Purification and characterization of neuraminidase from Clostridium perfringens

Neuraminidase (EC 3.2.1.18) has been purified from the culture medium of Clostridium perfringens ATCC 10543, through steps of gel filtration on Sephadex G-75 column, DEAE-cellulose DE 23 anion exchange chromatography, and isochromatofocusing. A homogeneous enzyme was obtained with a 7552-fold increase in specific activity to 295 units/mg protein. The yield was about 25%. The enzyme consists of a single polypeptide with a molecular weight of 69,000 as determined by SDS-polyacrylamide gel electrophoresis. Kinetic studies showed that Km is 1.5 mM for sialyllactose and Vmax is 0.41 mumole/min/ml at the enzyme concentration of 0.14 microgram/ml. The enzyme is stable at pH 5.2-8.0 with an optimal pH of 6.0. A concentrated solution of the purified enzyme was stable over one year at 4 degrees C. The purified enzyme hydrolyzed human alpha 1-acid glycoprotein completely; thus, it can be used in the clinical assay of N-acetylneuraminic acid in the serum.     

Structural and functional properties of a Ca2+-ATPase from human platelets

An antibody prepared against highly purified rabbit muscle Ca2+-ATPase from sarcoplasmic reticulum has been observed to cross-react with proteins in human platelet membrane vesicles. The antibody specifically precipitated Ca2+-ATPase activity from solubilized human platelet membranes and recognized two platelet polypeptides denatured in sodium dodecyl sulfate with Mr = 107,000 and 101,000. Ca2+-ATPase activity from Brij 78-solubilized platelet membranes was purified up to 10-fold. The purified preparation consisted mainly of two polypeptides with Mr approximately 100,000, and 40,000. The lower molecular weight protein appeared unrelated to Ca2+-ATPase activity. The Ca2+-ATPase in human platelet membrane vesicles exhibited "negative cooperativity" with respect to the kinetics of ATP hydrolysis. The apparent Km for Ca2+ activation of ATPase activity was 0.1 microM. Ca2+-dependent phosphorylation of platelet vesicles by [gamma-32P]ATP at 0 degrees C yielded a maximum of 0.2-0.4 nmol of PO4/mg of protein that was labile at pH 7.0 and 20 degrees C. This result suggests that only about 2-4% of the total protein in platelet membrane vesicles is the Ca2+-ATPase, which agrees with an estimate based on the specific activity of the Ca2+-ATPase in platelet membranes (20-50 nmol of ATP hydrolyzed/min/mg of protein at 30 degrees C). Calmodulin resulted in only a 1.6-fold stimulation of Ca2+-ATPase activity even after extensive washing of membranes with a calcium chelator or chlorpromazine. It is concluded that human platelets contain a Ca2+-ATPase immunochemically related to the Ca2+ pump from rabbit sarcoplasmic reticulum and that the enzymatic characteristics and molecular weight of the platelet ATPase are quite similar to those of the muscle ATPase.     

Affinity purification of human brain tissue factor utilizing factor VII bound to immobilized anti-factor VII

An efficient procedure for affinity purification of human tissue factor apoprotein that requires binding of only microgram quantities of human factor VII to anti-factor VII agarose is described. Factor VII was added to a 2% Triton X-100 extract of acetone brain powder in the presence of calcium. The resultant factor VII/tissue factor/calcium complex was bound to anti-factor VII-agarose, and the bound tissue factor was then eluted with EDTA. The eluate was passed through anti-goat IgG-agarose to remove contaminating goat IgG that leaks from the anti-factor VII column. Yield (units of activity) was 27%; specific activity was 2400 U/mg, which corresponds to that reported by others. The purified apoprotein migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 47,000. Immunostaining with a goat anti-tissue factor IgG raised against the purified material yielded a major band of the same apparent molecular weight. Factor VII remains bound to the column and, therefore, for subsequent use preincubation of tissue factor with factor VII and calcium is not required. Thus, the present purification procedure markedly reduces the amount of factor VII needed as affinity ligand to purify tissue factor apoprotein.     

