Purification and characterization of a bovine colostrum-derived growth factor

A growth factor in bovine colostrum was purified to homogeneity by a combination of acid extraction, boiling, cation exchange chromatography, isoelectric focusing, and reverse phase HPLC. The bovine colostrum growth factor (BCGF) had an isoelectric point of about 10, a native mol wt of about 30,000, was resistant to inactivation by boiling and exposure to pH 1, but was inactivated by dithiothreitol. BCGF appeared to be structurally related to human platelet-derived growth factor (PDGF) and competed with human PDGF in a radioreceptor assay. However, while human PDGF appeared to be a heterodimer of 17,000 and 14,000 mol wt subunits, BCGF appeared to be a homodimer of 20,000 mol wt subunits. Purified BCGF had a specific activity in stimulating 3T3 cell proliferation of about 3-6 U/ng and was active at about 1-2 ng/ml.     

Purification and characterization of bovine tissue factor

Tissue factor (tissue thromboplastin, factor III), an initiator of coagulation, has been purified 142,000-fold to homogeneity from bovine brain. The protein is an integral membrane glycoprotein with an apparent molecular weight of 43,000 as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The apoprotein was first purified by extraction with Triton X-100 and repeated preparative polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Antiserum was produced against a few micrograms of purified apoprotein and was used to construct an immunoadsorbent column. The column was then used for affinity purification of the apoprotein directly from the Triton X-100 extract, thereby significantly increasing the amount of purified protein produced. The purification scheme may be generally useful for the rapid and large scale purification of membrane proteins. Tryptic digestion of the apoprotein in Triton X-100 cleaved a peptide of approximately 3000 daltons without affecting the activity. The activity was recovered directly from stained SDS polyacrylamide gels, and the profile of recovered activity corresponded directly with the stained bands. The activity shifted along with the protein band following tryptic digestion, thus demonstrating that the protein observed on the gels is tissue factor. The coagulant activity of the purified apoprotein was reconstituted by the addition of phospholipid. Optimal activity was observed at phospholipid to protein ratios (w/w) greater than 450:1.     

Purification and characterization of elongation factor G from bovine liver mitochondria

The mitochondrial protein synthesis translocase elongation factor Gmt (EF-Gmt) from bovine liver has been purified to greater than 90% homogeneity by a combination of conventional gravity and high performance liquid chromatography. The purification scheme results in an approximate overall 14,000-fold purification with 2% total recovery of EF-Gmt activity. Gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicate that the mitochondrial factor is a single polypeptide with a molecular weight of 80,000. EF-Gmt displays similar levels of activity on its homologous mitochondrial ribosomes and on Escherichia coli ribosomes. The mitochondrial translocase is sensitive to temperatures above 37 degrees C, but the factor is partially protected from heat inactivation in the presence of GTP or GDP. The activity of EF-Gmt is inhibited by treatment of the factor with N-ethylmaleimide. In contrast to all other translocases tested to date, EF-Gmt is completely resistant to the inhibiting effect of fusidic acid when tested on its homologous ribosomes. It displays weak sensitivity to this antibiotic when assayed in the presence of heterologous E. coli ribosomes.     

Amino acid sequence of bovine osteoinductive factor

The complete amino acid sequence of bovine osteoinductive factor (OIF) was determined by automated Edman degradation of S-pyridylethylated bovine OIF and selected fragments. Cleavage with endoproteinase Lys-C, endoproteinase Glu-C, or endoproteinase Asp-N established all fragments in an unambiguous sequence. Bovine OIF contains 105 residues with a calculated molecular weight of 12,055. It is a single chain polypeptide containing two intramolecularly linked cysteines at residues 62 and 95. Two asparagine-linked glycosylation sites at positions 52 and 65 were found by comparing sequence data and peptide profiles of native and deglycosylated OIF fragments. The amino acid sequence of OIF has no homology to other reported proteins.     

