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PubMed1996

Solubilization, partial purification, and affinity labeling of the membrane-bound isoprenylated protein endoprotease.

A previously described [Ma, Y.-T., & Rando, R. R. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6275-6279] membrane-associated isoprenylated protein endoprotease is important in the processing of isoprenylated proteins terminating with CAAX. The enzyme is of substantial interest because specific inhibitors of it block the processing and functioning of ras in vivo. The enzyme appears to be an integral membrane protein, as it can only be removed from microsomal membranes with detergent. The enzyme is effectively solubilized by the detergent CHAPSO and can be partially purified (approximately 10-fold) by anion ion exchange and size exclusion chromatography. Attempts to further purify the enzyme by other column means, including affinity chromatography, were unsuccessful. The partially purified enzyme is very sensitive to thiol reagents but insensitive to other kinds of protease inhibitors, suggesting that the enzyme is a thiol protease. Potent and specific chloroketone containing affinity labeling agents have been developed. These novel inactivators owe their potency to an S-farnesylcysteine moiety which is recognized by the enzyme. Specific inhibitors of this type should allow for the identification and cloning of this protease, which is important for signal transduction.

Amino Acid Chloromethyl Ketones↗
PubMed1998

A nucleotide-binding domain of porcine liver annexin VI. Proteolysis of annexin VI labelled with 8-azido-ATP, purification by affinity chromatography on ATP-agarose, and fluorescence studies.

Porcine liver annexin VI (AnxVI) of Mr 68.000 is an ATP-binding protein as evidenced by specific and saturable UV-dependent labelling with 8-azido-[gamma-32P]ATP or the fluorescent analog of ATP, 2'-(or 3')-O-(2,4,6-trinitrophenyl)adenosine triphosphate and by binding of AnxVI to ATP-agarose. These characteristics of purified AnxVI were used to identify and characterize preliminary nucleotide-binding domain of the protein. AnxVI labelled with 8-azido-ATP was subjected to limited proteolysis and the proteolytic fragments of AnxVI that retained the covalently-bound nucleotide were separated by means of gel electrophoresis and visualized by exposure of the gel to a phosphor storage screen. It was found that the AnxVI proteolytic fragments of Mr 34-36.000 and smaller retained the nucleotide. In a reciprocal experiment, AnxVI was digested with proteolytic enzymes and in an ATP eluate from an ATP-agarose column protein fragments of similar Mr to these labelled with 8-azido-ATP were identified. The extent of AnxVI labelling with 8-azido-ATP and the distribution of proteolytic fragments varied upon calcium concentration. These results lead to the conclusion that there is a nucleotide-binding domain within the AnxVI molecule that is functionally similar to the nucleotide-binding domains of other nucleotide-binding proteins. The nucleotide-binding domain is located close to the tryptophan residue 343 of AnxVI and in close vicinity to the Ca2+- and phospholipid-binding sites of the protein. This is confirmed by the observation that the tryptophan fluorescence intensity of AnxVI decreases in the presence of a fluorescence analog of ATP in a calcium-dependent manner, due to the quenching properties of the nucleotide and/or fluorescence energy transfer from AnxVI tryptophan to fluorophore. Both processes were modulated by the presence of phospholipid molecules.

Adenosine Triphosphate↗
PubMed1980

Studies on human pregnancy-associated plasma protein A. Purification by affinity chromatography and structural comparisons with alpha 2-macroglobulin.

Pregnancy-associated plasma protein-A (PAPP-A) has been purified by a combination of methods including antibody-affinity chromatography. The resultant protein, obtained in 16% yield from maternal serum, appeared as a single major component on non-denaturing polyacrylamide and SDS/polyacrylamide gel electrophoresis. The protein showed a single component when analysed by isoelectric focusing under denaturing conditions in the presence and absence of reduction and had a pI of 4.34 and 4.42 respectively. These pI values were indistinguishable from those of alpha 2-macroglobulin (alpha 2M). The molecular weight of the PAPP-A polypeptide as shown by SDS/polyacrylamide-gel electrophoresis was 187000, with a minor component of mol.wt. 82500 that was attributed to proteolysis. Since native PAPP-A had a molecular weight on gel chromatography very similar to that of alpha 2M (620000--820000), it was concluded that PAPP-A was a homotetramer. In the absence of reduction, a high-molecular-weight (420000) protomer of PAPP-A was found. It was deduced that PAPP-A, like alpha 2M, is a dinner, whose protomers are composed of disulphide-linked polypeptide chains. It was found that the molecular weight of the PAPP-A polypeptide exceeded that of alpha 2M by 3.3%, but that the total carbohydrate content of PAPP-A exceeded that of alpha 2M by 10% and that its neutral carbohydrate content exceeded that of alpha 2M by between 7.4 and 9.0%. The significance of the estimated molecular weights of alpha 2M (181000) and its major tryptic fragments is discussed in the light of published values. A tryptic fragment alpha 2M (82500 mol.wt.) was apparently the same size as the major tryptic fragment of PAPP-A.

