Heterogeneity of purified mouse interferons.

A procedure for obtaining highly purified stable interferon from mouse L cells is described. Interferon with a specific activity of 2.5 times 10-8 reference units/mg of protein is composed of 10 to 11 polypeptides. They differ in their molecular size as determined by electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate. The molecular weight range is estimated to be from the smallest at 20, 000 to the largest at 32, 000. At least six of the polypeptides are glycoproteins and each of the polypeptides can be obtained as an apparent homogeneous entity and each has interferon activity.     

Purification and characterization of bovine brain glucocerebrosidase

Glucocerebrosidase was isolated from bovine brain by cholate extraction, ammonium sulfate fractionation, acid precipitation at pH 5.35, and hydrophobic chromatography. The purification is about 2400-fold with a specific activity of about 286,000 nmole/hr/mg protein. Molecular weight as determined by chromatography on Bio-Gel P-200 was 138,000. On SDS-polyacrylamide gel electrophoresis the enzyme protein resolved into two bands with apparent molecular weights of 63,000 and 56,000. These bands are cross-reactive to monospecific polyclonal antibody to homogeneous human placental glucocerebrosidase. The enzyme was found to be a complex glycoprotein based on its lectin binding specificity. Brain enzyme was found to be similar to placental glucocerebrosidase in its pH optima, heat stability at 52 degrees C, and substrate affinity. Enzyme kinetics were measured in the presence of conduritol-beta-epoxide, an irreversible inhibitor, and gluconolactone, a competitive inhibitor.     

Isolation and in vitro synthesis of trypsin from the biting fly,Stomoxys calcitrans

The synthesis of [3H]trypsinlike enzyme by the fat body was followed in Stomoxys calcitransin vitro using a radioimmunoassay (RIA) developed against mammalian trypsin. Using high specific activity [3H]valine, trypsinlike activity was followed in midgut epithelial cells, thoracic muscle, and fat body removed from sugar-fed flies. Excreta protease of S. calcitrans was partially purified using charge and hydroxylapatite gel chromatography. Seventy-five percent of the enzyme eluted from these gels was inhibited by tosyl-L-lysine chloromethyl ketone HCI (TLCK) and was classified as trypsinlike. Electrophoresis of the trypsinlike enzyme indicated that it was only 50% pure. Trypsinlike activity from S. calcitrans bound to α₁-globulin IV-I and formed a complex that was dissociated on a P-100 Bio-Gel column. Binding between the protease and the α₁-gobulin IV-I caused a 1.4-fold increase in the apparent molecular weight of the protease on the P-100 Bio-Gel column. Trypsinlike activity was characterized in the midgut and excreta by affinity binding to covalently linked TLCK and tosyl-L-lysine chloromethyl ketone HCI (TAME)Sepharose 4B gels. Between 50% and 55% of the excreta protease and 5669% of the midgut protease bound to the affinity gels and was trypsinlike. Protease activity that did not bind to the gels was not inhibited by TLCK and did not have the esterolytic activity of trypsin.     

Purification of a novel calmodulin binding protein from bovine cerebral cortex membranes

A new calmodulin (CaM) binding protein, designated P-57, has been purified to apparent homogeneity from bovine cerebral cortex membranes. In contrast to other calmodulin binding proteins, P-57 has higher affinity for calmodulin in the absence of bound Ca2+ than in its presence. The protein was purified by DEAE-Sephacel chromatography and two CaM-Sepharose affinity column steps. The first CaM-Sepharose column was run in the presence of Ca2+; the second was run in the presence of chelator in excess of Ca2+. P-57 was adsorbed by CaM-Sepharose only in the absence of bound Ca2+ and was eluted from the second column by buffers containing Ca2+. Sodium dodecyl sulfate (SDS)-polyacrylamide gels of the purified protein showed only one band at Mr 57 000. The major form of the protein on Bio-Gel A-1.5m and native polyacrylamide gradient gel electrophoresis ran with an apparent Stokes radius of 41 A. Photoaffinity labeling of P-57 with azido[125I]calmodulin yielded one cross-linked product on SDS gels with an Mr of 70 000. This interaction occurred only when excess ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid was present and was inhibited by the presence of Ca2+ in excess of chelator. It appears that P-57 has novel binding properties for calmodulin distinct from all other calmodulin binding proteins described thus far.     

