Biochemical analysis of the p30''s of N-, B-, and B leads to NB-tropic murine leukemia viruses of BALB/c origin.

Previous analysis of the virion proteins of an N- and a B-tropic type C virus of BALB/c mice, of 16 N-tropic recombinants (XLPN viruses) between these viruses, and of eight NB-tropic viruses derived from the B-tropic virus suggested that among these closely related viruses N-, B-, or NB-tropism was associated with the electrophoretic mobility of p30 on sodium dodecyl sulfate-polyacrylamide gels, and thus that p30 might determine this phenotype. To obtain further evidence for the association of structural markers of p30 with N-, B-, or NB-tropism, we have analyzed the p30's of these same viruses by using two-dimensional tryptic peptide mapping and slab gel isoelectric focusing. The results of these analyses suggest that (i) a single peptide unique to the N-tropic virus p30- is present in the p30 of all N-tropic recombinants; (ii) a single peptide unique to the B virus p30 is not present in p30's of the N-tropic recombinants, and this peptide is also absent in p30's of NB-tropic viruses derived from the B-tropic virus; and (iii) p30's of NB-tropic viruses possess a new tryptic peptide not found in the p30 of their B-tropic virus progenitors, and this new peptide is not found in the p30 of the N-tropic virus of BALB/c or the XLPN viruses. These results are consistent with the possibility that p30 may determine the N-, B-, or NB-tropism of murine leukemia viruses. In addition, these studies indicate that some of the N-tropic recombinants have experienced recombination within the p30 gene.     

Analysis of secondary modifications of mouse mammary tumor virus proteins by two-dimensional gel electrophoresis.

The structural proteins of mouse mammary tumor virus (MMTV) were analyzed by two-dimensional electrophoresis on isoelectric focusing and sodium dodecyl sulfate gels. Many of the viral proteins displayed heterogeneity in charge due to variable contents of carbohydrates (in particular, sialic acid) and phosphate residues. Neuraminidase treatment of the virions influenced the isoelectric pattern of the envelope glycoproteins. The glycoproteins of an MMTV variant which was attenuated by replication in feline kidney cells had different isoelectric points. This suggested that the acquisition of an altered carbohydrate configuration had changed the host range of the virus. The major MMTV structural core protein, p27, consisted of two species, which had identical iodinated tryptic peptide compositions but differed in phosphate contents. Another MMTV phosphoprotein, p21, was separated into four different phosphorylated species. Phosphorylation of p21 could be performed in vitro by the MMTV virion-associated protein kinase. This enzyme also has a high affinity for MMTV p30 as a substrate. Possible functions of this enzyme are discussed.     

α1-Acute-phase globulins of rats. Microheterogeneity after isoelectric focusing

1. Improved resolution of mixtures of alpha(1)-globulins was obtained by the use of isoelectric focusing. 2. Because material recovered after isoelectric focusing in polyacrylamide gels behaved in a manner which suggested interaction with components derived from the gel, isoelectric focusing when used for preparative purposes was done in a matrix of Sephadex G-75. 3. By this means material from the individual bands formed by isoelectric focusing in 6m-urea could be isolated. The stability of these substances was examined by further isoelectric focusing. 4. Analysis of material that had been shown to be homogenous by isoelectric focusing in the absence of urea and of that from several individual bands derived from the same sample by isoelectric focusing in 6m-urea showed different proportions of sialic acid but no change in amino acid composition. 5. In the presence of 6m-urea the isoelectric points found were increased by 0.14-0.25 pH unit. After removal of most of the sialic acid with neuraminidase the increase was 0.36-0.72 pH unit. After treatment with 0.025m-H(2)SO(4) at 80 degrees C for 1h, which removed all the sialic acid, the increase was 0.40-0.87 pH unit. 6. Because removal of all the sialic acid did not decrease the number of bands formed by isoelectric focusing the observed heterogeneity could not be caused entirely by the presence of various proportions of sialic acid.     

Chromatographic Separation and Antigenic Analysis of Proteins of the Oncornaviruses IV. Biochemical Typing of Murine Viral Proteins

Tryptic peptide maps were prepared for four purified structural proteins derived from several murine leukemia viruses (MuLV's). Analyses of these peptide maps reveal that the p30 proteins of Rauscher, Moloney, and Gross MuLV's are very similar to each other, as are the p10's obtained from these three viruses. In contrast, the peptide maps of the individual p15's and p12's from the same viruses establish that each of these polypeptides is highly strain specific. For all four polypeptides studied, unique peptides appear in the Rauscher MuLV and Moloney MuLV tryptic profiles that are not present in the corresponding Gross MuLV profile. By this method of analysis it was possible to distinguish the p30's of N-tropic and B-tropic MuLV's derived from the same BALB/c mouse.     

