Polyacrylamide Gel Electrophoresis In Presence Research Articles

Schinus terebinthifolia Raddi leaf lectin is an antiangiogenic agent for Coturnix japonica embryos.

Angiogenesis (budding of new blood vessels) is involved in several processes, including the development of embryos and growth of tumors. Schinus terebinthifolia leaves express an antitumor lectin (SteLL). This work hypothesized that SteLL can interfere with the formation of a vascular network from preexisting vessels. To test this hypothesis, the effect of SteLL on the angiogenesis process was assessed using an in vivo model of yolk sac membrane of Coturnix japonica embryos. SteLL was isolated with purification factor of 46.6. As expected, polyacrylamide gel electrophoresis (PAGE) for native basic proteins confirmed the homogeneity and PAGE in presence of dodecyl sodium sulphate revealed a single 14-kDa polypeptide band. The fractal analysis by box counting and information dimension measurements indicated that SteLL at 1.35 mg/mL significantly decreased by ca. 12% the angiogenesis within the C. japonica yolk sac membrane regarding the control. The inhibition of the vascular network formation in the yolk sac membrane resulted in decreased blood supply to the embryos. Consequently, the area of embryos was significantly reduced by 9.2% regarding the control, which corroborated with the antiangiogenic activity of SteLL. The findings implicate SteLL as an antiangiogenic agent and add to the panel of biological activities of this lectin.

IDENTIFICATION OF EPO-Fс FUSION PROTEIN BY MEANS OF POLYACRYLAMIDE GEL-ELECTROPHORESIS WITH ISOELECTROFOCUSING (IEF-PAGE)AND IN PRESENCE OF SODIUM DODECYLSULPHATE (SDS-PAGE)/ LAUROYLSARCOSINATE (SAR-PAGE) FOR THE PURPOSE OF ANTI-DOPING CONTROL

The article is devoted to develop of an approach for the identification of new stimulator of ematopoiesis, EPO-Fc fusion protein, which is banned by the World Anti-doping Agency (WADA) to use by athletes since it has become doping. Existing methods of qualitative determination of this substances in routine practice of antidoping laboratories such as polyacrylamide gelelectrophoresis in presence of sodium dodecylsulphate (SDS-PAGE) or lauroylsarcosinate (SARPAGE) are insufficiently specific. The article shows the principal possibility of identification of EPO-Fc fusion protein by means of IEF-PAGE in carrier ampholyte-based gels with a pH range 2-6 after Fc-fragment removal via fermentative hydrolysis.It has been shown that the removing of the crystallizable fragment leads to decrease of molecular weight of whole hybrid molecule and to increase its electrophoretic mobility that allows to detect this banned substances with high specificity by existing methods. During the study the enzyme for hydrolytic cleavage and optimum conditions of hydrolysis of EPO-Fc in serum samples were selected.

Congenital Dyserythropoietic Anemias: Molecular Diagnosis and Diagnostic Approach in a Cohort of Italian Patients

