Conformational destabilization of Bacillus licheniformis α-amylase induced by lysine modification and calcium depletion.

Bacillus licheniformis α-amylase (BLA) was chemically modified using 100-fold molar excess of succinic anhydride over protein or 0.66 M potassium cyanate to obtain 42 % succinylated and 81 % carbamylated BLAs. Size and charge homogeneity of modified preparations was established by Sephacryl S-200 HR gel chromatography and polyacrylamide gel electrophoresis. Conformational alteration in these preparations was evident by the larger Stokes radii (3.40 nm for carbamylated and 3.34 nm for succinylated BLAs) compared to 2.43 nm obtained for native BLA. Urea denaturation results using mean residue ellipticity (MRE) as a probe also showed conformational destabilization based on the early start of transition as well as ΔG(D)(H(2)O) values obtained for both modified derivatives and Ca-depleted BLA. Decrease in ΔG(D)(H(2)O) value from 5,930 cal/mol (for native BLA) to 3,957 cal/mol (for succinylated BLA), 3,336 cal/mol (for carbamylated BLA) and 3,430 cal/mol for Ca-depleted BLA suggested reduced conformational stability upon modification of amino groups of BLA or depletion of calcium. Since both succinylation and carbamylation reactions abolish the positive charge on amino groups (both α- and ε- amino), the decrease in conformational stability can be ascribed to the disruption of salt bridges present in the protein which might have released the intrinsic calcium from its binding site.

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Low Molecular Weight Trypsin-Plasmin Inhibitors Isolated from Papain Treated Urinary Trypsin Inhibitor

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657115 ◽

1982 ◽

Vol 47 (01) ◽

pp. 014-018 ◽

Cited By ~ 13

Author(s):

H Sumi ◽

N Toki ◽

S Takasugi ◽

S Maehara ◽

M Maruyama ◽

...

Keyword(s):

Trypsin Inhibitor ◽

Gel Electrophoresis ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Chromatography ◽

Urinary Trypsin Inhibitor ◽

Plasmin Activity ◽

Molecular Weights ◽

Plasmin Inhibitors

SummaryPapain treatment of human urinary trypsin inhibitor (UTI67; mol. wt. 43,000 by SDS-polyacrylamide gel electrophoresis, specific activity 1,897 U/mg protein) produced four new protease inhibitors, which were highly purified by gel chromatography on Sephadex G-100 and isoelectric focusing. The purified inhibitors (UTI26, UTI9-I, UTI9-II, and UTI9-III) were shown to be homogeneous by polyacrylamide disc gel electrophoresis, and had apparent molecular weights of 26,000, 9,000, 9,000, and 9,800, respectively, by sodium dodecyl sulfate gel electrophoresis. During enzymatic degradation of UTI67, the amino acid compositions changed to more basic, and the isoelectric point increased from pH 2.0 (UTI67) to pHs 4.4, 5.2, 6.6, and 8.3 (UTI26, UTI9-I, UTI9-II, and UTI9-III), respectively. Both the parent and degraded inhibitors had anti-plasmin activity as well as antitrypsin and anti-chymotrypsin activities. Much higher anti-plasmin/anti-trypsin and anti-plasmin/anti-chymotrypsin activities were observed in the degraded inhibitors than in the parent UTI67. They competitively inhibited human plasmin with Ki values of 1.13 X 10-7 - 2.12 X 10-6 M (H-D-Val-Leu-Lys-pNA substrate). The reactions were very fast and the active site of the inhibitors to plasmin was thought to be different from that to trypsin or chymotrypsin.

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Magnetic Resonance and Catalytic Studies of Pyruvate Kinase with Essential Sulfhydryl or Lysyl ε-Amino Groups Chemically Modified

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)43945-8 ◽

1973 ◽

Vol 248 (10) ◽

pp. 3419-3425

Author(s):

Michael Flashner ◽

Ilana Tamir ◽

Albert S. Mildvan ◽

H. Paul Meloche ◽

Minor J. Coon

Keyword(s):

Magnetic Resonance ◽

Pyruvate Kinase ◽

Amino Groups ◽

Chemically Modified

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Purification and partial characterization of cysteine-glutamate transaminase from rat liver

Canadian Journal of Biochemistry ◽

10.1139/o77-143 ◽

1977 ◽

Vol 55 (9) ◽

pp. 958-964 ◽

Cited By ~ 6

Author(s):

