Isolation of allergenically active glycoprotein from Prosopis juliflora pollen

An allergenically active glycoprotein was homogeneously isolated from the aqueous extract of Prosopis juliflora pollen by ConA-Sepharose affinity chromatography. The molecular weight of this glycoprotein was 20,000 dalton, determined by gel filtration and SDS-PAGE. This fraction showed a total carbohydrate concentration of 25%. The purified glycoprotein revealed immunochemically most antigenic or allergenic and demonstrated homogeneous after reaction with P. juliflora pollen antiserum, characterized by gel diffusion, Immunoelectrophoresis and Radioallergosorbent test.     

Isolation and characterization of two antigenic glycoproteins from the pollen of Prosopis juliflora

Two antigenically active glycoprotein fractions were isolated from crude extract of the pollen of Prosopis juliflora using DEAE-cellulose ion exchange chromatography. The glycoproteins gave single band on polyacrylamide gel electrophoresis. The molecular weight of these two glycoprotein was 20,000 and 10,000 as determined by gel filtration on Sephadex G-75. With the help of crossed immunoelectrophoresis and gel diffusion crude extract exhibited twelve and three precipitating antigens suggesting its heterogeneous nature; and the purified glycoprotein fractions however formed single precipitin band on gel diffusion test and immunoelectrophoresis. As tested by ELISA the polyclonal antisera raised in rabbit showed strong binding affinity with glycoprotein of MW 20,000. These result indicates that the two glycoprotein fractions are not antigenically identical.     

Purification and characterization of the glycoprotein allergen from Prosopis juliflora pollen

Highly active glycoprotein allergens have been isolated from pollen of Prosopis juliflora by a combination of Sephadex G-100 gel filtration and Sodium dodecyl sulphate-Poly-acrylamide gel electrophoresis. The glycoprotein fraction was homogeneous, and had molecular weight 20,000. The purified glycoprotein allergen contained 20% carbohydrate, mainly arabinose and galactose. Enzymatic digestion of glycoprotein with protease released glycopeptides of molecular weight ranging from less than 1,000 to more than 5,000 on Sephadex G-25 gel filtration. Antigenicity or allergenicity testing of these glycopeptides by immunodiffusion, immunoelectrophoresis, and radioallergosorbent test indicated complete loss of allergenic activity after digestion with protease whereas incubation with beta-D-galactosidase and periodate oxidation had little affect on the allergenic activity of the glycoprotein fraction. But incubation with alpha-D-glucosidase did not affect the allergenic activity significantly. All these tests indicated that protein played significant role in allergenicity of P. juliflora pollen.     

Prosopis juliflora pollen allergen induced hypersensitivity and anaphylaxis studies in guinea pigs

In vivo and in vitro allergenic activities of Prosopis juliflora pollen allergens were measured in guinea pigs. Intracutaneous skin test showed an early wheal flare response and a late erythema-redness, sensitized with various concentrations (100, 50, 25, 5 and 1.5 micrograms/ml) of Prosopis juliflora pollen extract after administration of a challenging dose. A 50 micrograms/ml sensitizing dose of Prosopis juliflora pollen allergen gave optimum skin response as both early and late effects. The nature of immunochemical reactivity between pollen allergens and reaginic antibodies were further characterized by histamine release test, gel diffusion test, radioallergosorbent test and passive cutaneous anaphylaxis test. These tests confirm allergenicity caused by Prosopis juliflora pollen allergens and showed the binding of allergens with reaginic antibody and its regulation in guinea pigs.     

Isolation and characterization of allergens of Prosopis juliflora pollen grains

Proteins and glycoproteins from Prosopis juliflora (Pj) pollen grains were separated by gel filtration, electrophoresis, DEAE cellulose chromatography and their molecular weight was determined by gel filtration and SDS-Polyacrylamide gel electrophoresis. The allergenic activity of different fractions were evaluated by in vivo skin prick test and in vitro gel diffusion test. It was found that fraction E of gel filtration and fraction III and IV of DEAE cellulose chromatography were most allergenic. This fraction E of gel filtration showed positive reaction with periodic acid Schiff's reagent as determined by SDS-gel electrophoresis.     

