Purification and characterization of testosterone-binding globulin of canine serum.

Testosterone-binding globulin (TeBG) of canine serum was purified to apparent homogeneity by affinity chromatography on testosterone-17 alpha-ethynylcarboxyaminoethyl-Sepharose 4B followed by hydroxyapatite column chromatography. Canine TeBG was a glycoprotein containing 5.5% carbohydrates. Equilibrium sedimentation analysis in the presence and absence of 6 M guanidine hydrochloride gave molecular weights of 40,000 and 76,000, respectively, suggesting that native TeBG consists of two subunits. Equilibrium dissociation constants at 0 degrees C for testosterone and dihydrotestosterone were estimated to be 5.58 x 10(-8) M and 1.43 x 10(-8) M, respectively, and the number of binding site per native molecule was approximately unity for both androgens. Canine TeBG had virtually no affinity for estradiol, progesterone, or cortisol. Canine TeBG did not cross-react with a rabbit antiserum raised against bovine TeBG.     

Molecular weights of estrogen and androgen binding proteins in the liver of Xenopus laevis.

[3-H]Estradiol-17beta and [3-H]dihydrotestosterone binding proteins in the cytosol fraction of liver from both male and female Xenopus laevis were characterized by electrophoresis on polyacrylamide gels. These binding proteins, which were indistinguishable based upon their mobilities on gels of different acrylamide concentrations, migrated as single components with a molecular weight of 2.0 x 10-4. Separation of native or sodium dodecyl sulfate denatured specific estrogen-binding components on dodecyl sulfate free acrylamide gels gave similar results, i.e., a single species of molecular weight 2.0-2.5 x 10-4. The same molecular weight also was obtained when cytosol was prepared in the presence of either diisopropyl fluorophosphate or phenylmethanesulfonyl fluoride, protease inhibitors. Evidence that the liver components binding either [3-H]estradiol-17-beta or [3-H]dihydrotestosterone were not plasma contaminants was provided by the observation that the plasma sex-steroid binding globulin of Xenopus had a different mobility when separated by polyacrylamide gel electrophoresis.     

Size isomers of testosterone-estradiol-binding globulin exist in the plasma of individual men and women

We isolated testosterone-estradiol-binding globulin TeBG rapidly and in high yield from pooled pregnancy plasma. It showed two bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). Both bands stained with three different monoclonal antibodies to TeBG, thus demonstrating their immunological similarity. Freshly drawn, individual sera, from men, women, and pregnant patients were submitted to microaffinity chromatography, a procedure which partially purifies TeBG in approximately 4 hr. The partially purified plasma was submitted to SDS PAGE, followed by immunoblotting. The blotted TeBG exhibited the same two bands seen in the isolated, purified protein. The size heterogeneity observed in TeBG purified to: proteolysis occurring during isolation; a peculiarity of pregnancy plasma; or heterogeneity attendant upon the use of pooled plasma for isolation.     

Isolation and characterization of bovine factor VII.

Factor VII (proconvertin) has been purified approximately 5 x 10(5)-fold from bovine plasma with an overall yield of 30%. The isolation procedure involves barium sulfate adsorption and elution, DEAE-Sephadex batchwise adsorption and elution, benzamidine-agarose column chromatography, heparin-agarose column chromatography, and preparative polyacrylamide gel disc electrophoresis. The final product was homogeneous when examined by gel electrophoresis in the presence of sodium dodecyl sulfate. A minimal molecular weight of 45,500 was determined by sedimentation equilibrium. The molecular weight estimated by sodium dodecyl sulfate gel electrophoresis was 54,000. Factor VII is composed of a single polypeptide chain possessing an amino-terminal sequence of Ala-Asn-Gly-Phe-Leu-. The amino acid and carbohydrate compositions of factor VII are also reported.     

Simultaneous ultramicroanalysis of both 17-keto-and 17beta-hydroxy androgens in biological fluids.

