Purification of the hepatic vasopressin receptor using a novel affinity column

A vasopressin receptor was purified, using a novel affinity column, from rat liver plasma membranes treated with guanosine 5'-(3-O-thio)triphosphate and solubilized with 0.8% cholate. Incubation of the membranes with the GTP analogue resulted in a dissociation of the receptor-guanine nucleotide regulatory protein complex. This manipulation, although resulting in a low-affinity state of the receptor, facilitated purification. The solubilized receptor was assayed using a new reconstitution procedure in which the soluble extracts were inserted into lipid vesicles composed of phosphatidylcholine and phosphatidylinositol. The receptor was purified by sequential chromatography on Q-Sepharose and hydroxyapatite. The use of a novel affinity column, a V1-vasopressin antagonist-agarose, resulted in a near-homogeneous preparation of a protein which exhibited an Mr = 58,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autoradiography of purified receptor, as well as crude membrane preparations cross-linked to [125I]arginine vasopressin, also revealed a protein band with an approximate Mr = 58,000. These findings indicate that V1-antagonist affinity chromatography should be useful for purifying adequate amounts of the receptor for studies of structure and function.     

Purification of individual tRNAs using a monoclonal anti-AMP antibody affinity column

A murine monoclonal anti-AMP antibody affinity matrix was used for isolation of individual species of amino acid transfer nucleic acids (tRNAs). The antibodies had been prepared using 5'-AMP covalently attached to bovine serum albumin as antigen and exhibited high affinity for 5'-AMP but greatly reduced affinity for 3'-AMP. Native uncharged tRNAs that terminate in a 5'-AMP group on the amino acid acceptor arm of the molecule bind tightly to the anti-AMP affinity matrix, whereas aminoacylated tRNAs are not retained. This allows separation of a particular tRNA species as its aminoacyl derivative from a complex mixture of uncharged tRNAs under very mild conditions.     

Purification of L-type pyruvate kinase from human liver by affinity chromatography on Blue-Dextran-Sepharose column

L-type pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase; EC 2.7.1.40) was purified from human liver by an original method. This purification included toluene extraction, a-monium sulphate fractionation, DEAE-Sephadex bactchwise, CM-Sephadex batchwise with elective elution by ATP and affinity chromatography on a Blud Dextran-Sepharose column with specific elution by fructose 1, 6-diphosphate. This purification procedure allowed us to obtain 6 mg protein with a specific activity of 420 IU/mg protein, i.e. 2,690-fold purification with an overall yield of 34%. This preparation was homogeneous as judged by immuno-diffusion, acrylamide and sodium dodecyl sulphate acrylamide-gel electrophoresis.     

Purification of guinea pig liver transglutaminase using a phenylalanine-sepharose 4B affinity column

Guinea pig liver transglutaminase has been purified utilizing an improved protocol. The new steps include QAE-Sephadex ion-exchange, hydroxylapatite adsorption, and affinity chromatography using a phenylalanine-Sepharose column. The overall yield for the enzyme was 31%. This new scheme should enable more laboratories to take advantage of the protein crosslinking and labeling properties of transglutaminase.     

Purification of a metallothionein-like protein from serum of mercury-treated rats using phosphorylcholine-Sepharose affinity column

A metallothionein-like protein (MLP) from the serum of mercury-treated rats was isolated while purifying an acute phase plasma protein, C-reactive protein, by its Ca2(+)-dependent affinity to phosphorylcholine (PC)-Sepharose column. The MLP was further purified by a single DEAE-Sephacel ion-exchange chromatography. The MLP is similar to mammalian hepatic Zn-metallothionein on the basis of its low molecular weight of approximately 7000, 7 g Zn atoms/molecule of MLP and an absorption maximum at 220 nm. The purity of the protein was confirmed by double immunodiffusion test against anti-MLP antiserum raised in a rabbit. Immunologically MLP was detected not only in the hepatic cytosol, serum and urine from Hg-treated rats but also in the serum and hepatic cytosol of untreated rats. Using PC-Sepharose column, MLP could not be purified from normal serum.     

Purification of adenosine deaminase from chicken-egg yolk by affinity column chromatography

Adenosine deaminase (adenosine aminohydrolase; E.C. 3.5.4.4) has been purified 4686-fold from egg yolk. The procedure developed was used to isolate the enzyme from eight chicken eggs. An easily prepared affinity column employing purine riboside was used as the final step in the purification. The method developed permits the rapid isolation and a high recovery of the protein. The specific activity of the enzyme preparation obtained is 81.4 mU/mg.     

Purification of guanosine triphosphate cyclohydrolase I and dihydrofolate reductase on a dihydrofolate-Sepharose affinity column.