Inhibition of DNA polymerase alpha activity by proteins from rat liver

We determined that there is a protein in rat liver capable of inhibiting DNA polymerase alpha. To assay for this inhibitor, DNA polymerase alpha was purified from R3230AC rat mammary tumor, a rich source of this enzyme. Protein fractions from Sephacryl S200 gel filtration of total soluble liver extract showing inhibition of DNA polymerase alpha were further chromatographed on DEAE-cellulose. This step revealed two inhibitor protein populations with the major form corresponding to a molecular weight of 143,000 dalton. Soluble extract from isolated rat liver nuclei also showed the presence of at least two inhibitors; the major form was 200,000+ dalton in molecular weight. Both the 143,000 and 200,000+ dalton inhibitor proteins were capable of inhibiting the R3230AC tumor DNA polymerase alpha in a dose-dependent manner. These inhibitors exhibited similar inhibition of nuclear matrix-associated DNA polymerase alpha from either the R3230AC tumor or from regenerating rat liver.     

Immunological study of cellular components of Pseudomonas aeruginosa. IV. Fractionation of Pseudomonas aeruginosa mucus by diafiltration and biological properties of isolated fractions]

In fractionation of Pseudomonas aeruginosa mucus (strains No. 8 and 1463) by means of diafiltration on the system of membranes Diaflo XM-300, XM-100A, PM-30, and PM-10 there was obtained a successive series of fractions differing by the molecular weight and chemical composition. According to the results of gel chromatography fractions with the mol wt of 100000 dalton and over apparently represented protein-polysaccharide components of mucus in the form of complexes; fractions with the mol wt of 30000 dalton and lower contained a considerable amount of free protein along with the protein-polysaccharide complex. The fractions obtained differed by biological properties: fractions with the mol wt of 100000 dalton and over were toxic for mice and possessed weak antigenic properties in the precipitation in agar test and immunoelectrophoresis; fractions with the mol wt lower than 30000 dalton expressed in the mentioned test distinct antigenic properties and proved to be practically nontoxic for mice. Thus the use of diafiltration method permitted to separate the antigenic, weakly toxic component of Ps. aeruginosa mucus from the toxic factor with weak antigenic properties.     

Cardiac myosin from pig heart ventricle. Purification and enzymatic properties.

A method is described for the preparation of high purity myosin from the left ventricle of pig heart. The purified myosin was free from nucleic acid, actin, tropomyosin, troponin, the 150,000 molecular weight protein and other contaminants. Analyses of subunits in the purified myosin were carried out on 3.5% acrylamide gel with 0.1% SDS. Of the total protein present in myosin, 11.3% was in the light chains; light chain 1 (LC1), 5.9% and light chain 2 (LC2), 5.4%. Urea gel electrophoresis of the purified myosin showed three closely spaced bands corresponding to the 20,000 dalton, the charge-modified 20,000 dalton and the phosphorylated 20,000 dalton components. The properties of the Ca2+-activated and K+-activated ATPases [EC 3.6.1.3] of the purified myosin were also studied. The Km values were 27 and 55 muM and the Vmax values were 0.263 and 0.317 mumole P1/mg/min for the Ca2+-activated and K+-activated ATPases, respectively. The pH-activity profiles and the effects of SH modification were of the skeletal myosin type except that the activities were lower.     