Purification and characterization of human factor II

Human plasma Factor II has been purified approximately 800-fold by a combination of barium citrate adsorption, ion-exchange chromatography and preparative polyacrylamide gel electrophoresis. The procedure is relatively simple and results in excellent yields of purified Factor II essentially free of Factor X activity. The purified factor behaved as a single component by analytical polyacrylamide gel disc electrophoresis at pH 8.9. No Factor V, VII or IX activity was detected in the purified Factor II. Its molecular weight was 7200±3000 as determined by analytical ultracentrifugation, electrophoresis in the presence of sodium dodecyl sulfate and gel filtration on Bio-Gel P-200. An apparent molecular weight of 90 000–100 000 was observed on calibrated columns of Sephadex G-100, G-150, and G-200. The specific activity of human factor II was approximately 1300 N.I.H. units/mg as determined by the two-stage assay and 7 Ortho units/mg by the one stage assay. The purified protein contained by weight 2.8% neutral hexose, 2.3% sialic acids and 3.1% hexosamines.     

High-molecular-weight cadmium-binding proteins in rat liver cytosol: isolation and partial characterization of an approximately 50-kDa protein

The time-dependent changes in the chromatographic pattern of subcutaneously injected cadmium associated with non-metallothionein cadmium-binding proteins were studied in the rat liver cytosol. Prior to the induction of cadmium-thionein (less than 3 h), cadmium appeared in three major peaks (P-1 with the void volume, P-2 and P-3) on Sephacryl S-300 column chromatography. Accompanied with the emergence of apo-metallothionein (about 3 h after administration), the amount of P-3 decreased and instead a cadmium-thionein peak (P-4) increased. Ion-exchange chromatography of P-3 with a combination of CM and DEAE Bio-Gel columns showed the existence of three major cadmium-binding proteins with molecular sizes of 46 kDa (in the CM Bio-Gel column eluate), 50 kDa (in the DEAE Bio-Gel column eluate), and 41 kDa (in the non-adsorbed fraction). The cadmium-binding protein in the CM Bio-Gel column eluate was purified to apparent homogeneity. The purified protein (CM-CdP) was 47 or 53 kDa in molecular size as determined by SDS-polyacrylamide gel electrophoresis or gel filtration chromatography, respectively. The apparent dissociation constant and maximum binding for cadmium were about 1 microM and 1 mol of the metal/mol of protein, respectively. The isoelectric point was estimated to be 8.8. The amino acid composition showed that the protein was relatively rich in glutamyl (including its amide) and alanyl residues. The N-terminal amino acid sequence was determined as Ala-Pro-Ile-Ala-Gly-Lys-Lys-Ala-Lys-Ala-Gly-Ile-Leu-Leu-Gly-. In-vitro experiments revealed that cadmium bound to CM-CdP could be easily transferred to apo-metallothionein, confirming that the affinity for the metal of the former protein was lower than that of the latter.     

Some Properties and Characterization of Rice Seed Hemagglutinin

The phytohemagglutinin of rice seed has been purified by a sequence of steps involving fractionation with ammonium sulfate and successive chromatography on DEAE-and eMcellulose and finally gel filtration on Bio-Gel P-100. The purified rice seed hemagglutinin was shown to be homogeneous by electrophoresis on polyacrylamide gel and its molecular weight was 10,000, calculated from both the Ve/Vo value of gel filtration on Bio-Gel P-100 and the sum of the individual constituents (amino acids, sugars and metals). In addition to amino acid, the rice seed hemagglutinin contained 26.8% covalently bound carbohydrate which was identified and quantitated by gas chromatography of the acetylated alditols. Glucose was the predominant sugar with lesser amounts of glucosamine, xylose, and mannose also being present. And the rice seed hemagglutinin contained 1 g atom of calcium per molecule. The molecular weight of the rice seed hemagglutinin is smallest compared with some of phytohemagglutinins isolated from leguminous seeds and other plant sources. The rice seed hemagglutinin has the blastogenetic activity for human peripheral lymphocytes as well as Phaseolus vulgaris phytohemagglutinins or concanavalin A, jack bean hemagglutinin.     

A toxic protein from Bryonia dioica Jacq. fruits: the brydiofin.

A toxic protein isolated from Bryonia dioica Jacq. fruits is described. The fruit extract, obtained in phosphate buffer saline, in mild conditions, showed a strongly toxic activity after intraperitoneal injection of 0.4 mg per Balb/c mouse that was killed in eighteen minutes. The extract, filtered through a Bio-Gel P-60 column, presented a main peak that showed similar toxicity. When the material from this peak was analyzed in polyacrylamide slab gels, a single band corresponding to a molecular weight of about 66,000 was observed. The lethal effect of B. dioica fruit could be attributed to a protein that we called Brydiofin. The extracts from roots and leaves that did not present this toxicity showed no band in this molecular weight region.     