Folate was coupled to AH-Sepharose 4B, the gel poured into small columns, and the Sepharose-bound folate reduced in situ to dihydrofolate by dithionite/ascorbate at pH 6 to 7. The dihydrofolate-Sepharose column was used to purify guanosine triphosphate cyclohydrolase I (EC 3.5.4.16) and dihydrofolate reductase (EC 1.5.1.3). All steps were carried out in the cold and in the presence of 20 mm mercaptoethanol. GTP cyclohydrolase I bound strongly to the dihydrofolate-Sepharose column and was purified several-hundred-fold in a single step. It did not bind to folate-Sepharose. Binding to dihydrofolate-Sepharose is assumed to reflect a physiological role of dihydrofolate. GTP cyclohydrolase II did not bind to either folate- or dihydrofolate-Sepharose. Dihydrofolate reductase from Escherichia coli B and from rat liver did not bind to folate-Sepharose under the test conditions, but could be well purified on the dihydrofolate-Sepharose column. This column is judged to beuseful for the purification of other folate-converting enzymes.     

Purification of the (Ca2+-Mg2+)-ATPase from human erythrocyte membranes using a calmodulin affinity column.

The (Ca2+-Mg2+)-ATPase from human erythrocyte membranes has been solubilized in Triton X-100 and purified on a calmodulin affinity chromatography column in the presence of phosphatidylserine, to limit the inactivation of the enzyme. The enzyme was purified at least 150 times when compared with the original ghosts and showed a specific activity of 3.8 mumol.mg-1.min-1. In sodium dodecyl sulfate-polyacrylamide gels, a single major band was visible at a position corresponding to a molecular weight of about 125,000; a minor band (11% of the total protein) was present at a position corresponding to Mr = 205,000. Upon incubation of the purified preparation with [32P]ATP, both bands were phosphorylated in proportion to their mass, suggesting that both were active forms of purified ATPase.     

Purification of two enolases from human brain using a chromatofocusing column

When a purified preparation of rat αγ enolase (2-phospho-D-glycerate hydrolyase, EC 4.2.1.11) was applied to a chromatofocusing column, the enolase was almost completely dissociated and recombined to form αα and γγ enolases, which were eluted at different fractions from the column. Using these phenomena, two homo-dimer forms (αα and γγ) of human brain enolase were purified from a crude preparation of the hybrid αγ enolase by the chromatofocusing, and subsequent chromatography on a QAE-Sephadex column (αα) or a DEAE-Sephadex column (γγ). Each purified preparation showed a single band on SDS-gel electrophoresis with a relative mobility corresponding to a molecular weight of about 50 000. Amino acid analysis, peptide mapping analysis with a limited proteolysis and immunochemical studies of the purified αα and γγ enolases revealed that the two subunits, α and γ, are distinct proteins. The antisera to human αα or γγ enolase cross-reacted with the respective form of rat enolase.     

Reaction of a cyclic hydroxamic acid from gramineae with thiols

An insect inhibitor isolated from maize extracts, 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), reacted with cysteine, mercaptoethanol, ethane     

Purification of GFP fusion proteins with high purity and yield by monoclonal antibody-coupled affinity column chromatography

GFP has often been used as a marker of gene expression, protein localization in living and fixed tissues as well as for protein targeting in intact cells and organisms. Monitoring foreign protein expression via GFP fusion is also very appealing for bioprocess applications. Many cells, including bacterial, fungal, plant, insect and mammalian cells, can express recombinant GFP (rGFP) efficiently. Several methods and procedures have been developed to purify the rGFP or recombinant proteins fused with GFP tag. However, most current GFP purification methods are limited by poor yields and low purity. In the current study, we developed an improved purification method, utilizing a FMU-GFP.5 monoclonal antibody (mAb) to GFP together with a mAb-coupled affinity chromatography column. The method resulted in a sample that was highly pure (more than 97% homogeneity) and had a sample yield of about 90%. Moreover, the GFP epitope permitted the isolation of almost all the active recombinant target proteins fused with GFP, directly and easily, from the crude cellular sources. Our data suggests this method is more efficient than any currently available method for purification of GFP protein.     

Nitroproteins from a human pituitary adenoma tissue discovered with a nitrotyrosine affinity column and tandem mass spectrometry

The aim of this study was to characterize endogenous nitroproteins, and those proteins that interact with nitroproteins, in a human pituitary nonfunctional adenoma so as to clarify the role of protein nitration in adenomas. A nitrotyrosine affinity column (NTAC) was used to preferentially enrich and isolate endogenous nitroproteins and nitroprotein-protein complexes from a tissue homogenate that was prepared from a human pituitary nonfunctional pituitary adenoma. The preferentially enriched endogenous nitroproteins and nitroprotein-protein complexes were subjected to trypsin digestion, desalination, and tandem mass spectrometry analysis. Nine nitroproteins (Rho-GTPase-activing protein 5, leukocyte immunoglobulin-like receptor subfamily A member 4 precursor, zinc finger protein 432, cAMP-dependent protein kinase type I-beta regulatory subunit, sphingosine-1-phosphate lyase 1, centaurin beta 1, proteasome subunit alpha type 2, interleukin 1 family member 6, and rhophilin 2) and three proteins (interleukin 1 receptor-associated kinase-like 2, glutamate receptor-interacting protein 2, and ubiquitin) that interacted with nitroproteins were discovered. The nitration site of each nitroprotein was located onto the functional domain where nitration occurred, and each nitroprotein was related to a corresponding functional system. Those data indicate that protein nitration might be an important molecular event in the formation of a human pituitary nonfunctional adenoma.     