Green Synthesis of Bimetallic Nanoparticles From Prosopis juliflora (Sw) DC., and Its Effect Against Cotton Mealybug,Phenacoccus solenopsis (Hemiptera: Pseudococcidae)

Phenacoccus solenopsis has been recognized as an aggressively invasive species on cotton plants in different countries. This study was conducted to investigate the effect of green synthesized Cu/Zn-nanoparticles using aqueous leaf extract of Prosopis juliflora (mezquite) against P. solenopsis. The scanning electron microscope (SEM) showed bimetallic nanoparticles of Cu/Zn-NPs with spherical shape with varying size of 74.33 nm to 59.46 nm. More than 30% mortality of P. solenopsis was observed with Cu/Zn-nanoparticles (100 ppm) at 96 hours after treatment. Negligible mortality of P. solenopsis was recorded with Cu/Zn solution (100 ppm) and aqueous P. juliflora extracts. The results of the viability test for Cu/Zn-nanoparticles of P. juliflora showed a significant reduction of the cell viability by 50% in insect exposed to Cu/Zn-nanoparticles-P.juliflora. Therefore studies about nanotoxicity of Cu/Zn-NPs of P.juliflora are needed to reveal the mechanism of toxicity this phytonanoparticles in P.solenopsis.     

Purification and characterization of allergens from Xanthium strumarium pollen

The allergenic components present in whole pollen extract of Xanthium strumarium were isolated by sequential ammonium sulphate precipitation, DEAE Sephadex A50 chromatography and gel filtration. The techniques of RAST inhibition and skin test were utilized to check the allergenicity of fractionated proteins revealing the presence of Xan Ib and Xan VIa as the important allergenic components. Xan Ib was found to be devoid of carbohydrate and had a molecular weight of 103,000 daltons. Xan VIa was a glycoprotein of molecular weight 17,000 daltons. The carbohydrate moiety of Xan VIa was found to be associated with allergenicity. The characteristic pattern of whole pollen extract on CIE and TLIEF showed 36 and 21 protein bands, respectively. The use of FPLC in isolation of partially purified allergens from Xanthium is discussed.     

Serotonin induced protein phosphorylation in the Aplysia eye

Serotonin (5-HT) increases the phosphorylation of two low molecular weight phosphoproteins of 23,000 and 15,000 daltons molecular weight and decreases the phosphorylation of a 20,000 dalton phosphoprotein in the isolated Aplysia eye. The cAMP analog 8-benzylthio cAMP increases and decreases the phosphorylation of the 23,000 and 20,000 dalton 5-HT sensitive phosphoproteins, respectively. The effect of 5-HT on protein phosphorylation is not affected by the phase of the circadian rhythm of spontaneous compound action potentials generated in the eye.     

Purification and characterization of allergens from Xanthium strumarium pollen

The allergenic components present in whole pollen extract of Xanthium strumarium were isolated by sequential ammonium sulphate precipitation, DEAE Sephadex A50 chromatography and gel filtration. The techniques of RAST inhibition and skin test were utilized to check the allergenicity of fractionated proteins revealing the presence of Xan Ib and Xan VIa as the important allergenic componenets. Xan Ib was found to be devoid of carbohydrate and had a molecular weight of 103 000 daltons. Xan VIa was a glycoprotein of molecular weight 17 000 daltons. The carbohydrate moiety of Xan Vla was found to be associated with allergenicity. The characteristic pattern of whole pollen extract on CIE and TLIEF showed 36 and 21 protein bands, respectively. The use of FPLC in isolation of partially purified allergens from Xanthium is discussed.     

Incorporation of the Ca2+ -binding component (g2) into heavy meromyosin and subfragment-1 of pig cardiac myosin.

A new protein component was found in heavy meromyosin and in subfragment-1 (S-1) prepared by chymotrypsin digestion of pig cardiac myosin in the presence of Ca2+. The molecular weight of this protein was estimated as 15,000 dalton. It was able to bind Ca2+ and showed a similar UV absorption spectrum to that of the g2 light chain. Heavy meromyosin and subfragment-1 which contained the 15,000 dalton component incorporated exogenous g2 and the 15,000 dalton component disappeared after such treatment. We concluded that the 15,000 dalton component was produced from g2 by limitted proteolysis. The subfragment-1 was separated into two protein fractions in equal yield by recycling the gel filtration. One contained the 15,000 dalton component and was able to bind Ca2+ while the other did not contain the component and was unable to bind Ca2+. According to analysis by SDS gel electrophoresis, the large polypeptide chain (the f component) of the first S-1 was approximately 5,000 dalton larger than the f component of the second S-1. The polypeptide corresponding to 5,000 dalton was designated polypeptide-C, because it was released from the C terminal of the f component. It seems to be essential for the attachment of the Ca2+-binding light chain g2. The location of g2 in myosin may thus be at the polypeptide-C which links the head to the tail of myosin.     

