Component A2 of the methylcoenzyme M methylreductase system from Methanobacterium thermoautotrophicum.

Component A2 of the methylcoenzyme M methylreductase system of Methanobacterium thermoautotrophicum has been purified 370-fold by liquid chromatography. Homogeneity was obtained by anaerobic preparative polyacrylamide gel electrophoresis. Component A2 is a colorless, air-stable protein consisting of a single polypeptide as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The relative molecular mass of the native protein was determined by high-performance, size exclusion chromatography to be Mr 52,000; on sodium dodecyl sulfate-polyacrylamide gel electrophoresis a value of Mr 59,000 was obtained. When cell extract was subjected to N6-ATP-agarose affinity chromatography the methylcoenzyme M methylreductase system was resolved into two fractions; one of them was component A2. This work provides a new operational definition for component A2, i.e., its characteristic chromatographic behavior on N6-ATP-agarose. However, its functional definition is its ability to reconstitute the methylreductase activity with components A1, A3, and C. Several attempts to assign a role to component A2 are reported.     

Human tumor necrosis factor. Production, purification, and characterization

Human tumor necrosis factor (TNF) was purified to homogeneity from serum-free tissue culture supernatants of the HL-60 promyelocytic leukemia cell line induced by 4 beta-phorbol 12-myristate 13-acetate. The purification scheme consisted of controlled-pore glass and DEAE-cellulose chromatography, Mono Q-fast-protein liquid chromatography, and reverse-phase high performance liquid chromatography. The purified protein was homogeneous by the criteria of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and NH2-terminal sequence analysis. The specific activity of purified tumor necrosis factor is approximately 10(8) units/mg. The protein has a molecular weight of approximately 17,000, an isoelectric point of 5.3, and contains two cysteines involved in a disulfide bridge. Approximately 50% homology between TNF and another cytolytic lymphokine, lymphotoxin, exists when the NH2-terminal 34 residues of TNF and internal sequence generated by tryptic, Staphylococcus aureus V8 protease, and chymotryptic digests of TNF are aligned with the complete amino acid sequence of lymphotoxin.     

Identification of a 50,000 Mr protein from rabbit brush border membranes that binds Clostridium perfringens enterotoxin

A protein that binds Clostridium perfringens enterotoxin was extracted with NP-40 from rabbit intestinal brush border membranes. This protein was partially purified by affinity chromatography on enterotoxin-coupled CNBr-activated Sepharose 4B. The molecular weight of this protein was approximately 50,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Affinity-purified samples containing this protein specifically inhibited biological activity of the enterotoxin on Vero (African green monkey kidney) cells. These studies suggest that this protein may be involved in the binding of the enterotoxin to rabbit intestinal epithelial cells.     

Purification and characterization of Saccharomyces cerevisiae uridine monophosphate kinase

The SOC8 gene was isolated as an extragenic suppressor of cdc8 mutant cells. It has been suggested that SOC8 is allelic with the URA6 gene which was originally identified as a uridine monophosphate kinase. In this article, we describe the purification of the uridine monophosphate kinase from a yeast Saccharomyces cerevisae strain that overproduces the activity 8-fold. The protein was purified through Fast-Flow Q-Separose, phosphocellulose, blue-agarose, and fast protein liquid chromatography Superose 12 columns, and appears homogeneous by sodium dodecyl sulfate-polyacrylamide gel analysis. The uridine monophosphate kinase contains a single polypeptide with a molecular weight of 25,000, as evidence by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration analysis. The amino acid composition has also been determined. Substrate specificity studies show that the relative activity of nucleoside monophosphates is in order of UMP greater than dUMP, and to a lesser extent, dTMP, GMP, and dGMP. The Km and Vm of UMP, dUMP, and dTMP have been determined.     

Isolation of a low molecular mass vasoactive intestinal peptide binding protein.

A vasoactive intestinal peptide (VIP) binding protein was purified in active form by detergent solubilization of lung membranes, gel filtration, VIP-Sepharose affinity chromatography, reverse phase high performance liquid chromatography, and anion exchange chromatography. The mass of this protein was estimated at 18 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 17 kDa by gel filtration. The binding of VIP by this protein was inhibited by Mg2+, covalent cross-linking of [Tyr10-125I]VIP to the protein produced two radioactive bands at 22 and 26 kDa identified by electrophoresis, and the purified protein exhibited saturable and high affinity binding of VIP and the related neuropeptide, rat growth hormone releasing factor.     

