Purification and partial characterization of a plasma inhibitor of tonin

A plasma inhibitor of tonin activity in the rat, was purified by ammonium sulfate precipitation, ion-exchange of chromatography, and gel filtration. Its purity was investigated by analytical electrophoresis on polyacrylamide gel and by ultracentrifugation sedimentation velocity. The molecular weight (360 000) of the purified inhibitor was determined by sodium dodecyl sulfate electrophoresis and its isoelectric point (4.5) by gel isoelectrofocusing. The Stokes radius (640 nm) was evaluated by gel filtration studies and a frictional ratio (f/fo) of 1.95 was calculated from the molecular weight and Stokes radius. Kinetic studies using angiotensin I as substrate showed that the inhibition of tonin by the purified inhibitor was noncompetitive and does not exceed 70%. Electrophoresis showed the same mobility for [125I]tonin bound to plasma proteins and for [125I]tonin bound to the purified inhibitor. The inhibitor may be a protein resembling half of the dimeric protease inhibitor rat alpha 1-macroglobulin or human alpha 2-macroglobulin.     

Evidence for naturally occurring hyaluronic acid binding protein in rat liver

Hyaluronic acid binding protein (HBP) was purified homogeneously from normal adult rat liver by hyaluronate-sepharose affinity chromatography. The molecular weight of this protein as determined by gel filtration was found to be 64,000 daltons. This protein HBP appeared as a single band in non-dissociating gel electrophoresis and has a subunit of molecular weight approximately 12,000 as determined by SDS-gel electrophoresis.     

Purification of a bovine counterpart to human prealbumin and its interaction with a bovine retinol-binding protein

A bovine counterpart to human prealbumin was purified from bovine serum by thiol-disulfide exchange chromatography on thiol-Sepharose 4B and affinity chromatography on human retinol-binding protein linked to Sepharose 4B. The bovine prealbumin had alpha1-mobility on agarose gel electrophoresis at pH 8.6. It has the same molecular weight as human prealbumin on gel filtration and consisted of subunits with a molecular weight of 12 500. This is compatible with a tetrameric structure for the bovine protein. Antiserum against human prealbumin cross-reacted with bovine prealbumin and vice versa. The bovine prealbumin formed at high ionic strength complexes with another bovine serum protein which were dissociated at low ionic strength. This property was used to isolate a protein from bovine serum, by chromatography on bovine prealbumin linked to Sepharose which cross-reacted with antiserum against human retinol-binding protein; had a molecular weight of 21 000 and alpha 2-mobility on agarose gel electrophoresis. It was concluded that the latter protein was a bovine retinol-binding protein.     

Isolation and characterization of allergens of Prosopis juliflora pollen grains

Proteins and glycoproteins from Prosopis juliflora (Pj) pollen grains were separated by gel filtration, electrophoresis, DEAE cellulose chromatography and their molecular weight was determined by gel filtration and SDS-Polyacrylamide gel electrophoresis. The allergenic activity of different fractions were evaluated by in vivo skin prick test and in vitro gel diffusion test. It was found that fraction E of gel filtration and fraction III and IV of DEAE cellulose chromatography were most allergenic. This fraction E of gel filtration showed positive reaction with periodic acid Schiff's reagent as determined by SDS-gel electrophoresis.     

The Isolation and Characterization of the Two Macromolecular Antitumor Agents from Streptomyces

Purification of the two antitumor macromolecules, A216 and A280 substances, from culture filtrates of Streptomyces is achieved by chromatography using ion-exchanged celluloses. The purified macromolecules appeared homogeneous are characterized as a protein from the chemical and biological properties by paper electrophoresis, paper chromatography and ultracentrifuge.

The simple method for approximation of molecular weight of a protein from distribution coefficient on gel filtration is proposed. The molecular weights of both macromolecules given by gel filtration are near to those given by ultracentrifugal analysis.     

A functional, thioester-containing alpha 2-macroglobulin homologue isolated from the hemolymph of the American lobster (Homarus americanus)

