ChemInform Abstract: ISOLATION OF TRANSFORMING DNA FROM BACILLUS SUBTILIS BY GEL FILTRATION ON SEPHAROSE 4B

Intracellular serine proteinase was isolated from sporulating cells of Bacillus subtilis Marburg 168 by gramicidin S-Sepharose 4B affinity chromatography. The enzymological characteristics, the amino acid composition and the 19 residues of the N-terminal sequence of the enzyme are reported. The isolated proteinase was closely related to, but not completely identical with, the intracellular serine proteinase of B. subtilis A-50. The divergence between these two intracellular enzymes was less than that between the corresponding extracellular serine proteinases (subtilisins) of types Carlsberg and BPN′!, produced by these bacterial strains. This may be connected with the more strict selection constraints imposed in intracellular enzymes during evolution.

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HUMAN AMYLOID PROTEIN: CHEMICAL VARIABILITY AND HOMOGENEITY

Journal of Histochemistry & Cytochemistry ◽

10.1177/19.1.1 ◽

1971 ◽

Vol 19 (1) ◽

pp. 1-15 ◽

Cited By ~ 62

Author(s):

M. HARADA ◽

C. ISERSKY ◽

P. CUATRECASAS ◽

D. PAGE ◽

H. A. BLADEN ◽

...

Keyword(s):

Amino Acid ◽

Amyloid Fibril ◽

Gel Filtration ◽

Protein S ◽

Amyloid Protein ◽

X Ray Diffraction ◽

Amyloid Proteins ◽

X Ray ◽

Chemical Variability ◽

Sepharose 4B

The morphology of the fibril of amyloid derived from different individuals is similar, but occasionally significant differences are noted. All human amyloid filaments have a "β-pleated sheet" conformation as revealed by x-ray diffraction, and those examined after orientation show a "cross-β" pattern. All amyloid fibril concentrates studied so far can be fractionated to obtain the major amyloid protein component(s) by sequential gel filtration with 5 M guanidine-HCl in 1 N acetic acid on Sepharose 4B and Sephadex G-100 or G-75 columns with the removal of over 28% of proteins representing minor constituents. The major amyloid protein(s) obtained from the spleen and/or liver of six patients is found to contain tryptophan, to be deficient in hydroxylysine and hydroxyproline and usually at least one commonly occurring amino acid and to have a high content of dicarboxylic acid and short chain amino acids and unreactive (blocked) NH2-terminal groups or aspartic acid-asparagine (Asx). However, the amyloid protein(s) from each individual differs from that of the others in molecular weight, in amino acid composition and in the presence or absence of specific tryptic peptides. Amyloid protein(s) from the liver and spleen of the same individual is identical. No chemical characteristics distinguish amyloid proteins derived from cases classified clinically as "primary" from those classified as "secondary." There is a striking chemical similarity between amyloid proteins and the NH2-terminal variable fragment of the light and heavy chain of immumoglobulin proteins.

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Purification Of Vasoactive Peptides From Human Fibrinogen Degraded By Human Leucocyte Elastase

10.1055/s-0038-1652102 ◽

1981 ◽

Author(s):

R Wallin ◽

M Belew ◽

K Ohlsson ◽

T Saldeen

Keyword(s):

Affinity Chromatography ◽

Vascular Permeability ◽

Gel Filtration ◽

Molar Ratio ◽

Significant Activity ◽

Small Peptides ◽

Human Fibrinogen ◽

Leucocyte Elastase ◽

Pm 10 ◽

Sepharose 4B

The presence of leucocytes around extravascular fibrin deposits suggests that the leucocyte elastases might be partly responsible for the extravascular degradation of fibrin. Our previous studies have shown that the degradation of fibrin(ogen) by plasmin leads to the release of 2 small peptides which markedly increase vascular permeability and induce oedema e.g. in the lungs. The results of this investigation show that small peptides released from fibrinogen after degradation by leucocytes elastases also increase vascular permeability.Human fibrinogen (Kabi, Grade L) was made plasminogenfree by affinity chromatography on Lysine-Sepharose 4B prior to use. The human leucocyte elastases were isolated from extracts of lysosome-like granules of human leukaemic myeloid cells by a combination of gel filtration, affinity chromatography and preparative agarose gel electrophoresis. The fibrinogen (0.5 %) and the leucocyte elastases (in a molar ratio of 100:1) were incubated together for 48 h at +37°C and at pH 8.5. The mixture was then cooled to +4°C to stop the lysis and ultrafiltrated on a DIAFLO PM 10 membrane until the retentate was approximately 10 % of the starting volume. The peptides in the diffusate accounted for about 20 % of the starting material as estimated from absorbance measurements at 280 nm. The diffusate was concentrated by lyophilization and fractionated by chromatography on a column of Bio-Gel P-6. At least 8 fractions were obtained of which only two showed a significant activity in their ability to increase vascular permeability in rat skin. The active peptides in these two fractions were further purified to homogeneity by column zone electrophoresis at various pHs and their amino acid compositions established.