Production and purification of human MG63 cell interferon

MG63 cells induced by Sendai virus were a convenient source of human interferon, which was of the beta type in antigenicity. When analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the interferon migrated as a single component with a molecular weight of 22,000 daltons. It was purified to apparent homogeneity by chromatography on Blue Sepharose, followed by SDS-PAGE, and estimated to have a specific activity of 4 x 10(8) international units/mg of protein.     

Single major polypeptide of a calicivirus: characterization by polyacrylamide gel electrophoresis and stabilization of virions by cross-linking with dimethyl suberimidate.

A calicivirus, San Miguel sea lion virus serotype 4, isolate 15FT, externally labelled with 125I, was shown by gel electrophoresis to possess a single major polypeptide. The polypeptide migrated anomalously upon electrophoresis in two sodium dodecyl sulfate (SDS) systems: more slowly than bovine serum albumin in a continuous phosphate-buffered system and more rapidly than bovine serum albumin in a discontinuous system. Estimated molecular weights in the two systems were approximately 71,000 and 64,000, respectively. There was no clear evidence for a minor virion polypeptide. Treatment of purified San Miguel sea lion virions with dimethyl suberimidate, a cross-linking reagent, preserved virion integrity during long-term storage at 4 degrees C. Oligomeric species of the polypeptide were observed upon electrophoresis of products from cross-linked virions. Based upon a preferred polypeptide molecular weight estimate of 71,000 and distribution of oligomeric species, a calicivirion model with 120 monomeric protein units is proposed as an alternative to a 180-unit model.     

Bovine osteogenic protein is composed of dimers of OP-1 and BMP-2A, two members of the transforming growth factor-beta superfamily

A bone-inductive protein has been purified from bovine bone and designated as osteogenic protein (OP). The purified OP induces new bone at less than 5 ng with half-maximal bone differentiation activity at about 20 ng/25 mg of matrix implant in a subcutaneous bone induction assay. The purified osteogenic protein is composed of disulfide-linked dimers that migrate on sodium dodecyl sulfate gels as a diffuse band with an apparent molecular weight of 30,000. Upon reduction, the dimers yield two subunits that migrate with molecular weights of 18,000 and 16,000. Both subunits are glycosylated. After chemical or enzymatic deglycosylation, the dimers migrate as a diffuse 27-kDa band that upon reduction yields two polypeptides that migrate at 16 kDa and 14 kDa, respectively. The carbohydrate moiety does not appear to be essential for biological activity since the deglycosylated proteins are capable of inducing bone formation in vivo. Amino acid sequences of peptides generated by proteolytic digestion show that the subunits are distinct but related members of the transforming growth factor-beta super-family. The 18-kDa subunit is the protein product of the bovine equivalent of the human OP-1 gene and the 16-kDa subunit is the protein product of the bovine equivalent of the human BMP-2A gene.     

Isolation of two distinct activator proteins for lipoprotein lipase from ovine plasma

Two distinct activator proteins for lipoprotein lipase (LPL) have been isolated in approximately equal amounts from ovine plasma. These low molecular weight proteins were readily separated from each other on the basis of size and charge. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated proteins of Mr about 8000 and 5000, with pI in urea-containing gels of about 5.1 and 4.8 respectively. Each of the ovine activator proteins was as effective as human apolipoprotein C-II (apo C-II) in activating LPL, with 1 microgram/ml giving near to maximum activation, and in lowering the apparent Km of LPL for triolein substrate. As the ratio of activator to triolein increased from 0.16 to 5.2 (micrograms/mg) the apparent Km fell from about 0.5 to 0.18 mM. Whereas human apo C-II and the two ovine activators were equally effective in stimulating the hydrolysis of triolein, differences were observed between the human and ovine activators when p-nitrophenylbutyrate was used as substrate. Unlike human apo C-II, which produced significant inhibition of p-nitrophenylbutyrate hydrolysis, the ovine activators were without effect. This suggests that the interaction between the ovine activators and LPL is different from that of human apo C-II.     