Purification and characterization of a unique alkaline elastase from Micrococcus luteus

Micrococcus luteus isolated from human skin secretes an alkaline protease which degrades elastin. M. luteus protease (MLP) was produced in the late logarithmic and stationary phases of growth. MLP, purified to homogeneity by a three-step process, had a molecular mass of 32,812 Da and an isoelectric point of 9.3. MLP was active and highly stable in solution for 24 h from pH 6.0 to 10.5; it had maximal activity at temperatures between 57 and 59 degrees C. The presence of calcium in the solution was essential for enzyme activity and to prevent autolysis. Optimal activity occurred between pH 9.0 and 9.5, with 60% maximal activity from pH 6.5 to 11.0. The enzyme was inhibited by the serine enzyme inhibitors phenylmethylsulfonyl fluoride and chymostatin but not by the metalloenzyme inhibitor 1,10-phenanthroline or sulfhydryl enzyme inhibitors. Casein, bovine serum albumin, ovalbumin, beta-lactoglobulin, and elastin were digested by the protease while collagen and keratin were resistant to digestion. MLP demonstrated both esterase and amidase activity on synthetic peptide substrates. MLP preferentially cleaved the Leu(15)-Tyr(16) and Phe(24)-Phe(25) bonds of the oxidized beta-chain of insulin. Longer digests of insulin and the pattern of activity against synthetic substrates suggest that MLP has a cleavage specificity for bulky, hydrophobic, or aromatic amino acids in the P(1) or P(1)' positions. Amino acid sequences from the N-terminus and internal peptides of MLP were unique.     

Purification and partial characterization of an acidic fibroblast growth factor from bovine pituitary

Isoelectric focusing has allowed us to fractionate pituitary extracts into basic (pI 8-9) and acidic (pI 4-5) fibroblast growth factor. The acidic fibroblast growth factor (a) is stable upon refocusing, (b) migrates as an acidic protein in urea-containing gel electrophoresis; (c) is not cell-specific, being active with fibroblasts, adrenal, and glial cells, and (d) is a heterogeneous protein fraction with active components of different pI values. The component of pI 4.7, purified to or near homogeneity by isoelectric focusing shows a single peak of activity (Mr = 12,000) in gel chromatography and a single protein band of apparent Mr = 15,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Maximal restimulation of DNA synthesis initiation on serum-deprived 3T3 fibroblasts is achieved at 1-2 ng/ml; activity with rat glial cells (C6-3D) is less pronounced than with 3T3 fibroblasts.     

Purification and characterization of a 2'-phosphodiesterase from bovine spleen

Interferon-induced 2',5'-oligoadenylates are transiently produced during viral infection and are believed to play a role in the interferon-mediated inhibition of replication of at least some viruses. 2',5'-Oligoadenylates must be catabolized but are resistant to degradation by most known ribonucleases. A 2'-phosphodiesterase that degrades 2',5'-oligoadenylates was purified 1500-fold from a low speed homogenate of bovine spleen by precipitation at pH 5.2, ammonium sulfate fractionation, differential ultrafiltration, and successive chromatography on DEAE-Sephacel, hydroxylapatite, and a fast protein liquid chromatography Mono P column. No other 2-5A-degrading activity was observed during the purification procedure. The molecular mass of the enzyme estimated by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 65,000. The enzyme is distinct from bovine spleen phosphodiesterase II. The 2'-phosphodiesterase cleaves 2',5'- and 3',5'-linked oligonucleotides, as well as branched oligoadenylate, A(2'pA)(3'pA), but appears to be most active on 3',5'-oligoribonucleotides. The enzyme cleaves 5'-AMP from the 2' terminus of 2',5'-oligoadenylates and appears to require a free 2' terminus and a 3'-oxygen on the penultimate nucleotide. Substrate length, 5'-phosphorylation, and base composition do not appear to be critical factors in determining enzyme activity. The effects of pH, Mg2+, Mn2+, EDTA, phosphate, 2-mercaptoethanol, and N-ethylmaleimide are also described. This enzyme may be involved in the catabolism of the interferon-induced 2',5'-oligoadenylates and other 2',5'-linked RNAs in the cell.     