An alpha 2-macroglobulin-like protease inhibitor was isolated from the cell-free hemolymph of the american lobster (Homarus americanus) by ion-exchange chromatography and gel filtration. Whereas the undissociated molecule has a molecular weight of 342,000 as determined by ultracentrifugation studies, under reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein has a subunit molecular weight of 180,000. On the basis of this and other evidence, we conclude that the lobster protein is a dimer consisting of two disulfide-bonded monomers. The purified protein inhibits proteolytic enzymes but protects the esterolytic activity of trypsin toward low molecular weight substrates from inactivation by soybean trypsin inhibitor. The methylamine sensitivity of this activity suggests the presence of an internal thioester bond. This was confirmed by the covalent incorporation of [14C]methylamine, by the formation of Mr 55,000 and 125,000 autolytic cleavage fragments in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and, more directly, by the amino acid sequence of a tryptic peptide containing the putative thioester region. Whereas the N-terminal amino acid sequence (22 residues) of the protein revealed an overall identity of only 18% when compared with the human protein, the sequence of the thioester-containing peptide was highly conserved, both with respect to human alpha 2-macroglobulin and to other proteins having a thioester bond. The protein showed the "slow to fast" conformational change typical in alpha 2-macroglobulins in nondenaturing gel electrophoresis after treatment with trypsin, but not after incubation with methylamine.     

Purification and characterization of primer recognition proteins from HeLa cells

We have purified to homogeneity the primer recognition proteins (PRP) from human HeLa cells. PRP is associated with DNA polymerase alpha complex in HeLa cells. Purified PRP is free of DNA polymerases alpha, beta, and delta, deoxyribonuclease, DNA primase, ATPase, topoisomerase, and DNA ligase activities. The protein structure of the PRP was defined by sodium dodecyl sulfate gel electrophoresis, which revealed two polypeptides of 36,000 Da (PRP 1) and 41,000 Da (PRP 2). The two polypeptides are associated in a complex in the native state. The Stokes radius of the PRP complex by gel filtration is 40.5 A and the sedimentation coefficient in glycerol gradients is 5.7 S. Purified PRP, which exhibits no DNA polymerase activity, completely restores the activity of DNA polymerase alpha on templates with low primer to template ratios such as heat-denaturated DNA, poly(dA)-oligo(dT), and singly primed M13 single-stranded DNA. Experiments using various amounts of PRP, DNA polymerase alpha, and DNA indicate that a concentration dependence exists between these components in the DNA replication process. Amino acid composition analysis indicates that the PRP is rich in hydrophobic amino acids.     

Cellular retinoic acid-binding protein from rat testis. Purification and characterization

Cellular retinoic acid-binding protein has been purified to homogeneity from rat testes. The procedures utilized in the purification included acid precipitation, gel filtration, and chromatography on DEAE-cellulose. The binding protein was purified approximately 12,000-fold, based on total soluble testicular protein. The protein is a single polypeptide chain with a molecular weight of 14,600, determined by information from gel filtration and sodium dodecyl sulfate-polyacrylamide electrophoresis. The protein binds retinoic acid with high affinity; the apparent dissociation constant was determined by fluorometric titration to be 4.2 X 10(-9) M.     

Purification of the secretor-type beta-galactoside alpha 1----2-fucosyltransferase from human serum.

The secretor-type beta-galactoside alpha 1----2-fucosyltransferase from human serum was purified by hydrophobic chromatography on phenyl-Sepharose, ion-exchange chromatography on sulfopropyl-Sepharose, and affinity chromatography on GDP-hexanolamine-Sepharose. Final purification of the enzyme was achieved by high pressure liquid chromatography gel filtration and resulted in a homogeneous protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the radiolabeled protein. The native enzyme appears as a molecule of apparent Mr 150,000 as determined by gel filtration high pressure liquid chromatography. The apparent Mr of the enzyme resolved in the presence of beta-mercaptoethanol by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was determined to be 50,000, indicating a multisubunit structure of the enzyme. Secretor-type alpha 1----2-fucosyltransferase is a glycoprotein as determined by WGA binding properties. A comparison of the Mr of the native blood group H gene encoded with the secretor-type beta-galactoside alpha 1----2-fucosyltransferases as well as comparison of subunit Mr for both enzymes suggests structural similarity. The alpha 1----2 linkage formed between alpha-L-fucose and terminal beta-D-galactose by the purified H- and secretor-type alpha 1----2-fucosyltransferases was determined by 1H NMR homonuclear cross-irradiation analysis of the oligosaccharide products. The substrate specificity and Km values calculated from the initial rate using various oligosaccharide acceptors showed that purified enzymes differ primarily in affinity for phenyl-beta-D-galactopyranoside and GDP-fucose as well as type 1 (Gal beta 1----3GlcNAc), 2 (Gal beta 1----4GlcNAc), and 3 (Gal beta 1----3GalNAc) oligosaccharide acceptors. The secretor-type alpha 1----2-fucosyltransferase shows significantly lower affinity than the H enzyme for phenyl-beta-D-galactopyranoside and GDP-fucose as well as for type 2 oligosaccharide acceptors. On the contrary, type 1 and 3 oligosaccharide acceptors are preferentially utilized by the secretor-type enzyme as compared with the H enzyme. The enzymes also differ in several physicochemical properties, implying nonidentity of the two enzymes (Sarnesto, A., K?hlin, T., Thurin, J., and Blaszczyk-Thurin, M. (1990) J. Biol. Chem. 265, 15067-15075).     