Characterization of bovine placental lactogen as a glycoprotein with N-linked and O-linked carbohydrate side chains

In a previous report we showed that purified bovine placental lactogen (bPL) exists in two isoforms in the 31,000-33,000 Mr range, each with at least five isoelectric variants differing in approximately 2 orders of magnitude in isoelectric points (pI) 4-6. The multiple isoelectric variants are unique to the bovine hormone. In an effort to determine the nature of these variants endo- and exoglycohydrolase digestions were conducted to determine if this hormone was glycosylated. Analysis of peptide/N-glycosidase F and endoglycosidase F digests of radioiodinated bPL on one-dimensional gel electrophoresis showed a Mr decrease from 31,000 to 24,000 and 33,000 to 26,000 for the two isoforms. Digestion with a mixture of neuraminidase plus mixed exoglycosidases resulted in a Mr decrease of 4,000. Digestion with neuraminidase resulted in a Mr decrease of 2,000. Further analysis of peptide/N-glycosidase F- and neuraminidase-treated bPL by two-dimensional gel electrophoresis showed the isoelectric variants shifted from pI 4.4-6.3 to 4.9-8.0. The sialic acid residues on the N-linkage are responsible for the pronounced acidic character of bPL, but do not account for the residual charge heterogeneity as the different isoelectric variants persist after sialic acid removal. The apparent Mr of the protein after removal of N-linked carbohydrate residues is similar to that of PRL and GH. These enzymatic digestion results demonstrate the presence of N-linked complex oligosaccharide residues attached to the beta-amide group of an asparagine residue. Analyses of the sugar content of the molecule were consistent with the presence of one biantennary N-linked and two O-linked carbohydrate chains.     

Identification of different charged species of a human monocyte derived neutrophil activating peptide (MONAP)

Lipopolysaccharide-stimulated human monocytes secrete a 10 kD peptide (MONAP) of high neutrophil, not however monocyte or eosinophil stimulating activity. By reversed phase HPLC MONAP could be distinguished from Interleukin 1. Analytic isoelecto-focusing of pure MONAP (single line upon sodiumdodecylsulfate polyacrylamide gel electrophoresis, single peak after RP-18-HPLC), obtained by size exclusion HPLC followed by two different reversed phase HPLC steps revealed charge heterogeneity giving major components with isoelectric points at 4.7, 4.9, 6.4 and 6.9, all of which exhibited chemotactic activity.     

Multiple forms of an extracellular cyclic-AMP phosphodiesterase from Dictyostelium discoideum.

The extracellular cyclic-AMP phosphodiesterase of a mutant of Dictyostelium discoideum which accumulates this enzyme was found to exist in multiple forms. Using the isoelectric focusing technique the phosphodiesterase activity was distributed into three peaks with isoelectric points of 4.6, 6.5 and 8.3, designated as p4, p6 and p8. Gel filtration and sucrose gradient analysis showed that the p4 activity consisted of two forms of different sedimentation coefficients. At high enzyme concentrations, the heavy form was favored. Dilution of enzyme activity shifted the equilibrium toward the light form. Direct analysis by sucrose gradient sedimentation of all isoelectric forms demonstrated that besides p4, p6 activity also existed as a mixture of the heavy (9.7 S) and the light (5.4 S) components. In contrast, the p8 activity displayed only the light form. The heterogeneity of the p4 and p6 isoelectric forms was also observed by polyacrylamide gel electrophoresis. A procedure for a partial purification of the extracellular enzyme to about 70-fold is presented.     

Genetic variability in charge microheterogeneity and polypeptide composition of phaseolin, the major storage protein of Phaseolus vulgaris; and peptide maps of its three major subunits

Molecular polymorphism of phaseolin, the major storage protein of Phaseolus vulgaris L., and structural homology of its subunits were investigated. A high degree of charge heterogeneity was evidenced by isoelectric focusing. Comparison among phaseolins isolated from seven different cultivars showed genetic variability both in isoelectric focusing pattern and subunit composition. However tryptic peptide maps of these phaseolins were very similar. The three phaseolin subunits isolated from cultivar Greensleeves were compared by peptide mapping after limited enzymatic digestion and had very similar primary structures.     