Sensitive methods for quantifying androgens were lacking. Therefore, a relatively simple procedure for separating steroids was combined with highly specific assay methods so that eight androgens could be measured with high accuracy, precision and sensitivity. Semi-automated separations on Sephadex LH-20 columns used heptane:methylene chloride:ethanol:water (50:50:1:0.12) and a flow rate of 17.0 min/ml. The six peaks eluted contained androstenedine; androsterone, epiandrosterone and dihydrotestosterone; testosterone and dehydroepiandrosterone; 3alpha-androstanediol; 3beta-androstanediol; and androstenediol. Androstenedione, dehydroepiandrosterone and androstenediol were quantified using specific antisera (sensitivity less than or equal to 75 pg). Testosterone and dihydrotestosterone were measured by competitive protein-binding assays using rabbit TeBG (sensitivity less than or equal to 150 pg). 3alpha- and 3beta-androstanediol were similarly assayed using human TeBG (sensitivity approximately 150 pg). Androsterone was reduced with NaBH4 and the resulting 3alpha-androstanediol was assayed using human TeBG (sensitivity approximately 200 pg). Inter- and intra-assay variations were less than 10% for radioimmunoassays and less than 16% for competitive protein-binding assays over the entire dose response curve.     

Measurement of the testosterone binding parameters for both testosterone-estradiol binding globulin and albumin in individual serum samples

This report describes a solid phase method for the characterization of testosterone binding to both albumin and testosterone-estradiol binding globulin (TeBG). TeBG is adsorbed from serum samples onto a solid phase matrix of concanavalin A covalently linked to 4B Sepharose. The binding of testosterone is then examined both in the presence and absence of the endogenous serum albumin. Analysis of the resulting Scatchard plots permits determination of the TeBG binding capacity, TeBG association constant and a parameter of albumin binding equivalent to the product of its affinity and capacity for binding testosterone. Results showed that the TeBG capacity was lower in men than in women (18.4 +/- 5.8 vs. 33.1 +/- 19.2 nM, p less than 0.01). The association constant was greater in men (1.59 +/- 0.35 vs. 1.19 +/- 0.32 x 10(9)M-1, 10(9)M-1, p less than 0.01). There was no difference in the albumin binding parameter (43.8 +/- 18.3 vs. 46.6 +/- 15.5, NS). These parameters can then be used to calculate the distribution of the circulating testosterone into albumin bound, TeBG bound and unbound fractions.     

Isolation of testosterone-binding globulin from bovine serum by affinity chromatography and its molecular characterization.

The testosterone-binding globulin (TeBG) from bovine serum was purified by affinity chromatography and hydroxylapatite chromatography. The affinity column used was prepared by coupling 17 alpha-carboxyethynyl-17-hydroxy-4-androsten-3-one to aminoethyl-Sepharose. The compound was replaceable by 17alpha-carboxyethynyl-17-hydroxy-5alpha-androstan-3-one, but not by testosterone 17-hemisuccinate, estradiol 17-hemisuccinate, or testosterone 3-(O-carboxymethyl)oxime. The TeBG isolated was homogeneous on analytical polyacrylamide gel electrophoresis and equilibrium centrifugation. The protein was a glycoprotein having a molecular weight of 89,500 and a carbohydrate content of 17%. The association constant (M-1) at 4 degrees C was 1.1 X 10(8) and the number of binding sites per molecule was 0.8. Treatment with guanidine-HCl dissociated the protein into subunits having a molecular weight of 28,400 (about one-third of that of the original molecule). SDS-gel electrophoresis showed that two of the three subunits were slightly larger than the other. The dissociation into subunits could also be accomplished by GEDTA treatment with concomitant loss of testosterone-binding activity. The activity and molecular size were reversibly restored by incubation with excess Ca2+.     

Structure of asparagine-linked oligosaccharides on human and rabbit testosterone-binding globulin