Characterization of the sex steroid binding protein of human pregnancy serum. Improvements in the purification procedure

The sex steroid binding protein (SBP) of human pregnancy serum was purified to homogeneity by the sequential use of ammonium sulfate precipitation, affinity chromatography on 5alpha-dihydrotestosterone-17beta-succinyldiaminoethyl-(1,4-butanediol diglycidyl ether)-agarose, and preparative polyacrylamide gel electrophoresis. The yield of pure SBP was improved from 5% as originally reported [Mickelson, K. E., and Petra, P. H. (1975), Biochemistry 14, 957] to 34%. Homogeneity of SBP was shown by equilibrium sedimentation ultracentrifugation in 6 M guanidine hydrochloride containing 0.1 M mercaptoethanol which yields a minimum molecular weight of 36 335 +/- 525. The protein is also homogeneous when examined by gel electrophoresis in the presence of sodium dodecyl sulfate. A value of 52 000 for the molecular weight is obtained by this method. SBP partially purified from Cohn fraction IV has also a molecular weight of 52 000 by gel electrophoresis in the presence of sodium dodecyl sulfate; that fraction is contaminated with another protein of molecular weight 90 000 which must be removed to obtain homogeneous SBP. The amino acid composition of SBP isolated from pregnancy serum is presented.     

Recombinant human erythropoietin: purification and analysis of carbohydrate linkage

Erythropoietin was purified to homogeneity from the culture medium of a baby hamster kidney cell line stably transfected with a human erythropoietin gene. A three-step procedure was used, which included affinity chromatography, ion-exchange chromatography, and reverse-phase chromatography. Purity of the protein was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino-terminal sequence analysis. Overall recovery was 35%. The biological activity of purified recombinant erythropoietin was similar to that of the native hormone in vitro. The purified recombinant hormone contained N-linked carbohydrate at residues 24, 38, and 83, and and O-linked carbohydrate at residue 126.     

Purification of a factor inhibiting differentiation from conditioned medium of nondifferentiating mouse myeloid leukemia cells

Mouse myeloid leukemic M1 cells are induced to differentiate by various differentiation inducers. Activity for inhibition of induction of differentiation of M1 cells (I-factor activity) was detected in conditioned medium of variant M1 cell clones that were resistant to differentiation inducers, and this I-factor activity was shown to be closely associated with resistance of the cells to differentiation inducers. In this work, the I-factor was purified to apparent homogeneity from conditioned medium of resistant M1 cells. The purification procedure consisted of ammonium sulfate precipitation, CM-Sepharose CL-6B, Sephadex G-200, reverse-phase high performance liquid chromatography on a C18 hydrophobic support, and high-performance liquid chromatography on a gel filtration column. The factor was analyzed by radioiodination, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography. The purified factor gave a single band of protein with a molecular weight of 68,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis which coincided with its biological activity. The concentration of I-factor required for 50% inhibition of dexamethasone-induced differentiation of M1 cells was 24 pM. At its effective concentration it had no effect on cell proliferation, and even at 1.2 nM it did not inhibit colony formation of normal bone marrow cells, suggesting that it was distinct from the inhibitor of normal precursors of macrophages and/or granulocytes.     

Soluble guanylate cyclase from rat lung exists as a heterodimer

The soluble form of guanylate cyclase (EC 4.6.1.2) from rat lung has been purified to homogeneity by a one-step immunoaffinity chromatographic procedure. The purified soluble guanylate cyclase has specific activities of 432 and 49.1 nmol of cyclic GMP formed per min/mg protein with manganese and magnesium ions as a cofactor, respectively. This represents a purification of approximately 2,000-fold with a 50% recovery. The native enzyme has a molecular weight of 150,000 and a Stokes radius of 4.8 nm as determined on Spherogel TSK-G3000SW gel permeation chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis results in two protein-staining bands with molecular weights of 82,000 and 70,000. The purified soluble guanylate cyclase was also subjected to native polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis, ion exchange chromatography, and GTP-agarose affinity chromatography. These additional purification procedures confirmed the presence of a single protein peak coincident with enzyme activity. The two subunits separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis were shown to have different primary structures by immunoblotting with monoclonal and polyclonal antibodies prepared against purified soluble guanylate cyclase and by peptide mapping with papain or Staphylococcus aureus V8 protease treatment. These data demonstrate that soluble guanylate cyclase purified from rat lung is a heterodimer composed of 82,000- and 70,000-dalton subunits with different primary structures.     