Secretion of DNA synthesis factor (DSF) by A431 cells that can grow in protein-free medium

A431 cells grew in protein-free Coon's modified Ham's F12 medium at a similar rate to that in medium supplemented with calf serum and secreted a growth factor capable of stimulating DNA synthesis in BALB/c3T3 cells. This factor had strong affinity for heparin and was partially purified from the conditioned medium by heparin-Sepharose affinity chromatography and molecular sieving on Bio-Gel P-60. The apparent molecular weight of the factor was 20-30K. Its activity was inhibited by heparin at concentrations of above 0.03 microgram/ml.     

Mycoplasma Phosphoenolpyruvate-Dependent Sugar Phosphotransferase System: Purification and Characterization of Enzyme I

The Mycoplasma phosphoenolpyruvate-dependent sugar phosphotransferase system consists of three components: a membrane-bound enzyme II, a soluble phosphocarrier protein (HPr), and a soluble enzyme I. The soluble enzyme I was purified by ammonium sulfate fractionation; Bio-Gel P-10 gel filtration; acid precipitation; diethylaminoethyl-Bio-Gel A; and Bio-Gel HTP column chromatography. The enzyme I was shown to be homogeneous by electrophoresis in a pH 8.9 non-sodium dodecyl sulfate gel and by isoelectric focusing. Whereas the protein moved as a single component in both the non-sodium dodecyl sulfate gel and isoelectric focusing, on sodium dodecyl sulfate gels, it moved as three subcomponents. The molecular weights of the three subunits, alpha, beta, and gamma, were 44,500, 62,000 and 64,500, respectively. The holoprotein moved as a single component, in the region of 220,000 daltons, in a Bio-Gel A 0.5-agarose column. The molar ratio of subunits was estimated to be 2alpha:1beta:1gamma. The elution characteristics on a diethylaminoethyl column at pH 7.4 and 6.8, acid precipitation data, and amino acid composition indicated that the protein is acidic. Isoelectric focusing occurred at pH 4.8. N-terminal amino acids determined by the dansyl chloride method indicated that glycine, alanine, and tyrosine are N-terminal amino acids of the three subunits. Although the protein was stable for at least 14 months at -20 degrees C, it was irreversibly inactivated by the thiol reagent N-ethyl-maleimide.     

Purification of the T lymphocyte growth factor interleukin-2 from culture media of human peripheral blood leukocytes (buffy coats)

Interleukin 2 was purified 100 000-fold to apparent homogeneity from the supernatants of mitogen-stimulated human blood leukocytes. A sequence of three purification steps was used: affinity chromatography on the bound dye cibacron blue, gel filtration on Ultrogel AcA44, and reversed-phase high-performance liquid chromatography on hexyl phase. The resulting interleukin 2 had a specific activity of 2 X 10(6) U/mg protein, and was free of pyrogenicity in the rabbit test. The final purification product showed two bands in sodium dodecyl sulfate/polyacrylamide gels with apparent molecular masses of 15 kDa and 17 kDa respectively. Both bands were biologically active.     

The proteolytic system of Penicillium roqueforti. V. -- Purification and properties of an alkaline aminopeptidase.

An alkaline aminopeptidase was isolated from the culture medium of Penicillium roqueforti. The enzyme was purified by ammonium sulfate precipitation, filtration on Bio-Gel P-100, chromatography on D.E.A.E.-cellulose and hydroxylapatite, filtration on Bio-Gel P-150 and electrofusing. The purified preparation was homogeneous on polyacrylamide gel electrophoresis at pH 8.5. The molecular weight of the enzyme was estimated to be about 35,000 daltons. The isoelectric point is 4.5. The optimum pH for L-leucine-p-nitroanilide hydrolysis is 8.0. At 35 degrees C the enzyme is stable between pH 6.0 and 7.0. Ethylenediamine tetraacetic acid and a sulfhydryl reagent (p-hydroxymercuribenzoate) inhibit the activity, but the enzyme is insensitive to diisopropylfluorophosphate. Hydrolysis of synthetic peptides shows that the enzyme releases apolar amino acids. Dipeptides are poorly hydrolyzed and Gly in penultimate or N-terminal position causes poor activity. The enzyme is able to cleave the N-terminal Arg-Pro bond of bradykinin.     