Identification, purification, and characterization of a protein activator (PA28) of the 20 S proteasome (macropain).

A protein that greatly stimulates the multiple peptidase activities of the 20 S proteasome (also known as macropain, the multicatalytic protease complex, and 20 S protease) has been purified from bovine red blood cells and from bovine heart. The activator protein was a single polypeptide with an apparent molecular weight of 28,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and had a native molecular weight of approximately 180,000. This protein, which we have termed PA28, regulated all three of the putatively distinct peptidase activities displayed by each of two functionally different forms of the proteasome. This regulation usually included both an increase in the maximal reaction velocity and a decrease in the concentration of substrate required for half-maximal velocity and indicated that PA28 acted as a positive allosteric effector of the proteasome. PA28 failed, however, to stimulate the hydrolysis of large protein substrates such as casein and lysozyme. These results suggested that the hydrolysis of protein substrates occurred at a site or sites distinct from those that hydrolyzed small peptides and that the regulation of the two processes could be uncoupled. Evidence for direct binding of PA28 to the proteasome was obtained by glycerol density gradient centrifugation. PA28 may play an important regulatory role in intracellular proteolytic pathways mediated by the proteasome.     

Production and Purification of d-Aminoacylase from Alcaligenes denitrificans and Taxonomic Study of the Strain

A d-aminoacylase-producing microorganism, strain DA181, isolated from soil was identified as Alcaligenes denitrificans subsp. denitrificans. This strain produced about 29,300 units (micromoles of product formed per hour) of d-aminoacylase and 2,300 units of l-aminoacylase per gram of cells (wet weight) when cultivated in a medium containing 1% N-acetyl-dl-leucine as the carbon source. The d-aminoacylase was purified 345-fold. The specific activity of the purified enzyme was 108,600 units per mg of protein when N-acetyl-d-methionine was used as a substrate. The apparent molecular weight was 58,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N-Acetyl-d-methionine was the favored substrate, followed by N-acetyl-d-phenylalanine. This enzyme had a high stereospecificity, and its hydrolysis of N-acetyl-l-amino acids was almost negligible.     

Synthesis and processing of sphingolipid activator protein-2 (SAP-2) in cultured human fibroblasts

Sphingolipid activator proteins (SAP) are relatively small molecular weight proteins that stimulate the enzymatic hydrolysis of sphingolipids in the presence of specific lysosomal hydrolases. SAP-2 has previously been demonstrated to activate the hydrolysis of glucosylceramide, galactosylceramide, and, possibly, sphingomyelin. Using monospecific rabbit antibodies against human spleen SAP-2, the synthesis and processing of SAP-2 were studied in cultured human fibroblasts. When [35S]methionine was presented in the medium to control human cells for 4 h, five major areas of radiolabeling were found. These had apparent molecular weights of 73,000, 68,000, 50,000, 12,000, and 9,000. Further studies indicated that the major extracellular product in normal cells given NH4Cl along with the [35S]methionine and in medium from cultures from patients with I cell disease had an apparent molecular weight of 73,000. The Mr = 68,000 and 73,000 species can be converted to a species with an apparent molecular weight of 50,000 by the action of endoglycosidase F. After labeling cells for 1 h followed by a 1-h chase, the Mr = 12,000 and 9,000 species appear. Treatment of the immunoprecipitated mixture with endoglycosidase F resulted in conversion of these species to one band with an apparent molecular weight of 7,600. These studies indicate that this relatively low molecular weight protein is rapidly synthesized from a relatively large molecular weight highly glycosylated precursor.     

Purification and characterization of rat angiotensinogen

1. Angiotensinogen (renin substrate) was purified from plasma of nephrectomized rats by a four step procedure using ammonium sulfate fractionation, chromatography on Blue Sepharose CL-6B and SP-Sephadex C-50, and gel filtration on Sephadex G-150. 2. The final preparation had a specific concentration of 9.3 microgram angiotensin I/mg (mean of six separate runs). The best preparation so far obtained contains 14.6 microgram angiotensin I/mg protein, which represents a purity of 62%. 3. By sodium dodecyl sulfate disc electrophoresis an apparent molecular weight of 56,400, and by isoelectric focusing an isoelectric point of 4.85 has been determined. These properties of rat angiotensinogen are similar to those reported for human angiotensinogen.     