Carbohydrate composition of rat hepatocyte nuclear membrane as compared to normal, Morris hepatoma 7800, and phenobarbital-induced microsomal membranes

The composition of the nonhistone nuclear proteins of rat liver, brain, thymus, and kidney has been analyzed by isoelectric focusing in polyacrylamide gel. Approximately 20–30 components were separated with a wide range of isoelectric points (pl) in the 3- to 10-pH region.Different extraction procedures applied to liver nuclei removed protein mixtures with similar components present in varying amounts. 8 m Urea 50 mm phosphate, pH 7.6, was the most successful and removed most of the nonhistone protein.The thiol groups of proteins extracted from the nuclei of liver, brain, thymus, and kidney with 8 M urea, 50 mM phosphate were labeled with [¹⁴C]N-ethylmaleimide. Although there was a slight variation in the overall thiol content of these tissue proteins, separation of the mixture by isoelectric focusing and SDS polyacrylamide gel electrophoresis showed complex patterns indicating greater heterogeneity than was apparent from the Coomassie blue dye binding.     

Detection of phosphorylated forms of Moloney murine leukemia virus major capsid protein p30 by immunoprecipitation and two-dimensional gel electrophoresis.

We detected phosphorylation of the major Moloney murine leukemia virus (M-MuLV) capsid polypeptide, p30, by using 32Pi-labeled virions. This was observed both on two-dimensional polyacrylamide gels directly or on one-dimensional gels of viral lysates that had been immunoprecipitated with monospecific goat anti-p30 serum. The phosphorylation event had been difficult to detect because pp12 the major virion phosphoprotein incorporates almost all of the 32P label added to infected cells (Y. Yoshinaka and R. B. Luftig, Virology 116:181-195, 1982). When immunoprecipitates from M-MuLV lysates labeled with 32Pi were compared with those labeled with [35S]methionine, it was calculated that the degree of phosphorylation at the p30 domain of Pr65gag was only 0.22 to 0.54% relative to phosphorylation at the p12 domain. Two-dimensional gel electrophoresis of the 32P-labeled p30 immunoprecipitates showed that there were three phosphorylated p30 forms with isoelectric points (pIs) of 5.7, 5.8, and 6.0. These forms were generally more acidic than the [35S]methionine-labeled p30 forms, which had pIs of 6.0, 6.1, 6.3 (the major constituent with greater than 80% of the label), and 6.6. The predominant phosphoamino acid of the major phosphorylated p30 form (pI 5.8) was phosphoserine. Further, tryptic peptide analysis of this p30 form showed that only one peptide was predominantly phosphorylated. Based on a comparison of specific labeling of p30 tryptic peptides with [14C]serine, [35S]methionine, and 32Pi, we tentatively assigned the phosphorylation site to a 2.4-kilodalton NH2-terminal peptide containing triple tandem serines spanning the region from amino acids 4 to 24.     

Predominant identification of RNA-binding proteins in Fas-induced apoptosis by proteome analysis

Proteome analysis of Jurkat T cells was performed in order to identify proteins that are modified during apoptosis. Subtractive analysis of two-dimensional gel patterns of apoptotic and nonapoptotic cells revealed differences in 45 protein spots. 37 protein spots of 21 different proteins were identified by peptide mass fingerprinting using matrix-assisted laser desorption/ionization mass spectrometry. The hnRNPs A0, A2/B1, A3, K, and R; the splicing factors p54(nrb), SRp30c, ASF-2, and KH-type splicing regulatory protein (FUSE-binding protein 2); and alpha NAC, NS1-associated protein 1, and poly(A)-binding protein 4 were hitherto unknown to be involved in apoptosis. The putative cleavage sites of the majority of the proteins could be calculated by the molecular masses and isoelectric points in the two-dimensional electrophoresis gel, the peptide mass fingerprints, and after translation by treatment with recombinant caspase-3. Remarkably, 15 of the 21 identified proteins contained the RNP or KH motif, the best characterized RNA-binding motifs.     

Interstrain Variation of the Major Internal Structural Component (p30gag) of Two Murine Oncornaviruses: Comparative Immunochemical, Biochemical, and Biophysical Analysis