Crystal structure of human macrophage elastase (MMP-12) in complex with a hydroxamic acid inhibitor

Human macrophage elastase (MMP-12) is a member of the family of matrix metalloproteinases (MMPs) that plays, like other members of the family, an important role in inflammatory processes contributing to tissue remodelling and destruction. In particular, a prominent role of MMP-12 in the destruction of elastin in the lung alveolar wall and the pathogenesis of emphysema has been suggested. It is therefore an attractive therapeutic target. We describe here the crystal structure of the catalytic domain of MMP-12 in complex with a hydroxamic acid inhibitor, CGS27023A. MMP-12 adopts the typical MMP fold and binds a structural zinc ion and three calcium ions in addition to the catalytic zinc ion. The enzyme structure shows an ordered N terminus close to the active site that is identical in conformation with the superactivated form of MMP-8. The S1'-specificity pocket is large and extends into a channel through the protein, which puts MMP-12 into the class of MMPs 3, 8 and 13 with large and open specificity pockets. The two crystallographically independent molecules adopt different conformations of the S1'-loop and its neighbouring loop due to differing crystal packing environments, suggesting that flexibility or the possibility of structural adjustments of these loop segments are intrinsic features of the MMP-12 structure and probably a common feature for all MMPs. The inhibitor binds in a bidentate fashion to the catalytic zinc ion. Its polar groups form hydrogen bonds in a substrate-like manner with beta-strand sIV of the enzyme, while the hydrophobic substituents are either positioned on the protein surface and are solvent-exposed or fill the upper part of the specificity pocket. The present structure enables us to aid the design of potent and selective inhibitors for MMP-12.     

Purification of human plasma haptoglobin by hemoglobin-affinity column chromatography

Haptoglobin (Hp) is an acute-phase protein; its plasma levels increase consistently in response to infection and inflammation. The concentration of human plasma Hp is ranged between 1 and 1.5 mg/ml. Similar to blood type, individual human Hp is classified as Hp 1-1, 2-1, or 2-2. The structural and functional analysis of the Hp, however, has not been studied in detail due to its difficult isolation procedure. Previously, we reported a single step for the purification of porcine Hp. In this study, we established a purification method using a high capacity hemoglobin-affinity column. Briefly, DEAE-purified human hemoglobin was first coupled to Sepharose 4B to prepare an affinity column in a 15-ml bed volume. Following a flow through of human plasma and an extensive wash, the bound material was eluted with a solution of 0.15 M NaCl, pH 11 (adjusted by ammonium), to remove low-affinity bound proteins. The high-affinity bound Hp was then eluted with 0.15 M NaCl containing 5 M urea, pH 11, and collected in tubes containing 100 microl of 1 M Tris buffer, pH 7.0. The biological activity of dialyzed Hp was retained as it formed a complex with hemoglobin on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Using this procedure, approximately 10 mg of Hp 1-1, with homogeneity greater than 96%, was obtained from 15 ml of human plasma. Affinity purified Hp 2-1 or 2-2, however, contained trace amounts of apoA-I with the similar approach. The Hp could be further purified by HPLC using a Superose 12 gel-permeation chromatography, if desired, to achieve 100% purity. All the phenotypes of purified Hp consisted of alpha and beta chains on SDS-PAGE in the presence of a reducing reagent, further confirmed by a Western blot analysis. We conclude that human hemoglobin-affinity column was most suitable for the isolation of Hp 1-1 in large quantities. Whereas, one additional step using a gel-permeation was necessary for that of Hp 2-1 and 2-2.     

Purification of human liver glycoprotein sialyltransferase by affinity chromatography

A simple procedure has been developed for purifying solubilized human liver glycoprotein sialyltransferase (EC 2.4.99.1) 16 000-fold in 4–5% yield. The procedure involves two centrifugation steps, affinity chromatography of the ultrasupernatant fluid on cytidine diphosphate-hexanolamine-agarose followed by gel filtration on Sephadex G-150. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that the purified sialyltransferase preparation contains approximately equivalent amounts of three protein bands (with apparent molecular weights of 61 000, 63 000 and 70 000) and is highly purified if not homogeneous.     

Purification of a putative Na+/D-glucose cotransporter from pig kidney brush border membranes on a phlorizin affinity column

Phlorizin, a potent inhibitor of the Na+/D-glucose cotransporter, was derivatised to 3-aminophlorizin and subsequently coupled to Affi-Gel 15. Affinity chromatography of pig kidney brush border membranes solubilised in Triton X-100 allowed the purification of a 60 kDa protein on this resin. We consider this protein to be the Na+/D-glucose cotransporter, or part of it, for the following reasons: (i) binding of this protein to Affi-Gel 15 specifically requires phlorizin covalently attached to the resin and is lowered when phlorizin is replaced by phloretin; (ii) binding of the 60 kDa protein to a phlorizin affinity column requires the presence of Na+; (iii) polyclonal as well as monoclonal antibodies against the 60 kDa protein inhibit binding of phlorizin to brush border membranes from rabbit and pig kidney.