Introduction. Congenital dyserythropoietic anemias (CDAs) are hereditary rare erythropoietic disorder characterized by distinct morphological abnormalities of the bone marrow cells, ineffective erythropoiesis and systemic iron overload. This conditions is characterized by a maturation arrest during erythropoiesis with a reduced reticulocyte production in contrast with erythroid hyperplasia in bone marrow. Three types of CDA are known: types 1, 2 and 3.The identification of their causative genes provided evidence that these conditions have different molecular mechanisms that induce abnormal cell maturation and division. We describe the clinical and laboratory manifestations, the diagnosis procedure, the therapeutic approaches and the clinical phenotype in some cases of congenital dyserythropoietic anemia (CDAs) in Italian patients. The molecular analysis allow us to identify several genetic variants some of which have never been described previously. This report highlights the importance of recognizing CDAI even in countries where thalassemia is common. Among the different tools genetic study of CDA-related genes remains the main approach for pursuing the right diagnosis.Methods. Peripheral blood samples from 100 normal adults and 20 patients with different types of anemia were collected. Blood samples were analyzed by EMA binding test and by polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate ( SDS-PAGE). SDS PAGE was carried out in a 3.5-17% exponential gradient gel in Fairbanks buffer Genomic DNA was extracted from mononuclear cells of peripheral blood samples, by using a phenol-chloroform method. Polymerase chain reaction (PCR) products were sequenced directly using Big-Dye terminator 3.1 cycle sequencing kit and run on ABI PRISM 3130 DNA analyzer. The bone marrow of all patients was analysed with light or electron microscopy. The diagnosis of CDAs was based on the presence of mild to moderate anemia ineffective erythropoiesis and bone marrow erythroblasts morphological abnormalities. SEC23B, CDAN1, KLF1, BCL11A gene sequencing analysis was performed to highlight the presence of nucleotide variations and their relationship with the clinical presentation.Results and Discussion. We collected blood samples from 20 Italian patients with suspect of CDAs and 100 samples belonging to the healthy control subjects.None mutation in the genes analyzed was found in the samples controls.We identified SEC 23 B mutations in 4 out of 20 patients analysed. In two patients with suspect of CDAII we found two nucleotide variations; SDS PAGE in these patients identified the presence of abnormal band 3 and the EMA binding test resulted pathologic. Bone marrow (BM) light microscopy revealed more than 10% mature binucleated erythroblasts. In the patients suspected of CDA1, several gene variants were found in the CDAN1 gene, some described in the literature as mutations or polymorphisms, others of uncertain significance. In a patient diagnosed with CDA1 two novel mutations was found. EMA binding test resulted normal in all patients with CDA1. Light microscopy of the BM of CDA1 patients showed erythroid hyperplasia, presence of internuclear bridges between intermediate erythroblasts and characteristic pattern of spongy "swiss cheese" hetrochromatin in electron microscopy. None KLF1 and BCL11A mutations correlated with atypical CDAs was identified. In this last patients the EMA binding test resulted normal. CDAs are rare clinical hereditary disorders whose correct diagnosis is often delayed to adolescence or adulthood, when significant iron overload and end organ damage may have been occurred. Patients are often misdiagnosed with other congenital haemolytic anaemias. Inaccurate diagnosis can lead to inappropriate therapies, such as iron supplements, aggressive transfusion or splenectomy. However even if the gold standard for the CDAs diagnosis is the electronic microscopy, the identification of the mutated genes involved in the majority of CDAs patients made in recent years has improved the possibility of detecting these diseases. DisclosuresNo relevant conflicts of interest to declare.

Quantitative analysis for estimating injury effects of metal-catalyzed oxidation on human erythrocytes

ObjectiveTo investigate whether human erythrocyte proteins were susceptible to oxidative effects of pharmacological doses of iron and whether resulting damages affect their structure. MethodsConformational changes in hemoglobin were indicated by spectrophotometric analysis from 300 to 650 nm. Carbonyl assay was performed for estimating the protein oxidation in erythrocytes. Oxidative injury in erythrocyte membrane was investigated by evaluation of the structural changes in cytoskeleton proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis in presence of 2-mercaptoethanol and staining with Coomassie briliant blue G-250. ResultsA significant increase in absorbance at 630 nm represented the formation of methemoglobin. Increase in absorbance at 340 nm was indicated by interaction between globin and heme group, which predicted for low oxygen affinity. A decrease in absorbance at 420 nm showed the conversion of oxygen hemoglobin to methemoglobin and significant decrease in oxygen hemoglobin concentration. There was marked elevation in hemichrome compared with control group. Of interest, a positive correlation was observed between iron concentration and hemoglobin absorbance at 340 nm. Elevated levels of carbonyl groups confirmed the oxidative damage to erythrocyte proteins. Analysis of membrane proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis, showed molecular aggregates in the range of 150 to 180 kDa and slight decrease in the intensity of α-spectrin band. ConclusionsIt is possible to predict the situation of everyone who exposed to oxidant agent via a simple blood analysis. In this way, contents of oxidative products in blood samples would be assessed by this method.

Purification, molecular and kinetic characterization of phosphofructokinase-1 from the yeast Schizosaccharomyces pombe: evidence for an unusual subunit composition.

Phosphofructokinase-1 (Pfk-1) from Schizosaccharomyces pombe was purified by 54-fold enrichment to homogeneity elaborating the following steps: (a) Disruption of the cells with glass beads; (b) fractionated precipitation with polyethylene glycol 6000; (c) affinity chromatography on Cibacron-Blue F3G-A-Sephadex G 100; (d) ion exchange chromatography on Resource Q. The native enzyme exhibits a mass of 790+/-30 kDa, as detected by sedimentation equilibrium measurements. The apparent sedimentation coefficient was found to be s(20,c)=20.2+/-0.3 S. No significant dependence of the s-value on the protein concentration was observed in the range 0. 07-0.7 mg/ml. Polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate and MALDI-TOF spectra showed that the enzyme is composed of subunits of identical size of 100+/-5 kDa, forming an octameric structure. The N-terminus of the enzyme was found to be blocked. Sequences of tryptic and chymotryptic peptides of the subunit coincide with the proposed amino acid sequence as deduced from the gene from the EMBL library. The Pfk-1 coding sequence of S. pombe was transformed into a Pfk-1 double deletion mutants of Saccharomyces cerevisiae resulting in glucose-positive cells with enzyme activity in the crude cell extract. The kinetic analysis revealed less cooperativity to fructose 6-phosphate (n(H)=1.6) and less inhibition by ATP as compared to the enzyme from baker's yeast. Fructose 2,6-bisphosphate (in micromolar range) and AMP (in millimolar range) were found to overcome ATP inhibition and to increase the affinity to fructose 6-phosphate.