M. P. C. Ip ◽

R. J. Thibert ◽

D. E. Schmidt Jr.

Keyword(s):

Molecular Weight ◽

Rat Liver ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Liver Homogenate ◽

Polyacrylamide Gel ◽

Gel Chromatography ◽

Labile Hydrogen ◽

Apparent Homogeneity

Cysteine-glutamate transaminase (cysteine aminotransferase; EC 2.6.1.3) has been purified 149-fold to an apparent homogeneity giving a specific activity of 2.09 IU per milligram of protein with an overall yield of 15%. The isolation procedures involve the preliminary separation of a crude rat liver homogenate which was submitted sequentially to ammonium sulfate fractionation, TEAE-cellulose column chromatography, ultrafiltration, and isoelectrofocusing. The final product was homogenous when examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). A minimal molecular weight of 83 500 was determined by Sephadex gel chromatography. The molecular weight as estimated by polyacrylamide gel electrophoresis in the presence of SDS was 84 000. The purified enzyme exhibited a pH optimum at 8.2 with cysteine and α-ketoglutarate as substrates. The enzyme is inactivated slowly when kept frozen and is completely inactivated if left at room temperature for 1 h. The enzyme does not catalyze the transamination of α-methyl-DL-cysteine, which, when present to a final concentration of 10 mM, exhibits a 23.2% inhibition of transamination of 30 mM of cysteine. The mechanism apparently resembles that of aspartate-glutamate transaminase (EC 2.6.1.1) in which the presence of a labile hydrogen on the alpha-carbon in the substrate is one of the strict requirements.

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Studies on human pregnancy-associated plasma protein A. Purification by affinity chromatography and structural comparisons with α2-macroglobulin

Biochemical Journal ◽

10.1042/bj1910799 ◽

1980 ◽

Vol 191 (3) ◽

pp. 799-809 ◽

Cited By ~ 41

Author(s):

R G Sutcliffe ◽

B M Kukulska-Langlands ◽

J R Coggins ◽

J B Hunter ◽

C H Gore

Keyword(s):

Molecular Weight ◽

Affinity Chromatography ◽

Plasma Protein ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Protein A ◽

Carbohydrate Content ◽

Polyacrylamide Gel ◽

Gel Chromatography ◽

Tryptic Fragment

Pregnancy-associated plasma protein-A (PAPP-A) has been purified by a combination of methods including antibody-affinity chromatography. The resultant protein, obtained in 16% yield from maternal serum, appeared as a single major component on non-denaturing polyacrylamide and SDS/polyacrylamide gel electrophoresis. The protein showed a single component when analysed by isoelectric focusing under denaturing conditions in the presence and absence of reduction and had a pI of 4.34 and 4.42 respectively. These pI values were indistinguishable from those of alpha 2-macroglobulin (alpha 2M). The molecular weight of the PAPP-A polypeptide as shown by SDS/polyacrylamide-gel electrophoresis was 187000, with a minor component of mol.wt. 82500 that was attributed to proteolysis. Since native PAPP-A had a molecular weight on gel chromatography very similar to that of alpha 2M (620000–820000), it was concluded that PAPP-A was a homotetramer. In the absence of reduction, a high-molecular-weight (420000) protomer of PAPP-A was found. It was deduced that PAPP-A, like alpha 2M, is a dinner, whose protomers are composed of disulphide-linked polypeptide chains. It was found that the molecular weight of the PAPP-A polypeptide exceeded that of alpha 2M by 3.3%, but that the total carbohydrate content of PAPP-A exceeded that of alpha 2M by 10% and that its neutral carbohydrate content exceeded that of alpha 2M by between 7.4 and 9.0%. The significance of the estimated molecular weights of alpha 2M (181000) and its major tryptic fragments is discussed in the light of published values. A tryptic fragment alpha 2M (82500 mol.wt.) was apparently the same size as the major tryptic fragment of PAPP-A.

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Contribution of Salt Bridges near the Surface of a Protein to the Conformational Stability†,‡

Biochemistry ◽

10.1021/bi000849s ◽

2000 ◽

Vol 39 (40) ◽

pp. 12375-12381 ◽

Cited By ~ 71

Author(s):

Kazufumi Takano ◽

Kimiko Tsuchimori ◽

Yuriko Yamagata ◽

Katsuhide Yutani

Keyword(s):

Conformational Stability ◽

Salt Bridges

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Purification and Partial Amino Acid Sequence of Pentocin 31-1, an Anti-Listeria Bacteriocin Produced by Lactobacillus pentosus 31-1

Journal of Food Protection ◽

10.4315/0362-028x-72.12.2524 ◽

2009 ◽

Vol 72 (12) ◽

pp. 2524-2529 ◽

Cited By ~ 20

Author(s):

JINLAN ZHANG ◽

GUORONG LIU ◽

NAN SHANG ◽

WANPENG CHENG ◽

SHANGWU CHEN ◽

...

