Pheromaxein, the pheromonal steroid-binding protein, is a major protein synthesized in porcine submaxillary salivary glands

1991 ◽  
Vol 128 (2) ◽  
pp. 205-212 ◽  
Author(s):  
W. D. Booth ◽  
K. I. von Glos
Keyword(s):  
Total Protein ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Submaxillary Gland ◽  
Major Protein ◽  
Sds Page ◽  
Gland Tissue ◽  
Steroid Binding ◽  
Steroid Binding Protein

ABSTRACT Submaxillary salivary gland tissue from large White, Göttingen miniature and Meishan (Chinese) breeds of pig, and European wild boars, was incubated with [35S]methionine. The radiolabelled amino acid was incorporated into protein in all incubations as demonstrated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Specifically [35S]methionine was predominantly incorporated into the α- and β-charge isomers of pheromaxein, a 16-androstene steroid-binding protein, as shown by SDS-PAGE in combination with vertical isoelectric focusing on polyacrylamide slab gels. The synthesis of pheromaxein occurred in submaxillary gland tissue from both sexes, including tissues stored frozen at −70 °C for long periods. There was little evidence for pheromaxein synthesis in parotid gland tissue or skeletal muscle. Total protein, pheromaxein and total 16-androstenes were determined in the submaxillary gland cytosols of six mature Göttingen miniature boars and a positive correlation was found between these glandular constituents. The amounts of endogenous pheromaxein relative to total protein in the submaxillary gland cytosols (range 10·3–18·0%), together with the predominant synthesis of this protein in vitro, indicate that pheromaxein is a major protein produced in porcine submaxillary glands, particularly in those of the male. Journal of Endocrinology (1991) 128, 205–212

The isolation, purification and some properties of pheromaxein, the pheromonal steroid-binding protein, in porcine submaxillary glands and saliva

1988 ◽  
Vol 118 (1) ◽  
pp. 47-NP ◽  
Author(s):  
W. D. Booth ◽  
C. A. White
Keyword(s):  
Liquid Chromatography ◽  
Affinity Chromatography ◽  
Sodium Dodecyl Sulphate ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Submaxillary Gland ◽  
Fast Protein Liquid Chromatography ◽  
Steroid Binding ◽  
Steroid Binding Protein

ABSTRACT Pheromaxein, the 16-androstene steroid-binding protein with a relative molecular mass of 15 000 was isolated in sub-milligram quantities from the submaxillary gland and saliva of the Gottingen miniature boar, after a fourfold purification involving the following methods: ultrafiltration for submaxillary gland cytosols and ethanol precipitation for saliva, Concanavalin-A-Sepharose affinity chromatography, sodium dodecyl sulphate polyacrylamide gel electrophoresis, 'Extractigel-D' affinity chromatography (to remove sodium dodecyl sulphate) and fast protein-liquid chromatography. Yields of purified pheromaxein obtained after fast protein-liquid chromatography represented 10–20% of total protein present in an ultrafiltrate of a submaxillary gland cytosol. Fast protein-liquid chromatography separated the α- and β-charge isomers of pheromaxein which were shown to have isoelectric points of 4·78 and 5·35 respectively on flat-bed isoelectric focusing. Some data are provided for the variable occurrence of the isomeric forms of pheromaxein in relation to different breeds of pig. Five 16-unsaturated steroids showed the highest binding to pheromaxein. Other steroids of the 5α- and 5β-androstane series also showed some binding to pheromaxein, i.e. 17β-hydroxy-5α-androstan-3-one (19·2%), with 5α-androstan-3-one, which has a similar urinous odour to 5α-androst-16-en-3-one, showing the greatest binding (42·6%) relative to 5α-androst-16-en-3-one (100%). J. Endocr. (1988) 118, 47–57

A metalloproteinase extracellularly released byCrithidia deanei

2003 ◽  
Vol 49 (10) ◽  
pp. 625-632 ◽  
Author(s):  
Claudia Masini d'Avila-Levy ◽  
Rodrigo F Souza ◽  
Rosana C Gomes ◽  
Alane B Vermelho ◽  
Marta H Branquinha
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Residual Activity ◽  
Major Protein ◽  
Motile Cells ◽  
Sds Page ◽  
Triton X