Purification and characterization of a protein-tyrosine kinase from bovine thymus

A protein-tyrosine kinase has been isolated from a soluble extract of bovine thymus based on its ability to phosphorylate the tyrosine-containing peptide angiotensin I. The purification procedure employs sequential chromatography on columns of DEAE-cellulose, heparin-agarose, casein-agarose, butyl-agarose, and Sephadex G-75. The purified enzyme (p40) is a monomer of Mr = 40,000. The p40 kinase contains an ATP-binding site as determined by photoaffinity labeling experiments and catalyzes an intramolecular autophosphorylation reaction that leads to its modification on tyrosine. Of several proteins tested only the cytoplasmic domain of the erythrocyte band 3 protein serves as a good substrate for p40 (Km = 12 microM). Increasing concentrations of NaCl stimulate the phosphorylation of angiotensin I, inhibit the phosphorylation of band 3, and have no effect on the autophosphorylation of p40. At low concentrations of NaCl, Mn2+ is the preferred divalent cation. Peptide mapping experiments indicate that p40 is distinct from pp60src and from the major phosphotyrosine containing proteins of T and B lymphocyte membranes.     

Purification and identification of a BMP-like factor from bovine serum

Myogenic differentiation is suppressed in vitro by unknown factors present in fetal bovine serum (FBS). We found that specific inhibitors of bone morphogenetic proteins (BMPs) stimulated myogenic differentiation even in the presence of 20% FBS, which in turn activated specific BMP signaling. Moreover, these specific BMP inhibitors blocked maturation of osteoblastic cells induced by FBS, indicating that BMP-like factor(s) in serum regulate both myogenic and osteoblastic differentiation. The factor identified had an apparent molecular weight (Mw) of over 100kDa on a Superdex 200 column for molecular sieving HPLC, but an apparent Mw of 33kDa on SDS-PAGE under non-reducing conditions. Analysis of a purified preparation from FBS (5L) by liquid chromatography-tandem mass spectrometry revealed the presence of an amino acid sequence conserved between mature human and murine BMP-4. This is the first study to show that BMP-4 is present in FBS as a large complex.     

Purification and partial characterization of bovine pituitary fibroblast growth factor

A purification procedure and partial characterization of bovine pituitary fibroblast growth factor (FGF) are described. The steps of the published methods [3,4] which yield inhomogeneous material, were retained, with modifications. The final isolation, with an additional purification of ～20-fold, was achieved by electro-phoresis in polyacrylamide gels at acid pH. The mitogenic peptide has a molecular weight of 14,500–15,00 as determined on SDS gels, chromatographs as a monomer in nondenaturing conditions, and is active at the picomolar level in effecting the incorporation of ³H-thymidine in Balb/c 3T3 cells. A preliminary amino acid composition is presented.     

Purification and partial characterization of a mitogenic factor from bovine liver: structural homology with basic fibroblast growth factor

Two mitogenic peptides in bovine liver extract were purified to apparent homogeneity by monitoring the purification steps with two in vitro bioassays; one based on stimulation of adult bovine aortic arch endothelial cell proliferation and the other incorporation of [3H]thymidine to mouse fibroblast 3T3 cells. The purification procedure involved cation-exchange chromatography followed by affinity chromatography on heparin-Sepharose and two steps of reversed-phase HPLC. The purified material showed the same biological activity as pituitary basic fibroblast growth factor (FGF). Amino acid analyses of the purified mitogen yielded a similar, but not identical composition to that of bovine pituitary basic FGF(1-146) reported previously. Gas-phase microsequencing identified two sequences in equal amounts in the purified preparation. Furthermore, the sequencing results are in accord with the theoretical data obtained when two truncated forms of basic FGF, corresponding to FGF(12-146) and (16-146), are being sequenced simultaneously. Basic FGF(12-146) is a novel truncated form of basic FGF which has not been isolated before although the (16-146) fragment has been found previously in kidney, corpus luteum, and adrenal. SDS-PAGE analysis could not separate the two forms and showed that both migrated as a protein of about 15,100 daltons, which is slightly smaller than intact basic FGF(1-146) (16,200 daltons). These results, taken together, indicate that at least some of the mitogenic activity in liver may be derived from basic FGF-related polypeptides.     