We have previously demonstrated, using two-dimensional polyacrylamide gel electrophoresis, that much of the microheterogeneity of human (h) and rabbit (rb) testosterone-binding globulin (TeBG) is due to differential glycosylation of a single protomer. Since glycosylation has been shown to be a physiologically important modification of proteins, we have examined the structure of the oligosaccharide chains attached to hTeBG and rbTeBG to facilitate future studies on the mechanisms of action of the proteins. The structures of the oligosaccharides attached to TeBG were determined by using serial lectin chromatography. About 10% of the TeBG from castrated male rabbits and about 20% of the TeBG from pregnant rabbits and from a human sample were not retained on a column of immobilized concanavalin-A (Con-A). This fraction would consist of TeBG with attached asparagine (Asn)-linked tri- and tetraantennary complex and serine/threonine (O)-linked oligosaccharides as well as non-glycosylated forms. None of the lectins used to subfractionate these species was effective. Forty to 50% of the TeBG applied to Con-A possessed biantennary complex oligosaccharides as indicated by the fact that it could be eluted with 10 mM 1-O-methyl-alpha-D-glucopyranoside and by its retention on wheat germ agglutinin (WGA). About 8% of the biantennary complex oligosaccharides on hTeBG and none of those on rbTeBG were fucosylated on the chitobiose core, as determined by chromatography on Lens culinaris lectin (LcH). Galactosylated oligosaccharides were also present on the TeBG in this fraction as indicated by its interaction with Ricinus communis-I (RCA-I). Thirty to 40% of the TeBG applied to Con-A was retained and could be eluted with 0.5 M methyl-alpha-D-mannopyranoside. This fraction contains TeBG possessing high mannose-type, hybrid-type, and complex galactosylated glycans as determined by chromatography on Con-A, WGA, and RCA-I. Evidence based on the binding of mannoside-eluted TeBG to Con-A, WGA, and RCA-I indicated that at least the TeBG in this fraction contained two glycosylation sites and that the sites were differentially glycosylated.     

Monomeric purine nucleoside phosphorylase from rabbit liver. Purification and characterization.

Rabbit liver purine nucleoside phosphorylase (purine nucleoside: orthophosphate ribosyltransferase EC 2.4.2.1.) was purified to homogeneity by column chromatography and ammonium sulfate fractionation. Homogeneity was established by disc gel electrophoresis in presence and absence of sodium dodecyl sulfate, and isoelectric focusing. Molecular weights of 46,000 and 39,000 were determined, respectively, by gel filtration and by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Product inhibition was observed with guanine and hypoxanthine as strong competitive inhibitors for the enzymatic phosphorolysis of guanosine. Respective Kis calculated were 1.25 x 10(-5) M for guanine and 2.5 x 10(-5) M for hypoxanthine. Ribose 1-phosphate, another product of the reaction, gave noncompetitive inhibition with guanosine as variable substrate, and an inhibition constant of 3.61 x 10(-4) M was calculated. The protection of essential --SH groups on the enzyme, by 2-mercaptoethanol or dithiothreitol, was necessary for the maintenance of enzyme activity. Noncompetitive inhibition was observed for p-chloromercuribenzoate with an inhibition constant of 5.68 x 10(-6)M. Complete reversal of this inhibition by an excess of 2-mercaptoethanol or dithiothreitol was demonstrated. In the presence of methylene blue, the enzyme showed a high sensitivity to photooxidation and a dependence of photoinactivation on pH, strongly implicating histidine as the susceptible group at the active site of the enzyme. The pKa values determined for ionizable groups of the active site of the enzyme were near pH 5.5 and pH 8.5 The chemical and kinetic evidences suggest that histidine and cysteine may be essential for catalysis. Inorganic orthophosphate (Km 1.54 x 10(-2) M) was an obligatory anion requirement, and arsenate substituted for phosphate with comparable results. Guanosine (Km 5.00 x 10(-5) M), deoxyguanosine (Km 1.00 x 10(-4)M) and inosine (Km 1.33 x 10(-4)M), were substrates for enzymatic phosphorolysis. Xanthosine was an extremely poor substrate, and adenosine was not phosphorylyzed at 20-fold excess of the homogeneous enzyme. Guanine (Km 1.82 x 10(-5)M),ribose 1-phosphate (Km 1.34 x 10(-4) M) and hypoxanthine were substrates for the reverse reaction, namely, the enzymatic synthesis of nucleosides. The initial velocity studies of the saturation of the enzyme with guanosine, at various fixed concentrations of inorganic orthophosphate, suggest a sequential bireactant catalytic mechanism for the enzyme.     