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IDENTIFICATION AND PURIFICATION OF A NEW PROTEIN FROM WATER-SOLUBLE EXTRACTS OF HUMAN ADRENAL GLAND

Journal of Endocrinology ◽

10.1677/joe.0.0820383 ◽

1979 ◽

Vol 82 (3) ◽

pp. 383-NP ◽

Cited By ~ 1

Author(s):

M. A. AL-AWQATI ◽

Y. B. GORDON ◽

T. CHARD

Keyword(s):

Molecular Weight ◽

Adrenal Gland ◽

Gel Filtration ◽

Water Soluble ◽

Double Immunodiffusion ◽

Molecular Weights ◽

Physicochemical Methods ◽

Two Peaks ◽

Sepharose 4B ◽

New Protein

An homogenate of human foetal adrenal gland was subjected to negative immunoabsorption by column chromatography using anti-whole human serum coupled to Sepharose 4B. Two peaks were eluted and used to immunize rabbits. The antisera produced were absorbed and tested for specificity by double immunodiffusion. Two antigens, which appeared to be specific to the adrenal gland, were identified having molecular weights of 25 000 and 65 000 as determined by gel filtration. The lower molecular weight antigen was isolated by physicochemical methods and found to be a protein. The amino acid composition is reported.

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Chromatography of transforming deoxyribonucleic acid on methylated albumin and by gel filtration

Biochemical Journal ◽

10.1042/bj1370543 ◽

1974 ◽

Vol 137 (3) ◽

pp. 543-546

Author(s):

G. R. Barker ◽

P. Hodges

Keyword(s):

Molecular Weight ◽

Bacillus Subtilis ◽

High Molecular Weight ◽

Deoxyribonucleic Acid ◽

Degradation Products ◽

Gel Filtration ◽

Agarose Gel ◽

Transforming Activity ◽

Different Strains ◽

Two Peaks

1. Native DNA from two strains of Bacillus subtilis was chromatographed by stepwise elution from MAK (methylated albumin on kieselguhr). 2. Transforming activity was confined to two out of the three main fractions, activity being distributed between the two peaks differently for DNA from the different strains. 3. Fractionation of DNA from both strains on 2% agarose gel gave two components. Approx. 75% of the material was eluted within the void volume of the column. Approx. 25% of the material consisted of degradation products of lower molecular weight. 4. Chromatography on MAK of the material of high molecular weight eluted from agarose gel gave a number of peaks differing in molecular weight, indicating that degradation of the DNA takes place during chromatography on MAK. 5. The distribution of transforming activity among the fractions from MAK suggests that degradation occurs preferentially in certain regions of the DNA.

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Extra Purified Exosomes from Human Placenta Contain an Unpredictable Small Number of Different Major Proteins

International Journal of Molecular Sciences ◽

10.3390/ijms20102434 ◽

2019 ◽

Vol 20 (10) ◽

pp. 2434 ◽

Cited By ~ 8

Author(s):

Evgeniya E. Burkova ◽

Alina E. Grigor’eva ◽

Dmitrii V. Bulgakov ◽

Pavel S. Dmitrenok ◽

Valentin V. Vlassov ◽

...

Keyword(s):

Interleukin 1 ◽

Gel Filtration ◽

Normal Pregnancy ◽

Maldi Mass Spectrometry ◽

2D Electrophoresis ◽

Cytoplasmic Actin ◽

Sds Page ◽

Two Peaks ◽

Sepharose 4B ◽

Different Sources

Exosomes are nanovesicles (30–100 nm) containing various RNAs and different proteins. Exosomes are important in intracellular communication, immune function, etc. Exosomes from different sources including placenta were mainly obtained by different types of centrifugation and ultracentrifugations and were reported to contain from a few dozen to thousands of different proteins. First crude exosome preparations from four placentas (normal pregnancy) were obtained here using several standard centrifugations but then were additionally purified by gel filtration on Sepharose 4B. Individual preparations demonstrated different gel filtration profiles showing good or bad separation of exosome peaks from two peaks of impurity proteins and their complexes. According to electron microscopy, exosomes before gel filtration contain vesicles of different size, ring-shaped structures forming by ferritin and clusters of aggregated proteins and their complexes. After filtration through 220 nm filters and gel filtration exosomes display typically for exosome morphology and size (30–100 nm) and do not contain visible protein admixtures. Identification of exosome proteins was carried out by MS and MS/MS MALDI mass spectrometry of proteins’ tryptic hydrolyzates after their SDS-PAGE and 2D electrophoresis. We have obtained unexpected results. Good, purified exosomes contained only 11–13 different proteins: CD9, CD81, CD-63, hemoglobin subunits, interleukin-1 receptor, annexin A1, annexin A2, annexin A5, cytoplasmic actin, alkaline phosphatase, serotransferin, and probably human serum albumin and immunoglobulins. We assume that a possible number of exosome proteins found previously using crude preparations may be very much overestimated. Our data may be important for study of biological functions of pure exosomes.