Purification and characterization of myosin from bovine retina

Myosin has been isolated from bovine retinae and characterised by its ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity, its mobility in sodium dodecyl sulphate polyacrylamide gels and by electron microscopy. The purified myosin shows high ATPase activity in the presence of EDTA or Ca²⁺ and a low activity in the presence of Mg²⁺. The Mg²⁺-dependent ATPase activity is stimulated by rabbit skeletal muscle actin. The presumptive retinal myosin possesses a major component which has a mobility in sodium dodecyl sulphate polyacrylamide gel electrophoresis similar to that of the heavy chain of bovine skeletal mucle myosin. Electron microscopy showed retinal myosin to form bipolar filaments in 0.1 M KCl. It is concluded that the retina possesses a protein with enzymic and structural properties similar to those of muscle myosin.     

Purification and characterization of an oxidase activating factor of 63 kilodaltons from bovine neutrophils

A 63-kDa protein, which behaves as an oxidase activating factor in bovine neutrophils, has been purified to electrophoretic homogeneity. The protein was isolated from the cytosol of resting bovine neutrophils after several steps, including ammonium sulfate precipitation and chromatography on AcA44, DE-52 cellulose, Mono Q, and Superose 12 in the presence of dithiothreitol. The oxidase activating potency of the protein was assayed with a cell-free system consisting of neutrophil membranes, GTP gamma S, arachidonic acid, and a complementary cytosolic fraction. The purification factor was 200 and the yield 3%. During the course of gel filtration on calibrated Superose 12, the 63-kDa protein eluted as a dimer. Its isoelectric point was 6.4 +/- 0.1. Antibodies raised in rabbits against the 63-kDa protein reacted with a protein of similar size in human neutrophils and in HL60 promyelocytic cells induced to differentiate into granulocytes. No immune reaction was observed in cytosol from undifferentiated HL60 cells, in extracts from bovine skeletal muscle, liver, and brain, or in cytosol prepared from neutrophils derived from a patient with an autosomal cytochrome b positive form of chronic granulomatous disease lacking the 67-kDa oxidase activating factor. Immunoblotting with the 63-kDa bovine protein antiserum demonstrated that activation of bovine neutrophil oxidase by phorbol myristate acetate induced the translocation of the 63-kDa protein from cytosol to the membrane.     

Purification and characterization of a protein tyrosine kinase from bovine spleen

Protein tyrosine kinase was purified extensively from a 30,000 X g particulate fraction of bovine spleen by a procedure involving four column chromatographies: DEAE-Sepharose, polyamino acids affinity, hydroxylapatite, and Sephacryl S-200 molecular sieving. The purification resulted in more than 3,000-fold enrichment in [Val5]angiotensin II phosphorylation activity (specific activity 202 nmol/min/mg). All column chromatography profiles showed single protein tyrosine kinase activity peaks with the exception of that of affinity chromatography, where about 50% of the enzyme activity appeared with the breakthrough fraction; only the bound enzyme was further purified. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of a purified sample phosphorylated in the presence of [gamma-32P]ATP revealed the presence of a single phosphorylated polypeptide of molecular weight 50,000 which represents about 40% of total protein. Analysis by polyacrylamide gel electrophoresis under nondenaturing conditions showed that protein tyrosine kinase activity co-migrated with the phosphoprotein. Stoichiometry of the phosphorylation of the 50-kDa polypeptide was found to be 1.0 mol/mol. The purified sample did not appear to contain phosphotyrosine protein phosphatase activity. Both casein and histone could be phosphorylated by the purified sample, and the phosphorylation occurred only at tyrosine residue, suggesting that there was no protein serine and threonine kinase contamination.     

Purification and characterization of a magnesium-dependent neutral sphingomyelinase from bovine brain

The magnesium-dependent, plasma membrane-associated neutral sphingomyelinase (N-SMase) catalyzes hydrolysis of membrane sphingomyelin to form ceramide, a lipid signaling molecule implied in intracellular signaling. We report here the biochemical purification to apparent homogeneity of N-SMase from bovine brain. Proteins from Nonidet P-40 extracts of brain membranes were subjected to four purification steps yielding a N-SMase preparation that exhibited a specific enzymatic activity 23,330-fold increased over the brain homogenate. When analyzed by two-dimensional gel electrophoresis, the purified enzyme presented as two major protein species of 46 and 97 kDa, respectively. Matrix-assisted laser desorption/ionization-mass spectrometry analysis of tryptic peptides revealed at least partial identity of these two proteins. Amino acid sequencing of tryptic peptides showed no apparent homologies of bovine N-SMase to any known protein. Peptide-specific antibodies recognized a single 97-kDa protein in Western blot analysis of cell lysates. The purified enzyme displayed a K(m) of 40 microM for sphingomyelin with an optimal activity at pH 7-8. Bovine brain N-SMase was strictly dependent on Mg(2+), whereas Zn(2+) and Ca(2+) proved inhibitory. The highly purified bovine N-SMase was effectively blocked by glutathione and scyphostatin. Scyphostatin proved to be a potent inhibitor of N-SMase with 95% inhibition observed at 20 microM scyphostatin. The results of this study define a N-SMase that fulfills the biochemical and functional criteria characteristic of the tumor necrosis factor-responsive membrane-bound N-SMase.     

