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PubMed1989

Digitalis-like activity in human plasma. Purification, affinity, and mechanism.

A factor having digitalis-like characteristics has been isolated from human plasma and its mechanism of action compared with the commonly used cardenolide, ouabain. The purification scheme involved dialysis of human plasma, lyophilization of dialysate, extraction of methanol-soluble components, and flash evaporation, followed by preparative, semipreparative, and analytical scale reverse-phase chromatography. One peak of biologically active material was obtained and shown to possess digitalis-like activity in assays of sodium pump activity, receptor binding, and Na,K-ATPase activity. Results from (i) the determination of the ligand conditions supporting binding, (ii) kinetics of association and dissociation from the Na,K-ATPase, (iii) affinity titration, (iv) selectivity, and (v) competition studies, when taken together, show that the endogenous digitalis-like factor is a specific inhibitor of the sodium pump that stabilizes the E2P form of the enzyme in a manner analogous to ouabain. The endogenous digitalis-like factor binds competitively in or near the receptor site for cardiac glycosides with an apparent affinity 8-20-fold greater than any known cardioactive steroid. The presence of digitalis-like activity in the circulation of individuals with no known intake of these compounds suggests that the material characterized here is an endogenous counterpart to the cardenolides. This factor may regulate sodium pump activity and provide a rationale for the existence of gene and tissue-specific forms of the Na,K-ATPase having distinct sensitivity to the cardenolides.

Binding, Competitive↗
PubMedDate not supplied

Optimized single-step affinity purification with a self-cleaving intein applied to human acidic fibroblast growth factor.

To reduce the number of recovery steps during downstream processing and to overcome the limitations of present fusion-based affinity separations, a controllable self-splicing protein element in the form of a mini-intein was used to optimize the recovery of proteins for both batch and flow purification strategies. The ability to recover purified proteins was demonstrated using a tripartite fusion consisting of a maltose binding domain, a truncated intein as a controllable linker molecule, and a protein of interest. To characterize expression level, solubility, cleavage rates, pH and temperature controllability, and protein activity, recombinant human acidic fibroblast growth factor (aFGF) was used as a model protein. A simple mass transport model, based on cleavage reaction-limited mass transfer and constant dispersion, was successfully used to predict product concentration and peak shape in relation to critical process parameters (with no fitting parameters). Insight into the nature of the cleavage reaction and its regulation was obtained via temperature- and pH-dependent kinetic data.

Chromatography, Affinity↗
PubMed1988

Affinity purification of human deoxycytidine kinase: avoidance of structural and kinetic artifacts arising from limited proteolysis.

Homogeneous deoxycytidine kinase has been isolated from leukemic human T-lymphoblasts by affinity chromatography based on a multisubstrate analog, deoxycytidine 5'-adenosine 5"'-P1,P4-tetraphosphate (dCp4A). Chromatography of extract treated with protease inhibitors yielded a monomeric polypeptide, inasmuch as the Mr of the native protein, 59,300, is comparable to the value of 52,000 from sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isoelectric pH was 6.1. But, enzyme isolated without protease inhibitors exhibited two fragments of Mr = 30,000 and 33,000, suggesting that proteolytic cleavage of the parental polypeptide had occurred during affinity chromatography. Both the parental and proteolyzed enzymes phosphorylated deoxyadenosine and deoxyguanosine, as well as deoxycytidine. However, the proteolyzed enzyme had an increased apparent Km for deoxycytidine. In consequence of this, a mixture of the two forms produced bimodal kinetic plots, whereas linear kinetics were displayed by each form alone.

Chromatography, Affinity↗
PubMed2001

Sephadex-binding RNA ligands: rapid affinity purification of RNA from complex RNA mixtures.