The major internal structural protein (p30(gag)) of the Moloney leukemia virus and the endogenous Y-1 murine oncornavirus was examined for biochemical and biophysical manifestations of interstrain antigenic variation. Although the two viral proteins share murine group-specific antigenic determinants, the Y-1 virus p30 appeared to have both a lower relative number of such determinants and a decreased affinity at the cross-reactive sites for Moloney virus p30 monospecific antibodies. Further, immunological analysis indicated the presence of unique antigenic sites on the Moloney virus p30 not shared by the analogous Y-1 virus molecule. The two polypeptides copurified and had similar isoelectric points (pH 6.2 to 6.3) and sedimentation coefficients (2.47S). However, equilibrium sedimentation yielded a significant mass difference between the two proteins, 28,300 +/- 600 and 31,000 +/- 300 daltons for the Moloney and Y-1 virus molecules, respectively. Amino acid analysis indicated a concomitant increase in total residues for the Y-1 virus p30, although a number of residues appeared to have been conserved between the two viral proteins. Conformational studies and hydrodynamic calculations demonstrated marked secondary and tertiary structural differences; with the Y-1 virus p30 being an asymmetric prolate ellipsoid containing 27 to 28% alpha-helix and Moloney virus p30 being somewhat more spherical and possessing an alpha-helical content of 50 to 55%. Two-dimensional mapping of (125)I-labeled tryptic peptides of each p30 suggested that considerable sequence heterogeneity is responsible for many of the biophysical, biochemical, and immunochemical differences in these two analogous structural proteins.     

Purification and characterization of an immunomodulatory Peptide from bovine placenta water-soluble extract

An immunomodulatory peptide was isolated from bovine placenta water-soluble extract and purified by consecutive chromatography on DEAE Sepharose CL-6B, Sephadex G-25, and Sephasil C18 column using lymphocyte proliferation assay to identify the active fractions. The immunomodulatory peptide showed a dose-dependent stimulating effect on lymphocyte proliferation. The isoelectric point of the immunodulatory peptide was determined to be 3.82 by capillary isoelectric focusing electrophoresis. The molecular mass of the immunomodulatory peptide was determined to be 2133.52 Da by mass spectrometry. The first 10 amino acid sequence of the immunomodulatory peptide was Tyr-X-Phe-Leu-Gly-Leu-Pro-Gly-X-Thr. This immunomodulatory peptide showed no significant homology with other immunomodulatory peptides. Additionally, it was thermostable, retaining about 60% of immune activity after incubating at 80 degrees C for 30 min.     

Scrapie PrP 27-30 is a sialoglycoprotein.

The major scrapie prion protein, designated PrP 27-30, exhibited both charge and size heterogeneity after purification from infected hamster brains. Eight or more discrete charge isomers of PrP 27-30 with isoelectric points ranging from approximately pH 4.6 to 7.9 were found by using non-equilibrium pH gradient electrophoresis in the first dimension followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. The charge isomers were detected by silver staining as well as by radioiodination. The procedures used to disaggregate PrP 27-30 before electrophoresis in the first dimension do not appear to be responsible for the charge heterogeneity. However, heating PrP 27-30 to 100 degrees C for 15 min in 0.1 N NaOH or 0.1 N HCl resulted in modification of the protein and alteration of its electrophoretic pattern. A PrP 27-30 fragment (molecular weight, 17,100 to 21,900) obtained by cyanogen bromide cleavage also exhibited charge and size heterogeneity. Periodic acid-Schiff staining of PrP 27-30 electrophoresed into sodium dodecyl sulfate-polyacrylamide gels demonstrated that carbohydrate residues are attached to the protein. Digestion of PrP 27-30 with neuraminidase and endo-beta-N-acetylglucosaminidase H resulted in significant changes in the isoelectric pH of PrP 27-30 isomers, whereas digestion with alkaline phosphatase had no effect. Our results demonstrate that PrP 27-30 is a sialoglycoprotein; this is consistent with several properties of this protein and of the scrapie prion.     

The charge heterogeneity of soluble human galactosyltransferases isolated from milk, amniotic fluid and malignant ascites.

UDP-galactose: N-acetylglucosamine galactosyltransferase was isolated from pooled human milk, pooled amniotic fluid and from two different individual samples of malignant ascites. The purification procedure involving two successive affinity chromatography steps on N-acetylglucosamine--agarose and alpha-lactalbumin--agarose yielded an enzyme preparation homogeneous by size. Under non-denaturing conditions the ascites and amniotic fluid enzymes had identical electrophoretic mobility, but they moved faster than the milk enzyme. Isoelectric analysis in the presence and absence of urea resolved the milk enzyme into at least 13 different forms, nine of which had the same isoelectric points after refocusing. All enzyme forms showed similar activity when free N-acetylglucosamine, ovalbumin, sialic-acid-free ovine submaxillary mucin and glucose, in the presence of alpha-lactalbumin, were used as acceptor substrates. Comparative isoelectric focusing of the three galactosyltransferases revealed identical patterns of the amniotic and ascites enzymes, but only partial overlap with the milk enzyme, which was less negatively charged. Neuraminidase treatment of ascites and milk galactosyltransferases produced very similar focusing patterns. The possible structural basis for this charge heterogeneity is briefly discussed.     