Purification, molecular and kinetic characterization of phosphofructokinase-1 from the yeastSchizosaccharomyces pombe: evidence for an unusual subunit composition

Phosphofructokinase-1 (Pfk-1) from Schizosaccharomyces pombe was purified by 54-fold enrichment to homogeneity elaborating the following steps: (a) Disruption of the cells with glass beads; (b) fractionated precipitation with polyethylene glycol 6000; (c) affinity chromatography on Cibacron-Blue F3G-A-Sephadex G 100; (d) ion exchange chromatography on Resource Q. The native enzyme exhibits a mass of 790+/-30 kDa, as detected by sedimentation equilibrium measurements. The apparent sedimentation coefficient was found to be s(20,c)=20.2+/-0.3 S. No significant dependence of the s-value on the protein concentration was observed in the range 0. 07-0.7 mg/ml. Polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate and MALDI-TOF spectra showed that the enzyme is composed of subunits of identical size of 100+/-5 kDa, forming an octameric structure. The N-terminus of the enzyme was found to be blocked. Sequences of tryptic and chymotryptic peptides of the subunit coincide with the proposed amino acid sequence as deduced from the gene from the EMBL library. The Pfk-1 coding sequence of S. pombe was transformed into a Pfk-1 double deletion mutants of Saccharomyces cerevisiae resulting in glucose-positive cells with enzyme activity in the crude cell extract. The kinetic analysis revealed less cooperativity to fructose 6-phosphate (n(H)=1.6) and less inhibition by ATP as compared to the enzyme from baker's yeast. Fructose 2,6-bisphosphate (in micromolar range) and AMP (in millimolar range) were found to overcome ATP inhibition and to increase the affinity to fructose 6-phosphate.

Analytic and Immunologic Characterization of Chickpea (Cicer arietinum) Protein Hydrolysates Obtained by Bromelain and α-Chymotrypsin

A water soluble concentrate of chickpea (Cicer arietinum) proteins was hydrolyzed by bromelain and α-chymotrypsin. Hydrolysis was verified by polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate. The use of ELISA in an inhibition system, conducted with chickpea protein antiserum raised in rabbits, showed that enzymatic hydrolysis resulted in a considerable reduction in the antigenic character of the proteins. Thus, the percentage inhibition by the α-chymotrypsin of hydrolysate was 58 ± 2.3% and that by the bromelain hydrolysate was 45 ± 4.5%. The peptides were measured by size exclusion chromatography. Peptides with molar mass lower than 1000 Da could be fractionated into fraction F1 containing peptides with molar mass higher than 500 Da, fraction F2 containing peptides with molar mass included between 200 and 500 Da, essentially small peptides containing 2, 3, or 4 amino acids, and fraction F3 containing free amino acids. The purified fractions were quantified with the TNBS method (2,...

Gradient polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate: a practical approach to muscle contractile and regulatory proteins.

Two gradient systems for polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS) are described, with emphasis on improvements accumulated over two decades of studies on contractile proteins and regulatory enzymes from smooth muscle. The first "big slab" system utilizes 18 x 20 x 0.1 cm3 gels and a 10-18% acrylamide gradient, optimized for a high resolution of 10 to 500 kDa polypeptides. Eight (or more) gels are cast simultaneously with a gradient formation from "bottom to top" and 20% glycerol is added to the 18% acrylamide solution. The second "minislab" system represents an improved version of the system of Matsudaira and Burgess (Anal. Biochem. 1978, 87, 386-396), with 8 x 10 x 0.05 cm3 gels and 5-15% or 9-18% acrylamide gradient ranges. They are cast from "top to bottom" in 28-piece batches also with the addition of glycerol for improved gradient formation. Both types of gels can also be cast individually using a specially designed pestle-type gradient maker. For gel destaining, a convenient continuous hydrodynamic destainer is also described.