Sephadex-binding RNA ligands (aptamers) were obtained through in vitro selection. They could be classified into two groups based on their consensus sequences and the aptamers from both groups showed strong binding to Sephadex G-100. One of the highest affinity aptamers, D8, was chosen for further characterization. Aptamer D8 bound to dextran B512, the soluble base material of Sephadex, but not to isomaltose, isomaltotriose and isomaltotetraose, suggesting that its optimal binding site might consist of more than four glucose residues linked via alpha-1,6 linkages. The aptamer was very specific to the Sephadex matrix and did not bind appreciably to other supporting matrices, such as Sepharose, Sephacryl, cellulose or pustulan. Using Sephadex G-100, the aptamer could be purified from a complex mixture of cellular RNA, giving an enrichment of at least 60 000-fold, compared with a non-specific control RNA. These RNA aptamers can be used as affinity tags for RNAs or RNA subunits of ribonucleoproteins to allow rapid purification from complex mixtures of RNA using only Sephadex.

Acrylic Resins↗
PubMed1983

Pertussis toxin. Affinity purification of a new ADP-ribosyltransferase.

Pertussis toxin, the major toxin produced by Bordetella pertussis, catalyzes the ADP-ribosylation of a specific membrane polypeptide which appears to be involved in regulation of the catalytic subunit of adenylate cyclase. In the current study, a rapid purification procedure has been developed for the preparation of pertussis toxin in high yields. Through the sequential use of the affinity matrices Affi-Gel blue and fetuin-Sepharose 4B, milligram quantities of apparently homogeneous toxin can be prepared from the culture supernatants of B. pertussis strain 165. Structural, amino acid, and immunologic analyses indicate that toxin prepared from strain 165 is indistinguishable from toxin prepared from other strains. Activation of the ADP-ribosyltransferase activity requires treatment of the toxin with a thiol reducing agent. This activation appears to be associated with the reduction of intrachain disulfide bonds present in the catalytic subunit. Activated toxin preparations catalyzed ADP-ribosylation of a protein (Mr = 40,000) present in cell membrane preparations obtained from human red blood cells and platelets, rat adipocytes, and cyc- S49 cells which are deficient in the adenylate cyclase regulatory component which is the substrate for cholera toxin.

Adenylate Cyclase Toxin↗
PubMed1991

Affinity purification of transcription factor IIA from HeLa cell nuclear extracts.

One of the general transcription factors, TFIIA, was purified to homogeneity from HeLa cell nuclear extracts by yeast TFIID affinity chromatography. Human TFIIA had a molecular weight of approximately 38 kd. It was able to associate with the complex formed by yeast TFIID and the TATA elements of the adenovirus E4 and ML promoters, and the HSP70 promoter. The association extended the protected region on each TATA element by yeast TFIID from DNase I digestion. Affinity-purified TFIIA was also able to stimulate transcription from the E4 and ML promoters in in vitro reconstituted systems.

Adenoviridae↗
PubMed1992

Design of hydropathically complementary peptides for Big Endothelin affinity purification.

Molecular recognition between Big Endothelin (Big ET) and a computer generated peptide hydropathically complementary to Big ET[16-29] sequence has been studied by analytical high performance liquid affinity chromatography (HPLAC), circular dichroism (CD) and nuclear magnetic resonance (NMR) experiments. Specific binding was observed between solid support immobilized complementary peptide and Big ET[1-38], [1-32], and [16-32], but not with Big ET fragments [1-21], [16-21], [22-32], and [22-38], obtained by chymotrypsin proteolytic degradation. Selectivity in the recognition process was clearly demonstrated by the ability of complementary peptide affinity column to purify the Big ET molecule from complex peptide mixtures, even when present in very low concentrations. Similar selectivity was evidenced with the Big ET fragment [16-32], [NH2-HLDIIWVNTPEHIVPYG-COOH] containing the entire hydropathically complementary sequence. Binding was followed by marked spectroscopic changes, as monitored by circular dichroism and one- and two-dimensional nuclear magnetic resonance experiments. The NMR spectra of the complementary peptides 1:1 mixture showed variations in the chemical shifts of proton resonances in several residues, both in the main chain (amide protons) and in the side chains (aliphatic and aromatic protons). These data support the hypothesis of a multilocalized type of interaction between complementary peptides, where many residues along the peptide chains participate in co-operative stabilizing contacts in the forming complex.