Keyword(s):

Molecular Mass ◽

Specific Activity ◽

Consensus Sequence ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Mass Spectrometry Analysis ◽

Gel Chromatography ◽

Original Activity ◽

Lactobacillus Pentosus ◽

Spectrometry Analysis

Pentocin 31-1, an anti-Listeria bacteriocin produced by Lactobacillus pentosus 31-1 from the traditional Chinese fermented Xuan-Wei ham, was successfully purified by the pH-mediated cell adsorption-desorption method and then purified by gel chromatography with Sephadex G-10. The purification resulted in a 1,381.9-fold increase in specific activity with a yield of 76.8% of the original activity. Using Tricine–sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), the molecular mass of the purified peptide was found to be between 3,500 and 6,400 Da, and bacteriocin activity was confirmed by overlayer techniques. When subjected to mass spectrometry analysis, the protein was highly pure and its molecular mass was 5,592.225 Da. The partial N-terminal sequence of pentocin 31-1 was the following: NH2-VIADYGNGVRXATLL. Compared with the sequence of other bacteriocins, pentocin 31-1 has the consensus sequence YGNGV in its N-terminal region, and therefore it belongs to the class IIa of bacteriocins.

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Affinity purification of cellular retinoic acid-binding protein on 14-carboxy-13-cis-retinamide-sepharose 4B

Biochemical Journal ◽

10.1042/bj2620917 ◽

1989 ◽

Vol 262 (3) ◽

pp. 917-922 ◽

Cited By ~ 2

Author(s):

R K Singh ◽

B P Sani ◽

M I Dawson ◽

Y F Shealy

Keyword(s):

Retinoic Acid ◽

Carboxy Group ◽

Binding Proteins ◽

Binding Protein ◽

Polyacrylamide Gel Electrophoresis ◽

Biologically Active ◽

Amide Bond ◽

Gel Chromatography ◽

Fatty Acid Binding ◽

Sepharose 4B

A biologically active bifunctional retinoid, ethyl 14-carboxyretinoate, has been synthesized and shown to bind cellular retinoic acid (RA)-binding protein (CRABP) via its free carboxy group. We describe herein the synthesis of 14-carboxy-13-cis-retinamide-Sepharose 4B, which is an affinity matrix bearing an all-trans-RA moiety, and thus was used to purify and characterize CRABP from chick-embryo skin. An amide bond was first formed between the free carboxy group of the retinoid and a primary amino group of aminohexyl-Sepharose 4B, by reaction with carbodi-imide, and the ester group of the resin-bound retinoid was then hydrolysed in an alkaline medium. Polyacrylamide-gel electrophoresis and f.p.l.c. Superose column-chromatographic analysis demonstrated that the affinity-purified CRABP (Mr 15,000) was close to electrophoretic homogeneity (greater than 90%) and specifically interacts with RA. By using affinity gel chromatography, conversion of holo-CRABP into apo-CRABP by treatment with p-hydroxymercuribenzoate and a possible involvement of a thiol group in RA binding to CRABP were established. This affinity procedure provides several advantages: (i) 14-carboxy-13-cis-retinamide-Sepharose exhibited high efficiency and selectivity for RA-binding protein (i.e. retinol- or fatty-acid-binding proteins did not bind); (ii) the presence of the amide linkage between the ligand and the matrix makes this affinity resin relatively stable to cytosolic enzymes; and (iii) other RA-binding proteins, e.g. nuclear receptor(s), may be purified.

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Partial characterization of an abnormal lactate dehydrogenase isoenzyme, LDH-1ex, in serum from a patient with hepatocellular carcinoma.