Genetic variants of serum alpha-1-antitrypsin (Pi types) in a Breton population, the Bigoudens

Pi typing was carried out by high resolution isoelectric focusing in 397 Bigoudens and in 100 non-Bigouden Bretons. Gene frequencies were computed by the gene counting method. No difference between the two groups could be demonstrated, neither was there a deviation from the expected Hardy-Weinberg distribution nor a heterogeneity between the Bigouden villages. The results were significantly different (p less than 0.02) from those reported in Normans.     

Production of chitinases by Aphanocladium album grown on crystalline and colloidal chitin

Abstract The deuteromycete Aphanocladium album produced two endochitinases (EC 3.2.1.14) with apparent isoelectric points of 7.1 and 7.6 and seven exochitinases (EC 3.2.1.30) with apparent isoelectric points ranging from 3.8 to 6.4 when grown on a colloidal chitin preparation. With crystalline chitin as carbon source, two endochitinases (p I 7.1 and 7.6) and only one exochitanase (p I 4.9) were detected. The exochitinase p I 4.9, which was produced with both substrates, has a relative molecular mass of 44 000. The different chitinases could be separated by chromatofocusing and their specific activities were determined.     

Efficient fractionation and improved protein identification by peptide OFFGEL electrophoresis

The sample fractionation steps conducted prior to mass detection are critically important for the comprehensive analysis of complex protein mixtures. This paper illustrates the effectiveness of OFFGEL electrophoresis with the Agilent 3100 OFFGEL Fractionator for the fractionation of peptides. An Escherichia coli tryptic digest was separated in 24 fractions, and peptides were identified by reversed-phase liquid chromatography on a microfluidic device with mass spectrometric detection. About 90% of the identified individual peptides were found in only one or two fractions. The distribution of the calculated isoelectric points for the peptides identified in each fraction was especially narrow in the acidic pH range. Standard deviations approached the size of the pH segment covered by the respective fraction. The experimental peptide isoelectric point measured by OFFGEL electrophoresis was used as an additional filter for validation of peptide identifications.     

Isolation and comparative biochemical properties of the major internal polypeptides of equine infectious anemia virus.

We describe procedures for the large-scale production of equine infectious anemia virus (EIAV) and for the isolation of the four major non-glycosylated virion proteins, designated p26, p15, p11, and p9. Comparisons of the purified proteins by peptide mapping procedures and by enzyme-linked immunosorbent assays demonstrated the unrelatedness of the four proteins. The characteristic properties of each purified protein were examined by determining isoelectric points and amino acid compositions. We found that EIAV p26 and p9 focus at pI values of 6.2 and 5.0, respectively, and that these proteins contain no unusual amino acids. In contrast, EIAV p15 reproducibly displayed a heterogeneous isoelectric focusing pattern, with major pI values ranging from 5.7 to 8.3. This charge variation evidently correlated with different levels of phosphorylated serine or threonine or both, which could be detected by an amino acid analysis of purified p15. EIAV p11 apparently focused at a pI of greater than 10, reflecting its high content of basic amino acids. Moreover, localization experiments indicated that all four nonglycosylated proteins constitute the internal components of the virus, with all of the virion p11 closely associated with the viral RNA genome. Thus, our results demonstrated that EIAV, a lentivirus, contains structural polypeptides which are analogous to the structural polypeptides described previously in prototype C oncoviruses.     

Purification and Characterization of an Immunomodulatory Peptide from Bovine Placenta Water‐Soluble Extract

Abstract

An immunomodulatory peptide was isolated from bovine placenta water‐soluble extract and purified by consecutive chromatography on DEAE Sepharose CL‐6B, Sephadex G‐25, and Sephasil C18 column using lymphocyte proliferation assay to identify the active fractions. The immunomodulatory peptide showed a dose‐dependent stimulating effect on lymphocyte proliferation. The isoelectric point of the immunodulatory peptide was determined to be 3.82 by capillary isoelectric focusing electrophoresis. The molecular mass of the immunomodulatory peptide was determined to be 2133.52 Da by mass spectrometry. The first 10 amino acid sequence of the immunomodulatory peptide was Tyr‐X‐Phe‐Leu‐Gly‐Leu‐Pro‐Gly‐X‐Thr. This immunomodulatory peptide showed no significant homology with other immunomodulatory peptides. Additionally, it was thermostable, retaining about 60% of immune activity after incubating at 80°C for 30 min.