Isolation and purification of cytokinin binding protein from tobacco leaves by affinity column chromatography

Cytokinin binding protein from tobacco leaves was isolated and purified to a single protein by means of affinity chromatography on benzyladenine-linked Sepharose column combined with polyacrylamide gel electrophoresis. In vitro binding of this protein to [¹⁴C] benzyladenine was inhibited remarkably by cold benzyladenine and kinetin and slightly by adenine, but not adenosine. The molecular weight of the protein was determined to be about 4,000 daltons by gel filtration and SDS polyacrylamide gel electrophoresis.     

A novel glycoprotein that binds to hyaluronic acid

Hyaluronic acid binding protein (HBP) has been purified to homogeneity from normal rat brain by using Hyaluronate-Sepharose affinity chromatography. It appears as a single band in non-dissociating gel electrophoresis. The molecular weight of native protein, as determined by gel filtration is found to be 68,000 daltons, and has a single subunit of molecular weight approximately 13,500 as determined under denaturing conditions in polyacrylamide gel electrophoresis, indicating that this protein is apparently composed of five identical subunits. Amino acid analysis shows the purified HBP to be rich in glycine and glutamic acid content, and is distinct from fibronectin, link proteins, and gelatin binding proteins which are known to bind to hyaluronic acid. This protein is further characterised as sialic acid containing glycoprotein.     

Purification and partial characterization of protein phosphatases from rat thymus

Protein phosphatases assayed with phosphorylase alpha are present in the soluble and particulate fractions of rat thymocytes. Phosphorylase phosphatase activity in the cytosol fraction was resolved by heparin-Sepharose chromatography into type-1 and type-2A enzymes. Similarities between thymocyte and muscle or liver protein phosphatase-1 included preferential dephosphorylation of the beta subunit of phosphorylase kinase, inhibition by inhibitor-2 and retention by heparin-Sepharose. Similarities between thymocyte and muscle or liver protein phosphatase-2A included specificity for the alpha subunit of phosphorylase kinase, insensitivity to the action of inhibitor-2, lack of retention by heparin-Sepharose and stimulation by polycationic macromolecules such as polybrene, protamine and histone H1. Protein phosphatase-1 from the cytosol fraction of thymocytes had an apparent molecular mass of 120 kDa as determined by gel filtration. The phosphatase-2A separated from the cytosol of thymocytes may correspond to phosphatase-2A0, since it was completely inactive (latent) in the absence of polycation and had activity only in the presence of polycations. The apparent molecular mass of phosphatase-2A0 from thymocytes was 240 kDa as determined by gel filtration. The catalytic subunit of thymocyte type-1 protein phosphatase was purified with heparin-Sepharose chromatography followed by gel filtration and fast protein liquid chromatography on Mono Q column. The purified type-1 catalytic subunit exhibited a specific activity of 8.2 U/mg and consisted of a single protein of 35 kDa as judged by SDS-gel electrophoresis. The catalytic subunit of type-2A phosphatase from thymocytes appearing in the heparin-Sepharose flow-through fraction was further purified on protamine-Sepharose, followed by gel filtration. The specific activity of the type-2A catalytic subunit was 2.1 U/mg and consisted of a major protein of 34.5 kDa, as revealed by SDS-gel electrophoresis.     

A homologue of alpha 2-macroglobulin purified from the hemolymph of the horseshoe crab Limulus polyphemus

A high molecular weight protease inhibitor has been purified from the cell-free plasma of the horseshoe crab Limulus polyphemus using high speed centrifugation, polyethylene glycol precipitation, and gel filtration. The inhibitor is sensitive to mild acidification, methylamine treatment, and inhibits the proteolytic activity of a variety of endopeptidases. The molecule does not inhibit trypsin-mediated hydrolysis of low molecular weight substrates and protects the active site of trypsin from inactivation by soybean trypsin inhibitor. These properties are diagnostic of the alpha 2-macroglobulin (alpha 2M) class of protease inhibitors found in vertebrates. Like vertebrate alpha 2M the Limulus alpha 2M molecule is composed of subunits of molecular weight 180,000-185,000 as determined by polyacrylamide gel electrophoresis under reducing conditions. The apparent native molecular weight for the Limulus molecule as determined by both gel filtration and gel electrophoresis under nonreducing conditions is 500,000-550,000, compared to a native molecular weight of 700,000-750,000 for human alpha 2M, determined in parallel under identical conditions. These results suggest that alpha 2M appeared in evolution at least 550 million years ago before the divergence of the lineages that gave rise to present-day arthropods and mammals.     