Plasma testosterone transport in primates

All primate species, including Old and New World primates and prosimians have a plasma testosterone-estradiol binding globulin (TeBG), which is a glycoprotein and has a similar mobility in polyacrylamide gel electrophoresis. In New World primates the TeBG binding capacity for [3H]testosterone was higher and its affinity lower than in Old World primates. These changes were associated with high unbound plasma testosterone concentrations in these species. Binding parameters of TeBG in prosimian species varied markedly. Thus, in primate evolution TeBG was conserved despite marked differences in binding characteristics. In New World primates changes are associated with high total and unbound testosterone, a finding concordant with alterations of other steroid hormones concentration in these species with "generalized steroid hormone resistance".     

Transfection of Escherichia coli Spheroplasts I. General Facilitation of Double-Stranded Deoxyribonucleic Acid Infectivity by Protamine Sulfate

The addition of 25 mug of protamine sulfate per ml to lysozyme-ethylenediamine-tetraacetic acid spheroplasts of Escherichia coli stimulates transfection not only for T1 phage deoxyribonucleic acid (DNA; Hotz and Mauser, 1969) but also for the following phage DNA species: lambda, 10,000-fold to an efficiency of 10(-3) infective centers per DNA molecule; phiX174 replicative form, 300-fold to an efficiency of 5 x 10(-2); fd replicative form, 300-fold to 10(-6); T7, 300-fold to 3 x 10(-7). Three native phage DNA species were not infective at all in the absence of protamine sulfate but were infective in the presence of protamine sulfate with the following efficiencies: T4, 10(-5); T5, 3 x 10(-6); and P22, 3 x 10(-9). The effect of protamine sulfate is specific for double-stranded DNA. The application of infectivity assays to the study of phage DNA replication, recombination, prophage integration, prophage excision, and interspecies transfection are discussed.     

Detection of genetic variation with radioactive ligands. V. Genetic variants of testosterone-binding globulin in human serum

We examined genetically determined polymorphisms in testosterone-binding beta-globulin (TeBG) by polyacrylamide gel electrophoresis (PAGE). Four electrophoretic variants were identified, which we suggest are the result of combinations of three alleles. Gene frequencies were calculated for the three alleles in white American, black American, and Japanese-American populations. There was good agreement between observed and expected phenotype numbers. Distribution of phenotypes among offspring of several crosses was consistent with simple Mendelian inheritance of an autosomal gene.     

Rabbit liver growth hormone receptor and serum binding protein. Purification, characterization, and sequence

A putative growth hormone receptor from detergent-solubilized rabbit liver membranes and the growth hormone binding protein from rabbit serum have been purified 59,000- and 400,000-fold, respectively, primarily by affinity chromatography. Both purified proteins exhibit high affinity binding for human growth hormone; K alpha = 9-30 x 10(9) M-1 for the liver receptor and K alpha = 6 x 10(9) M-1 for the binding protein. The apparent molecular weight of the liver receptor is 130,000 by reduced sodium dodecyl sulfate gel electrophoresis, while that of the binding protein is 51,000. Both contain N-linked carbohydrate. The amino-terminal sequences of the liver growth hormone receptor and the serum binding protein were found to be the same, indicating that the binding protein corresponds to the extracellular domain of the liver receptor. Ubiquitin was found covalently linked to the liver receptor but not to the serum binding protein. The amino acid sequences of several peptides from the liver receptor were also determined after tryptic and V8 protease digestion.     

Prenyltransferase from Saccharomyces cerevisiae. Purification to homogeneity and molecular properties.

Prenyltransferase (EC 2.5.1.1) has been purified to homogeneity from the supernatant fraction of yeast by ammonium sulfate fractionation, diethylaminoethyl-cellulose and hydroxylapatite chromatography, and column isoelectric focusing techniques. The active enzyme from isoelectric focusing columns emerged as a single symmetrical peak with specific activities 15- to 35-fold higher than previously reported preparations. The enzyme was found to be homogeneous by continuous polyacrylamide gel electrophoresis at pH 8.4 and discontinuous polyacrylamide gel electrophoresis at pH 6.9 as well as sodium dodecyl sulfate polyacrylamide electrophoresis at pH 7.0. By means of gel chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis, the protein was shown to be a dimer with a molecular weight of 84,000 plus or minus 10%. The isoelectric point of the enzyme was determined to be 5.3. The enzyme synthesizes farnesyl and geranylgeranyl pyrophosphates from dimethylallyl, geranyl, and farnesyl pyrophosphates. Michaelis constants for the enzyme were 4, 8, and 14 mu M for isopentenyl, dimethylallyl, and geranyl pyrophosphates, respectively.     

