Biochemical and immunochemical study of antigen preparations of Rickettsia prowazekii

The biochemical and immunochemical study of the qualitative composition of R. prowazekii antigenic preparations, isolated and purified by different methods, indicates that these preparations contain high-molecular polypeptide 3 (133600 D), morphologically linked with the cell membrane of R. prowazekii. The method of adsorption chromatography on calcium phosphate permits obtaining specific rickettsial antigens with a greater degree of purification from ballast admixtures than the methods of acid precipitation and gel filtration on Sephadex G-200.     

A gel filtration study of the fractional composition of tetanus antitoxic sera

The fraction composition of tetanus antitoxic sera has been studied by the method of gel filtration in columns packed with Sephadex G-200. Differences in the fraction composition of preparations manufactured by different enterprises have been revealed. The specific activity of antitoxic sera has been found to depend on the degree of their fragmentation.     

Staphylococcal L-asparaginase: purification and properties of enzymic protein

Staphylococcal L-asparaginase has been purified 400-fold with 40% recovery. The procedure involves ammonium sulphate precipitation and a column chromatography on Sephadex G-200 gel filtration). The enzyme is composed of not identical subunits. protein (pI 4.4) with the approximate molecular weight of 125,000 (estimated by Sephadex G-200 gel filtration). The enzyme is composed of not identical subunits. The polyacrylamide-SDS gel electrophoresis indicated two subunits with molecular weight 18,000 and 22,000.     

Purification and chemical characterization of human hexosaminidases A and B.

N-Acetyl-beta-hexosaminidases A and B were purified to homogeneity from human placenta. In the initial step of purification, the enzymes were adsorbed on concanavalin A-Sepharose 4B and eluted from the column with alpha-methyl D-mannosides. Subsequent purification steps included DEAE-cellulose column chromatography, QAE-Sephadex [diethyl-(2-hydroxypropyl)aminoethyl-Sephadex] column chromatography, Sephadex G-200 gel filtration and preparative disc polyacrylamide-gel electrophoresis, followed by another QAE-Sephadex chromatography for the hexosaminidase A preparation, and DEAE-cellulose column chromatography, calcium phosphate gel chromatography, Sephadex G-200 gel filtration, QAE-Sephadex chromatography and CM-cellulose chromatography for the hexosaminidase B preparation. The purified preparations, particularly hexosaminidase A, had significantly higher specific enzyme activities than previously reported. The preparations moved on polyacrylamide-gel electrophoresis as single protein bands, which also stained for enzyme activity. Sedimentation-equilibrium centrifugation indicated homogenous dispersion of the enzymes, and the molecular weight was estimated as about 110000 for both enzymes. Complete amino acid and carbohydrate compositions of the two isoenzymes were determined, and, in contrast with previous suggestions, no sialic acid was found in the enzymes.     

Extraction of acid alpha-glucosidase from human spleen]

An efficient method for isolation of acid alpha-glucosidase from human spleen is developed. The method involves chromatography of the enzyme on rho-aminophenyl-alpha-D-glucopyranoside covalently bound to CH-Sepharose 4B, with subsequent gel filtration on Sephadex G-200. The enzyme was homogeneous by the polyacrylamide gel electrophoresis data; it was purified about 1500-fold, as compared with the crude extract (the total yield 12.5%). Besides acid alpha-glucosidase, the preparations of alpha-L-fucosidase, alpha-D-galactosidase and beta-N-acetylglucosaminidase were isolated and purified 200-, 130- and 280-fold, respectively. The nature of interaction between acid alpha-glucosidase and immobilized rho-aminophenyl-alpha-glucopyranoside is discussed.     

The choice of a method of isolating human IgG F(ab)2 fragments for determining antibodies to staphylococcal protein A]

The comparative study of two methods of the proteolysis of IgG with the aim of obtaining F(Ab)2-fragments from the blood sera of patients and healthy donors has been made. Different methods for the isolation of F (ab)2-fragments, such as gel filtration on Sephadex G-200, affinity chromatography on protein A-BrCN-sepharose, reprecipitation with zinc sulfate, have been analyzed. For further work the method of the peptic decomposition of whole serum with subsequent salting out with ammonium sulfate and gel filtration on Sephadex G-200 has been chosen.     