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Structural studies on the glycoproteins from bovine cervical mucus

Biochemical Journal ◽

10.1042/bj1730941 ◽

1978 ◽

Vol 173 (3) ◽

pp. 941-947 ◽

Cited By ~ 10

Author(s):

G P Roberts

Keyword(s):

Physical Properties ◽

Gel Electrophoresis ◽

Gel Filtration ◽

Cervical Mucus ◽

Cysteine Residues ◽

Structural Studies ◽

Disulphide Bonds ◽

Structural Differences ◽

Sepharose 4B

The depolymerization of bovine cervical glycoprotein resulting from cleavage of disulphide bonds. Pronase digestion and both procedures sequentially was assessed by using gel filtration. Cleavage of disulphide bonds followed by Pronase digestion produced more extensive depolymerization than did either treatment alone, and gel filtration of the products resulted in two major peaks of glycosylated material on Sepharose CL-2B and Sepharose 4B. The glycopolypeptides in both peaks had similar sugar and sulphate compositions, but they migrated to different extents on gel electrophoresis. Electrophoretic studies indicated that both glycopolypeptides were derived from the same glycoprotein molecule and not from a mixture of two similar glycoproteins. Pronase digestion of glycoproteins in which the disulphide bonds had been labelled with iodo-[1-14C]acetamide revealed that most of the cysteine residues were situated in regions susceptible to Pronase. The results show the presence of two types of structural regions in bovine cervical glycoprotein, namely ‘naked’ peptide or non-glycosylated regions and glycopolypeptide subunit regions in which glycopolypeptides of two different sizes predominate. Comparison of the cervical glycoproteins isolated from mucus secreted during oestrus and pregnancy, by the methods outlined above, did not reveal any structural differences in the glycoproteins to explain the different physical properties of the mucus secreted under these conditions.

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Purification and Characterization of Kallikrein from Plasma of Patients with Acute Pancreatitis

Clinical Science ◽

10.1042/cs0600199 ◽

1981 ◽

Vol 60 (2) ◽

pp. 199-205 ◽

Cited By ~ 6

Author(s):

Naotika Toki ◽

Hiroyuki Sumi ◽

Sumiyoshi Takasugi

Keyword(s):

Acute Pancreatitis ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Apparent Molecular Weight ◽

Purification And Characterization ◽

Α2 Macroglobulin ◽

Sepharose 4B

1. A kallikrein-like enzyme in plasma of patients with acute pancreatitis was further purified by successive hydroxyapatite/cellulose and Sepharose-4B column chromatography. 2. By these procedures 0.26 mg of purified enzyme with a specific activity of 215 S-2266 chromozyme units/mg of protein was obtained from 10 ml of original plasma. 3. The purified material was homogeneous as ascertained by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and had an apparent molecular weight of 31 000 as measured by gel filtration on Sephadex G-200. 4. It was confirmed immunologically that this enzyme was pancreatic kallikrein, which is distinct from plasma kallikrein, and that it could combine with α2-macroglobulin only in the presence of trypsin.

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Carcinoembryonic antigen-like substances of human urothelial carcinomas. Isolation of components from pathological urine and comparison with colorectal carcinoma antigens

Biochemical Journal ◽

10.1042/bj1390431 ◽

1974 ◽

Vol 139 (2) ◽

pp. 431-440 ◽

Cited By ~ 9

Author(s):

R. Nery ◽

A. L. Barsoum ◽

H. Bullman ◽

A. M. Neville

Keyword(s):

Carcinoembryonic Antigen ◽

Gel Filtration ◽

Molecular Size ◽

Density Gradient Ultracentrifugation ◽

Colorectal Carcinomas ◽

Net Charge ◽

Molecular Weights ◽

Urothelial Carcinomas ◽

Sepharose 4B ◽

Mean Molecular Weight

Urines of several patients with urothelial carcinomas contain inhibitors of the immunoreaction between carcinoembryonic antigen derived from human colorectal carcinomas and monospecific goat antiserum raised against the antigen. These inhibitors range in approximate molecular weights from less than 1000 to several millions, and two have been isolated by a combination of extraction, gel filtration and electrophoretic procedures. These are respectively a macromolecular aggregate, component UCEA-3, which is excluded by Sepharose 4B, and a glycoprotein(s) component, UCEA-1, with mean molecular weight (2×105) similar to that of carcinoembryonic antigen. Comparison of the properties of component UCEA-1 and carcinoembryonic antigen on gel filtration, electrophoresis, immunoelectrophoresis and density gradient ultracentrifugation indicates that these substances of similar molecular size and net charge differ in some immunochemical properties.

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