about 32% in terms of binding activity. Characterization of the SR by crossed immunoelectrophoresis, sodium dodecyl sulphate- or 4-30%-native-gradient-polyacrylamide-gel electrophoresis showed that the receptor is a single polypeptide chain of Mr approx. 74000. Native or denaturated forms of the SR adsorbed on to a solid support, such as nitrocellulose, are recognized by monoclonal antibody LO SM2, and both forms are still able to bind the ligand, saliva.

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.

Purification and Characterization of an Unusual Aminopeptidase From Pea Seeds

1980 ◽  
Vol 7 (2) ◽  
pp. 131 ◽  
Author(s):  
JB Caldwell ◽  
LG Sparrow
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sulfhydryl Group ◽  
Purification And Characterization ◽  
Apparent Homogeneity ◽  
Pea Seeds ◽  
Hydrolysis Of

An aminopeptidase with specificity for N-terminal glutamic and aspartic acid residues has been purified to apparent homogeneity from pea seeds (Pisum sativum cv. Greenfeast). It also catalyses the hydrolysis of the glutaryl-phenylalanine bond of the synthetic chymotrypsin substrate glutaryl- L-phenylalanine p-nitroanilide. The native enzyme, which has a molecular weight of approximately 500 000, gives a single band on polyacrylamide gel electrophoresis but two major bands when subjected to electrophoresis in the presence of sodium dodecyl sulfate after reduction. Its behaviour with various inhibitors suggests that a sulfhydryl group is important for its activity.

Purification and characterization of an exoglucanase from Streptomyces flavogriseus

1984 ◽  
Vol 30 (9) ◽  
pp. 1171-1178 ◽  
Author(s):  
C. R. Mackenzie ◽  
D. Bilous ◽  
K. G. Johnson
Keyword(s):  
Isoelectric Focusing ◽  
Gel Electrophoresis ◽  
Extracellular Enzymes ◽  
Main Product ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Permeation Chromatography ◽  
Purification And Characterization ◽  
Gel Permeation

Streptomyces flavogriseus, a mesophilic actinomycete, produces high levels of extracellular enzymes capable of hydrolyzing cellulose and xylan. One such enzyme, an exoglucanase, has been purified to molecular homogeneity by a sequence involving DEAE Bio-Gel A chromatography, gel permeation chromatography on Bio-Gel P-60, preparative isoelectric focusing, and concanavalin A affinity chromatography. This purification sequence disclosed the presence of several distinct endoglucanase and xylanase fractions. Homogeneity of the purified enzyme was demonstrated by analytical isoelectric focusing and sodium dodecyl sulphate – polyacrylamide gel electrophoresis. The purified enzyme had a molecular weight of approximately 45 000 and an isoelectric point of 4.15. The enzyme demonstrated negligible activity with carboxymethylcellulose as the substrate. It was able to extensively hydrolyse acid-swollen cellulose; the main product of enzyme action was cellobiose.

Isolation and characterization of goat serum α1-globulin protease inhibitors

1982 ◽  
Vol 60 (1) ◽  
pp. 28-35 ◽  
Author(s):  
Albert Hercz
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Protease Inhibitors ◽  
Isoelectric Focusing ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Isolation And Characterization ◽  
Sds Page