Isolation and characterization of human brain aminopeptidase which hydrolysis enkephalin-containing peptides

A human brain aminopeptidase, which hydrolyses low molecular weight enkephalin-containing peptides (ECPs), was purified to apparent homogeneity from the homogenate of human brain. The enzyme purification involved DEAE-cellulose chromatography and preparative polyacrylamide gel electrophoresis. The purified aminopeptidase hydrolyses only the Tyr 1Gly 2 bond of enkephalines and of ECPs. The rate of hydrolysis of Met-enkephalin and Leu-enkephalin was higher than the rate of hydrolysis of ECPs containing 7 to 13 aminoacid residues. Large ECPs such as peptide E and β-endorphin were not hydrolysed. The molecular weight of this enzyme is about 100,000 daltons, as determined by gel filtration on Sephadex G-200 and by polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate. The enzyme has an isoelectric point of pH 4.9, is activated by dithiothreitol (DTT) and inhibited by puromycin, bacitracin, p-mercuryacetate, Zn ++, Cu ++ and Ni ++. The optimum pH for enzyme activity is 7.5.

Accumulation of large VLDL in cyclophosphamide treated rabbits. Relationship with lipoprotein lipase deficiency

Rabbit very low density lipoproteins (VLDL) have been fractionated by heparin sepharose chromatography into two subpopulations : an unretained fraction (UR) and a retained fraction (R). The separation profiles of VLDL from cyclophosphamide treated rabbits differed from those obtained in normal animals : UR fraction was far more important in treated rabbits than in control animals. Comparative studies of the two VLDL subfractions isolated from treated rabbits have been performed. Polyacrylamide gel electrophoresis in presence of urea showed a similar distribution in both fractions of apolipoproteins X, a group of low molecular weight apolipoproteins detected after antimitotic therapy. SDS - polyacrylamide electrophoresis revealed the presence of one form of apolipoprotein B : apo B100 in the VLDL from treated rabbits giving evidence of their hepatic origin. Relative to the R-fraction, the UR-fraction was characterized by an increased triacylglycerol content and a larger diameter as observed by electron microscopy. In vitro incubations with lipoprotein lipase and reisolation of postlipolysis particles suggest that both VLDL fractions can undergo metabolic conversion to LDL. A decrease of lipoprotein lipase activity after treatment, as previously observed, may thus explain the accumulation of the large VLDL.

Mannitol-1-phosphate dehydrogenase of Escherichia coli Chemical properties and binding of substrates

Mannitol-1-phosphate dehydrogenase was purified to homogeneity, and some chemical and physical properties were examined. The isoelectric point is 4.19. Amino acid analysis and polyacrylamide-gel electrophoresis in presence of SDS indicate a subunit Mr of about 22,000, whereas gel filtration and electrophoresis of the native enzyme indicate an Mr of 45,000. Thus the enzyme is a dimer. Amino acid analysis showed cysteine, tyrosine, histidine and tryptophan to be present in low quantities, one, three, four and four residues per subunit respectively. The zinc content is not significant to activity. The enzyme is inactivated (greater than 99%) by reaction of 5,5'-dithiobis-(2-nitrobenzoate) with the single thiol group; the inactivation rate depends hyperbolically on reagent concentration, indicating non-covalent binding of the reagent before covalent modification. The pH-dependence indicated a pKa greater than 10.5 for the thiol group. Coenzymes (NAD+ and NADH) at saturating concentrations protect completely against reaction with 5,5'-dithiobis-(2-nitrobenzoate), and substrates (mannitol 1-phosphate, fructose 6-phosphate) protect strongly but not completely. These results suggest that the thiol group is near the catalytic site, and indicate that substrates as well as coenzymes bind to free enzyme. Dissociation constants were determined from these protective effects: 0.6 +/- 0.1 microM for NADH, 0.2 +/- 0.03 mM for NAD+, 9 +/- 3 microM for mannitol 1-phosphate, 0.06 +/- 0.03 mM for fructose 6-phosphate. The binding order for reaction thus may be random for mannitol 1-phosphate oxidation, though ordered for fructose 6-phosphate reduction. Coenzyme and substrate binding in the E X NADH-mannitol 1-phosphate complex is weaker than in the binary complexes, though in the E X NADH+-fructose 6-phosphate complex binding is stronger.