Rapid and efficient separation and identification of the two molecular forms of human adenosine deaminase by thin-layer gel filtration chromatography

Two molecular forms of adenosine deaminase have been found in human tissues. The column gel filtration method has been used for the separation of the two enzyme forms. Routine separation and analysis of the enzyme forms based on the molecular size difference can be achieved by thin-layer gel filtration on Sephadex G-200 superfine gel. The thin-layer method has been found to be more rapid and efficient than the column method. Enzymes in crude preparations can be studied effectively with the thin-layer method.     

Isolation and some properties of NAD+ reductase of the green photosynthetic bacterium Prosthecochloris aestuarii.

NAD+ reductase of the green photosynthetic bacterium Prosthecochloris aestuarii was isolated and purified by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and Sephadex G-200 gel filtration. This enzyme is an FAD-containing flavoprotein and has absorption maxima at 485 (shoulder0 452, 411, and 385 nm (the 411 nm band is due to cytochrome). The molecular weight of the enzyme as determined by gel filtration using Sephadex G-200 is 119,000. The enzyme catalyzes the reduction of NAD+ and NADP+ by photoreduced spinach ferredoxin or reduced benzyl viologen...     

Purification and Partial Characterization of Hypocalcemic Factor from Human Saliva

This study reports the isolation and partial purification of a polypeptide from human saliva which causes a significant serum calcium lowering when administered to mice. Purification was achieved by preparative electrophoresis, dialysis, two gel filtration steps on Sephadex G-150, and ion exchange chromatography on DEAE-cellulose. Homogeneity was determined by poly-acrylamide electrophoresis. Blood sampling was carried out by puncture of the orbital venous plexus and serum analyzed for calcium. The most active preparations lower serum calcium from 10–27% of initial value, producing tetany and convulsions in some cases. The molecular weight of this polypeptide was estimated to be 4, 260 by the use of a calibrated Sephadex G-75 column. This is a much smaller molecular weight than that expected from its initial exclusion from Sephadex G-150, and suggests that this hypocalcemic factor is associated with larger molecules through most of the purification procedure up to and including DEAE-cellulose chromatography. A second gel filtration on Sephadex G-150 separates two minor salivary protein contaminants (IgA and IgG immunoglobulin) in the excluded fraction from the smaller, hypocalcemically active polypeptide.

No hypocalcemia activity could be detected or isolated in a preliminary investigation on the saliva of a dysgammaglobuli-nemic (IgA deficient) patient.

The hypocalcemia induced does not differ significantly from that observed after administration of calcitonin to mice in that: 2) minimum values are reached in 1.5–2 hours and return to normal in 5–6 hours, b) magnitude of hypocalcemia response is dose dependent. The salivary hypocalcemia factor isolated in this study has the properties of a protein, in that its activity is destroyed by the proteolytic enzyme trypsin, it yields amino acids upon acid hydrolysis and it behaves on electrophoresis, gel filtration and ion exchange chromatography as a typical protein.     

An immunoglobulin preparation as a calibrating agent for the gel filtration method

The fractional composition of commercial immunoglobulin was studied by the method of gel filtration on ultragel AcA-34 and the possibility of its use for the calibration of a chromatographic column was shown. The fractionation of a specimen of IgG revealed the presence of 4 fractions. Their molecular weights corresponded to dimers, monomers, Fab fragments and low-molecular peptides. The qualitative fractional composition of the preparation was shown to be stable during 3 years of storage, as well as after keeping the preparation at 37 degrees C for 1 month. The attested characteristic of each IgG fraction, the distribution coefficient (Kav), was established. The use of a specimen of immunoglobulin obtained with the use of the modified Cohn's method--as calibrant will make it possible to calibrate the columns for a shorter period and to control the correctness of the analysis.     