α1-Globulin-type protease inhibitors were isolated from goat serum by two methods, namely preparative isoelectric focusing and preparative electrophoresis in polyacrylamide gel. The fractions obtained by the first method showed varying isoprotein compositions by analytical isoelectric focusing. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) revealed the presence of one protein in the fractions with the same velocity of migration as purified human α1-antitrypsin and a second protein with a slightly higher migration velocity. The ratios of trypsin-inhibiting to chymotrypsin-inhibiting capacities in all the fractions were the same and both inhibitors were stable upon storage. The reaction of the inhibitors with trypsin and chymotrypsin was also demonstrated by analytical isoelectric focusing.The fractions obtained by preparative gel electrophoresis (the second method) contained the same proteins but their proportions varied widely in different fractions as demonstrated by analytical electrofocusing in the presence of urea and by SDS–PAGE. The early fractions, which consisted predominantly of α1-antitrypsin, showed a high inhibiting capacity for trypsin and none or only negligible capacity for chymotrypsin. Conversely, in the late fractions, the proportions of the proteins and inhibiting capacities were reversed. At 4 °C the trypsin-inhibiting capacity was stable for weeks but the chymotrypsin-inhibiting capacity of the preparation rapidly decreased.These observations indicate that the inhibition of proteases by goat α1-globulins is due to at least two closely associated but distinguishable proteins. One of these, corresponding to human α1-antitrypsin, would have an appreciable capacity to inhibit trypsin, but unlike the latter, little or no capacity for chymotrypsin inhibition. The inhibition of chymotrypsin is due to the second, unidentified α1-globulin.

Purification and characterization of 2-ethoxyphenol-induced cytochrome P450 fromCorynebacteriumsp. strain EP1

1999 ◽  
Vol 45 (10) ◽  
pp. 833-839 ◽  
Author(s):  
Naoko Kawahara ◽  
Hisayoshi Ikatsu ◽  
Hiroshige Kawata ◽  
Shin-ichi Miyoshi ◽  
Ken-ichi Tomochika ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Enzyme Purification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Metabolic Reaction ◽  
Difference Spectra ◽  
Original Activity ◽  
Apparent Homogeneity ◽  
Corynebacterium Sp

A soluble cytochrome P450 (P450EP1A) induced by 2-ethoxyphenol was purified to apparent homogeneity from Corynebacterium sp. strain EP1. The P450EP1Ashowed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of about 45 kDa. The CO-reduced difference spectra of P450EP1Ahad a Soret maximum at 447.6 nm. The substrate difference spectra with 2-ethoxyphenol showed an absorption maximum at 394.0 nm. The purified P450EP1Adegraded 2-ethoxyphenol in an assay system composed of spinach ferredoxin-NADP+oxidoreductase and NADPH. The reaction activity decreased to 1.4% of its original activity by addition of CO. The existence of catechol in the reaction mixture was confirmed after the metabolic reaction, indicating that P450EP1Acatalyzes O-dealkylation of 2-ethoxyphenol. In addition to 2-ethoxyphenol, the P450EP1Ametabolized 2-methoxyphenol, 1,1,1-trichloroethane, carbon tetrachloride, benzene, and toluene.Key words: cytochrome P450, Corynebacterium sp., 2-ethoxyphenol, enzyme purification, biodegradation.

scholarly journals Purification and characterization of chicken erythrocyte ferrochelatase

1984 ◽  
Vol 222 (3) ◽  
pp. 695-700 ◽  
Author(s):  
J W Hanson ◽  
H A Dailey
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
High Rate ◽  
Chicken Erythrocyte ◽  
Purification And Characterization ◽  
First Order ◽  
Apparent Homogeneity ◽  
Mammalian Liver ◽  
Erythrocyte Enzyme

Ferrochelatase (EC 4.99.1.1) was purified 2000-fold to apparent homogeneity from isolated chicken erythrocyte mitochondria. The purified enzyme yields a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with an apparent Mr of 42 000. The enzyme utilizes proto-, meso- and deutero-porphyrin with Km values of 37, 51 and 80 microM respectively. The disubstituted porphyrins 2,4-bisglycol deutero-porphyrin and 2,4-disulphonic deuteroporphyrin were not substrates. Mn2+, Hg2+, Pb2+ and Co2+ were strong inhibitors of the purified enzyme. Palmitic acid and oleic acid stimulated activity, whereas linoleic acid inhibited and phospholipids had variable effects. Chicken ferrochelatase was inhibited by N-ethylmaleimide and iodoacetamide. Inhibition by iodoacetamide was pseudo-first-order, but inhibition by N-ethylmaleimide appeared to be biphasic in nature with an initial high rate followed by a much lower rate of inactivation. The characteristics of the chicken erythrocyte enzyme are compared with those previously reported for mammalian liver ferrochelatase.

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