A simple method to characterize γ-crystallin synthesized in vitro

Investigation of the migration of in vitro synthesized γ-crystallin which has not been heat denatured on polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate indicates that this class of proteins behaves in an anomalous manner. While other in vitro synthesized lens proteins under these conditions migrate as theoretically expected, γ-crystallin is retarded, having a mobility comparable to a 92 000 dalton component. Only upon heat denaturation does this protein fraction migrate as a 20 000 dalton species. Since other in vitro synthesized lens proteins have molecular weights of approximately 20 000 daltons, it is only with this methodology that γ-crystallin synthesis can be unequivocally followed in a simple, rapid, one-dimensional fractionation system.

Isolation and partial characterization of a α2- β1-glycoprotein from horse plasma

1. 1. A α 2- β 1-glycoprotein was isolated from horse plasma by classical methods. The final product appeared homogeneous by agarose gel and pore limit SDS polyacrylamide gel electrophoresis, immunoelectrophoresis and crossed immunoelectrophoresis. 2. 2. The protein moved in agarose gel electrophoresis just above the β 1 region and seemed composed of a single polypeptide chain. A highly heterogenic banding pattern, focused between pH 5.1 and 6.5 was revealed by isoelectric focusing. 3. 3. The molecular weights determined by gel filtration on Sephadex G100 and by a pore limit polyacrylamide gel electrophoresis in presence of SDS were 65,000 and 82,300 dalton, respectively. 4. 4. No serological relation was found between the horse α 2- β 1-glycoprotein and human and bovine plasma proteins.

75] Maltose-binding protein from Escherichia coli

Publisher Summary This chapter describes the assay method, the purification procedure, and the properties of maltose-binding protein from Escherichia coli . The maltose-binding protein is a periplasmic protein of Escherichia coli that is involved in the transport of maltose and maltodextrins across the bacterial envelope and in the chemotaxis toward these sugars. Maltose binding is measured by equilibrium dialysis using [ 14 C]maltose. The chapter presents two purification procedures for maltose-binding protein—growth of cell and osmotic shock treatment, and QAE-Sephadex chromatography. The alternative purification procedure is affinity chromatography. Both methods yield a protein that is more than 90% pure as estimated by electrophoresis on polyacrylamide gels in different conditions. A molecular weight of 40,000 to 44,000 of the protein is determined by polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate. The maltose binding protein is coded by gene malE , one of the five cistrons of the malB region involved in the transport of maltose and maltodextrins across the envelope of E.coli.

Isolation and characterization of an acid-stable proteinase inhibitor from amniotic fluid

A trypsin/chymotrypsin inhibitor was isolated from human amniotic fluid in a homogenous form by chromatography on DEAE-cellulose, Sephadex G-100, and Con A—Sepharose. The molecular weight of the inhibitor was found to be 18,000 based on the data on polyacrylamide gel electrophoresis in presence of SDS and mercaptoethanol. The inhibitor was stable over a wide pH range (1–10.5) and was inactivated only under highly alkaline conditions. The inhibitor was found to be a glycoprotein. The mode of inhibition of trypsin as well as α-chymotrypsin was noncompetitive. The binding sites on the inhibitor for trypsin and α-chymotrypsin are different. Arginine residues formed part of the reactive center for trypsin binding.

Protein composition of Bdellovibrio bacteriovorus and Escherichia coli membranes during their interaction

A comparative study of membrane proteins of Bdellovibrio bacteriovorus and host-bacteria Escherichia coli was performed by polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate. Infection of E. coli cells by bdellovibrions resulted in the loss of some high-molecular proteins and appearance of new ones in the host-bacteria membranes. The possible role of parasite proteases in degradation of host-bacteria membrane proteins is discussed.

Comparison of Naja n. naja and Naja h. haje Cobra-Venom Factors: Correlation between Binding Affinity for the Fifth Component of Complement and Mediation of its Cleavage

Two cobra-venom factors, one from Naja n. naja (CVF n), the other from Naja h. haje venom (CVF h), have been purified and compared, functionally and structurally. Both factors interacted with human factors B and xxxD to form a potent C3 convertase, CVFBb. However, while the convertase formed with CVF n did also efficiently cleave C5, CVF hBb had very little C5-cleaving potency only, in particular when human C5 was used as substrate. Studies with agarose-linked CVF preparations indicated that CVF h has only low binding affinity for C5 gp and C5 hu whereas CVF n binds to both C5 species with much higher affinity. Since C5-binding (to CVF or to C3b) is a prerequisite for its cleavage by C3/C5 convertases, the difference in binding potency explains the different C5-cleaving activity of the two CVF preparations. When a ligand for C5, surface-fixed C3b, is present, CVF hBb is also capable of cleaving C5. The difference in activities of CVF n and CVF h is reflected in their different potency to interfere with immune haemolysis and in causing indirect lysis by their complexes with activated factor B. By gel chromatography of the CVF preparations in C5-containing medium, a stoichiometric complex CVF n-C5 (1 + 1) could be demonstrated. An analogous complex of C5 was neither found with CVF h, nor with C3 hu or soluble C3b hu. Structural differences between CVF n and CVF h were revealed by immunodiffusion analysis and by polyacrylamide-gel electrophoresis in presence of SDS. The data available so far provide, however, no clear information about the structure of the C5 binding site.