Isolation and various characteristics of tryptophan deaminase from Proteus vulgaris

Tryptophan deaminase was isolated from Proteus vulgaris and purified. The procedure for enzyme purification included the cell destruction on USD-1, fractionation by ammonium sulphate, gel chromatography on ultragel AcA34, ion exchange chromatography on DEAE-cellulose. A degree of the enzyme purification--95, yield--5.7%. The pH optimum was 7.5, the temperature optimum--47 degrees C. The enzyme molecular weight (105 kD) was estimated by gel chromatography on Sephadex G-200, Km--5.0 mM in the K-phosphate buffer (pH 7.5). The SH groups are supposed to be present in the active site of the enzyme. The enzyme does not accelerate oxidation deamination of phenylalanine and tyrosine.     

Separation of lymphocyte mitogen from lymphotoxin and experiments on the production of lymphotoxin by lymphoid cells stimulated with the partially purified mitogen: a possible amplification mechanism of cellular immunity and allergy.

A mitogenic factor (MF) from guinea pig lymph node cells (LNC), which was produced by stimulating immune LNC with the specific antigen, ovalbumin, was partially purified by the sequential use of Sephadex G-200 gel filtration. CM-cellulose ion exchange chromatography, DEAE-cellulose ion exchange chromatography, and polyacrylamide disc gel electrophoresis. The product was purified at least 100-fold with regard to protein content. In addition, the purified MF was functionally pure with respect to the absence fo lymphotoxin (LT), another guinea pig lymphokine.     

Starch phosphorylase from tapioca leaves: Absence of pyridoxal phosphate

Starch phosphorylase from tapioca leaves has been purified to homogeneity, using the technique of ammonium sulfate fractionation, heat treatment, DEAE-cellulose chromatography, filtration through Sephadex G-100 and Sephadex G-200, and DEAE-Sephadex chromatography. The enzyme has a molecular weight of 450,000, as determined by gel filtration through Sephadex G-200 and contains 22 sulfhydryl groups per mole of the enzyme protein. Several types of evidence indicate the absence of pyridoxal 5′-phosphate as a prosthetic group of the enzyme. The kinetic data show a sequential type of the reaction mechanism. The enzyme activity is inhibited by tyrosine (K_i = 2.15 mm).     

Purification and properties of a fibrinolytic enzyme from Bacillus subtilis

A fibrinolytic enzyme obtained from B. subtilis was purified, using DEAE-cellulose column chromatography, and gel filtration on Sephadex G-100. The preparation was homogeneous as tested by gel filtration on Sephadex G-200, and disc electrophoresis. The molecular weight of this enzyme was 29.400 estimated by gel filtration on Sephadex G-100. The optimum pH for enzyme activity was 7.2 Copper ions significantly increased enzyme activity, while Zn++ and Mn++ caused marked inhibition.     

ACETYLCHOLINESTERASE IN MOUSE BRAIN, ERYTHROCYTES AND MUSCLE

Abstract— The properties of the enzyme acetylcholinesterase (EC 3.1.1.7) from mouse brain, erythrocytes and muscle were investigated. The enzymes were examined by gel filtration in Sephadex G-200, polyac-rylamide gel electrophoresis, immunological reactions, active-site labelling with tritiated di-isopropyl-phosphorofluoridate and also their kinetic properties were compared. All three enzymes appeared to have a single active small mol. wt. component of 80,000 to 82,000 which produced higher mol. wt. forms by aggregation. The partial purification of the enzyme from brain was achieved by affinity chromatography and this product was used to prepare antibodies. The purified immunoglobulin was shown to react with all three enzymes.     