Carbohydrates↗
PubMed1984

Purification by affinity chromatography and immunological characterization of a 110kDa component of the chick oviduct progesterone receptor.

A 110kDa component of the chick oviduct progesterone receptor (PR) has been purified to homogeneity according to electrophoretic criteria and specific activity (assuming one progestagen-binding site/110kDa). The procedure involved affinity chromatography of 0.3 M-KCl-prepared cytosol, followed by DEAE-Sephacel chromatography (elution at 0.2 M-KCl). The final yield was about 12% in terms of binding activity. Properties of the 110kDa component indicate that it is identical with the 'B' subunit described previously [Stokes radius approximately 6.1 nm; sedimentation coefficient, (S20, w) approximately 4S; frictional ratio approximately 1.77]. It reacted with the IgG-G3 polyclonal antibody, but not with BF4 monoclonal antibody raised against the 8S molybdate-stabilized chick oviduct PR and reacting with its 90kDa component. Another progesterone-binding component, corresponding to the 'A' subunit, also previously described, was eluted from the DEAE-Sephacel column at approximately 0.08 M-KCl, and contained a peptide of molecular mass approx. 75-80kDa, which had S20, w approximately 4S in a sucrose gradient. This component was also recognized by IgG-G3, but not by BF4; it was very unstable in terms of hormone-binding activity.

Animals↗
PubMed1984

Human plasma alpha-cysteine proteinase inhibitor. Purification by affinity chromatography, characterization and isolation of an active fragment.

Human plasma alpha-cysteine proteinase inhibitor (alpha CPI) was purified by a two-stage method: affinity chromatography on S-carboxymethyl-papain-Sepharose, and high-resolution anion-exchange chromatography. The protein was obtained as a form of Mr about 64 000 and material of higher Mr (about 100 000). In sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with reduction, both forms showed a major component of Mr 64 000. An antiserum was raised against alpha CPI, and 'rocket' immunoassays showed the mean concentration in sera from 19 individuals to be 35.9 mg/dl. Both low-Mr and high-Mr forms of alpha CPI were confirmed to be sialoglycoproteins by the decrease in electrophoretic mobility after treatment with neuraminidase. alpha CPI was shown immunologically to be distinct from antithrombin III and alpha 1-antichymotrypsin, two serine proteinase inhibitors from plasma with somewhat similar Mr values. alpha CPI was also distinct from cystatins A and B, the two intracellular low-Mr cysteine proteinase inhibitors from human liver. Complexes of alpha CPI with papain were detectable in immunoelectrophoresis, but dissociated to free enzyme and intact inhibitor in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The stoichiometry of binding of papain was close to 1:1 for both low-Mr and high-Mr forms. alpha CPI was found to be a tight-binding inhibitor of papain and human cathepsins H and L (Ki 34 pM, 1.1 nM and 62 pM respectively). By contrast, inhibition of cathepsin B was much weaker, Ki being about 35 microM. Dipeptidyl peptidase I also was weakly inhibited. Digestion of alpha CPI with bromelain gave rise to an inhibitory fragment of Mr about 22 000, which was isolated.

Blood Proteins↗
PubMed1971

Partial purification by affinity chromatography of tyrosine aminotransferase-synthesizing ribosomes from hepatoma tissue culture cells.

This report describes a cell-free protein-synthesizing system derived from hepatoma tissue culture cells in which the synthesis of tyrosine aminotransferase is enhanced in relation to total protein synthesis. The enhancement was achieved by using affinity chromatography to select a specific fraction of ribosomes in which the proportion of tyrosine aminotransferase synthesis is higher than it is in unfractionated ribosomes.

Adsorption↗
PubMed1972

Identification of the cardiac beta-adrenergic receptor protein: solubilization and purification by affinity chromatography.

A protein that binds catecholamines with a specificity parallel to that of their in vivo effects on cardiac contractility (isoproterenol > epinephrine or norepinephrine > dopamine > dihydroxyphenylalanine) was solubilized from a microsomal fraction of canine ventricular myocardium. The binding protein was purified 500 to 800-fold by solubilization and subsequent affinity chromatography with conjugates of norepinephrine linked to agarose beads. Purified beta-adrenergic binding protein exists in two forms, corresponding to molecular weights of 40,000 and 160,000. The purified material has a single association constant, 2.3 x 10(5) liters/mol (as compared to two association constants, 10(7) and 10(6) liters/mol, for the binding protein in particulate form) but retains the identical binding specificity for beta-adrenergic drugs and antagonists.