Expression and purification of hexahistidine-tagged human glutathione S-transferase P1-1 in Escherichia coli

The bacterial expression and purification of human pi class glutathione S-transferase (hGST P1-1) as a hexahistidine-tagged polypeptide was performed. The expression plasmid for hGST P1-1 was constructed by ligation of the cDNA which codes for the protein into the expression vector pET-15b. The expressed protein was purified by either glutathione or metal (Co(2+)) affinity column chromatography, which produced the pure and fully active enzyme in one step with a yield of more than 30 mg/liter culture. The activity of the purified protein was 130 units mg(-1) from the GSH affinity column and 112 units mg(-1) from the Co(2+) affinity column chromatography. The purity of the protein was assessed by electrospray ionization mass spectrometry and size-exclusion chromatography. It showed that the real molecular weight of the hexahistidine-tagged hGST P1-1 polypeptide chain agreed with the calculated value and that the purified protein eluted as an apparent homodimer on the gel filtration column. Our expression system allows the expression and purification of active hexahistidine-tagged hGST P1-1 in high yield with no need of removal of the hexahistidine tag and gives pure protein in one purification step allowing further study of this enzyme.     

Isolation and characterization of thromboxane synthase from human platelets as a cytochrome P-450 enzyme

Thromboxane synthase from human platelets was purified to apparent homogeneity by conventional chromatographic techniques. A 423-fold enrichment over the specific content in the 100,000 X g sediment from platelet homogenates was obtained. The enzyme gave a single band on sodium dodecyl sulfate-gel electrophoresis corresponding to a monomeric molecular weight of 58,800. One heme per polypeptide chain was present, and by optical and EPR spectroscopy a close analogy to the group of cytochrome P-450 proteins was established. From its substrate prostaglandin H2, the stable end product thromboxane B2 is formed with a specific activity of 24.1 mumol min-1 mg of protein-1 which corresponds to a molecular activity of 1628 min-1. The enzyme formed 12L-hydroxy-5,8,10-heptadecatrienoic acid together with thromboxane B2 in a 1:1 ratio. Both products were identified by gas chromatography-mass spectrometry analysis. As reported previously for platelet microsomes (Ullrich, V., and Haurand, M. (1983) Adv. Prostaglandin Thromboxane Leukotriene Res. 11, 105-110), the pure hemoprotein spectrally interacts with pyridine- or imidazole-based inhibitors and for the potent inhibitor imidazo-(1,5-a)pyridine-5-hexanoic acid a stoichiometric binding to the heme was shown. Substrate analogs with a methylene group replacing the oxygen in either the 9- or 11-position caused difference spectra showing spectral shifts towards 387 and 407 nm, respectively. The identification of thromboxane synthase as a P-450 protein suggests that the heme-thiolate group of the enzyme is required to split and activate the endoperoxide bond of prostaglandin H2.     

Identification of the bombesin receptor on murine and human cells by cross-linking experiments

The bombesin receptor present on the surface of murine and human cells was identified using 125I-labeled gastrin-releasing peptide as a probe, the cross-linking agent disuccinimidyl suberate, and sodium dodecyl sulfate gels. A clone of NIH-3T3 cells which possesses approximately 80,000 bombesin receptors/cell with a single binding constant of approximately 1.9 X 10(-9) M was used in these studies. In addition, we used Swiss 3T3 cells and a human glioma cell line which possesses approximately 100,000 and approximately 55,000 bombesin receptors/cell, respectively. Under conditions found optimal for binding, it is demonstrated that 125I-labeled gastrin-releasing peptide can be cross-linked specifically to a glycoprotein of apparent molecular mass of 65,000 daltons on the surface of the NIH-3T3 cells. Similar results were obtained when the cross-linked product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing or non-reducing conditions. Moreover, the cross-linking reaction is specific and saturable and the 65,000-dalton polypeptide is not observed when the cross-linking experiments were performed with a NIH-3T3 cell line which is devoid of bombesin receptors. Interestingly, glycoproteins with apparent molecular weights of 75,000 were labeled specifically by 125I-labeled gastrin-releasing peptide when similar experiments were performed with Swiss 3T3 cells and with human glioma cell line GM-340. These different molecular weights may indicate differential glycosylation as treatment with the enzyme N-glycanase reduced the apparent molecular weight of the cross-linked polypeptide to 45,000. On the basis of these results it is concluded that the cross-linked polypeptides represent the bombesin receptor or the ligand-binding subunit of a putative larger bombesin receptor expressed on the surface of these cells.     