Characterization of the A Component of Streptococcus zymogenes Lysin

By utilizing conventional techniques of pressure ultrafiltration, gel filtration chromatography, diethylaminoethyl cellulose chromatography, and preparative polyacrylamide electrophoresis, the A component of the group D lysin produced by Streptococcus zymogenes has been purified to a state of apparent homogeneity when determined by the techniques of anionic and cationic disc gel electrophoresis. The A component was found to be a protein possessing a molecular weight of 27,000, a sedimentation coefficient approximating 3.2S, and a net negative charge at physiological pH.     

Purification and characterization of a novel, oligomeric, plasminogen kringle 4 binding protein from human plasma: tetranectin

Purification of alpha 2-plasmin inhibitor (alpha 2PI) from human plasma by affinity chromatography on plasminogen-Sepharose resulted in copurification of a contaminating protein with Mr 17,000 as judged by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. This contaminating protein could not be removed from the purified alpha 2-PI preparation by several types of gel chromatography applied. The use of the kringle 1-3 part of plasminogen, K(1 + 2 + 3), bound to Sepharose for affinity chromatography, instead of plasminogen-Sepharose, resulted in an alpha 2PI preparation without this contaminant. The contaminating protein was found to interact specifically with the kringle 4 part of plasminogen (K4) and not with K(1 + 2 + 3) or miniplasminogen. The K4-binding protein was purified by ammonium sulphate precipitation, affinity chromatography on K4-Sepharose, ion-exchange chromatography and gel filtration on AcA 34. The relative molecular mass of the protein (Mr 68 000) was estimated by gel filtration. This suggests a tetrameric protein composed of four subunits (Mr 17,000), that are dissociated by 1% sodium dodecyl sulphate. Dissociation into subunits was also demonstrated by gel filtration in the presence of 6 M guanidine hydrochloride. A specific antibody was raised in rabbits against the purified protein and this antibody was shown not to react with any known fibrinolytic components. The pI of the K4-binding protein was found to be 5.8. The first three N-terminal amino acids were determined to be Glu-Pro-Pro. The concentration of the protein in plasma was estimated to be 0.20 +/- 0.03 microM (15 +/- 2 mg/l). The electrophoretic mobility of the K4-binding protein was shown by crossed immunoelectrophoresis to be influenced by the presence of Ca2+, EDTA and heparin. The protein was found to enhance plasminogen activation catalyzed by tissue-type plasminogen activator (t-PA) in the presence of poly(D-lysine). The protein appeared to be a novel plasma protein tentatively called 'tetranectin'.     

Proteins and glycoproteins of the erythrocyte membrane

Erythrocyte membranes from several species were prepared by three different methods of hypotonic hemolysis and examined for variations in protein and glycoprotein content by acrylamide gel electrophoresis in sodium dodecyl sulfate. Significant variations were noted in morphology of the membranes prepared by the different methods without attendant variations in protein patterns of the major membrane proteins for most cases observed, which show a similar pattern of nine common bands for all of the species observed. The significant difference in protein pattern which was noted was attributed to proteolytic digestion of membranes which were fragmented during preparation. Failure to remove white blood cells from membrane preparations was shown to be a significant source of the problem with proteolytic digestion. Glycoproteins were analyzed by acrylamide gel electrophoresis or by column chromatography. Each species appears to have a different major glycoprotein (or group of closely related glycoproteins). Molecular weights of glycoproteins calculated from acrylamide gel electrophoresis were found to vary with the percentage of acrylamide in the gel, indicating that these proteins do not behave in a normal fashion in this electrophoresis system. The molecular weight calculated from gel filtration data for the human membrane glycoproteins (26,000) was quite disparate from those calculated from gel electrophoresis (88,000 to 62,000 in 5 to 10% gels).     

Quaternary structure of pyruvate carboxylase from Pseudomonas citronellolis.