Amination↗
PubMed1979

Eukaryotic mRNA cap binding protein: purification by affinity chromatography on sepharose-coupled m7GDP.

A 24,000-dalton polypeptide that binds strongly and can be specifically crosslinked to the 5'-terminal cap structure m7GpppN in eukaryotic mRNAs has been detected in protein synthesis initiation factor preparations [Proc. Natl. Acad. Sci. USA (1978) 75, 4843--4847]. This polypeptide has been purified to apparent homogeneity by one chromatographic passage through an affinity resin prepared by coupling the levulinic acid O2',3'-acetal of m7GDP to AH-Sepharose 4B. Translation, in HeLa cell extracts, of capped mRNAs including Sindbis virus, reovirus, and rabbit globin mRNAs was stimulated by the cap-binding protein under conditions that did not increase translation of noncapped RNAs of encephalomyocarditis virus and satellite tobacco necrosis virus.

Animals↗
PubMed2001

Identification of endogenous SsrA-tagged proteins reveals tagging at positions corresponding to stop codons.

The SsrA.SmpB quality control system adds a C-terminal degradation peptide (AANDENYALAA) to nascent chains on stalled ribosomes, thereby freeing the ribosome and ensuring proteolysis of the tagged protein. An SsrA mutant with the tag sequence AANDEHHHHHH was used to slow degradation and facilitate Ni2+-nitrilotriacetic acid affinity purification. Display of affinity-purified Escherichia coli proteins on two-dimensional gels revealed small quantities of a diverse set of SsrA-H6-tagged proteins, and mass spectroscopy identified LacI repressor, lambda cI repressor, YbeL, GalE, RbsK, and a SlyD-kan(R) fusion protein as members of this set. For lambda repressor and YbeL, the SsrA-H6 tag was added after the natural C terminus of the protein, suggesting that tagging occurred while the ribosome idled at the termination codon of these genes. Potential causes of tagging for the other proteins include interference from translation of downstream reading frames, rare codons, and gene disruption. These and previous results support a broad role for the SsrA.SmpB system in freeing stalled ribosomes and in directing degradation of the products of these frustrated protein synthesis reactions.

Amino Acid Sequence↗
PubMed2000

Ribonuclease from cobra snake venom: purification by affinity chromatography and further characterization.

A ribonuclease from cobra snake venom was isolated and purified to homogeneity using antibody-affinity chromatography, increasing the yield fourfold. The purified enzyme showed cytidylic acid specificity, as reported earlier. Further, the effects of temperature, pH, metal ions, inhibitors, and urea on the enzyme activity were studied. Snake venom RNase exhibited salt-dependent reversible association-dissociation behaviour. Immunological studies indicate that this enzyme shares one of the antigenic sites of RNase A. The partial N-terminal sequence of the enzyme showed considerable homology with phospholipases from snake venom; however, the enzyme itself did not show any phospholipase activity.

Amino Acid Sequence↗
PubMed1977

Distribution of neuraminidase in Arthrobacter and its purification by affinity chromatography.

Neuraminidase [sialidase, EC 3.2.1.18] was found to be widely distributed in bacteria belonging to Arthrobacter. Among these bacteria, Arthrobacter ureafaciens, A. oxydans, and A. aurescens produced relatively potent neuraminidase activities. For the production of this enzyme, not only colominic acid, a homopolymer of N-acetylneuraminic acid, but also N-acetylneuraminic acid, the reaction product of this enzyme, are effective as sources of carbon. An affinity adsorbent specific for neuraminidase was prepared by cross-linking colominic acid with soluble starch by means of epichlorohydrin. Neuraminidase from A. ureafaciens could be purified on this affinity column. The purified neuraminidase was shown to be free from protease, N-acetylneuraminic acid aldolase, phospholipase C, and glycosidases. Aminoff's assay procedure for sialic acid was modified to avoid the centrifugation step. The modified procedure gave a higher molecular extinction coefficient.

Arthrobacter↗
PubMed1980

Purification by affinity chromatography and physicochemical properties of the guanine-specific ribonuclease of Fusarium moniliforme.

Ribonuclease F1, the guanine-specific ribonuclease of Fusarium moniliforme, was purified to homogeneity by a combination of ethanol fractionation, affinity chromatography and DEAE-cellulose column chromatography. The adsorbent for the affinity chromatography was synthesized by the coupling of periodate-oxidized guanosine 5'-monophosphate to aminohexyl agarose followed by sodium borohydride reduction. Ribonuclease F2, the minor component, was also purified to near homogeneity by the same procedure. Ribonucleases F1 and F2 had the same molecular weight (about 11,000) as determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. They also showed the same amino acid composition and differed only in the isoelectric point: 4.10 for F1 and 3.96 for F2.

Amino Acids↗