Purification of rosmarinic acid synthase from cell cultures of Coleus blumei Benth

Rosmarinic acid synthase from cell cultures of Coleus blumei Benth. was purified to apparent homogeneity by fractionated ammonium sulfate precipitation (60–80% saturation), hydrophobic interaction chromatography, affinity chromatography and gel filtration. This purification procedure resulted in a 225-fold-enriched specific enzyme activity with a yield of 9%. The protein preparation was apparently pure according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis. The apparent molecular mass determined by gel filtration and SDS-PAGE was 77 kDa, indicating that rosmarinic acid synthase is a monomeric enzyme.Abbreviations DTT dithiothreitol - HIC hydrophobic interaction chromatography - RA rosmarinic acid - RAS rosmarinic acid synthase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis The financial support of the Deutsche Forschungsgemeinschaft is gratefully acknowledged. Two-dimensional gel electrophoresis was done with the help of Dr. Guy Bauw, University of Gent, Belgium.     

Purification and some physicochemical properties of staphylococcal enterotoxin D.

A method was developed for the isolation of staphylococcal enterotoxin D in highly purified form from cultures of Staphylococcus aureus strain 1151m. The method involves removal of the toxin from the culture supernatant fluid with the ion-exchange resin CG-50 followed by chromatography on carboxymethylcellulose (twice) and by gel filtration on Sephadex G-75 (twice). The purified toxin is homogeneous by polyacrylamide gel and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and double gel diffusion tests. It is a simple, colorless, antigenic protein with an isoelectric point of 7.4 as determined by isoelectric focusing. Its molecular weight was determined to be 27 300 +/- 700 by molecular sieve chromatography on Sephadex G-100 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its serological activity is stable over a wide range of pH values (1.2--10.7). The enterotoxin consists of 236 amino acid residues and contains no free sulfhydryl groups. End-group analysis showed serine to be the NH2-terminal amino acid and lysine to be the COOH-terminal amino acid.     

Purification and characterization of formyl-coenzyme A transferase from Oxalobacter formigenes.

Formyl-coenzyme A (formyl-CoA) transferase was purified from Oxalobacter formigenes by high-pressure liquid chromatography with hydrophobic interaction chromatography and by DEAE anion-exchange chromatography. The enzyme was a single entity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel permeation chromatography (Mr, 44,000). It had an isoelectric point of 4.7. The enzyme catalyzed the transfer of CoA from formyl-CoA to either oxalate or succinate. Apparent Km and Vmax values, respectively, were 3.0 mM and 29.6 mumols/min per mg for formyl-CoA with an excess of succinate. The maximum specific activity was 2.15 mumols of CoA transferred from formyl-CoA to oxalate per min per mg of protein.     

Purification of transforming growth factor type e

Transforming growth factor type e (TGFe) is a heat- and acid-stable polypeptide with an apparent molecular weight of 22,000, which stimulates the proliferation of certain epithelial and mesenchymal cells in monolayer and soft agar. TGFe has been purified to homogeneity. Initial acid-ethanol extraction of bovine kidney was followed by batch ion-exchange chromatography utilizing Bio Rex 70 resin. The activity eluted from the Bio Rex 70 resin was concentrated and diafiltered using an Amicon concentrator equipped with an S1Y10 spiral membrane, then was further purified by Bio-Gel P-60 molecular sieve chromatography. Active fractions from molecular sieve chromatography were pooled and purified by heparin-Sepharose affinity chromatography, followed by reverse-phase high-performance liquid chromatography using a microbore C-8 column. The final purification step involved electro-elution of TGFe separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Purity of TGFe was assessed to be greater than 90%.     

Purification and biochemical characterization of alpha 2-adrenergic receptor from the rat adrenocortical carcinoma

The alpha 2-adrenergic receptor was purified from rat adrenocortical carcinoma 494 by an affinity chromatographic step using a novel para-aminoclonidine-sepharose resin followed by a gel-permeation high performance liquid chromatographic step. The iodinated receptor protein was homogeneous as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by high performance liquid chromatography. Both SDS-PAGE and high performance liquid chromatographic studies revealed that Mr of the protein was 64,000, suggesting the monomeric nature of the receptor protein. The purified protein showed the typical binding characteristics of alpha 2-adrenergic receptor.     

Purification and reconstitution of a 75-kilodalton protein identified as a component of the renal Na+/glucose symporter

A 75-kilodalton (kDa) protein was purified from solubilized renal brush border membranes by using high-pressure liquid chromatography (HPLC) and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Functional and immunological properties identified the 75-kDa protein as a component of the Na+/glucose symport system. The purified protein was specifically recognized by a monoclonal antibody that functionally interacts with the Na+/glucose symporter. Na+-dependent phlorizin binding activity was associated with fractions containing the 75-kDa protein during HPLC fractionation on the anion exchanger Mono-Q and was greatly increased after reconstitution into egg yolk phosphatidylcholine vesicles. The final purified preparation contained glucosamine and a blocked N-terminus.     

Cross-linking of iodine-125-labeled, calcium-dependent regulatory protein to the Ca2+-sensitive phosphodiesterase purified from bovine heart.