Physical-chemical studies of pyruvate carboxylase from Pseudomonas citronellolis demonstrate that the enzyme has an alpha 4 beta 4 structure. The individual polypeptides, alpha (Mr = 65,000) and beta (Mr = 54,000), were separated and isolated by preparative gel electrophoresis. Analysis of the relationship between Coomassie blue staining and protein quantity for each polypeptide indicated that the alpha and beta subunits are present in a 1:1 stoichiometry in the native enzyme. Determinations of the molecular weight of the protein by sedimentation equilibrium (Mr = 454,000), gel filtration analysis (Mr = 510,000), disc gel electrophoresis (Mr = 530,000), and mass measurement from the Scanning Transmission Electron Microscope (Mr = 530,000) are consistent with the proposed alpha 4 beta 4 structure. Disc gel electrophoresis studies revealed that under certain circumstances the enzyme may dissociate to a smaller molecular weight species (Mr = 228,000). This dissociation phenomenon could explain the earlier reported observation of Taylor et al. ((1972) J. Biol. Chem 22, 7388-8390) that the enzyme had a molecular weight of 265,000. Evidence from electron microscopic studies shows that the three-dimensional structure of this enzyme is quite distinct from other species of pyruvate carboxylase. The enzyme does not show the typical rhombic appearance which has been noted for chicken liver, sheep liver, and yeast pyruvate carboxylase.     

Purification and characterization of amphiphilic trehalase from rabbit small intestine

Rabbit intestinal trehalase (alpha,alpha-trehalose glucohydrolase, EC 3.2.1.28) was solubilized with Triton X-100 and purified in the presence of EDTA. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis in the presence of Triton X-100 or SDS. It showed amphiphilic properties on gel filtration. polyacrylamide gel electrophoresis, charge-shift electrophoresis and phenyl-Sepharose chromatography. Its molecular weight was estimated to be about 330 000 by gel filtration under nondenaturing conditions and in the presence of Triton X-100, the value being in satisfactory agreement with the sum of the weight of one Triton X-100 micelle and twice the molecular weight (105 000) of purified hydrophilic trehalase which had been deprived of the anchor segment. The two purified trehalases gave almost the same molecular weights (about 75 000) on SDS-polyacrylamide gel electrophoresis. These results suggest that intestinal trehalase consists of two subunits with a molecular weight of 75 000 and that its anchor segment is small (less than 5000). Triton X-100 extracts freshly prepared from intestinal microvilli essentially showed one form of trehalase, which behaved on phenyl-Sepharose and Con A-Sepharose chromatography in the same manner as purified amphiphilic trehalase.     

R plasmid dihydrofolate reductase with subunit structure.

Dihydrofolate reductase, specified by the type II plasmid of a trimethoprim-resistant Escherichia coli, was purified 40-fold to homogeneity using a combination of gel filtration, DEAE-Sephacel chromatography, and hydrophobic chromatography. The final product shows a single protein band on polyacrylamide gel electrophoresis and has a specific activity of 1.0 unit/mg. The molecular weight of the purified enzyme is 36,000 as determined both by gel filtration and Ferguson analysis of polyacrylamide gel electrophoresis. In contrast, a single polypeptide with a molecular weight of 8,500 was observed on sodium dodecyl sulfate-gel electrophoresis. These experiments suggest that, unlike any bacteria or vertebrate dihydrofolate reductase previously examined, the type II R plasmid reductase is a tetramer composed of four identical subunits. A partial amino acid sequence determination shows no heterogeneity of the subunits and also no clear homology with any reductase sequence previously reported.     

Latent transforming growth factor-beta in serum. A specific complex with alpha 2-macroglobulin

The biological latency of serum transforming growth factor-beta (TGF-beta) was shown to be due to the interaction of TGF-beta with a specific serum binding protein. This binding protein was affinity labeled with 125I-TGF-beta, and its Mr and subunit structure were determined using sodium dodecyl sulfate-gel electrophoresis and gel filtration chromatography. Its Mr is reminiscent of that of the serum protease inhibitor, alpha 2-macroglobulin (alpha 2M). Immunoprecipitation of the 125I-TGF-beta-binding protein complex by a specific anti-alpha 2M antibody, and the formation of identical complexes between 125I-TGF-beta and purified alpha 2M, confirmed that alpha 2M is the TGF-beta-binding protein in serum. Immunoblot analysis showed that endogenous serum TGF-beta is also bound to alpha 2M. However, in contrast to added 125I-TGF-beta, the majority of the endogenous TGF-beta is linked to alpha 2M covalently. Alpha 2M and acid-activated TGF-beta co-eluted from a Superose 6 fast protein liquid chromatography column, confirming that the interaction of TGF-beta with alpha 2M accounts for the latency of serum TGF-beta. It is proposed that alpha 2M may serve an important multifunctional role at sites of inflammation by scavenging both active peptides and proteases that are released by platelets at the site of injury.