Photoaffinity labelling of gonadotropin releasing hormone binding sites in human epithelial ovarian carcinomata

A photoaffinity labelled derivative of [D-Lys6]-GnRH was prepared with a bifunctional photolabile reagent (4-azidobenzoyl)-N-hydroxysuccinimide. In rat pituitary membranes, this analog retained high binding affinity (Ka = 0.12 x 10(9) M-1) consistent with a single class of receptors. The analog was iodinated and used for the identification of GnRH binding sites in human epithelial ovarian carcinomata. By sodium dodecyl sulfate electrophoresis in 10% polyacrylamide gel the presence of two labelled components could be demonstrated: a high molecular weight component of 63,200 and a smaller component of 46,000. Competition experiments with unlabelled ligand suggest that it is the high molecular weight component which specifically binds GnRH.     

Progesterone binding components of chick oviduct. X. Purification by affinity chromatography.

Cytoplasmic progesterone receptors of chick oviduct have been purified in 8% yield by steroid affinity and ion exchange chromatography. The affinity resin, deoxycorticosterone-bovine serum albumin-Sepharose, binds progesterone receptors with high affinity (KD equals 8 times 10-minus 10 M) and its use resulted in a greater than 2000-fold purification over the starting material in a single step. DEAE-Sephadex A-50 chromatography was then used to achieve final purification. NA dodecyl-SO4 gel electrophoresis and DEAE-cellulose chromatography showed that the purified receptors contained both of the previously described 4 S progesterone binding components in near equal amounts. Na dodocyl-SO4 gel electrophoresis also showed that these components consisted of single polypeptide chains with molecular weights of 110, 000 (A component) and 117, 000 (B component). There was no evidence for subunits of lower molecular weight. The purified materials have identical hormone-binding kinetics and steroid specificity to crude cytosol receptors. The isolated receptors retain the three biologically important properties exhibited by progesterone binding components present in cruder preparations: they bind specifically to (a) nuclei (KD equals 1.1 times 10-minus 9 M, 10, 000 sites per nucleus); (b) chromatin (KD equals 3 times 10-minus 9 M, 2000 sites per pg of DNA-);and (C) DNA.     

Isolation, characterization and localization of the proteinase B inhibitor IB3 from Saccharomyces carlsbergensis.

A heat-stable polypeptide has been detected in Saccharomyces carlsbergensis which inhibits specifically proteinase B from yeast. This proteinase B inhibitor IB3 differs substantially in chemical, physical and antigenic properties from the earlier described proteinase B inhibitors IB1 and IB2 from yeast. The inhibitor IB3 has been purified from S. carlsbergensis and appears to be homogeneous by disc gel electrophoresis and sodium dodecyl sulfate gel electrophoresis. The molecular weight has been estimated at 11 500, with no evidence for the existence of subunits. The amino acid analysis shows the absence of tryptophan. No compounds other than amino acids could be detected. The isoelectric point is 4.6. The inhibitor is not affected by incubation with proteinase B but is inactivated by proteinase A and carboxypeptidase Y from yeast and by trypsin from bovine pancreas. The proteinase B inhibitor association constant was calculated to be 3.3 x 10(9) M-1 and the enzyme inhibitor complex is stable at 25 degrees C in the pH range 5--10. The inhibitor does not exhibit immunological cross-reactivity with IB1 and IB2. After centrifugal fractionation at 40 000 x g of a metabolic lysate from spheroplasts the inhibitor was found to be localized in the supernatant, i.e. the extravacuolar soluble fraction.     

兔的一种新病毒：Ⅱ.一株兔出血症...