Amino Acid Sequence↗
PubMed1984

Heparin affinity: purification of a tumor-derived capillary endothelial cell growth factor.

A tumor-derived growth factor that stimulates the proliferation of capillary endothelial cells has a very strong affinity for heparin. This heparin affinity makes it possible to purify the growth factor to a single-band preparation in a rapid two-step procedure. The purified growth factor is a cationic polypeptide, has a molecular weight of about 18,000, and stimulates capillary endothelial cell proliferation at a concentration of about 1 nanogram per milliliter.

Angiogenesis Inducing Agents↗
PubMed1986

Affinity purification of staphylococcal toxic shock syndrome toxin 1 and its pathologic effects in rabbits.

Toxic shock syndrome toxin 1 (TSST-1) was purified to apparent homogeneity by chromatofocusing and affinity chromatography. The amino acid composition of the toxin was very similar to that reported for TSST-1 by other investigators. The amino-terminal amino acid was serine. A partial specific volume of 0.73 ml/g was calculated for the toxin from the amino acid data, and a molecular weight of 19,200 +/- 1,300 was determined by hydrodynamic methods. New Zealand white rabbits of both sexes were equally susceptible to the lethal effects of the toxin; however, older rabbits (greater than 12 months) were far more susceptible than young adults or weanlings. The 50% lethal dose of TSST-1 in older rabbits was 50 to 60 micrograms/kg when injected subcutaneously and 20 to 30 micrograms/kg when injected intravenously. Enhancement of lethal endotoxin shock by TSST-1 could not be demonstrated when both toxins were injected subcutaneously; however, lethal shock did occur when endotoxin (10 micrograms/kg) was injected intravenously after TSST-1 had been injected by either the subcutaneous (50 to 60 micrograms/kg) or the intravenous (20 to 30 micrograms/kg) route. Endotoxin alone was not lethal at a dose of 500 micrograms/kg of body weight when injected subcutaneously. When injected intravenously, endotoxin at a dose of 500 micrograms/kg was not lethal in weanling males or in females in any age group; however, young (6 to 7 months) and adult (greater than 12 months) males were killed by endotoxin doses as low as 45 to 50 micrograms/kg. Histopathologic studies of rabbits by both sexes which died as a result of TSST-1 alone or in combination with endotoxin showed extensive damage to organs rich in lymphoid and mononuclear phagocytic cells such as the thymus, mesenteric lymph nodes, liver, and spleen. Severe congestion of these organs as well as erythrophagocytosis and lymphoid depletion in the spleen and mesenteric lymph nodes were noted. Congestion and hemorrhage were also found in the heart, lungs, trachea, and thymus. The systemic pathology produced by TSST-1 was strikingly similar to that seen in humans who had died of toxic shock syndrome and in rabbits with subcutaneous chamber inoculated with toxic shock case strains of Staphylococcus aureus. Rabbits that were not killed by the toxin suffered a very rapid and severe leukopenia followed by leukocytosis with a left shift. Lymphopenia was also noted as was a mild but persistent anemia. With the exception of the early leukopenia, very similar hematologic findings have been noted in humans with toxic shock syndrome.

Amino Acids↗
PubMed1996

Affinity purification of N-acetylglucosamine specific lectins. Purification and partial charactersation of triticale lectin.

Seeds of Triticale contain a lectin that agglutinates rabbit erythrocytes whose activity is inhibited by N-Acetylglucosamine and its oligomers. An affinity method has been developed that efficiently binds the lectin activities from Triticale seeds and wheatgerm. Purified Triticale lectin is a glycoprotein with 3% carbohydrate and migrates as a single band corresponding to Mr. 15000 on SDS-PAGE. Antibodies raised to purified wheat germ agglutinin do not cross-react with purified Triticale lectin. Leucine/Isoleucine is the only terminal residue in the Triticale lectin. Presence of inhibitory sugar induces spectral changes in the protein in the UV region. Triticale lectin does not agglutinate human ABO erythrocytes and does not inhibit fungal growth.