Molecular properties of the Factor V-activating enzyme from Russell's viper venom.

The protease from Russell's viper venom that activates Factor V was purified by gel filtration on Sephadex G-150 and ion exchange column chromatography on sulfopropyl (SP)-Sephadex C-50. The purified enzyme is a glycoprotein containing 6% carbohydrate. It migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 29,000. A minimum molecular weight of 27,200 was determined by sedimentation equilibrium in the presence of 6 M guanidine hydrochloride. The enzyme is composed of a single polypeptide chain possessing an NH2-terminal sequence of Val-Val-Gly-Gly-Asp-Glu-Cys-Asn-Ile-Asn-Glu-His-Pro-Ile. The specific activity of the Factor V activator toward tosyl-L-arginine methyl ester and D-phenylalanyl-L-pipecolyl-L-arginyl-p-nitroanilide was 380 and 11 nmol/min/mg, respectively. The esterase and coagulant activities of the enzyme were readily inhibited by diisopropyl fluorophosphate. The enzyme was not inhibited by bovine antithrombin III in the presence or absence of heparin. The amino acid and carbohydrate compositions of the enzyme are also reported.     

Purification, and properties of a bovine uricase

Uricase from bovine kidney, purified to homogeneity level, had a molecular weight of 70 kDa. The apparent K(m) and V(max) values for uric acid hydrolysis were 0.125 mM and 102 IU mg(-1) protein respectively. The activation energy requirement for uric acid hydrolysis by uricase and inactivation of enzyme were 11.6 and 14.5 kJ/M respectively. Both enthalpy (Delta H*) and entropy of activation (Delta S*) for uricase activity were lower than those reported for some thermostable enzymes.     

A comparison of the mouse versus human aryl hydrocarbon (Ah) receptor complex: effects of proteolysis.

The differences in the molecular properties of the nuclear aryl hydrocarbon (Ah) receptor from human Hep G2 and mouse Hepa 1c1c7 cells were investigated by time-dependent partial proteolysis with chymotrypsin or trypsin followed by column chromatographic and velocity sedimentation analysis. The sedimentation coefficients, Stokes radii and apparent molecular weights of the untreated human and mouse Ah receptor complexes were similar. Treatment of the nuclear Ah receptor complexes from both cell lines with chymotrypsin for 10 or 60 min gave lower molecular weight proteolytic products which also exhibited comparable molecular properties and salt gradient elution profiles from Sepharose columns linked to DNA. Treatment of the human and mouse nuclear Ah receptor complexes with trypsin (5 micrograms/mg protein) for 10 or 60 min gave a minor low molecular weight (29.7- or 25.7-kDa) proteolysis product which was detected only with the mouse Hepa 1c1c7 Ah receptor complex. The time- and concentration-dependent proteolytic digest maps of the human and mouse Ah receptor were determined using receptor preparations which were photoaffinity labeled with [125I]7-iodo-2, 3-dibromodibenzo-p-dioxin. The human Ah receptor was significantly more resistant to proteolysis by trypsin or chymotrypsin than the mouse Ah receptor. At a low concentration of chymotrypsin (1 microgram/mg protein) the Hepa 1c1c7 receptor was degraded to two lower molecular weight fragments with apparent M(r) values at 71- and 48-kDa whereas the Hep G2 Ah receptor was relatively stable under these conditions. Although the human Ah receptor was more slowly hydrolyzed than the mouse receptor by trypsin, the major photolabeled breakdown products for the Ah receptor from both cell lines were observed at M(r) 48- and 45-kDa. The results of this study demonstrate that there were subtle but significant differences in the human and mouse Ah receptor complex; however, the proteolysis studies suggest that there are common structural features in their ligand binding sites.     