Purification and characterization of a phosphatidylinositol 4-kinase from bovine uteri

Phosphatidylinositol 4-kinase has been purified 10,148-fold to a specific activity of 2.7 mumol/mg/min from bovine uteri. This purification was accomplished by detergent extraction of an acetone powder, ammonium sulfate precipitation, and chromatography on MonoQ, S-Sepharose, MonoP, and hydroxylapatite columns. The purified enzyme has a molecular mass of 55 kDa and appears to be monomeric. Kinetic analyses of the enzymatic activity demonstrated apparent Km values of 18 microM and 22 micrograms/ml (approximately 26 microM) for ATP and phosphatidylinositol, respectively, optimal activity in the pH range of 6.0-7.0, and a sigmoidal dependence of enzymatic activity on [Mg2+]. Ca2+ inhibited the enzyme at nonphysiological concentrations with 50% inhibition observed at a free [Ca2+] of approximately 300 microM. The purified enzyme efficiently utilized both ATP and 2'-deoxy-ATP as phosphoryl donors and specifically phosphorylated phosphatidylinositol on the fourth position. No phosphatidylinositol-4-phosphate 5-kinase activity was observed in the purified enzyme preparations. To our knowledge, this is the first reported purification of a phosphatidylinositol-specific phosphatidylinositol 4-kinase.     

Purification and characterization of human and bovine platelet factor 4.

Platelet antiheparin, platelet factor 4, was isolated from freeze-thaw lysates of fresh bovine and outdated human platelet concentrates by a single step affinity chromatographic procedure. The yields of PF4 were 93 microgram and 142 microgram/ml of human and bovine platelets respectively. Antiheparin activity of the products were 558 units/mg for the bovine isolate and 489 units/mg for the human material. The bovine product is a single chain polypeptide with an apparent molecular weight of 12,300. Amino acid composition indicates 107-109 residues compared to the smaller human product which has an apparent molecular weight of 8,000 for a 70 residue polypeptide. The intact polypeptide was resistant to enzymatic hydrolysis as opposed to the reduced-alkylated derivative which was susceptible to hydrolysis in the presence and absence of heparin.     

Purification of a fibroblast growth factor from bovine pituitary.

The purification from bovine pituitary gland of a growth factor responsible for the control of animal cell division in tissue culture is reported. This growth factor is a polypeptide of 13,300 molecular weight and is homogeneous when analyzed by polyacrylamide gel electrophoresis, carboxymethyl-Sephadex gradient elution chromatography, and Sephadex G-50 chromatography. The yield of growth factor is 5 mg per kg of pituitary. It is active in stimulating DNA synthesis in 3T3 cells at concentrations as low as 2 times 10-13 M with saturation at 1 times 10-10 M.     

Purification of bovine glia maturation factor and characterization with monoclonal antibody

Glia maturation factor (GMF) is purified 100 000-fold to apparent homogeneity from bovine brains by a procedure consisting of ammonium sulfate precipitation, column chromatography with diethylaminoethyl-Sephacel, Sephadex G-75, and hydroxylapatite, and a final step using C4 reverse-phase high-performance liquid chromatography. The product shows a single protein band in sodium dodecyl sulfate-polyacrylamide gel. It has a molecular weight of 14 000 and an isoelectric point of pH 5.2. Purified GMF stimulates cultured astroblasts to proliferate and to grow out cell processes with half-maximal activity at 8 ng/mL. A monoclonal antibody raised against partially purified GMF adsorbs the activity of pure GMF and immunologically binds the putative GMF protein band.