The calcium-dependent regulatory protein (CDR).Ca2+ sensitive cyclic nucleotide phosphodiesterase was purified to apparent homogeneity from bovine heart by using ammonium sulfate fractionation, DEAE-ceelulose chromatography, and CDR-Sepharose affinity chromatography. The enzyme was purifed 13 750-fold with a 10% yield and a specific activity of 275 mumol of cAMP min-1 mg-1. The purified enzyme ran as a single band during sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 57 000. Phosphodiesterase activity was stimulated 10-fold by Ca2+ and CDR with half-maximal activation occurring at 9 ng/assay. [125I]CDR was cross-linked to the purified phosphodiesterase by using dimethyl suberimidate Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cross-linked products revealed a number of discrete 125I-labeled bands. The molecular weights of the cross-linked products indicate that the stoichiometry of the phosphodiesterase complex is A2C2, where A is the phosphodiesterase catalytic subunit and C is the calcium-dependent regulatory protein.     

Purification of human interleukin 1 from human monocyte culture supernatants and identity of thymocyte comitogenic factor, fibroblast-proliferation factor, acute-phase protein-inducing factor, and endogenous pyrogen

Human interleukin 1 (IL-1) in lipopolysaccharide and silica-stimulated human peripheral blood monocyte culture supernatants was purified to apparent homogeneity by sequential chromatography using DEAE-Sephacel, Sephacryl S-200, CM-high-performance liquid chromatography (HPLC), and hydroxyapatite-HPLC. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) yielded only one band detectable by silver staining with an apparent molecular weight (MW) of 19,000 under nonreducing conditions. IL-1 activity was eluted from a single site from PAGE performed in the absence of SDS. About 4.4 micrograms of IL-1 was purified from 5.0 liters of culture supernatant of lipopolysaccharide- and silica-stimulated human peripheral blood monocytes, with 46.6% recovery of biological activity. The specific activity of the purified IL-1 was 4.3 X 10(7) U/mg protein. Amino acid composition analysis of the purified human IL-1 was similar to that previously described for murine IL-1. The purified IL-1 exhibited the biological activities previously attributed to IL-1, including thymocyte comitogenic activity, fibroblast proliferation activity, acute-phase protein (haptoglobin)-inducing activity, and endogenous pyrogen activity.     

C-terminal truncation of bovine protein disulfide isomerase increases its activity.

Protein disulfide isomerase as usually purified by the method of Lambert and Freedman (Biochem. J., 1983, 213, 225-234) although appeared homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis can be separated into two major components on a size-exclusion high performance liquid chromatography column or by polyacrylamide gel electrophoresis. These two components have the same N-terminal sequences but the C-terminal sequences are different, suggesting that one is the C-terminal slightly truncated protein. The shortened protein is more active in both the isomerase and the thiol-protein oxidoreductase activities.     

The primary structure of glycoprotein III from bovine adrenal medullary chromaffin granules. Sequence similarity with human serum protein-40,40 and rat Sertoli cell glycoprotein.

Glycoprotein III (GpIII) was purified from the soluble fraction of bovine chromaffin granules, the secretory vesicles of the adrenal medulla, by chromatography using wheat germ agglutinin-Sepharose followed by reverse-phase high performance liquid chromatography (HPLC). Characterization of this glycoprotein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reverse-phase HPLC, amino acid analysis and partial NH2-terminal sequence analysis indicated that GPIII was a disulfide-linked heterodimer with 37-kDa subunits. Analysis of in vitro translation products of adrenal medullary poly(A)+ RNA by immunoprecipitation using an anti-GpIII serum and sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested that both subunits are synthesized from a single precursor. Partial NH2-terminal sequence analysis allowed construction of oligonucleotides which were used as primers for a polymerase chain reaction to generate a GpIII-specific DNA probe. This probe was used to isolate a cDNA clone encoding the GpIII precursor from a bovine adrenal medullary cDNA library. The predicted amino acid sequence of GpIII has greater than 80% similarity to human serum protein-40,40, a protein implicated in the complement system, and to a major secretory product of Sertoli cells, glycoprotein 2, which is thought to play a role in spermatogenesis. Northern blot analysis confirmed that RNA encoding GpIII is also abundant in liver, testis, and brain.     

Overproduction and purification of McrC protein from Escherichia coli K-12.

The McrC protein, encoded by one of the two genes involved in the McrB restriction system, was produced in Escherichia coli cells by using a T7 expression system. Following sequential DEAE-Sepharose and hydroxylapatite column chromatography, the protein was purified to apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of the purified McrC protein agreed exactly with the one deduced from the DNA sequence by Ross et al. (J. Bacteriol. 171:1974-1981, 1989).