Isolation, purification and characterization of bovine epidermal transglutaminase.

A crosslinking enzyme, epidermal transglutaminase, was isolated from soluble proteins of glabrous cow snout epidermis. This enzyme stabilized fibrin clots rendering them insoluble in 2% acetic acid. It also catalyzed the incorporation of the fluorescent amine, dansyl cadaverine, into casein. Epidermal transglutaminase was purified by chromatography upon DEAE-Sephadex A-50, zone electrophoresis in Pevikon, and Sephadex G-200 gel permeation chromatography. The highly purified substance, which had a specific activity of 3267 amine-incorporating units/mg per h and a molecular weight of 55000, behaved as a single molecular species in the analytical ultracentrifuge. It had a sedimentation coefficient of 4.4 S and migrated as a gamma-globulin at pH 8.6; it displayed anomalous migration in polyacrylamide gels containing sodium dodecyl sulfate. The enzyme was dependent upon free calcium ions and a reduced sulfhydryl group for activity. The apparent Km for dansyl cadaverine was 1.2 - 10(-4) at pH 7.5. Monospecific antiserum to bovine epidermal transglutaminase precipitated with the enzyme in agar. The antiserum prevented fibrin crosslinking but enhanced incorporation of dansyl cadaverine into casein by the enzyme. The epidermal enzyme differed biochemically and immunochemically from bovine plasma transglutaminase (Factor XIII).     

The enzyme glutamine synthetase I of Drosophila melanogaster is associated with a modified RNA

Glutamine synthetase I (L-glutamate:ammonia ligase, ADP forming; EC 6.3.1.2) was purified from Drosophila melanogaster larvae. The complete enzyme has an apparent molecular weight of 380,000. The subunit of the active enzyme has an apparent molecular weight of 43,000 after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Routine preparations yield enzymes which have at least another polypeptide component of apparent molecular weight of 64,000. Several factors suggest that the 64,000-dalton polypeptide might be a transformation product of the 43,000-dalton subunit which occurs in association with enzyme inactivation. Distinct from its protein subunit, from pure glutamine synthetase I a material can be extracted which can be labeled with 32P-labeled gamma-ATP using polynucleotide kinase. After alkaline hydrolysis the majority of the radioactivity is recovered as 5'2' and 5'3' ribonucleotide diphosphates, and after venom phosphodiesterase digestion as 5' ribonucleotide. We therefore conclude that the native glutamine synthetase I enzyme contains, or at least is reproducibly associated with, an RNA component. Several characteristics of the labeled material indicate that the RNA is small in size and is bound to polymer molecules different from RNA.     

Biochemical and immunological studies of purified mouse beta-galactosidase.

Beta-Galactosidase (EC 3.2.1.23) has been purified from the livers of C57BL/6J mice. The enzyme migrated as a single band of protein on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weight of the denatured and reduced enzyme was 63,000. The native form of beta-galactosidase appeared to be a tetramer of 240,000 at pH 5.0, which was reversibly dissociated at alkaline pH to a dimer with apparent molecular weight of 113,000. Multiple charge isomers of beta-galactosidase were resolved by polyacrylamide gel electrophoresis and ion exchange chromatography. Treatment of beta-galactosidase with neuraminidase markedly reduced its electrophoretic mobility. Purified enzyme as well as crude liver extract hydrolyzed p-nitrophenyl-beta-D-fucoside at one-tenth the rate of hydrolysis of the beta-galactoside. Antiserum to the purified enzyme precipitated the major portion of beta-galactosidase activity of mouse liver, brain, and kidney. This antiserum cross-reacts with beta-galactosidases from rat and Chinese hamster, but not with human, porcine, or bovine beta-galactosidase.