Changes in the electroencephalographic and gamma-aminobutyric acid transaminase and succinic semialdehyde dehydrogenase in the allergen induced rat brain

Electroencephalographic activity and gamma-Aminobutyric acid Transaminase together with Succinic semialdehyde dehydrogenase activity changes produced by sensitization with Prosopis juliflora pollen allergen were studied in the cerebral cortex and hypothalamus of the rat brain. Electrical activity of EEG recording begins to appear on 3rd day after sensitization with maximum increase in activity was found on day 9 and decreased after that. A sudden increase in electrical activity was produced in 9th day sensitized rat with 10 min after giving challenging dose intravenously. The measurement of enzymatic activity of GABA-T and SSA-DH showed decrease and increase in 3, 9, 15 and 30 days sensitized rat hypothalamus and cerebral cortex whole homogenate and mitochondrial fractions. A maximum changes in enzymatic activity was found in 9th day sensitized rat with significant alterations after giving sudden stress as challenging dose. These changes in EEG activity and GABA-ergic neurotransmitter in allergenic rats showed the immunoregulatory role of nervous system mediated via GABA shunt.     

Absence of a 23 kd protein in testes of testicular feminization rat

Cryptorchid testes of testicular feminization rats are very low in zinc in spite of normal zinc status of the animals. Analysis of the cytosol of the cryptorchid testes by gel permeation chromatography showed decreased zinc binding by proteins eluted at fractions corresponding to 30,000 dalton. Further analysis by sodium dodecylsulphate polyacrylamide gel electrophoresis indicated the absence of a protein with molecular weight of 23,000.     

Reconstitution of the sodium channel with partially solubilized lobster nerve membrane

Reconstitution experiments were carried out with particles obtained from lobster nerve plasma membrane preparations by detergent treatment, differential centrifugation and ammonium sulfate fractionation. The NA channel activity of the three fractions obtained, which have different amounts of the same peptides present in the original membrane, appears related to their content in a large component which does not enter the 9% polyacrylamide gel and in peptides with 220,000 and 110,000 apparent molecular weight. Other reconstitution experiments made with two fractions obtained by detergent treatment, differential centrifugation and gel exclusion chromatography, revealed that the Na channel active fraction contains the material which does not enter the gel in addition to the 220,000 and 110,000 molecular weight peptides. The other fraction was inactive and does not contain those components. The 220,000 dalton peptide has a molecular weight similar to those determined for the tetrodotoxin-saxitoxin receptor and the scorpion toxin receptor of the Na channel. Whether any of the other peptides is a Na channel constituent is unknown at present.     

Characterization of prolamellar bodies, from dark-grown seedlings of Scots pine, containing light- and NADPH-dependent protochlorophyllide oxidoreductase