Immobilization of proteins on aldehyde-activated polyacrylamide supports

A method has been developed for the immobilization of proteins on derivatized polyacrylamide gels. Aminoethyl Bio-Gel P-150 was converted to its stable N-2,3-dihydroxypropyl derivative by borohydride reduction of the Schiff base formed with glyceraldehyde. Periodate oxidation of the modified gel provided a reactive aldehyde, which was subsequently coupled to protein by reductive amination with sodium cyanoborohydride. Coupling efficiencies were found to be >90% for concanavalin A and bovine serum albumin, and the gels contained as much as 5 and 20 mg of protein/ml of gel, respectively. Immobilized concanavalin A retained 89% of its binding capacity and was demonstrated to be chemically stable with variations in pH, and changes in concentrations of Triton X-100 and sodium dodecyl sulfate (at concentrations <0.1%). Bovine β-hexosaminidase and β-glucuronidase, higher molecular weight proteins, were also bound with retention of activity, but with less efficiency. This procedure provides an efficient method for the covalent immobilization of proteins.     

Purification of the beta-glucosidase from Sclerotinia sclerotiorum

A beta-glucosidase (EC 3.2.1.21) has been isolated from culture filtrates of the fungus Sclerotinia sclerotiorum. The protein was purified by gel filtration on a column of Bio-Gel P-300 and by ion exchange chromatography on DEAE-Bio-Gel A. The molecular weight, determined by gel filtration, was 240,000. Km values for the enzyme towards p-nitrophenyl-beta-D-glucoside and cellobiose were respectively 0.10 mM and 1.23 mM. The beta-glucosidase activity was found to be strongly associated with a beta-xylosidase (EC 3.2.1.37) activity, suggesting that both activities could be represented in a single protein complex.     

alpha-Amylase inhibitor from fungus Cladosporium herbarum F-828

A strain of fungus Cladosporium herbarum extracellularly produced an inhibitor specific for mammalian alpha-amylase. The inhibitor was purified 81-fold by freeze-thawing, heat treatment, and column chromatography on DEAE-cellulose, Sephadex G-75, DEAE-Sephacel, and Bio-Gel P-100. An apparent molecular weight of approximately 18,000 was estimated for the inhibitor using Bio-Gel P-100 filtration. The purified inhibitor preparation was a glycoprotein containing about 10% carbohydrate. The amino acid analysis of the inhibitor showed abundances of Gly, Asp, Glu, Ser, Ala, and Thr residues. The inhibitor was stable between pH 5 and 12 at 4 degrees C, and below 80 degrees C at pH 7.0. A binary complex formation out of equimolar amounts of the inhibitor and alpha-amylase, was demonstrated by polyacrylamide gel electrophoresis, and Bio-Gel P-100 chromatography. Kinetic studies exhibited that the inhibitor noncompetitively inhibited the enzyme reaction with a Ki value of 2.3 approximately 4.8 x 10(-10) M, by combining with the enzyme molecule at a different site from the substrate binding site.     

Bone sialoprotein II synthesized by cultured osteoblasts contains tyrosine sulfate

Isolated mouse osteoblasts that retain their osteogenic activity in culture were incubated with [35S] sulfate. Two radiolabeled proteins, in addition to proteoglycans, were extracted from the calcified matrix of osteoblast cultures. All the sulfate label in both proteins was in the form of tyrosine sulfate as assessed by amino acid analysis and thin layer chromatography following alkaline hydrolysis. The elution behavior on DEAE-Sephacel of the major sulfated protein and the apparent Mr on sodium dodecyl sulfate gels were characteristic of bone sialoprotein II extracted from rat. This protein was shown to cross-react with an antiserum raised against bovine bone sialoprotein II, indicating that bone sialoprotein II synthesized by cultured mouse osteoblasts is a tyrosine-sulfated protein. The minor sulfated protein was tentatively identified as bone sialoprotein I or osteopontin based on its elution properties on DEAE-Sephacel and anomalous behavior on sodium dodecyl sulfate gels similar to those reported for rat bone sialoprotein I.     