Acetylglucosamine↗
PubMedDate not supplied

Development of mammalian serum albumin affinity purification media by peptide phage display.

Several phage isolates that bind specifically to human serum albumin (HSA) were isolated from disulfide-constrained cyclic peptide phage-display libraries. The majority of corresponding synthetic peptides bind with micromolar affinity to HSA in low salt at pH 6.2, as determined by fluorescence anisotropy. One of the highest affinity peptides, DX-236, also bound well to several mammalian serum albumins (SA). Immobilized DX-236 quantitatively captures HSA from human serum; mild conditions (100 mM Tris, pH 9.1) allow release of HSA. The DX-236 affinity column bound HSA from human serum with a greater specificity than does Cibacron Blue agarose beads. In addition to its likely utility in HSA and other mammalian SA purifications, this peptide media may be useful in the proteomics and medical research markets for selective removal of mammalian albumin from serum prior to mass spectrometric and other analyses.

Amino Acid Sequence↗
PubMed1999

Affinity-purification of a TMV-specific recombinant full-size antibody from a transgenic tobacco suspension culture.

A TMV-specific full-size murine IgG-2b/K antibody (mAb24) was expressed in a Nicotiana tabacum cv. Petite Havana SR1 suspension culture (P9s), which was derived from a stably transformed transgenic plant (P9). The integration of an N-terminal murine leader peptide directed the assembled immunoglobulin for secretion. However, in suspension culture, the full-size recombinant antibody, rAb24, was retained by the plant cell wall and was not present in the culture medium. rAb24 expression reached a basal level of 15 microg per gram wet cell weight, corresponding to 0.3% of the total soluble plant cell protein. The level of rAb24 could be increased three-fold by amino acid supplementation of the culture medium. For purification of the recombinant antibody from batch-cultured tobacco suspension cells, the primary plant cell wall was partially digested by enzymatic treatment. This resulted in a total release of recombinant full-size rAb24 into the extraction buffer. A three-step procedure was used to purify the immunoglobulins, starting with cross-flow filtration (step 1) followed by protein A affinity chromatography (step 2) and gel filtration as a final purification step (step 3). This procedure gave a recovery of more than 80% of the expressed rAb24 from plant cell extracts. SDS-PAGE, IEF and immunoblot analyses demonstrated a high degree of homogeneity for the affinity-purified rAb24. An ELISA procedure demonstrated that the specificity and affinity of the protein A affinity purified antibody was indistinguishable from its murine counterpart, indicating the potential of plant cell suspension cultures as bio-reactors for the production of recombinant antibodies.

Animals↗
PubMed1984

Inactive proenzyme to tissue-type plasminogen activator from human melanoma cells, identified after affinity purification with a monoclonal antibody.

The human 66 000 mol. wt. plasminogen activator (HPA66; tissue-type plasminogen activator) has been purified from melanoma cells by a one-step affinity method with a monoclonal antibody. HPA66 purified in this way consists mainly of a one-polypeptide chain form with small amounts (15%) of a form containing two polypeptide chains held together by one or more disulphide bridges. The one-chain form was converted to the two-chain form by catalytic amounts of plasmin. During the conversion, the enzyme activity of HPA66, as measured by an [125I]plasminogen conversion assay and with a chromogenic substrate, increased linearly with the percentage of the two-chain form. A linear regression analysis showed that all enzyme activity could be accounted for by the two-chain form, while the one-chain form had no measurable enzyme activity (detection limit approximately 5% of the activity of the two-chain form). Together with previous findings of inactive proenzymes to murine and human approximately 50 000 mol. wt. (urokinase-type) plasminogen activators, these findings indicate that plasminogen activators are generally formed from inactive one-chain proenzymes which are converted to active two-chain enzymes by limited proteolysis, thus demonstrating a third step in a cascade reaction leading to extracellular proteolysis.

Antibodies, Monoclonal↗