Clinical Chemistry ◽

10.1093/clinchem/35.5.844 ◽

1989 ◽

Vol 35 (5) ◽

pp. 844-848

Author(s):

D L Kalpaxis ◽

E E Giannoulaki

Keyword(s):

Hepatocellular Carcinoma ◽

Lactate Dehydrogenase ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Chromatography ◽

Heat Stable ◽

Single Polypeptide ◽

Polypeptide Chains

Abstract Serum from a patient with hepatocellular carcinoma contained an abnormal isoenzyme of lactate dehydrogenase (LDH; EC 1.1.1.27), LDH-1ex, that on electrophoresis on 10-g/L agarose gel migrated anodally to the LDH-1 band. This isoenzyme was partly purified by ultrafiltration and preparative electrophoresis. Gel chromatography and sodium dodecyl sulfate/polyacrylamide gel electrophoresis studies of the resulting LDH-1ex preparation suggested that this isoenzyme is probably a tetramer made up of four single polypeptide chains (monomers), each having a molecular mass of about 32,000 Da. LDH-1ex was heat stable and reacted more readily with 2-hydroxybutyrate than did the slower migrating LDH-4 and LDH-5 isoenzymes. LDH-1ex showed no activity when lactate was omitted from the substrate solution or replaced by ethanol.

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Isolation and characterization of a T lymphocyte-derived differentiation inducing factor for the myeloid leukemic cell line HL- 60

Blood ◽

10.1182/blood.v63.3.510.510 ◽

1984 ◽

Vol 63 (3) ◽

pp. 510-517 ◽

Cited By ~ 65

Author(s):

IL Olsson ◽

MG Sarngadharan ◽

TR Breitman ◽

RC Gallo

Keyword(s):

Cell Line ◽

T Lymphocyte ◽

Polyacrylamide Gel Electrophoresis ◽

Gel Filtration ◽

Periodate Oxidation ◽

Morphological Characteristics ◽

Exchange Chromatography ◽

Gel Chromatography ◽

Isolation And Characterization ◽

Sds Page

Abstract Mitogen-stimulated mononuclear blood cells produce differentiation inducing factors (DIFs) for the promyelocytic cell line HL-60. We report that DIF is produced constitutively by a malignant T lymphocyte line HUT-102. DIF was purified 7,000-fold from HUT-102 conditioned media by utilizing ion-exchange chromatography with DEAE-Sepharose, gel chromatography, Blue-Sepharose chromatography, and preparative SDS- polyacrylamide gel electrophoresis (SDS-PAGE). The final preparation is susceptible to protease treatment, has a molecular weight of 46,000, as determined by SDS-PAGE and approximately 55,000 by gel filtration, has an isoelectric point of approximately 5.2, does not adhere to lectin- Sepharose and is resistant to periodate oxidation, and is free of colony-stimulating factor. DIF induced maturation of HL-60 into phagocytizing nitro blue tetrazolium reducing cells with the morphological characteristics of myelomonocytic or monocyte-like cells. An activity, co-chromatographing with DIF, acts synergistically with retinoic acid to induce maturation not only of HL-60, but also of the monoblast-like cell line U-937 (measured as percentage of cells reducing NBT).

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PURIFICATION OF "INHIBIN" FROM HUMAN OVARIAN FOLLICULAR FLUID

Acta Endocrinologica ◽

10.1530/acta.0.0900157 ◽

1979 ◽

Vol 90 (1) ◽

pp. 157-166 ◽

Cited By ~ 48

Author(s):

S. Chari ◽

C. R. N. Hopkinson ◽

E. Daume ◽

G. Sturm

Keyword(s):

Follicular Fluid ◽

Polyacrylamide Gel Electrophoresis ◽

Gel Filtration ◽

Deae Cellulose ◽

Exchange Chromatography ◽

Male Rats ◽

Acetone Powder ◽

Gel Chromatography ◽

Apparent Homogeneity ◽

Ovarian Follicular Fluid

ABSTRACT Following the earlier demonstration of inhibin-like activity in human ovarian follicular fluid a method for its purification to apparent homogeneity is described. The fluid was converted to acetone powder and subjected sequentially to ammonium sulphate fractionation, gel chromatography on Sephadex G-200, continuous gradient ion-exchange chromatography on DEAE-cellulose, first with a pH gradient from 8.0 to 4.0 and then with a NaCl gradient to 1 m at pH 5.2. The active fraction from this step was subjected to gel filtration on Sephadex G-100 and finally passed through an Amicon Centriflo membrane CF-25 (cut off point: 25 000 m.w.). The ultrafiltrate was homogeneous by SDS-polyacrylamide gel electrophoresis, had a molecular weight of the order of 23 000 and was capable of suppressing serum gonadotrophin levels in the castrated male rats in as low a dose as 25 μg/rat.

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