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 of human midcycle cervical mucin and characterization of its oligosaccharides with respect to size, composition, and microheterogeneity

Human cervical mucin was solubilized from the gel phase of pooled midcycle cervical mucus using 6 M guanidine hydrochloride and 10 mM dithiothreitol and was then alkylated with iodoacetamide. Mucin was then purified by gel filtration on Bio-Gel A-50m resin in buffer containing 0.1% sodium dodecyl sulfate. The purified mucin gave a single band upon electrophoresis in either 5% acrylamide or 1% agarose gels. Protein comprised 21% of the glycoprotein by weight and amino acid analysis revealed a high content of Ser and Thr. Saccharide analysis yielded approximate molar ratios of Fuc:Gal:GlcNAc:GalNAc:NeuAc = 1:2:1:1:0.5. Inorganic sulfate, 1% by weight, was detected, but mannose was absent. Reductive alkali treatment of mucin resulted in release of oligosaccharides with concomitant conversion of 77% of GalNAc to its reduced derivative N-acetylgalactosaminitol (GalNAcol) thus demonstrating O-glycosidic linkage of GalNAc to protein. Reduced oligosaccharides were purified by ion exchange chromatography on DEAE-cellulose, paper chromatography, and high resolution gel filtration on Bio-Gel P-2 resin. A total of 16 reduced oligosaccharides were identified by thin layer chromatography. These included neutral, sialylated, and sulfated oligosaccharides and they varied in size from a disaccharide to a nonasaccharide. The major neutral oligosaccharide isolated (21% of recovered GalNAcol) was a tetrasaccharide, Gal:GlcNAc:GalNAcol = 2:1:1, and the major acidic oligosaccharide isolated (11% of recovered GalNAcol) was a trisaccharide, Gal:GalNAcol:NeuAc = 1:1:1.     

Purification of beta-glucuronidase from urine of androgen-stimulated female mice

β-Glucuronidase (EC 3.2.1.31) has been purified from the urine of androgen-treated A/J female mice. This is a convenient starting material as the enzyme comprises 0.5 to 1.0% of total urinary proteins. Weekly injections of testosterone enanthate increased β-glucuronidase excretion from kidney into urine by approximately 300-fold. Unexpected urinary enzyme activity declined after six weekly treatments, but returned after the testosterone injections were discontinued. These observations suggest that testosterone influences not only the rate of β-glucuronidase synthesis but also the excretion of the enzyme into the urine. Other hormone regimens for achieving β-glucuronidase synthesis and excretion into urine are discussed. After concentrating the urine 10- to 12-fold, β-glucuronidase was isolated using two chromatography steps. Gel filtration on Bio-Gel A-0.5m resulted in a 16-fold purification of the enzyme and removed most of the major urinary proteins. Anion exchange on diethylaminoethyl-cellulose resulted in a further purification of β-glucuronidase by 12-fold. These two chromatography steps gave 190- to 200-fold increases of β-glucuronidase activity per milligram of protein and the enzyme electrophoresed as a single band in two native gels. However, analysis on sodium dodecyl sulfate gels revealed traces of protein smaller than the 70,000 molecular weight subunit of the enzyme. The β-glucuronidase isolated from urine had the same physical properties as the lysosomal form of the enzyme in mouse kidney.     

Partial Purification and Some Properties of the Staphylococcus aureus Cytoplasmic Nitrate Reductase

The cytoplasmic nitrate reductase in heme mutant H-14 of Staphylococcus aureus was partially purified by steps which included ammonium sulfate fractionation and chromatography on Bio-Gel A 1.5m and ion-exchange columns. The active fractions from the ion-exchange columns showed two forms of the enzyme upon electrophoresis in nondenaturing gels of polyacrylamide; these corresponded to proteins of R(f) 0.16 and 0.28. Each form contained a predominant polypeptide of molecular weight 140,000, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The R(f) 0.16 form contained another major polypeptide of molecular weight 57,000, but the R(f) 0.28 form contained several other polypeptides. The sedimentation properties of the enzyme were examined after partial purification on Bio-Gel A 1.5m. In sucrose gradients containing Triton X-100 the enzyme sedimented as a homogeneous peak with an estimated molecular weight of 225,000; without detergent a heterogeneous profile was observed of molecular weight greater than 250,000. Treatment of the enzyme with trypsin increased the specific activity, and the enzyme sedimented as a homogeneous peak in sucrose gradients without Triton X-100, with an estimated molecular weight of 202,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that trypsin treatment converted the polypeptide of molecular weight 140,000 to a polypeptide of molecular weight 112,000. We conclude that the cytoplasmic nitrate reductase of S. aureus has a large subunit of molecular weight 140,000, which can be modified by trypsin to a polypeptide of molecular weight 112,000 without loss of catalytic activity.