Actively motile cells from a cured strain of Crithidia deanei released proteins in phosphate buffer (pH 7.4). The molecular mass of the released polypeptides, which included some proteinases, ranged from 19 to 116 kDa. One of the major protein bands was purified to homogeneity by a combination of anion-exchange and gel filtration chromatographs. The apparent molecular mass of this protein was estimated to be 62 kDa by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE). The incorporation of gelatin into SDS–PAGE showed that the purified protein presented proteolytic activity in a position corresponding to a molecular mass of 60 kDa. The enzyme was optimally active at 37 °C and pH 6.0 and showed 25% of residual activity at 28 °C for 30 min. The proteinase was inhibited by 1,10-phenanthroline and EDTA, showing that it belonged to the metalloproteinase class. A polyclonal antibody to the leishmanial gp63 reacted strongly with the released C. deanei protease. After Triton X-114 extraction, an enzyme similar to the purified metalloproteinase was detected in aqueous and detergent-rich phases. The detection of an extracellular metalloproteinase produced by C. deanei and some other Crithidia species suggests a potential role of this released enzyme in substrate degradation that may be relevant to the survival of trypanosomatids in the host.Key words: endosymbiont, trypanosomatid, extracellular, proteinase.

Identification of the Poly(C) Binding Protein in the Complex Associated With the 3′ Untranslated Region of Erythropoietin Messenger RNA

Blood ◽  
1999 ◽  
Vol 93 (6) ◽  
pp. 2111-2120 ◽  
Author(s):  
Maria F. Czyzyk-Krzeska ◽  
Amy C. Bendixen
Keyword(s):  
Mrna Stability ◽  
Messenger Rna ◽  
Untranslated Region ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Mobility Shift ◽  
Protein Factor ◽  
Ribonucleoprotein Complexes ◽  
Sds Page

Hypoxia regulates expression of erythropoietin (EPO), a glycoprotein that stimulates erythrocytosis, at the level of transcription and also possibly at the level of messenger RNA (mRNA) stability. A pyrimidine-rich region within the EPO mRNA 3′ untranslated region was implicated in regulation of EPO mRNA stability element and shown to bind protein factors. In the present study we wished to identify the protein factor binding to the pyrimidine-rich sequence in the EPO mRNA stability element. Using mobility shift assays, ultraviolet light cross-linking, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and electroelution of protein factors from the gel slices corresponding to the ribonucleoprotein complexes, we found that two isoforms of a 40 kD poly(C) binding protein (PCBP, also known as CP or hnRNPE), PCBP1, and PCBP2 are present in that complex. In Hep3B or HepG2 cells hypoxia induces neither expression of PCBP nor formation of the ribonucleoprotein complex associated with EPO mRNA that involves PCBP.

Distinct regional localization of neurocalcin, a Ca2+-binding protein, in the bovine adrenal gland

1993 ◽  
Vol 138 (2) ◽  
pp. 283-NP ◽  
Author(s):  
A. Nakano ◽  
M. Terasawa ◽  
M. Watanabe ◽  
K. Okazaki ◽  
S. Inoue ◽  
...  
Keyword(s):  
Adrenal Gland ◽  
Dissociation Constant ◽  
Adrenal Medulla ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Physiological Role ◽  
Zona Glomerulosa ◽  
Zona Fasciculata ◽  
Sds Page