小鼠腹水型肝癌H22a细胞浆胞内磷蛋白磷酸酶的纯化和性质

 磷蛋白磷酸酶是磷酸化/脱磷酸化作用中重要的调节酶。本文建立了小鼠腹水型肝癌细胞胞浆内磷蛋白磷酸酶(PrP)的纯化方法。用~(32)P-酪蛋白为底物测定活力。经纯化的酶纯度提高1380倍,聚丙烯酰胺梯度凝胶电泳检查,只有一条泳带。用凝胶过滤法和聚丙烯酰胺梯度凝胶电泳法测得该酶分子量为200,000。该酶催化~(32)P-酪蛋白脱磷酸化反应的最适pH7.2,对热不稳定。     

Antioxidant Properties of the Peptides Isolated From Ganoderma lucidum Fruiting Body

Ganoderma lucidum is widely used as traditional medicine for centuries particularly in China, Japan and Korea. Many bioactive metabolites isolated from G. lucidum were therapeutically active against various diseases. The peptide isolated from water extract of G. lucidum was purified by employing Sephadex G-25, Sephadex G-50 and reverse phase HPLC column chromatography. The antioxidant property of the peptide fractions was determined by various in vitro methods. All fractions obtained from Sephadex G-25 and fraction G from Sephadex G-50 are effective antioxidants and comparably fraction C has the highest antioxidant activity. The molecular weight of purified peptide determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration chromatography and Matrix-assisted laser desorption ionization time-of-flight-mass spectrometer was found to be 2.8, 3.34 and 3.35?kDa respectively. The amino acid composition of the peptide was rich in phenylalanine, aspartic acid, proline, histidine and isoleucine. Peptide isolated in the present investigation suggests that has beneficial antioxidant properties may be due to its low molecular weight and specific amino acid composition.     

Isolation and characterization of calcitonin from pericardium and esophagus of eel.

Calcitonin was extracted from the pericardium and esophagus of eel in quantities sufficient to permit purification and chemical characterization. Homogeneous calcitonin could be isolated by a six-step fractionation starting from acetone powder of the organs. The fractionation procedure consisted of acid extraction, gel filtration on Sephadex G-75, chromatography on SP-Sephadex C-25, gel filtration on the Sephadex G-50, chromatography on carboxymethylcellulose, and gel filtration on Sephadex G-50. Fractionation of the hormone was monitored by assay of its biological activity and from its behaviour on thin layer chromatography and polyacrylamide gel disc electrophoresis. The hormone contained 32 amino acid residues, like calcitonins from other species of animals, but its amino acid composition was different from those of previously characterized hormones. Eel calcitonin possessed almost the same, or higher, biological activity as the salmon or chicken hormone, which show the highest specific activity among calcitonins so far isolated.     

A comparative study of horseradish peroxidase conjugates prepared with a one-step and a two-step method.

In this study we compared horseradish peroxidase (HRP)-labeled rabbit antihuman immunoglobulin G (IgG) conjugates, prepared by a one-step and a two-step method. Glutaraldehyde was used as a cross-linking agent. Two methods were used for removing unconjugated HRP: Sephadex G-200 gel chromatography and ammonium sulfate precipitation. The conjugates were characterized immunologically, immunochemically and enzymatically. The immunohistoenzymic properties of the conjugates were tested on unfixed cryostat sections of the skin of patients with chronic discoid lupus erythematosus. The influence of the presence of unconjugated HRP and unconjugated IgG was studied. Optimal results were obtained with conjugates prepared by a two-step method. Removing unconjugated HRPimproved the immunohistoenzymic properties of the conjugates. Conjugated and unconjugated IgG could be separated by Sephadex G-200 gel chromatography.     

An oviducal enzyme isolated by affinity chromatography which acts upon the vitelline envelope of Bufo arenarum coelomic oocytes.

A trypsin-like oviducal proteinase acting upon the vitelline envelope of Bufo arenarum coelomic oocytes has been purified to apparent homogeneity by gel filtration on Sephadex G-200 and by affinity chromatography on a column of Sepharose 4-B containing covalently bound concanavlin A (Con A). The biologically active molecule migrated as a single band of protein upon SDS polyacrylamide gel electrophoresis.