In this paper a strain of Rabbit Hemorrhagic Disease Virus (RHDV) was isolated and purified from the diseased rabbit livers with a method of using chloroform, two-phase of polyethylene-glycol-dextran sulfate sodium and sucrose density gradient centrifugation. Purified virus was nonenveloped, icosahedeal symmetry with a triangulation number of 3, and 33-37 nm in diameter. The capsid was composed of 32 capsomeres with central holes in an outer diameter of about 9nm. Two types of viral particles having different sedimentation coefficient, 130s and 166s could be identified after sucrose density gradient centrifugation. Probably no less than four virion proteins with molecular weight of 66.4, 65.0, 63.5, 41.0 x 10(3) dalton were detected by SDS-polyacrylamide gel electrophoresis. Viral nucleic acid was extracted from purified virus by using SDS-proteinase K-phenol. Tests with diphenylamine, formaldehyde, and staining with acridine orange as well as the curves of thermal denaturation showed that this kind of virus had a single-stranded DNA. The molecular weight of the ssDNA was approx 2.1 x 10(6) dalton as determined by electron microscopy. Data indicate that the RHDV may like the parvovirus of the family Parvoviridae.     

Production and Purification of Extracellular D-Xylose Isomerase from an Alkaliphilic, Thermophilic Bacillus sp

An alkaliphilic, thermophilic Bacillus sp. (NCIM 59) produced extracellular xylose isomerase at pH 10 and 50 degrees C by using xylose or wheat bran as the carbon source. The distribution of xylose isomerase as a function of growth in comparison with distributions of extra- and intracellular marker enzymes such as xylanase and beta-galactosidase revealed that xylose isomerase was truly secreted as an extracellular enzyme and was not released because of sporulation or lysis. The enzyme was purified to homogeneity by ammonium sulfate precipitation followed by gel filtration, preparative polyacrylamide gel electrophoresis, and ion-exchange chromatography. The molecular weight of xylose isomerase was estimated to be 160,000 by gel filtration and 50,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating the presence of three subunits. The enzyme is most active at pH 8.0 and with incubation at 85 degrees C for 20 min. Divalent metal ions Mg, Co, and Mn were required for maximum activity of the enzyme. The K(m) values for D-xylose and D-glucose at 80 degrees C and pH 7.5 were 6.66 and 142 mM, respectively, while K(cat) values were 2.3 x 10 s and 0.5 x 10 s, respectively.     

An adenosine 3':5'-monophosphate-adenosine binding protein from mouse liver.

A cyclic AMP-adenosine binding protein from mouse liver has been purified to apparent homogeneity as judged by polyacrylamide gel electrophoresis in the absence and presence of sodium dodecyl sulfate and by analytical ultracentrifugation. The binding protein had a Stokes radium of 48 A based on gel chromatography. Both the purified binding protein and the binding activity in fresh cytosol sedimented as 9 S on sucrose gradient centrifugation. The homogeneous protein had a sedimentation coefficient (S20, w) of 8.8 x 10-13 s, as calculated from sedimentation velocity experiments. By use of the Stokes radius and S20, w', the molecular weight was calculated to be 180,000. The protein was composed of polypeptides having the same molecular weight of 45,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and thus appeared to consist of four subunits of equal size. The isoelectric point, pI = 5.7. The binding capacity for cyclic AMP increased by preincubating the receptor protein in the presence of Mg2+ ATP. This process, tentatively termed activation, was studied in some detail and was shown not be be be accompanied by dissociation, aggregation, or phosphorylation of the binding protein. Cyclic AMP was bound to the protein with an apparent dissociation constant (Kd) of 1.5 x 10-7 M. The binding of cyclic AMP was competitively inhibited by adenosine, AMP, ADP, and ATP whose inhibition constants were 8 x 10-7 M, 1.2X 10-6 M, 1.5 X 10-6 M, and higher than 5 x 10-6 M respectively. A hyperbolic Scatchard plot was obtained for the binding of adenosine to the activated binding protein, indicating more than one site for adenosine. The binding of adenosine to the site with the highest affinity (Kd=2 x 10-7 M) for this nucleoside was not suppressed by excess cyclic AMP and was thus different from the aforementioned cyclic AMP binding site. Cyclic GMP, GMP, guanosine, cyclic IMP, IMP, and inosine did not inhibit the binding of either cyclic AMP or adenosine. The binding protein had no cyclic AMP phosphodiesterase, adenosine deaminase, phosphofructokinase, or protein kinase activities, nor does it inhibit the catalytic subunit of the cyclic AMP-dependent protein kinase.