ABSTRACT Neurocalcin (molecular weight 23 000 and 24 000) is a Ca2+-binding protein with three putative Ca2+-binding domains and is present in large amounts in nervous tissues. Neurocalcin isoproteins separated by C18 reverse-phase column chromatography are insoluble in buffer solution and it is impossible to determine the dissociation constant of neurocalcin with Ca2+. To overcome this difficulty, recombinant neurocalcin was synthesized, based on one of the cDNAs of the neurocalcin isoproteins. Stoichiometric titration experiments, using recombinant neurocalcin, indicated that this protein bound 2 mol Ca2+/mol protein and that the apparent dissociation constant for Ca2+ was 2·2 μmol/l, suggesting that neurocalcin plays a physiological role in cellular function. Immunoblotting showed that neurocalcin is present in the bovine adrenal gland in addition to the nervous tissues. Neurocalcin, identified by immunoblotting, was purified from the bovine adrenal gland. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of neurocalcin from the bovine brain showed 23 kDa and 24 kDa double bands, while SDS-PAGE of neurocalcin from the adrenal gland showed a single band of apparently 24 kDa, suggesting that the expression of neurocalcin isoproteins differs from tissue to tissue. The content of neurocalcin in the adrenal gland was 10 μg protein/100 g wet tissue. Immunohistochemical analysis showed the occurrence of neurocalcin in zona glomerulosa and adrenal medulla but not in zona fasciculata or zona reticularis. The restricted localization of neurocalcin in the adrenal gland suggests that a similar Ca2+ signal pathway may be present in zona glomerulosa and the adrenal medulla. Journal of Endocrinology (1993) 138, 283–290

scholarly journals Purification of a Zinc Binding Protein from Xylem of Citrus jambhiri

2002 ◽  
Vol 127 (5) ◽  
pp. 718-723 ◽  
Author(s):  
Kathryn C. Taylor ◽  
Danielle R. Ellis ◽  
Luciano V. Paiva
Keyword(s):  
Total Protein ◽  
Proteinase Inhibitors ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sulfhydryl Group ◽  
Size Exclusion ◽  
Binding Studies ◽  
Citrus Jambhiri ◽  
Citrus Rootstock

Zinc in xylem and phloem of the citrus rootstock, rough lemon [Citrus jambhiri (L.)] was associated with a Zn-binding protein, designated citrus vascular Zn-binding protein (CVZBP). The apparent molecular mass of the CVZBP was 19.5 kDa after nondenaturing size exclusion chromatography and 21.8 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Ion exchange chromatography demonstrated that CVZBP was anionic, requiring 0.43 n NaCl for elution from quaternary aminoethyl Sepharose. Antiserum to the protein cross-reacted more with total protein extracts from leaf midveins than with total protein from the rest of the leaf lamina, further suggesting a vascular location of the Zn-binding protein. Quantitative analysis indicated that ≈2 to 3 mol of Zn were associated with 1 mol of native protein. Binding studies with the partially purified CVZBP demonstrated a capacity to bind several divalent cations: Cd, Ni, Pb, and Zn. Reaction with Ellman's reagent suggested that the protein has significant sulfhydryl group content that may be involved in metal binding. N-terminal sequencing demonstrates identity with papaya latex trypsin inhibitor, sporamin, or other Kunitz soybean proteinase inhibitors.

scholarly journals Human C4-binding protein. I. Isolation and characterization

1978 ◽  
Vol 148 (1) ◽  
pp. 207-222 ◽  
Author(s):  
J Scharfstein ◽  
A Ferreira ◽  
I Gigli ◽  
V Nussenzweig
Keyword(s):  
Complement System ◽  
Binding Protein ◽  
Divalent Cations ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Electrophoretic Patterns ◽  
Isolation And Characterization ◽  
Sds Page ◽  
Edta Plasma ◽  
The Complement System

C4-binding protein (C4-bp), a new component of the complement system, was isolated from human plasma by precipitation with polyethyleneglycol, followed by chromatography on ion exchangers. C4-bp was identified on sodium dodecyl- sulfate polyacrylamide gel electrophoresis (SDS-PAGE) by two independent criteria: its ability to bind to C4b, and immunoprecipitation with a monospecificantiserum. Purified C4-bp is a 10.7 s glycoprotein. It consists of several disulfide bonded subunits of mol wt 70,000 daltons. Under nonreducing conditions, its mol wt has been estimated on SDS-PAGE as 540- 590,000 daltons. C4-bp moves as a slow B-globulin at pH 8.6 in the absence of free divalent cations, but when the buffers contain Ca(++)-lactate, C4-bp is a gamma globulin. Purified C4-bp binds to purified C4b. The reaction proceeds in the presence or absence of divalent cations and is not inhibited by diisopropylfluorophosphate. The C4b/C4-bp complexes have sedimentation coefficients between 15 and 17 s on sucrose gradient ultracentrifugation, and can be readily identified by crossed immunoelectrophoresis (CIE). The complexes move faster toward the anode than either protein. C4-bp is multivalent. Saturation is reached at molecular ratios of C4b/C4- bp of between 4 and 5. The interaction between C4b and C4-bp may complicate the electrophoretic patterns of these proteins in normal human serum, if the complement system is activated before or during the run. However, in EDTA-plasma, native C4 and C4-bp do not form stable complexes and can be identified in separate peaks after CIE.