Cotyledons of conifers have a light-independent pathway for chlorophyll biosynthesis. To investigate whether the prolamellar body of Scots pine ( Pinus sylveslris L.) is similar to the better known prolamellar body of wheat, etioplast membrane fractions were isolated from cotyledons of dark-grown Scots pine. Dark-grown cotyledons contained both chlorophyll and protochlorophyllide, 158 and 10 nmol (g fresh weight)'respectively, and had a chlorophyll a to b ratio of 4.2. The content of glyco- and phospholipids was 7.1 μmol (g fresh weight)¹. About 40 mol % of these lipids were the specific plastid lipids – monogalactosyl diacylglycerol. digalactosyl diacylglycerol and sulfoquinovosyl diacylglycerol in the relative amounts 50, 35 and 7 mol %. The mol ratio of monogalactosyl diacylglycerol to digalactosyl diacylglycerol was 1.7. Low temperature fluorescence emission spectra of intact cotyledons and homogenate showed maxima at 633, 657, 686, 696 nm and a broad peak at 725–735 nm. The maxima at 633 and 657 nm represented different forms of protochlorophyllide and the other emission maxima represented chlorophyll protein complexes. The 657 nm form of protochlorophyllide was phototransformable both in vivo and in the isolated membranes. The phototransformable protochlorophyllide was substantially enriched in the prolamellar body fraction.
The specific activity of light dependent protochlorophyllide oxidoreductase in the prolamellar body fraction was found to be 2 nmol chlorophyllide formed [(mg protein)⁻¹ min⁻¹]. The molecular weight of the enzyme polypeptide was determined as 38 000 dalton with sodium dodecylsulphate-polyacrylamide gel electrophoresis.     

Analysis of Histones from the Endosymbiont Nucleus of a Binucleate Dinoflagellate1,2

Histones were prepared from chromatin of the eukaryotic (endosymbiont) nucleus of Peridinium balticum (Levander) Lemmermann. The amino acid composition of whole histone was rich in lysine and similar to that of Olisthodiscus luteus and Euglena gracilis. Electropheretic analysis of these proteins in acidic-urea disc gels revealed four major bands: one with a mobility slightly lower than that of calf thymus HI; and three others which comigrated with calf H2B, H2A, and H4, respectively. The low mobility band was soluble in 5% perchloric acid and was sensitive to FeCl₃ destaining. Electrophoresis in slab gels containing 0.1% SDS revealed five major components, with approximate molecular weights of 23,000, 20,000, 15,000, 13,000, and 11,000, respectively. The 15,000 and 11,000 dalton histones had mobilities identical to those of calf H3 and H4, respectively. The two highest molecular weight components were soluble in 5% perchloric acid. No bands were found to comigrate with calf H2A or H2B but a band was present that migrated to a position intermediate between calf H2A and H4 (13,000 dalton histone). Two-dimensional gels consisting of acidic-urea gels in the first dimension and SDS gels in the second dimension revealed that the 20,000 dalton component and the 13,000 dalton component are not resolved in the acidic-urea gel. As a working hypothesis, it is suggested that two of the five bands seen in SDS gels represent an H1-like doublet, and two are analagous to H3 and H4, respectively. The remaining histone may replace H2A and H2B.     

Low molecular weight renin as a storage form in renin granules of the dog

The molecular weight of renin extracted from isolated renin granules of the dog was estimated by gel filtration, using tetradecapeptide as substrate, and was approximately 43,000 daltons. Neither big renin nor big big renin was demonstrable. On the other hand, crude extract of kidney cortex showed angiotensin I generating enzymes other than 43,000 dalton form of renin, whose molecular weight were over 100,000 and around 70,000 daltons. They seemed nonspecific proteases, since they hydrolyzed tetradecapeptide but not plasma angiotensinogen. Therefore renin is stored in the renin granules as a low molecular weight form.     

Nitrile hydratase of Brevibacterium R312--purification and characterization

Nitrile hydratase was purified and crystallized from the crude extract of Brevibacterium R312 and found to be homogeneous by the results of disc gel electrophoresis, analytical ultracentrifuge and double diffusion in agarose. The enzyme has a molecular mass of about 85,000 Da and contains approximately 3 g atoms iron/mol enzyme. The enzyme was composed of two kinds of subunits, of which molecular masses were 26,000 Da and 27,500 Da. The concentrated solution of the enzyme had a pronounced greyish green color and exhibited a broad absorption in visible range with a absorption maxima at 712 nm. The enzyme was active toward various aliphatic nitriles.