Unimpaired histone synthesis in human fibroblasts infected by human cytomegalovirus.

Serum-starved human foreskin fibroblasts were infected by human cytomegalovirus (Towne strain) that is thought to induce DNA replication in host cells during lytic infection. At various times postinfection, the cultures were pulse labeled with either 3H-thymidine or 14C-thymidine and 3H-lysine to examine DNA synthesis and histone synthesis, respectively. Isopycnic centrifugation of labeled DNA in CsCl revealed that precursor incorporation into host-cell DNA was enhanced over the control around 24 h postinfection and decreased after onset of viral DNA synthesis which reached a peak around 72 h postinfection. For analysis of histones 3H-lysine-labeled proteins of lysates of unfractionated cells and of chromatin preparations were subjected to polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate and subsequent fluorography. Comparison of the fluorograms from the various pulses postinfection suggested that 3H-lysine incorporation into histones exhibited no major variations concurrent with the changes of host-cell DNA synthesis. In contrast, herpes simplex virus type 1 was found progressively to extinguish histone synthesis in the course of the cellular infection. Furthermore, histone synthesis in phosphonoacetic acid-treated cytomegalovirus-infected cultures was not enhanced over that in mock-infected controls. These observations do not support the view that human cytomegalovirus induces host-cell DNA replication under the conditions used.

Purification of human α-fetoprotein

By employing complex and highly specialized immunochemical methods, several investigators have achieved purification of human α-fetoprotein (AFP) found in fetal serum and/or sera of patients with hepatoma. The present report describes a simpler method which results in the isolation of homogeneous preparation of AFP from human cord serum. AFP was purified by sequential use of Affi-Gel Blue affinity, DE-52 diethylaminoethyl cellulose ion-exchange, immunoadsorption with anti-albumin covalently coupled to Sepharose 4B, and Sephacryl S-200 molecular sieve chromatographic techniques. The homogeneity of the purified AFP was established by subjecting it to polyacrylamide gel electrophoresis, analytical isoelectric focusing, molecular sieve chromatography and immunological techniques. The purified AFP has a molecular weight of approximately 68,000 as determined by polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate and molecular sieve chromatography, and upon isoelectric focusing yielded a single band p I = 4.8. In addition, the purified AFP gives a single precipitin line when tested against rabbit antiserum to whole human hepatoma serum proteins, and no line(s) of precipitin when tested against rabbit antiserum to normal serum proteins.

In vitro cleavage of avian retrovirus gag proteins by viral protease p15

Avian myeloblastosis virus contains a proteolytic activity that can cleave in vitro the viral precursor polypeptide Pr76 gag. This substrate was prepared by radioactive labeling in vivo followed by immune precipitation, polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate, and elution from the gel. The major products of this reaction include the mature virion proteins p27 and p15, as well as an unstable fragment containing both of these proteins. Several other fragments are also formed, but mature p12 and the major p19 species are not. The cleavage of undenatured Pr76 bound to antibodies and formallin-fixed Staphylococcus yields similar fragments. The viral proteolytic enzyme is indistinguishable from the structural protein p15. Cleavage of Pr76 by p15 is optimal in the pH range 4–7 and is stimulated by salt. The activity of the enzyme is not inhibited by reagents specific for proteases with serine at their active sites, but is partially inhibited by reagents specific for thiols. Proteolysis is highly specific. Under the conditions used for Pr76 cleavage, p15 does not introduce breaks into mixtures of cellular proteins eluted in parallel to Pr76 from SDS-containing gels. However, it does fragment proteins that contain all or parts of the amino acid sequence of Pr76. These proteins include the precursor polypeptide for viral reverse transcriptase (Pr180 gag-pol), a virus-related protein found in uninfected gs + chick cells (P120), viral proteins from cells infected with avian erythroblastosis virus (P75) or with avian myelocytomatosis virus MC29 (P110).