PROFIL PROTEIN VAKSIN Aeromonas hydrophila DAN Streptococcus agalactiae HASIL INAKTIVASI DENGAN FORMALIN: DIUJI MENGGUNAKAN Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis

2014 ◽  
Vol 9 (3) ◽  
pp. 449
Author(s):  
Desy Sugiani ◽  
Angela Mariana Lusiastuti ◽  
Sukenda Sukenda ◽  
Enang Harris
Keyword(s):  
Streptococcus Agalactiae ◽  
Sodium Dodecyl Sulphate ◽  
Total Protein ◽  
Aeromonas Hydrophila ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Sds Page ◽  
Neutral Buffer Formalin

Vaksin bakterin dalam bentuk protein merupakan salah satu tipe vaksin yang telah banyak dikembangkan. Protein digunakan sebagai vaksin biasanya dibuat dengan teknik inaktivasi formalin-killed. Vaksin ini biasanya lebih mudah dibuat, lebih murah, lebih stabil, dan mampu disimpan dalam waktu lama. Akan tetapi masih sedikit informasi mengenai efek perlakuan tersebut terhadap profil protein. Pada penelitian ini, untuk mengevaluasi profil protein, dilakukan inaktivasi sediaan vaksin dari isolat bakteri Aeromonas hydrophila AHL0905-2 dan Streptococcus agalactiae N14G dengan menambahkan 0,5% formalin dan 3% neutral buffer formalin (NBF) ke dalam biakan plasebo bakterin dan diinkubasi selama 24 jam. Kualitas produk vaksin ditentukan berdasarkan uji karakterisasi protein menggunakan metode Bradford dan SDS-PAGE. Hasil uji menunjukkan bahwa sediaan vaksin A. hydrophila dan S. agalactiae yang diinaktivasi dengan 3% NBF memiliki profil protein lebih variatif dibandingkan dengan sediaan vaksin yang diinaktivasi dengan 0,5% formalin. Akan tetapi, inaktivasi vaksin A. hydrophila dan S. agalactiae dengan 3% NBF menghasilkan berat total protein yang lebih rendah jika dibandingkan dengan dengan sediaan vaksin yang diinaktivasi dengan 0,5% formalin.

scholarly journals Morphological, Molecular, Biochemical and Nutritional Characterization of Three Major Thais Species from the Sindh Coast of Pakistan

2018 ◽  
Vol 10 (1) ◽  
pp. 33-45
Author(s):  
Syed Abid Ali ◽  
Fozia Humayun ◽  
Iqra Munir ◽  
Shakil Ahmad ◽  
Zarrien Ayub ◽  
...  
Keyword(s):  
Total Protein ◽  
Biochemical Characterization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Nutritional Composition ◽  
The Body ◽  
Size Exclusion ◽  
High Concentration ◽  
Sds Page

Objective: The present study was conducted to investigate the biomass assessment, morphological and molecular identification, nutritive status and biochemical characterization of three major Thais species (T. bufo, T. hippocastanum and T. rudolphi) from the Sindh Coast, Pakistan. Methods: Samples were collected from Buleji and Paradise Point at the Sindh Coast. Species were identified morphologically as well as genetically by amplifying two mitochondrial 16S rDNA & Cytochrome Oxidase I (COI) and one nuclear (Histone H3) genes. Shell microstructure and chemistry were also studied by scanning electron microscopy and Energy Dispersive X-ray spectrometry (EDX). The body muscle was dissected and used for nutritional composition determination such as estimation of total protein, carbohydrates, lipids, protein fingerprinting by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Size-Exclusion - Fast Protein Liquid Chromatography (SEC-FPLC), amino acid and fatty acid analysis. Results: Nutritionally, the total protein was found to be the major content followed by carbohydrate and lipid in the three Thais sp. The presence of medicinally important hemocyanin as abundant hemolymph protein was confirmed via SDS-PAGE and SEC FPLC. Nine different types of fatty acids and a high concentration of essential amino acids were also determined. Conclusion: Our findings suggest that Thais sp. are nutritionally rich and can be consumed as a valuable marine resource to overcome the malnutrition problem in developing countries.

A Sensitive Peroxidase Staining Immunoblotting Method for Measuring Total Protein S in Human Plasma

1993 ◽  
Vol 69 (04) ◽  
pp. 331-334 ◽  
Author(s):  
Xiangan Li ◽  
Kaoru Hatanaka ◽  
Ling Guo ◽  
Motoo Tsushima ◽  
Yukihiko Kitamura ◽  
...  
Keyword(s):  
Human Plasma ◽  
Total Protein ◽  
Gel Electrophoresis ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Protein S ◽  
Polyacrylamide Gel ◽  
Free Form

SummaryIn plasma, protein S is found in its free form and as a complex with C4b-binding protein. After 125I-protein S was added tonormal human plasma and applied to SDS-8% polyacrylamide gel electrophoresis, the autoradiogram of the gel showed only one single band at free protein S position. Applying this evidence, we have developed a peroxidase staining Western Blotting method to quantitate total protein S in human plasma which consists of sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by blotting to nitrocellulose membrane and a sensitive avidin-biotinylated peroxidase staining method (ABC technique). The measurement of protein S by the immunoblotting was reproducible and the coefficient of variation was 7%. As little as 1 ng of protein S could be detected. C4b-binding protein did not affect the measurement of protein S. Compared to other immunoassays, this peroxidase staining immunoblotting method has the advantage of directly estimating the apparent molecular weight of protein of interest, eliminating nonspecific stain and having high sensitivity without using radioisotope.

scholarly journals Plasma protein concentrations of the young and adult Amazona brasiliensis parrots

2017 ◽  
Vol 71 (1) ◽  
pp. 44-51
Author(s):  
Elizabeth Schmidt ◽  
Patricia Serafini ◽  
Elenise Sipinski ◽  
Antonio Paulillo
Keyword(s):  
Plasma Protein ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Clinical Pathology ◽  
Specific Protein ◽  
Major Protein ◽  
Plasma Protein Concentration ◽  
Protein Gel ◽  
Sds Page

Introduction. The Red-tailed Amazon parrot (Amazona brasiliensis) is an endangered species of the Psittacine family, and for which various data are important for a comprehensive preservation plan. Data about plasma protein gel electrophoresis of Amazon parrot blood are scarce. The purpose of this study was to determine plasma protein concentrations and concentrations of major protein bands in blood of young and adult Red-tailed Amazon parrot (Amazona brasiliensis). Materials and Methods. Blood samples from eight young and eight adult healthy free-living parrots were obtained. Plasma protein concentration and fractions were determined using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Mann-Whitney U test was used to compare variables. Results and Conclusions. Six major protein bands with the following molecular weights were identified by SDS-PAGE: 170 kDa, 117 kDa, 85 kDa (putative ovotransferrin), 60 kDa, 45 kDa and 23 kDa. Adult parrots had significantly higher concentrations of total proteins, albumin and other proteins with similar mobility (around 60 kDa). Young birds had significantly higher levels of 23kDa proteins. The concentration of putative ovotransferrin (85 kDa) was not different between young and adult parrots. Plasma protein gel electrophoresis patterns in Red-tailed Amazon parrots are similar between young and adult animals, but specific protein bands differ in their absolute concentrations. This finding should be taken into consideration when clinical pathology data are analysed.

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